Science.gov

Sample records for hamster ovary cells

  1. Internalization of ricin in Chinese hamster ovary cells.

    PubMed Central

    Ray, B; Wu, H C

    1981-01-01

    Internalization of ricin into Chinese hamster ovary cells has been investigated. Combined treatment with galactose and pronase at 0 degrees C resulted in a complete release of surface-bound [125I]ricin into the media. Galactose-pronase-resistant cell-bound [125I]ricin represents internalized ricin molecules inside the cells. The internalization process is time, temperature, and concentration dependent. The pH optimum of internalization of ricin is about pH 7. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis has revealed that intact ricin molecules are internalized. Neither reduction nor proteolytic processing of ricin is required for the entry of ricin into Chinese hamster ovary cells. PMID:6965107

  2. Quantitative mutagenesis and mutagen screening with Chinese hamster ovary cells

    SciTech Connect

    Hsie, A.W.; San Sebastian, J.R.; Tan, E.L.

    1980-01-01

    A summary is presented on the development of a specific gene mutation assay, the Chinese hamster ovary cells/hypoxanthine-guanine phosphoribosyl transferase (CHO/HGPRT) system, and the utilization of this system to study structure-activity relationship affecting cytotoxicity and gene mutation by various carcinogens. Then, preliminary development and validation of a Multiplex CHO System for the simultaneous determination of chromosome aberration, sister chromatid exchange in addition to cytotoxicity and gene mutation is presented. The potential use of a CHO/human cell hybrid system for measuring chromosomal deletion and loss is discussed.

  3. Cell engineering and cultivation of chinese hamster ovary (CHO) cells.

    PubMed

    Omasa, Takeshi; Onitsuka, Masayoshi; Kim, Wook-Dong

    2010-04-01

    Mammalian cell lines are important host cells for the industrial production of pharmaceutical proteins owing to their capacity for correct folding, assembly and post-translational modification. In particular, Chinese hamster ovary (CHO) cells are the most dependable host cells for the industrial production of therapeutic proteins. Growing demand for therapeutic proteins promotes the development of technologies for high quality and productivity in CHO expression systems. The following are fundamentally important for effective production. 1) Construction of cultivation process. The CHO-based cultivation process is well established and is a general platform of therapeutic antibody production. The cost of therapeutic protein production using CHO cells is equivalent to that using microbial culture. 2) Cell line development. Recent developments in omics technologies have been essential for the development of rational methods of constructing a cell line. 3) Cell engineering for post-translational steps. Improvement of secretion, folding and glycosylaiton is an important key issue for mammalian cell production systems. This review provides an overview of the industrial production of therapeutic proteins using a CHO cell expression system.

  4. Glutamine protects Chinese Hamster Ovary cells from radiation killing

    SciTech Connect

    Winters, R.; Matthews, R.; Ercal, N.; Krishnan, K. )

    1994-01-01

    Chinese Hamster Ovary (CHO) cells were propagated in vitro and exposed to varying doses of ionizing radiation. The surviving fraction of cells was determined, being found to be a function of the radiation dose. The cell survival curves obtained as a function of radiation dose were modified by the inclusion of varying doses of glutamine in the medium, with glutamine demonstrating a radioprotective effect. The radioprotectant effect of glutamine for CHO cells was more pronounced at higher radiation doses. These results support the idea that glutamine protects body systems such as the gut more directly as a radioprotector as opposed to a more indirect route, such as preventing bacterial translocation from the gut. 16 refs.

  5. Superoxide Mediates the Toxicity of Paraquat for Chinese Hamster Ovary Cells

    NASA Astrophysics Data System (ADS)

    Bagley, Ann C.; Krall, Judith; Lynch, Robert E.

    1986-05-01

    The roles of superoxide and H2O2 in the cytotoxicity of paraquat were assessed in Chinese hamster ovary cells. Neither catalase nor superoxide dismutase inhibited the loss of ability to form colonies when added to the medium. When introduced into the cells, superoxide dismutase but not catalase inhibited the toxicity of paraquat. That superoxide dismutase acted by its known catalytic action is shown by the loss of inhibition when the enzyme was inactivated by H2O2 before being introduced into the cells. The lack of inhibition by catalase, by dimethyl sulfoxide, and by desferoxamine suggests that the toxicity is not mediated by a reaction between H2O2 and superoxide to engender the hydroxyl radical. Exposure of Chinese hamster ovary cells to paraquat may be a suitable means to determine the effects of superoxide anion in cultured cells and the ways in which cells can resist this toxic action.

  6. Chinese hamster ovary cell lysosomes retain pinocytized horseradish peroxidase and in situ-radioiodinated proteins

    SciTech Connect

    Storrie, B.; Sachdeva, M.; Viers, V.S.

    1984-02-01

    We used Chinese hamster ovary cells, a cell line of fibroblastic origin, to investigate whether lysosomes are an exocytic compartment. To label lysosomal contents, Chinese hamster ovary cells were incubated with the solute marker horseradish peroxidase. After an 18-h uptake period, horseradish peroxidase was found in lysosomes by cell fractionation in Percoll gradients and by electron microscope cytochemistry. Over a 24-h period, lysosomal horseradish peroxidase was quantitatively retained by Chinese hamster ovary cells and inactivated with a t 1/2 of 6 to 8 h. Lysosomes were radioiodinated in situ by soluble lactoperoxidase internalized over an 18-h uptake period. About 70% of the radioiodine incorporation was pelleted at 100,000 X g under conditions in which greater than 80% of the lysosomal marker enzyme beta-hexosaminidase was released into the supernatant. By one-dimensional electrophoresis, about 18 protein species were present in the lysosomal membrane fraction, with radioiodine incorporation being most pronounced into species of 70,000 to 75,000 daltons. After a 30-min or 2-h chase at 37 degrees C, radioiodine that was incorporated into lysosomal membranes and contents was retained in lysosomes. These observations indicate that lysosomes labeled by fluid-phase pinocytosis are a terminal component of endocytic pathways in fibroblasts.

  7. A cowpox virus gene required for multiplication in Chinese hamster ovary cells.

    PubMed Central

    Spehner, D; Gillard, S; Drillien, R; Kirn, A

    1988-01-01

    Cowpox virus, in contrast to vaccinia virus, can multiply in Chinese hamster ovary cells. To study the genetic basis for this difference in host range, recombinants between vaccinia and cowpox viruses were isolated and their DNA restriction patterns were examined. The ability to multiply in Chinese hamster ovary cells could be correlated with the conservation of cowpox virus sequences mapping at the left end of the genome. This was further demonstrated by marker rescue of the host range phenotype with restricted cowpox virus DNA. Marker rescue with cloned restriction fragments of decreasing size enabled the fine localization of the host range function to a 2.3-kilobase-pair fragment. Nucleotide sequencing revealed that the fragment encoded a single major polypeptide of approximately 77,000 daltons. It is suggested that the role of the host range gene from cowpox virus is to prevent the early and extensive shutoff of protein synthesis that normally occurs in Chinese hamster ovary cells infected by vaccinia virus. Images PMID:2831390

  8. Chinese hamster ovary cells contain transcriptionally active full-length type C proviruses.

    PubMed

    Lie, Y S; Penuel, E M; Low, M A; Nguyen, T P; Mangahas, J O; Anderson, K P; Petropoulos, C J

    1994-12-01

    We have isolated a genomic locus from Chinese hamster ovary (CHO) cells that contains a full-length provirus. Nucleotide sequence analysis indicates that it is a defective member of the rodent type C retrovirus family with an env region that is similar to those of mouse amphotropic retrovirus and subgroup B feline leukemia virus. We were able to demonstrate that this provirus is a member of a closely related family of full-length proviruses in CHO cells and Chinese hamster liver. Hybridization probes generated from this genomic clone were used to characterize type C retrovirus RNA expression in CHO cells. Full-length genomic RNA and subgenomic envelope mRNA were detected in CHO cell lines but not in the human-derived 293 cell line. Interestingly, we discovered that the site of retrovirus integration lies within a G repeat sequence belonging to the short interspersed element family of retroposons.

  9. Mutation Detection in an Antibody-Producing Chinese Hamster Ovary Cell Line by Targeted RNA Sequencing

    PubMed Central

    Zhang, Siyan; Hughes, Jason D.; Murgolo, Nicholas; Levitan, Diane; Chen, Janice; Liu, Zhong

    2016-01-01

    Chinese hamster ovary (CHO) cells have been used widely in the pharmaceutical industry for production of biological therapeutics including monoclonal antibodies (mAb). The integrity of the gene of interest and the accuracy of the relay of genetic information impact product quality and patient safety. Here we employed next-generation sequencing, particularly RNA-seq, and developed a method to systematically analyze the mutation rate of the mRNA of CHO cell lines producing a mAb. The effect of an extended culturing period to mimic the scale of cell expansion in a manufacturing process and varying selection pressure in the cell culture were also closely examined. PMID:27088091

  10. Changes in protein phosphorylation during the cell cycle of Chinese hamster ovary cells

    SciTech Connect

    Westwood, J.T.; Church, R.B.; Wagenaar, E.B.

    1985-08-25

    The phosphorylation patterns of proteins were examined during the cell cycle of Chinese hamster ovary cells. This was accomplished by labeling synchronized cells at various times with (TSP)orthophosphate and separating the proteins by both isoelectric focusing and nonequilibrium pH gradient two-dimensional gel electrophoresis. The most dramatic changes occurred during late G2/M when approximately eight proteins (including vimentin, lamin B, and histones 1 and 3) showed increased phosphorylation. Ten other proteins appeared to be uniquely phosphorylated during late G2/M. Of these 10 proteins, seven were no longer phosphorylated shortly after mitosis. There is also at least one protein which showed a relative decrease in phosphorylation during late G2/M.

  11. Recent progress with the DNA repair mutants of Chinese hamster ovary cells

    SciTech Connect

    Thompson, L.H.; Salazar, E.P.; Brookman, K.W.; Collins, C.C.; Stewart, S.A.; Busch, D.B.; Weber, C.A.

    1986-04-02

    Repair deficient mutants of Chinese hamster ovary (CHO) cells are being used to identify human genes that correct the repair defects and to study mechanisms of DNA repair and mutagenesis. Five independent tertiary DNA transformants were obtained from the EM9 mutant. In these clones a human DNA sequence was identified that correlated with the resistance of the cells to CldUrd. After Eco RI digestion, Southern transfer, and hybridization of transformant DNAs with the BLUR-8 Alu family sequence, a common fragment of 25 to 30 kb was present. 37 refs., 4 figs., 3 tabs.

  12. Methylglyoxal-induced DNA-protein cross-links and cytotoxicity in Chinese hamster ovary cells.

    PubMed

    Brambilla, G; Sciabà, L; Faggin, P; Finollo, R; Bassi, A M; Ferro, M; Marinari, U M

    1985-05-01

    The technique of alkaline elution was applied to study the capacity of methylglyoxal to induce DNA damage and repair in Chinese hamster ovary cells. DNA cross-linking was observed after a 90-min exposure to a subtoxic dose (1.5 mM), and the cross-links were fully repaired by 24 h. The cross-linking appeared to be DNA-protein in nature, since proteinase treatment removed the effect. When the same cells were exposed to methylglyoxal in the presence of a rat liver metabolic system, both cytotoxicity and cross-linking frequency were significantly reduced.

  13. Genotoxicity studies of methyl isocyanate in Salmonella, Drosophila, and cultured Chinese hamster ovary cells

    SciTech Connect

    Mason, J.M.; Zeiger, E.; Haworth, S.; Ivett, J.; Valencia, R.

    1987-01-01

    The genotoxic effects of methyl isocyanate (MIC) were investigated using four short-term tests: the Salmonella reversion assay (Ames test), the Drosophila sex-linked recessive lethal assay, and the sister chromatic exchange (SCE) and chromosomal aberration assays in cultured Chinese hamster ovary (CHO) cells. No evidence was found for the induction of mutations in either Salmonella or Drosophila. MIC did, however, induce SCEs and chromosomal aberrations in CHO cells both in the presence and absence of Aroclor-induced rat liver S-9.

  14. Formation of N epsilon-(gamma-glutamyl)-lysine isodipeptide in Chinese-hamster ovary cells.

    PubMed Central

    Fesus, L; Tarcsa, E

    1989-01-01

    N epsilon-(gamma-Glutamyl)-lysine isodipeptide was detected in a protein-free fraction of Chinese-hamster ovary cells and their culture fluid by using radioactive lysine as a tracer. The identity of the isodipeptide was established by its separation on ion-exchange chromatography, analysis by h.p.l.c. after derivatization, recovery of lysine after acidic hydrolysis or after cleavage by a specific enzyme, namely gamma-glutamylamine cyclotransferase. The amount of isodipeptide was raised (460 pmol/10(7) cells and 61 pmol/ml of culture fluid were observed as highest values) as the cell density increased. Effects of inhibitors of intracellular protein degradation have shown that the isodipeptide derives from cross-linking N epsilon-(gamma-glutamyl)-lysine bonds formed by tissue transglutaminase. Estimated half-life values of cross-linked proteins were about 3 h. gamma-Glutamylamine cyclotransferase, which may split the isodipeptide formed during the continuous turnover of cross-linked proteins, was also found in Chinese-hamster ovary cells. Isodipeptide may have been accumulated when either its generated amount is beyond the capacity of gamma-glutamylamine cyclotransferase or it is generated in cell compartments where this enzyme is not present. PMID:2574570

  15. Observation of Chinese Hamster Ovary Cells retained inside the non-woven fiber matrix of the CellTank bioreactor.

    PubMed

    Zhang, Ye; Chotteau, Véronique

    2015-12-01

    This data article shows how the recombinant Chinese Hamster Ovary (CHO) cells are located in the interstices of the matrix fibers of a CellTank bioreactor after completion of a perfusion culture, supporting the article entitled "Very high cell density perfusion of CHO cells anchored in a non-woven matrix-based bioreactor" by Zhang et al. [1]. It provides a visualization of the cell distribution in the non-woven fiber matrix in a deeper view.

  16. Observation of Chinese Hamster Ovary Cells retained inside the non-woven fiber matrix of the CellTank bioreactor

    PubMed Central

    Zhang, Ye; Chotteau, Véronique

    2015-01-01

    This data article shows how the recombinant Chinese Hamster Ovary (CHO) cells are located in the interstices of the matrix fibers of a CellTank bioreactor after completion of a perfusion culture, supporting the article entitled “Very high cell density perfusion of CHO cells anchored in a non-woven matrix-based bioreactor” by Zhang et al. [1]. It provides a visualization of the cell distribution in the non-woven fiber matrix in a deeper view. PMID:26958613

  17. CD147 overexpression promotes tumorigenicity in Chinese hamster ovary cells.

    PubMed

    Yong, Yu-Le; Liao, Cheng-Gong; Wei, Ding; Chen, Zhi-Nan; Bian, Huijie

    2016-04-01

    CD147 overexpresses in many epithelium-originated tumors and plays an important role in tumor migration and invasion. Most studies aim at the role of CD147 in tumor progression using tumor cell models. However, the influence of abnormal overexpression of CD147 on neoplastic transformation of normal cells is unknown. Here, the role of CD147 in malignant phenotype transformation in CHO cells was investigated. Three CHO cell lines that stably overexpressed CD147 (CHO-CD147), EGFP-CD147 (CHO-EGFP-CD147), and EGFP (CHO-EGFP) were generated by transfection of plasmids containing human CD147, EGFP-human CD147, and EGFP genes into CHO cells. Cell migration and invasion were detected by wound healing and transwell matrix penetration assay. Trypan blue exclusion, MTT, cell cycle analysis, and BrdU cell proliferation assay were used to detect cell viability and cell proliferation. Annexin V-FITC analysis was performed to detect apoptosis. We found that CD147 overexpression promoted the migration and invasion of CHO cells. CD147 accelerated the G1 to S phase transition and enhanced the CHO cell proliferation. Overexpression of CD147 inhibited both early- and late-stages of apoptosis of CHO-CD147 cells, which is caused by serum deprivation. CHO-EGFP-CD147 cells showed an increased anchorage-independent growth compared with CHO-EGFP cells as detected by soft-agar colony formation assay. The tumors formed by CHO-CD147 cells in nude mice were larger and coupled with higher expression of proliferating cell nuclear antigen and Ki-67 than that of CHO cells. In conclusion, human CD147 overexpression induces malignant phenotype in CHO cells.

  18. Temporal order of gene replication in Chinese hamster ovary cells.

    PubMed Central

    Taljanidisz, J; Popowski, J; Sarkar, N

    1989-01-01

    To investigate the molecular basis of the regulatory mechanisms responsible for the orderly replication of the mammalian genome, we have developed an experimental system by which the replication order of various genes can be defined with relative ease and precision. Exponentially growing CHO-K1 cells were separated into populations representing various stages of the cell cycle by centrifugal elutriation and analyzed for cell cycle status flow cytometry. The replication of specific genes in each elutriated fraction was measured by labeling with 5-mercuri-dCTP and [3H]dTPP under conditions of optimal DNA synthesis after cell permeabilization with lysolecithin. Newly synthesized mercurated DNA from each elutriated fraction was purified by affinity chromatography on thiol-agarose and replicated with the large fragment of Escherichia coli DNA polymerase I by using [alpha-32P]dATP and random primers. The 32P-labeled DNA representative of various stages of the cell cycle was then hybridized with dot blots of plasmid DNA containing specific cloned genes. From these results, it was possible to deduce the nuclear DNA content at the time each specific gene replicated during S phase (C value). The C values of 29 genes, which included single-copy genes, multifamily genes, oncogenes, and repetitive sequences, were determined and found to be distributed over the entire S phase. Of the 28 genes studied, 19 had been examined by others using in vivo labeling techniques, with results which agreed with the replication pattern observed in this study. The replication times of nine other genes are described here for the first time. Our method of analysis is sensitive enough to determine the replication time of single-copy genes. The replication times of various genes and their levels of expression in exponentially growing CHO cells were compared. Although there was a general correlation between transcriptional activity and replication in the first half of S phase, examination of specific

  19. Structure of the dihydrofolate reductase gene in Chinese hamster ovary cells.

    PubMed

    Carothers, A M; Urlaub, G; Ellis, N; Chasin, L A

    1983-04-11

    Overlapping recombinant lambda 1059 phages carrying regions of the dhfr locus from the amplified Chinese hamster ovary (CHO) cell clone MK42 have been isolated. In addition, dhfr cDNAs from this cell line have been cloned into plasmid pBR322. Restriction analysis of these recombinant molecules has led to a map of the Chinese hamster dhfr gene. This gene has a minimum size of 26 kb and contains six exons as defined by hybridization to a combination of mouse and CHO cDNA probes. The latter probes reveal 3' exonic sequences that are not present in mouse cDNA. The CHO dhfr gene thus extends about 700 bp further 3' than in the mouse, consistent with the larger size of the hamster mRNA. At least five intervening sequences are present, of approximate sizes: 0.3, 2.5, 8.6, 2.6 and 9.4 kb. Four sequences from highly repeated families are situated in introns within the dhfr gene. The overall structure of this gene is strikingly similar to that of the mouse. Evolutionary conservation of interrupted gene structure among mammals thus extends to genes that code for household enzymes as well as specialized or structural proteins.

  20. Membrane proteins of dense lysosomes from Chinese hamster ovary cells

    SciTech Connect

    Chance, S.C.

    1987-01-01

    In this work membrane proteins from lysosomes were studied in order to gain more information on the biogenesis and intracellular sorting of this class of membrane proteins. Membrane proteins were isolated from a purified population of lysosomes. These proteins were then examined for various co- and post-translational modifications which could serve as potential intracellular sorting signals. Biochemical analysis using marker enzymatic activities detected no plasma membrane, Golgi, endoplasmic reticulum, peroxisomes, mitochondria, or cytosol. Analysis after incorporation of ({sup 3}H)thymidine or ({sup 3}H)uridine detected no nuclei or ribosomes. A fraction containing integral membrane proteins was obtained from the dense lysosomes by extraction with Triton X-114. Twenty-three polypeptides which incorporated both ({sup 35}S)methionine and ({sup 3}H)leucine were detected by SDS PAGE in this membrane fraction, and ranged in molecular weight from 30-130 kDa. After incorporation by cells of various radioactive metabolic precursors, the membrane fraction from dense lysosomes was examined and was found to be enriched in mannose, galactose, fucose, palmitate, myristate, and sulfate, but was depleted in phosphate. The membrane fraction from dense lysosomes was then analyzed by SDS PAGE to determine the apparent molecular weights of modified polypepties.

  1. Stable Expression of the Motor Protein Prestin in Chinese Hamster Ovary Cells

    NASA Astrophysics Data System (ADS)

    Iida, Koji; Konno, Kazuaki; Oshima, Takeshi; Tsumoto, Kouhei; Ikeda, Katsuhisa; Kumagai, Izumi; Kobayashi, Toshimitsu; Wada, Hiroshi

    Mammalian hearing sensitivity relies on a mechanical amplification mechanism involving the outer hair cells (OHCs), which rapidly alter their longitudinal length in response to changes in their membrane potential. The molecular basis of this mechanism is thought to be a motor protein embedded in the lateral membrane of the OHCs. Recently, this motor protein was identified and termed prestin. Since then, prestin has been researched intensively to elucidate the behavior of the OHCs. However, little progress in the study of prestin at the molecular level has been made because no method of obtaining an adequate amount of prestin has been established. In this study, therefore, an attempt was made to construct a stable expression system of prestin using Chinese hamster ovary (CHO) cells. The expression of prestin in the transfected CHO cells and the activity of prestin on CHO cells were confirmed by immunofluorescence and whole-cell patch-clamp measurements, respectively.

  2. Dielectric model for Chinese hamster ovary cells obtained by dielectrophoresis cytometry

    PubMed Central

    Salimi, E.; Braasch, K.; Butler, M.; Thomson, D. J.

    2016-01-01

    We present a dielectric model and its parameters for Chinese hamster ovary (CHO) cells based on a double-shell structure which includes the cell membrane, cytoplasm, nuclear envelope, and nucleoplasm. Employing a dielectrophoresis (DEP) based technique and a microfluidic system, the DEP response of many single CHO cells is measured and the spectrum of the Clausius-Mossotti factor is obtained. The dielectric parameters of the model are then extracted by curve-fitting to the measured spectral data. Using this approach over the 0.6–10 MHz frequency range, we report the values for CHO cells' membrane permittivity, membrane thickness, cytoplasm conductivity, nuclear envelope permittivity, and nucleoplasm conductivity. The size of the cell and its nuclei are obtained using optical techniques. PMID:26858823

  3. Amplification and loss of dihydrofolate reductase genes in a Chinese hamster ovary cell line

    SciTech Connect

    Kaufman, R.J.; Schimke, R.T.

    1981-12-01

    During stepwise increases in the methotrexate concentration in culture medium, the authors selected Chinese hamster ovary cells that contained elevated dihydrofolate reductase levels which were proportional to the number of dihydrofolate reductase gene copies (i.e., gene amplification). The authors studied the dihydrofolate reductase levels in individual cells that underwent the initial steps of methotrexate resistance by using the fluorescence-activated cell sorter technique. Such cells constituted a heterogeneous population with differing dihydrofolate reductase levels, and they characteristically lost the elevated enzyme levels when they were grown in the absence of methotrexate. The progeny of individual cells with high enzyme levels behaved differently and could lose all or variable numbers of the amplified genes.

  4. Microthermometry of laser-heated Chinese hamster ovary cells and sperm cells

    NASA Astrophysics Data System (ADS)

    Liu, Yagang; Sonek, Gregory J.; Chapman, Curtis F.; Tromberg, Bruce J.; Patrizio, Pasquale; Tadir, Yona; Berns, Michael W.

    1995-05-01

    Microthermometric measurements on optically-trapped Chinese Hamster Ovary (CHO) cells and sperms cells re reported, using a noninvasive microfluorometric detection technique. Within an optical tweezer system that has been outfitted with a spectral fluorescence excitation and detection capability, the changes in temperature induced by the process of sample confinement by a focused laser beam has been quantified over micron-sized spatial regions of both motile and immotile cells. Our measurement technique is based on the use of environmentally sensitive fluorophores that can be incorporated into the cell membrane and used to sense local changes in temperature when the cell membrane is perturbed optically or via other environmental stress factors. Using a cw 1.064 micrometers Nd:YAG laser for trapping CHO and human sperm cells, a temperature increase of approximately equals 1°C per 100 mW laser power was observed. At this infrared wavelength, cellular heating as result of laser confinement appears to be mainly due to radiation absorption by water.

  5. Cell culture and gene transcription effects of copper sulfate on Chinese hamster ovary cells.

    PubMed

    Qian, Yueming; Khattak, Sarwat F; Xing, Zizhuo; He, Aiqing; Kayne, Paul S; Qian, Nan-Xin; Pan, Shih-Hsie; Li, Zheng Jian

    2011-07-01

    This study reports the effects of varying concentrations of copper sulfate on the metabolic and gene transcriptional profile of a recombinant Chinese hamster ovary (CHO) cell line producing an immunoglobulin G (IgG)-fusion protein (B0). Addition of 50 μM copper sulfate significantly decreased lactate accumulation in the cultures while increasing viable cell density and protein titer. These changes could be seen from day 6 and became increasingly evident with culture duration. Reducing the copper sulfate concentration to 5 μM retained all the above beneficial effects, but with the added benefit of reduced levels of the aggregated form of the B0 protein. To profile the cellular changes due to copper sulfate addition at the transcriptional level, Affymetrix® CHO microarrays were used to identify differentially expressed genes related to reduced cellular stresses and facilitated cell cycling. Based on the microarray results, down-regulation of the transferrin receptor and lactate dehydrogenase, and up-regulation of a cytochrome P450 family-2 polypeptide were then confirmed by Western blotting. These results showed that copper played a critical role in cell metabolism and productivity on recombinant CHO cells and highlighted the usefulness of microarray data for better understanding biological responses on medium modification.

  6. Antimutagenic activities of naturally occurring polyamines in Chinese hamster ovary cell in vitro

    SciTech Connect

    Cozzi, R.; Perticone, P.; Bona, R.; Polani, S. )

    1991-01-01

    Spermine and spermidine, ubiquitous polyamines present in bacteria and animal cells, are also involved in cell growth. Since they interact with the double helix, they can stabilize the DNA molecule. Recent evidence of the antimutagenic and anticarcinogenic capacity of spermine has focused attention on the he mechanism(s) by which such agents can protect cells from induced damages. In the present paper the authors show the ability of spermine and spermidine to decrease the level of sister chromatid exchanges induced in Chinese hamster ovary cells cultivated in vitro, by treating them with Psoralen + UVA irradiation (able to induce mainly monoadducts and DNA cross-links). Two different mechanisms of polyamine action can be invoked to explain the preservative activity of this class of agents.

  7. Cytotoxicity of refractory ceramic fibres to Chinese hamster ovary cells in culture.

    PubMed

    Hart, G A; Newman, M M; Bunn, W B; Hesterberg, T W

    1992-07-01

    The toxicity/oncogenicity of refractory ceramic fibres have been tested in chronic inhalation studies in rodents. Because these studies are time consuming and expensive, there is a need to develop and validate short-term models to screen fibres for their toxicological potential. In the present study, the toxic effects of four different compositions of refractory ceramic fibres were determined using Chinese hamster ovary cells grown in culture. These refractory ceramic fibres were the same size-selected fibres that had been used in animal inhalation studies, thus facilitating a direct comparison of findings in the two systems. Chinese hamster ovary cells were treated with refractory ceramic fibres 24 hr after seeding into 60-mm culture dishes in Ham's F12 medium with 10% serum. Inhibition of cell proliferation and colony formation were determined after 3-5 days of fibre exposure. Crocidolite and chrysotile asbestos were used as positive controls. Concentration-dependent inhibition of both cell proliferation and colony formation was observed after treatment with refractory ceramic fibres. The LC(50) for the different refractory ceramic fibres ranged from 10 to 30 mug/cm(2). The LC(50)s for crocidolite and chrysotile were 5 mug/cm(2) and 1 mug/cm(2), respectively. To assess the genotoxic potential of these fibres, fibre-exposed Chinese hamster ovary cell cultures were stained with acridine orange and scored for the incidence of micronuclei and other nuclear abnormalities. The incidence of nuclear abnormalities for refractory ceramic fibres at 20 mug/cm(2) ranged from 20 to 40%. Toxic endpoints of the in vitro studies were compared with those of the chronic animal inhalation studies. The latter included induction of lung fibrosis and pleural and airway tumours. A correlation was observed between the in vitro and in vivo toxicological potencies of the respective four refractory ceramic fibres: the fibres that were most toxic in vitro were also the most toxic in the

  8. Isolation of Chinese hamster ovary cell mutants requiring the continuous presence of taxol for cell division

    PubMed Central

    1983-01-01

    Chinese hamster ovary (CHO) cell mutants resistant to the cytotoxic effects of taxol and requiring the drug for normal growth were isolated in a single step. One of these mutant cell lines, Tax-18, fails to divide in the absence of taxol; instead, the cells become larger, rounder, flatter, and multinucleated. Analysis by flow cytometry indicates that during taxol deprivation there is an accumulation of cells in G2 + M phase but that the cells are able to leak through the block in the absence of cell division and further increase their DNA content beyond the tetraploid amount. This interpretation is confirmed by karyotype analysis and by time-lapse studies that show cells rounded for mitosis two to five times longer than in wild-type cultures or in Tax-18 cultures grown in taxol. The cells finally attempt to undergo cytokinesis, fail, and spread out again, but as larger cells than before. Tax-18 has a normal growth rate and morphology when grown in taxol even at concentrations three to five times below the selecting concentration of the drug. The cells, however, have increased sensitivity to microtubule-disrupting drugs such as colcemid, griseofulvin, and D2O. The mutation for taxol auxotrophy behaves recessively in somatic cell hybridization experiments, and the phenotypic reversion rate is approximately 10(-5) in a nonmutagenized population. Both alpha- and beta-tubulin are present in apparently normal amounts and with normal electrophoretic mobilities on two- dimensional gels. The results suggest that Tax-18 lacks a factor necessary for mitosis and that taxol may be able to substitute for this factor. PMID:6134736

  9. Adaptation of Chinese hamster ovary cells to low culture temperature: cell growth and recombinant protein production.

    PubMed

    Yoon, Sung Kwan; Hong, Jong Kwang; Choo, Seung Ho; Song, Ji Yong; Park, Hong Woo; Lee, Gyun Min

    2006-04-20

    Recombinant Chinese hamster ovary (rCHO) cells producing erythropoietin (EPO) and rCHO cells producing follicle-stimulating hormone (FSH) showed a significant increase in specific productivity (q) when grown at 32 degrees C compared to 37 degrees C. However, low culture temperature suppressed cell growth, and therefore, did not increase volumetric productivity as much as q. In an attempt to increase the volumetric productivity through improvement of hypothermic growth, EPO producing rCHO (CHO-EPO) cells and FSH producing rCHO (CHO-FSH) cells were adapted at 32 degrees C in a repeated batch mode using spinner flasks. Cell growth of both CHO-EPO and CHO-FSH gradually improved during adaptation at 32 degrees C. Specific growth rates of CHO-EPO and CHO-FSH cells at 32 degrees C, through adaptation, were increased by 73% and 20%, respectively. During adaptation at 32 degrees C, mRNA levels of cold-inducible RNA-binding protein (CIRP) of both rCHO cell lines did not change significantly, suggesting that CIRP expression may not be the only cause for growth suppression at low culture temperature. Unlike cell growth, the recombinant protein production of both rCHO cell lines was not increased during adaptation due to decreased specific productivities. The specific EPO productivity and specific FSH productivity were decreased by 49% and 22%, respectively. Southern blot analyses showed that the decreased specific productivities were not due to the loss of foreign gene copies. Taken together, improvement of hypothermic cell growth by adaptation does not appear to be applicable for enhanced recombinant protein production, since specific productivity decreases during adaptation to the low culture temperature.

  10. Effect of glutamine limitation on the death of attached Chinese hamster ovary cells

    SciTech Connect

    Sanfeliu, A.; Stephanopoulos, G. )

    1999-07-05

    The effect of glutamine depletion on the death of attached Chinese hamster ovary (CHO) cells was investigated. Experiments were performed using an anchorage dependent CHO cell line expressing [gamma]-IFN and a second cell line obtained by transfection of that cell line with the human bcl-2 (hbcl-2). Either cell line could grow in media devoid of glutamine with minimal cell death due to endogenous glutamine synthetase activity that allowed cells to synthesize glutamine from glutamic acid in the medium. However, compared to control cultures in glutamine-containing media, the cell growth rate in glutamine-free media was slower with an increased fraction of cells distributed in the G[sub 0]/G[sub 1] phase. The slower rate of cell cycling apparently protected the cells from entering apoptosis when they were stimulated to proliferate in an environment devoid of other protective factors, such as serum or over-expressed hbcl-2. The depletion of both glutamine and glutamic acid did cause cell death, which could be mitigated by hbcl-2 over-expression.

  11. Enhancement of ricin cytotoxicity in Chinese hamster ovary cells by depletion of intracellular K+: evidence for an Na+/H+ exchange system in Chinese hamster ovary cells

    PubMed Central

    1985-01-01

    Depletion of intracellular K+ has been reported to result in an arrest of the formation of coated pits in human fibroblasts (Larkin, J.M., M.S. Brown, J.L. Goldstein, and R.G.W. Anderson, 1983, Cell, 33:273- 285). We have studied the effects of K+ depletion on the cytotoxicities of ricin, Pseudomonas exotoxin A, and diphtheria toxin in Chinese hamster ovary (CHO) cells. The cytotoxicities of ricin and Pseudomonas toxin were enhanced in K+-depleted CHO cells whereas the cytotoxicity of diphtheria toxin was reduced by K+ depletion. The effects of NH4Cl on the cytotoxicities of ricin, Pseudomonas toxin, and diphtheria toxin were found to be similar to those of K+ depletion, and there were no additive or synergistic effects on ricin cytotoxicity by NH4Cl in K+- depleted medium. The enhancement of ricin cytotoxicity by K+ depletion could be completely reversed by the addition of K+, Rb+, and partially by the addition of Cs+, before the ricin treatment, whereas Li+ was ineffective. These protective effects of K+ or Rb+ requires a functional Na+/K+ ATPase. CHO cells grown in K+-depleted media were found to contain 6.3-fold increase in intracellular Na+ level, concomitant with a 10-fold reduction in intracellular K+ level. The enhanced cytotoxicity of ricin in K+-free medium and the increased uptake of Na+ could be abolished by amiloride or amiloride analogues, which are known to be potent inhibitors of the Na+/H+ antiport system. Our results suggest that a depletion of intracellular K+ results in an influx of Na+, which is accompanied by the extrusion of H+. Consequently, there is an alkalinization of the cytosol and the ricin- containing endosomes. As a result, ricin is more efficiently released from the endosomes in-K+-depleted cells. Results from the studies of the binding, internalization, and degradation of 125I-ricin, and the kinetics of inhibition of protein synthesis by ricin in K+-depleted cells are consistent with this working hypothesis. PMID:2991297

  12. Coupling of human delta-opioid receptor to retinal rod transducin in Chinese hamster ovary cells.

    PubMed

    Varga, E V; Stropova, D; Kim, T; Wang, M; Roeske, W R; Yamamura, H I

    2000-01-01

    Reverse transcription-polymerase chain reaction was used to identify the pertussis toxin (Ptx)-sensitive G protein alpha-subunit pool in Chinese hamster ovary (CHO) and mouse fibroblast (B82) cells. We detected the presence of mRNA for G(ialpha2), G(ialpha3), and G(oalpha) in both cell lines. G(ialpha1) and G(alphaz) mRNAs were not detected. We also found a homolog of the retinal rod transducin (G(talpha1)) in CHO, and the mouse cone transducin (G(talpha2)) in B82 cells. The presence of the transducin alpha-subunit proteins in CHO and B82 cells was confirmed by immunoprecipitation with specific antibodies. To test the interaction of heterologously expressed receptors with transducin in CHO cells, a Ptx-insensitive (C347S) rod transducin mutant was transfected into a CHO cell line stably expressing the human delta-opioid receptor (hDOR/CHO). (+)-4-[(alphaR)-alpha-((2S,2R)-4-allyl-2, 5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide, a selective delta-opioid receptor agonist, stimulated guanosine-5'-O-(3-[(35)S]thio)triphosphate binding by 293 +/- 36% after Ptx pretreatment in the mutant cell line with an EC(50) value of 54 +/- 32 nM, showing that transducin can functionally couple to the human delta-opioid receptors in these cells.

  13. Improved antibody production in Chinese hamster ovary cells by ATF4 overexpression.

    PubMed

    Haredy, Ahmad M; Nishizawa, Akitoshi; Honda, Kohsuke; Ohya, Tomoshi; Ohtake, Hisao; Omasa, Takeshi

    2013-12-01

    To improve antibody production in Chinese hamster ovary (CHO) cells, the humanized antibody-producing CHO DP-12-SF cell line was transfected with the gene encoding activating transcription factor 4 (ATF4), a central factor in the unfolded protein response. Overexpression of ATF4 significantly enhanced the production of antibody in the CHO DP-12-SF cell line. The specific IgG production rate of in the ATF4-overexpressing CHO-ATF4-16 cells was approximately 2.4 times that of the parental host cell line. Clone CHO-ATF4-16 did not show any change in growth rate compared with the parental cells or mock-transfected CHO-DP12-SF cells. The expression levels of mRNAs encoding both the antibody heavy and light chains in the CHO-ATF4-16 clone were analyzed. This analysis showed that ATF4 overexpression improved the total production and specific production rate of antibody without affecting the mRNA transcription level. These results indicate that ATF4 overexpression is a promising method for improving recombinant IgG production in CHO cells.

  14. [COMPARATIVE CYTOGENETIC ANALYSIS OF MONOLAYER AND SUSPENSION CHINESE HAMSTER OVARY CELL LINES CHO(dhfr-)].

    PubMed

    Stefanova, V N; Yartseva, N M; Petrov, A V

    2015-01-01

    The karyotypes of CHO(dhfr-) and CHO(dhfr-)/susp Chinese hamster ovary cell lines were investigated with the use of GTG-staining. Modal chromosome set consists of 20 and 18 chromosomes respectively. The karyotypes of both cell lines were stable with constant ratio of normal chromosomes and chromosomes with structural rearrangements. Monosomy for chromosomes 1, 2, 4, 5, 8 was observed in both cell lines and for chromosome 9 in CHO(dhfr-)/susp cell line. The differences between CHO(dhfr-) cell lines studied by us consists of inclusion of part of chromosome 7 in der(6)t(1;6), rearrangement of del(5) and monosomy of chromosome 9. It was shown that in karyotypes of all CHO cell lines studied up today there are 5 common structurally chromosome rearrangements: del(2), inv(3), add(6), del(9) and mar1. In both CHO(dhfr-) cell lines investigated by us three unique chromosome rearrangements: del(1), der(6)t(1,6) and mar3 were revealed. Necessity of simultaneous GTG and FISH analysis of chromosomes rearrangements in the CHO cell lines under study is discussed.

  15. Enhanced sialylation of recombinant erythropoietin in genetically engineered Chinese-hamster ovary cells.

    PubMed

    Jeong, Yeon Tae; Choi, One; Son, Young Dok; Park, Seung Yeol; Kim, Jung Hoe

    2009-04-01

    Sialic acid, the terminal sugar in N-linked complex glycans, is usually found in glycoproteins and plays a major role in determining the circulatory lifespan of glycoproteins. In the present study we attempted to enhance the sialylation of recombinant EPO (erythropoietin) in CHO (Chinese-hamster ovary) cells. To enhance EPO sialylation, we introduced human alpha2,3-ST (alpha2,3-sialyltransferase) and CMP-SAS (CMP-sialic acid synthase) into recombinant human EPO-producing CHO cells. The sialylation of EPO was increased by the expression of alpha2,3-ST alone. Although the co-expression of alpha2,3-ST and CMP-SAS did not further increase sialylation, an increase in the intracellular pool of CMP-sialic acid was noted. On the basis of these observations, it was postulated that the transport capacity of CMP-sialic acid into the Golgi lumen was limited, thereby causing the reduced availability of CMP-sialic acid substrate for sialylation. Therefore, we co-expressed human alpha2,3-ST and CMP-SAS, as well as overexpress Chinese hamster CMP-sialic acid transporter (CMP-SAT) in CHO cells, which produced recombinant human EPO. When alpha2,3-ST, CMP-SAS, and CMP-SAT were overexpressed in CHO cells, there was a corresponding increase in sialylation compared with the co-expression of alpha2,3-ST and CMP-SAS. The present study provides a useful strategy for enhancing the sialylation of therapeutic glycoproteins produced in CHO cells.

  16. The DNA methylation landscape of Chinese hamster ovary (CHO) DP-12 cells.

    PubMed

    Wippermann, Anna; Rupp, Oliver; Brinkrolf, Karina; Hoffrogge, Raimund; Noll, Thomas

    2015-04-10

    Chinese hamster ovary (CHO) cells represent the most commonly used production cell line for therapeutic proteins. By recent genome and transcriptome sequencing a basis was created for future investigations of genotype-phenotype relationships and for improvement of CHO cell productivity and product quality. In this context information is missing about DNA cytosine methylation as a crucial epigenetic modification and an important element in mammalian genome regulation and development. Here, we present the first DNA methylation map of a CHO cell line in single-base resolution that was generated by whole genome bisulfite sequencing combined with gene expression analysis by CHO microarrays. We show CHO DP-12 cells to exhibit global hypomethylation compared to a majority of mammalian methylomes and hypermethylation of CpG-dense regions at gene promoters called CpG islands. We also observed partially methylated domains that cover 62% of the CHO DP-12 cell genome and contain functional clusters of genes. Gene expression analysis showed these clusters to be either highly or weakly expressed with regard to CHO-specific characteristics and hence proves DNA methylation in CHO cells to be an important link between genomics and transcriptomics.

  17. Versatile microscale screening platform for improving recombinant protein productivity in Chinese hamster ovary cells

    PubMed Central

    Hansen, Henning Gram; Nilsson, Claes Nymand; Lund, Anne Mathilde; Kol, Stefan; Grav, Lise Marie; Lundqvist, Magnus; Rockberg, Johan; Lee, Gyun Min; Andersen, Mikael Rørdam; Kildegaard, Helene Faustrup

    2015-01-01

    Chinese hamster ovary (CHO) cells are widely used as cell factories for the production of biopharmaceuticals. In contrast to the highly optimized production processes for monoclonal antibody (mAb)-based biopharmaceuticals, improving productivity of non-mAb therapeutic glycoproteins is more likely to reduce production costs significantly. The aim of this study was to establish a versatile target gene screening platform for improving productivity for primarily non-mAb glycoproteins with complete interchangeability of model proteins and target genes using transient expression. The platform consists of four techniques compatible with 96-well microplates: lipid-based transient transfection, cell cultivation in microplates, cell counting and antibody-independent product titer determination based on split-GFP complementation. We were able to demonstrate growth profiles and volumetric productivity of CHO cells in 96-half-deepwell microplates comparable with those obtained in shake flasks. In addition, we demonstrate that split-GFP complementation can be used to accurately measure relative titers of therapeutic glycoproteins. Using this platform, we were able to detect target gene-specific increase in titer and specific productivity of two non-mAb glycoproteins. In conclusion, the platform provides a novel miniaturized and parallelisable solution for screening target genes and holds the potential to unravel genes that can enhance the secretory capacity of CHO cells. PMID:26657798

  18. Intracellular trafficking of recycling apolipoprotein E in Chinese hamster ovary cells.

    PubMed

    Braun, Nicole A; Mohler, Peter J; Weisgraber, Karl H; Hasty, Alyssa H; Linton, MacRae F; Yancey, Patricia G; Su, Yan Ru; Fazio, Sergio; Swift, Larry L

    2006-06-01

    We have investigated apolipoprotein E (apoE) recycling in Chinese hamster ovary (CHO) cells, a peripheral cell that does not produce lipoproteins or express apoE. Using a pulse-chase protocol in which cells were pulsed with 125I-apoE-VLDL and chased for different periods, approximately 30% of the apoE internalized during the pulse was resecreted within a 4 h chase in a relatively lipid-free state. The addition of lysosomotropic agents or brefeldin A had no effect on apoE recycling. Unlike previous results with hepatocytes and macrophages, neither apoA-I nor upregulation of ABCA1 stimulated apoE recycling. However, cyclodextrin, which extracts cholesterol from plasma membrane lipid rafts, increased recycling. Confocal studies revealed that apoE, internalized during a 1 h pulse, colocalizes with early endosomal antigen-1, Rab5, Rab11a, and lysobisphosphatidic acid but not with lysosomal-associated membrane protein-1. Colocalization of apoE and Rab11a persisted even after cells had been chased for 1 h, suggesting a pool of apoE within the endosomal recycling compartment (ERC). Our data suggest that apoE recycling in CHO cells is linked to cellular cholesterol removal via the ERC and phospholipid-containing acceptors in a pathway alternative to the ABCA1-apoA-I axis.

  19. Xbp1-based engineering of secretory capacity enhances the productivity of Chinese hamster ovary cells.

    PubMed

    Tigges, Marcel; Fussenegger, Martin

    2006-05-01

    A variety of successful transcription and translation engineering strategies implemented during the past decade have driven the specific productivity of mammalian cells to an apparent limit. Restricted post-translation competence has since been considered the major bottleneck preventing mammalian cells from fully exploiting their physiologic production capacity in a biopharmaceutical manufacturing scenario. Through ectopic expression of the human transcription factor Xbp1 (X-box-binding-protein 1), evolved to manage plasma cell differentiation and coordinate the unfolded protein response, we have specifically expanded the endoplasmic reticulum and the Golgi of transgenic Chinese hamster ovary (CHO-K1)-derived cell lines with a resulting increase in overall production capacity. Xbp-1-based engineering of secretory bottlenecks was compatible with a variety of different promoter–product gene configurations suggesting that Xbp-1 induces generic production increases in CHO-K1 cell derivatives. Secretion engineering, illustrated here by Xbp1-based reprogramming of the post-translational processing machinery, provides a first insight into mastering a major system bottleneck which impacts biopharmaceutical manufacturing of secreted protein therapeutics.

  20. Endogenous TRPM4-like channel in Chinese hamster ovary (CHO) cells

    SciTech Connect

    Yarishkin, Oleg V.; Hwang, Eun-Mi; Park, Jae-Yong; Kang, Dawon; Han, Jaehee; Hong, Seong-Geun

    2008-05-02

    Chinese hamster ovary (CHO) cells used in many transfection studies have been found to endogenously express channels permeable to monovalent cations, but not to divalent cations. In the presence of intracellular Ca{sup 2+}, 23-pS channel with a linear current-voltage (I-V) relationship could be frequently observed in inside-out patches but not in cell-attached patches. The open probability was voltage-dependent, which is higher at positive potentials. The channel was dose-dependently activated by relatively high level of Ca{sup 2+} (EC{sub 50} = 1.04 {+-} 0.08 mM), and sensitively inhibited by 100 {mu}M ATP, ADP, AMP, and 1 mM spermine. However, ruthenium red (2 {mu}M) had no effect. Reverse transcript polymerase chain reaction (RT-PCR) supported the presence of mRNA encoding TRPM4b channel protein. Western blot assay finally confirmed the presence of this channel protein in membrane fraction of CHO cells. These results provide evidence that CHO cells express an endogenous TRPM4b-like channel, and thereby can be used as a tool to study de novo regulation/modulation of TRPM4 channel.

  1. Gene silencing by DNA methylation and dual inheritance in Chinese hamster ovary cells.

    PubMed

    Paulin, R P; Ho, T; Balzer, H J; Holliday, R

    1998-06-01

    Chinese hamster ovary (CHO) cells strain D422, which has one copy of the adenine phosphoribosyl transferase (APRT) gene, were permeabilized by electroporation and treated with 5-methyl deoxycytidine triphosphate. Cells with a silenced APRT gene were selected on 2, 6-diaminopurine. Colonies were isolated and shown to be reactivated to APRT+ by 5-aza-cytidine and by selection in medium containing adenine, aminopterin and thymidine. Genomic DNA was prepared from eight isolates of independent origin and subjected to bisulphite treatment. This deaminates cytosine to uracil in single-stranded DNA but does not deaminate 5-methyl cytosine. PCR, cloning and sequencing revealed the methylation pattern of CpG doublets in the promoter region of the APRT- gene, whereas the active APRT gene had nonmethylated DNA. CHO strain K1, which has two copies of the APRT+ gene, could also be silenced by the same procedure but at a lower frequency. The availability of the 5-methyl dCTP-induced silencing, 5-aza-CR and a standard mutagen, ethyl methane sulphonate, makes it possible to follow concomitantly the inheritance of active, mutant or silenced gene copies. This analysis demonstrates "dual inheritance" at the APRT locus in CHO cells.

  2. Molecular engineering of exocytic vesicle traffic enhances the productivity of Chinese hamster ovary cells.

    PubMed

    Peng, Ren-Wang; Fussenegger, Martin

    2009-03-01

    A complex vesicle trafficking system manages the precise and regulated distribution of proteins, membranes and other molecular cargo between cellular compartments as well as the secretion of (heterologous) proteins in mammalian cells. Sec1/Munc18 (SM) proteins are key components of the system by regulating membrane fusion. However, it is not clear how SM proteins contribute to the overall exocytosis. Here, functional analysis of the SM protein Sly1 and Munc18c suggested a united, positive impact upon SNARE-based fusion of ER-to-Golgi- and Golgi-to-plasma membrane-addressed exocytic vesicles and increased the secretory capacity of different therapeutic proteins in Chinese hamster ovary cells up to 40 pg/cell/day. Sly1- and Munc18c-based vesicle traffic engineering cooperated with Xbp-1-mediated ER/Golgi organelle engineering. Our study supports a model for united function of SM proteins in stimulating vesicle trafficking machinery and provides a generic secretion engineering strategy to improve biopharmaceutical manufacturing of important protein therapeutics.

  3. Isolation and characterization of a Chinese hamster ovary cell mutant with altered regulation of phosphatidylserine biosynthesis

    SciTech Connect

    Hasegawa, K.; Kuge, O.; Nishijima, M.; Akamatsu, Y. )

    1989-11-25

    We have screened approximately 10,000 colonies of Chinese hamster ovary (CHO) cells immobilized on polyester cloth for mutants defective in (14C)ethanolamine incorporation into trichloroacetic acid-precipitable phospholipids. In mutant 29, discovered in this way, the activities of enzymes involved in the CDP-ethanolamine pathway were normal; however, the intracellular pool of phosphorylethanolamine was elevated, being more than 10-fold that in the parental CHO-K1 cells. These results suggested that the reduced incorporation of (14C)ethanolamine into phosphatidylethanolamine in mutant 29 was due to dilution of phosphoryl-(14C)ethanolamine with the increased amount of cellular phosphorylethanolamine. Interestingly, the rate of incorporation of serine into phosphatidylserine and the content of phosphatidylserine in mutant 29 cells were increased 3-fold and 1.5-fold, respectively, compared with the parent cells. The overproduction of phosphorylethanolamine in mutant 29 cells was ascribed to the elevated level of phosphatidylserine biosynthesis, because ethanolamine is produced as a reaction product on the conversion of phosphatidylethanolamine to phosphatidylserine, which is catalyzed by phospholipid-serine base-exchange enzymes. Using both intact cells and the particulate fraction of a cell extract, phosphatidylserine biosynthesis in CHO-K1 cells was shown to be inhibited by phosphatidylserine itself, whereas that in mutant 29 cells was greatly resistant to the inhibition, compared with the parental cells. As a conclusion, it may be assumed that mutant 29 cells have a lesion in the regulation of phosphatidylserine biosynthesis by serine-exchange enzyme activity, which results in the overproduction of phosphatidylserine and phosphorylethanolamine as well.

  4. Isolation and characterization of Chinese hamster ovary cell lines sensitive to mitomycin C and bleomycin

    SciTech Connect

    Robson, C.N.; Harris, A.L.; Hickson, I.D.

    1985-11-01

    Seven Chinese hamster ovary K1 cell lines exhibiting sensitivity to anticancer drugs have been isolated by a replica-plating technique. Five of the mutants are hypersensitive to the DNA cross-linking agent mitomycin C. Of these, one is also appreciably sensitive to UV light. Significant variations in their cross-sensitivity to cis-platinum(II) diammine dichloride, chlorambucil, and Adriamycin have also been observed. Two additional mutants have been isolated on the basis of sensitivity to the radiomimetic agent bleomycin. One of these shows greater than 6-fold sensitivity to bleomycin, while the other is approximately 14 times more sensitive than the parental strain to bleomycin and is also hypersensitive to a number of other DNA-damaging agents, including cis-platinum(II) diammine dichloride, chlorambucil, X-rays, and UV light. Both bleomycin-sensitive mutants also exhibit some degree of sensitivity to Adriamycin. In all cases, the cell lines have been grown in continuous culture for 3 months without evidence of reversion and should act as suitable recipients in DNA transfection experiments aimed at identifying human DNA repair genes.

  5. Expression of human extracellular superoxide dismutase in Chinese hamster ovary cells and characterization of the product

    SciTech Connect

    Tibell, L.; Hjalmarsson, K.; Edlund, T.; Skogman, G.; Engstroem, A.; Marklund, S.L.

    1987-10-01

    A complementary DNA clone from human placenta, encoding human extracellular superoxide dismutase, has recently been isolated and characterized. An expression plasmid, based on the EC-SOD complementary DNA, was transfected into Chinese hamster ovary cells (CHO-K1). The transfected cells secreted human EC-SOD to the culture medium. The secreted recombinant (r) EC-SOD was isolated in high yield with a three-step procedure beginning with immobilized monoclonal anti-EC-SOD antibodies. The properties of the rEC-SOD were compared with native (n) EC-SOD isolated from human umbilical cords. The specific activities and amino-terminal amino acid sequences were identical. The amino acid compositions were virtually identical and very similar to the composition deduced from the complementary DNA sequence. Both rEC-SOD and nEC-SOD contained 4 Cu and 4 Zn atoms per molecule, and the presence of Zn in EC-SOD is thus now established. The rEC-SOD produced is type C, since its affinity for heparin-Sepharose was identical to that of nEC-SOD type C. Both enzymes bound to concanavalin A, lentil lectin, and wheat germ lectin and are thus glycoproteins. rEC-SOD and nEC-SOD seem to have the same subunit structure and composition as analyzed by polyacrylamide gel electrophoresis and gel chromatography.

  6. Production and characterization of neurosecretory protein GM using Escherichia coli and Chinese Hamster Ovary cells

    PubMed Central

    Masuda, Keiko; Furumitsu, Megumi; Taniuchi, Shusuke; Iwakoshi-Ukena, Eiko; Ukena, Kazuyoshi

    2015-01-01

    Neurosecretory protein GL (NPGL) and neurosecretory protein GM (NPGM) are paralogs recently discovered in birds and in mammals. The post-translational products of NPGL and of NPGM genes include a signal peptide sequence, a glycine amidation signal, and a dibasic amino acid cleavage site. This suggests that the mature forms of NPGL and of NPGM are small proteins secreted in the hypothalamus and containing an amidated C-terminus. However, endogenous NPGL and NPGM have not yet been identified. Chicken NPGL and NPGM have two highly conserved Cys residues that are likely to form a disulfide bond, while mammalian NPGM has one additional Cys residue located between the two conserved Cys residues and the correct disulfide bond pattern is unclear. In this study, we prepared rat NPGM to elucidate the structure of its mature form. We first expressed the predicted mature NPGM, containing an extra C-terminal Gly, in Escherichia coli SHuffle cells, which are engineered to promote the formation of native disulfide bridges in recombinant proteins. We observed the presence of a disulfide bond between the N-terminal Cys residue and the second Cys residue, while the C-terminal Cys residue was free. Secondly, we transfected a construct containing the entire NPGM open reading frame into Chinese Hamster Ovary cells, and observed that NPGM was cleaved immediately after the signal peptide and that it was secreted into the medium. Furthermore, the protein presented a disulfide bond at the same location observed in recombinant NPGM. PMID:26587371

  7. Effects of selenocystine on lead-exposed Chinese hamster ovary (CHO) and PC-12 cells

    SciTech Connect

    Aykin-Burns, Nukhet; Ercal, Nuran . E-mail: nercal@umr.edu

    2006-07-15

    Lead is a pervasive environmental toxin that affects multiple organ systems, including the nervous, renal, reproductive, and hematological systems. Even though it is probably the most studied toxic metal, some of the symptoms of lead toxicity still cannot be explained by known molecular mechanisms. Therefore, lead-induced oxidative stress has recently started to gain attention. This in vitro study confirms the existence of oxidative stress due to lead exposure. Administration of lead acetate (PbA) to cultures of Chinese hamster ovary cells (CHO) had a concentration-dependent inhibitory effect on colony formation and cell proliferation. This inhibition was eliminated by 5 {mu}M selenocystine (SeCys). In order to evaluate the nature of SeCys's effect, we measured glutathione (GSH), its oxidized form glutathione disulfide (GSSG), malondialdehyde (MDA), catalase, and GSH peroxidase (GPx) activities in lead-exposed CHO cells both in the presence and absence of SeCys. Increases in MDA, catalase, and GPx activities were observed in cultures that received only PbA, but supplementation with SeCys returned these measures to pretreatment levels. The ratio of GSH to GSSG increased in lead-exposed cells incubated in SeCys-enhanced media but declined in cultures treated with PbA only. In order to determine whether SeCys also reverses lead-induced neurotoxicity, a neuronal cell line, PC-12 cells, was used. Lead's inhibition on neurite formation was significantly eliminated by SeCys in PC-12 cells. Our results suggest that SeCys can confer protection against lead-induced toxicity in CHO cells and neurotoxicity in PC-12 cells.

  8. RNA synthesis in the ultrastructural and biochemical components of the nucleolus of Chinese hamster ovary cells

    PubMed Central

    1975-01-01

    A correlated autoradiographic and biochemical study of RNA synthesis in the nucleoli of chinese hamster ovary cells has been made. Quantitative analysis of the labeling indicates that the fibrillar ribonucleoprotein (RNP) component is labeled faster than 80S RNP and 45S RNA molecules, but approaches simultaneously a steady-state 3H to 14C ratio or grains/mum2 after 30 min of [3H]uridine incorporation. On the other hand, the 55S RNP, the 36S + 32S RNA, and the granular RNP components have the same kinetic of labeling with [3H]uridine. These results suggest that the fibrillar and granular RNP components of the nucleolus are the ultrastructural substratum of, respectively, the 80S RNP (45S RNA) and 55S RNP (36S + 32S RNA). The possibility that precursors to 80S RNP exist also in the fibrillar region of the nucleolus is strongly suggested by the rapid labeling of the fibrils on the autoradiographs. PMID:1171872

  9. Propolis-induced genotoxicity and antigenotoxicity in Chinese hamster ovary cells.

    PubMed

    Tavares, Denise Crispim; Mazzaron Barcelos, Gustavo Rafael; Silva, Lívia Ferreira; Chacon Tonin, Conception Cortez; Bastos, Jairo Kenupp

    2006-10-01

    Propolis has been used in folk medicine since ancient times and is known for its antimicrobial, antiparasitic, antiviral, anti-inflammatory, antitumoral and antioxidant properties. In view of the great therapeutic interest in propolis and the small number of studies regarding its mechanism of action, the aim of the present study was to evaluate the mutagenic and antimutagenic effects of propolis using Chinese hamster ovary cells. Parameters such as the frequency of chromosome aberrations and mitotic index were analyzed. The results showed that, on one hand, the highest propolis tested concentration displayed a small but significant increase in the frequency of chromosome aberrations, and on the other hand, it was observed that the lowest tested concentration significantly reduced the chromosome damage induced by the chemotherapeutic agent doxorubicin. The present results indicate that propolis shows the characteristic of a "Janus" compound, i.e., propolis is genotoxic at higher concentrations, while at lower concentrations it display a chemopreventive effect on doxorubicin-induced mutagenicity. Flavonoids may be the components of propolis responsible for its both mutagenic and antimutagenic effects, once these compounds may act either as pro-oxidant or as free radicals scavenger, depending on its concentration.

  10. Metabolic analysis of antibody producing Chinese hamster ovary cell culture under different stresses conditions.

    PubMed

    Badsha, Md Bahadur; Kurata, Hiroyuki; Onitsuka, Masayoshi; Oga, Takushi; Omasa, Takeshi

    2016-07-01

    Chinese hamster ovary (CHO) cells are commonly used as the host cell lines concerning their ability to produce therapeutic proteins with complex post-translational modifications. In this study, we have investigated the time course extra- and intracellular metabolome data of the CHO-K1 cell line, under a control and stress conditions. The addition of NaCl and trehalose greatly suppressed cell growth, where the maximum viable cell density of NaCl and trehalose cultures were 2.2-fold and 2.8-fold less than that of a control culture. Contrariwise, the antibody production of both the NaCl and trehalose cultures was sustained for a longer time to surpass that of the control culture. The NaCl and trehalose cultures showed relatively similar dynamics of cell growth, antibody production, and substrate/product concentrations, while they indicated different dynamics from the control culture. The principal component analysis of extra- and intracellular metabolome dynamics indicated that their dynamic behaviors were consistent with biological functions. The qualitative pattern matching classification and hierarchical clustering analyses for the intracellular metabolome identified the metabolite clusters whose dynamic behaviors depend on NaCl and trehalose. The volcano plot revealed several reporter metabolites whose dynamics greatly change between in the NaCl and trehalose cultures. The elastic net identified some critical, intracellular metabolites that are distinct between the NaCl and trehalose. While a relatively small number of intracellular metabolites related to the cell growth, glucose, glutamine, lactate and ammonium ion concentrations, the mechanism of antibody production was suggested to be very complicated or not to be explained by elastic net regression analysis.

  11. Phosphatidylserine biosynthesis in cultured Chinese hamster ovary cells. II. Isolation and characterization of phosphatidylserine auxotrophs

    SciTech Connect

    Kuge, O.; Nishijima, M.; Akamatsu, Y.

    1986-05-05

    Chinese hamster ovary (CHO) cell mutants that required exogenously added phosphatidylserine for cell growth were isolated by using the replica technique with polyester cloth, and three such mutants were characterized. Labeling experiments on intact cells with /sup 32/Pi and L-(U-/sup 14/C)serine revealed that a phosphatidylserine auxotroph, designated as PSA-3, was strikingly defective in phosphatidylserine biosynthesis. When cells were grown for 2 days without phosphatidylserine, the phosphatidylserine content of PSA-3 was about one-third of that of the parent. In extracts of the mutant, the enzymatic activity of the base-exchange reaction of phospholipids with serine producing phosphatidylserine was reduced to 33% of that in the parent; in addition, the activities of base-exchange reactions of phospholipids with choline and ethanolamine in the mutant were also reduced to 1 and 45% of those in the parent, respectively. Furthermore, it was demonstrated that the serine-exchange activity in the parent was inhibited approximately 60% when choline was added to the reaction mixture whereas that in the mutant was not significantly affected. From the results presented here, we conclude the following. There are at least two kinds of serine-exchange enzymes in CHO cells; one (serine-exchange enzyme I) can catalyze the base-exchange reactions of phospholipids with serine, choline, and ethanolamine while the other (serine-exchange enzyme II) does not use the choline as a substrate. Serine-exchange enzyme I, in which mutant PSA-3 is defective, plays a major role in phosphatidylserine biosynthesis in CHO cells. Serine-exchange enzyme I is essential for the growth of CHO cells.

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

    PubMed

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

    2015-01-01

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

  13. Application of a nonradioactive method of measuring protein synthesis in industrially relevant Chinese hamster ovary cells.

    PubMed

    Dadehbeigi, Nazanin; Dickson, Alan James

    2013-01-01

    Due to the high medical and commercial value of recombinant proteins for clinical and diagnostic purposes, the protein synthesis machinery of mammalian host cells is the subject of extensive research by the biopharmaceutical industry. RNA translation and protein synthesis are steps that may determine the extent of growth and productivity of host cells. To address the problems of utilization of current radioisotope methods with proprietary media, we have focused on the application of an alternative method of measuring protein synthesis in recombinant Chinese hamster ovary (CHO) cells. This method employs puromycin as a nonradioactive label which incorporates into nascent polypeptide chains and is detectable by western blotting. This method, which is referred to as SUnSET, successfully demonstrated the expected changes in protein synthesis in conditions that inhibit and restore translation activity and was reproducibly quantifiable. The study of the effects of feed and sodium butyrate addition on protein synthesis by SUnSET revealed an increase following 1 h feed supplementation while a high concentration of sodium butyrate was able to decrease translation during the same treatment period. Finally, SUnSET was used to compare protein synthesis activity during batch culture of the CHO cell line in relation to growth. The results indicate that as the cells approached the end of batch culture, the global rate of protein synthesis declined in parallel with the decreasing growth rate. In conclusion, this method can be used as a "snapshot" to directly monitor the effects of different culture conditions and treatments on translation in recombinant host cells.

  14. Phosphorylation of 3-deazaguanosine by nicotinamide riboside kinase in Chinese hamster ovary cells.

    PubMed

    Saunders, P P; Tan, M T; Spindler, C D; Robins, R K

    1989-12-01

    The growth inhibitory activity of 3-deazaguanosine toward a mutant line (TGR-3) of Chinese hamster ovary cells deficient in hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) was substantially reversed by the simultaneous addition of nicotinamide riboside. The activities of most other ribonucleoside analogues tested were unaffected. The formation of cellular 3-deazaGMP and 3-deazaGTP from the ribonucleoside analogue, as measured by high-pressure liquid chromatography, was inhibited by the presence of nicotinamide riboside. The inhibition was dependent on concentration of 3-deazaguanosine and could also be demonstrated by following the metabolism of 3-deazaguanosine, labeled with 14C in the ribose moiety, to [14C]3-deazaGTP. In the presence of 100 microM nicotinamide riboside formation of the labeled triphosphate derivative of 3-deazaguanosine was undetectable. A 3-deazaguanosine phosphorylating activity was separated from other cellular kinases by DEAE-cellulose chromatography. Contaminating purine nucleoside phosphorylase (EC 2.4.2.1) was subsequently removed by sucrose density gradient centrifugation. The resulting enzyme preparation demonstrated the greatest activities with nicotinamide riboside and 3-deazaguanosine and, in addition, could also phosphorylate tiazofurin and guanosine to lesser, but significant, degrees. These and other observations suggest that 3-deazaguanosine, and perhaps other agents such as tiazofurin, may, at least in part, be phosphorylated by a nicotinamide ribonucleoside kinase in these cells. If so, it is possible that the activity of this agent in other types of cells in vivo could be dependent upon the presence of this enzyme and that it could be influenced by cellular concentrations of the natural pyridine nucleoside.

  15. DNA and chromosome breaks induced by iodine-123-labeled estrogen in Chinese hamster ovary cells

    SciTech Connect

    Schwartz, J.L. |; Mustafi, R.; Hughes, A.; DeSombre, E.R.

    1996-08-01

    The effects of the Auger electron-emitting isotope {sup 123}I, covalently bound to estrogen, on DNA single- and double-strand breakage and on chromosome breakage was determined in estrogen receptor-positive Chinese hamster ovary (CHO-ER) cells. Exposure to the {sup 123}I-labeled estrogen induced both single- and double-strand breaks with a ratio of single- to double-strand breaks of 2.8. The corresponding ratio with {sup 60}Co {gamma} rays was 15.6. The dose response was biphasic, suggesting either that receptor sites are saturated at high doses, or that there is a nonrandom distribution of breaks induced by the {sup 123}I-labeled estrogen. The {sup 123}I-labeled estrogen treatment induced chromosome aberrations with an efficiency of about 1 aberration for each 1000 disintegrations per cell. This corresponds to the mean lethal dose of {sup 123}I-labeled estrogen for these cells, suggesting that the lethal event induced by the Auger electron emitter bound to estrogen is a chromosome aberration. Most of the chromosome-type aberrations were dicentrics and rings, suggesting that {sup 123}I-labeled estrogen-induced chromosome breaks are rejoined. The F ratio, the ratio of dicentrics to centric rings, was 5.8 {+-} 1.7, which is similar to that seen with high-LET radiations. Our results suggest that {sup 123}I bound to estrogen is an efficient clastogenic agent, the cytotoxic damage produced by {sup 123}I bound to estrogen is very like damage induced by high-LET radiation, and the {sup 123}I in the estrogen receptor-DNA complex is probably in proximity to the sugar-phosphate backbone of the DNA. 40 refs., 7 figs.

  16. Glycoengineering of Chinese hamster ovary cells for enhanced erythropoietin N-glycan branching and sialylation.

    PubMed

    Yin, Bojiao; Gao, Yuan; Chung, Cheng-Yu; Yang, Shuang; Blake, Emily; Stuczynski, Mark C; Tang, Juechun; Kildegaard, Helene F; Andersen, Mikael R; Zhang, Hui; Betenbaugh, Michael J

    2015-11-01

    Sialic acid, a terminal residue on complex N-glycans, and branching or antennarity can play key roles in both the biological activity and circulatory lifetime of recombinant glycoproteins of therapeutic interest. In order to examine the impact of glycosyltransferase expression on the N-glycosylation of recombinant erythropoietin (rEPO), a human α2,6-sialyltransferase (ST6Gal1) was expressed in Chinese hamster ovary (CHO-K1) cells. Sialylation increased on both EPO and CHO cellular proteins as observed by SNA lectin analysis, and HPLC profiling revealed that the sialic acid content of total glycans on EPO increased by 26%. The increase in sialic acid content was further verified by detailed profiling of the N-glycan structures using mass spectra (MS) analysis. In order to enhance antennarity/branching, UDP-N-acetylglucosamine: α-1,3-D-mannoside β1,4-N-acetylglucosaminyltransferase (GnTIV/Mgat4) and UDP-N-acetylglucosamine:α-1,6-D-mannoside β1,6-N-acetylglucosaminyltransferase (GnTV/Mgat5), was incorporated into CHO-K1 together with ST6Gal1. Tri- and tetraantennary N-glycans represented approximately 92% of the total N-glycans on the resulting EPO as measured using MS analysis. Furthermore, sialic acid content of rEPO from these engineered cells was increased ∼45% higher with tetra-sialylation accounting for ∼10% of total sugar chains compared to ∼3% for the wild-type parental CHO-K1. In this way, coordinated overexpression of these three glycosyltransferases for the first time in model CHO-K1 cell lines provides a mean for enhancing both N-glycan branching complexity and sialylation with opportunities to generate tailored complex N-glycan structures on therapeutic glycoproteins in the future.

  17. Impact of graphene oxide on viability of Chinese hamster ovary and mouse hepatoma MH-22A cells.

    PubMed

    Batiuskaite, Danute; Grinceviciute, Nora; Snitka, Valentinas

    2015-08-01

    The evaluation of the cyto- and bio-compatibility is a critical step in the development of graphene oxide (GO) as a new promising material for in vivo biomedical applications. In this study, we report the impact of GO, with and without the addition of bovine serum albumin, on healthy (Chinese hamster ovary) and a cancer (mouse hepatoma MH-22A) cells viability and the estimation of the intracellular distribution of GO inside the cells in vitro. The viability tests were performed using a colony formation assay. The intracellular distribution of GO was estimated using Raman spectroscopy and imaging. The viability of both cell lines decreased with increasing concentration of graphene oxide (12.5-50.0 μg/ml): in the case of Chinese hamster ovary cells viability decreased from 44% to 11%, in the case of mouse hepatoma MH-22A cells--from 22% to 3%. These cell lines significantly differed in their response to GO and GO-BSA formulations. The results of viability tests correlate with results of atomic force microscopy and Raman spectroscopy and imaging findings. The GO influence on cell morphology changes, cell structure, cells colony growth dynamics and GO accumulation inside the cells was higher in the case of mouse hepatoma MH-22A cells.

  18. Understanding Transcriptional Enhancement in Monoclonal Antibody-Producing Chinese Hamster Ovary Cells

    NASA Astrophysics Data System (ADS)

    Nicoletti, Sarah E.

    With the demand for monoclonal antibody (mAB) therapeutics continually increasing, the need to better understand what makes a high productivity clone has gained substantial interest. Monoclonal antibody producing Chinese hamster ovary (CHO) cells with different productivities were provided by a biopharmaceutical company for investigation. Gene copy numbers, mRNA levels, and mAb productivities were previously determined for two low producing clones and their amplified progeny. These results showed an increase in mRNA copy number in amplified clones, which correlated to the observed increases in specific productivity of these clones. The presence of multiple copies of mRNA per one copy of DNA in the higher productivity clones has been coined as transcriptional enhancement. The methylation status of the CMV promoter as well as transcription factor/promoter interactions were evaluated to determine the cause of transcriptional enhancement. Methylation analysis via bisulfite sequencing revealed no significant difference in overall methylation status of the CMV promoter. These data did, however, reveal the possibility of differential interactions of transcription factors between the high and low productivity cell clones. This finding was further supported by chromatin immunoprecipitations previously performed in the lab, as well as literature studies. Transcription activator-like effector (TALE) binding proteins were constructed and utilized to selectively immunoprecipitate the CMV promoter along with its associated transcription factors in the different CHO cell clones. Cells were transfected with the TALE proteins, harvested and subjected to a ChIP-like procedure. Results obtained from the TALE ChIP demonstrated the lack of binding of the protein to the promoter and the need to redesign the TALE. Overall, results obtained from this study were unable to give a clear indication as to the causes of transcriptional enhancement in the amplified CHO cell clones. Further

  19. Radiation-induced division delay in Chinese hamster ovary fibroblast and carcinoma cells: dose effect and ploidy. [X-ray

    SciTech Connect

    Kimler, B.F.; Leeper, D.B.; Schneiderman, M.H.

    1981-02-01

    The mitotic selection procedure for cell cycle analysis was utilized to investigate the G/sub 2/ transition point for and the duration of radiation-induced division delay in diploid and tetraploid Chinese hamster ovary (CHO) fibroblasts and in Chinese hamster ovarian carcinoma cells. The location of the radiation-induced division delay transition point was dose independent at high doses and located approximately 42 min before division. At lower doses only an estimate of the point of blockade was possible; but the G/sub 2/ transition point appeared to be earlier in the cell cycle. The duration of radiation-induced division delay was dose dependent. This response is consistent with a sensitive population of cells in late G/sub 2/ that define the location of the transition point and the length of division delay. There was no difference observed in the dose response for radiation-induced division delay between the pseudotetraploid cell line of CHO and the pseudodiploid parent strain. However, in the cell line derived from a spontaneous Chinese hamster ovarian carcinoma the division delay was 39 +- 4 min/Gy. Therefore, radiation-induced division delay is independent of chromosome ploidy, but can show intraspecies cell line specificity.

  20. Viable cell recycle with an inclined settler in the perfusion culture of suspended recombinant Chinese hamster ovary cells.

    PubMed

    Searles, J A; Todd, P; Kompala, D S

    1994-01-01

    The perfusion culture of suspended mammalian cells requires a cell retention device, the best of which will retain all viable cells and reject all nonviable cells and debris. The inclined settler is a passive, simple, inexpensive, and easy-to-maintain device that has been shown in the past to selectively remove single nonviable cells of hybridoma cultures. In this work, we have demonstrated the preferential return of viable recombinant Chinese hamster ovary (CHO) cells through the use of a three-port settler maintained at lower temperatures and vibrated to reduce cell attachment and enhance cell return to the bioreactor. The residence time of CHO cells in the cooled, vibrated settler was determined by flow-cytometric discrimination of tracer recombinant CHO cells. Cells returning to the bioreactor through the underflow had an average residence time of 1.46 h in the settler. During perfusion cultures with cell densities above 10(6) cells/mL, cells seen to be stalled within the settler were easily dislodged by periodic air bubbling using a simple back-flushing procedure in which headspace gas was brought through the settler underflow port. The resuspended cells were returned to the bioreactor within an average of 32 min after bubbling. This study demonstrates that inclined sedimentation technology can be utilized to selectively recycle viable recombinant CHO cells with only a short retention time in an inclined settler.

  1. Transfer of human genes conferring resistance to methylating mutagens, but not to UV irradiation and cross-linking agents, into Chinese hamster ovary cells

    SciTech Connect

    Kaina, B.; Van Zeeland, A.A.; Backendorf, C.; Thielmann, H.W.; Van de Putte, P.

    1987-05-01

    Chinese hamster ovary cells were transfected by human DNA ligated to the bacterial gpt (xanthine-guanine-phosphoribosyltransferase) gene which was used either in its native form or after partial inactivation with methylnitrosourea. The gpt+ transfectants were screened for resistance to high doses of N-methyl-N'-nitro-N-nitrosoguanidine. Using this approach, we showed that Chinese hamster ovary cells can acquire N-methyl-N'-nitro-N-nitrosoguanidine resistance upon transfection with DNA from diploid human fibroblasts, that this resistance is transferable by secondary transfection and is specific for methylating mutagens, and that it is not caused by increased removal of O6-methylguanine, 3-methyladenine, and 7-methylguanine from DNA.

  2. Effects of preventing O-glycosylation on the secretion of human chorionic gonadotropin in Chinese hamster ovary cells

    SciTech Connect

    Matzuk, M.M.; Krieger, M.; Corless, C.L.; Boime, I.

    1987-09-01

    Human chorionic gonadotropin (hCG) is a member of a family of heterodimeric glycoprotein hormones that have a common ..cap alpha.. subunit but differ in their hormone-specific ..beta..-subunits. The ..beta.. subunit of hCG (hCG..beta..) is unique among the ..beta.. subunits in that it contains four mucin-like O-linked oligosaccharides attached to a carboxyl-terminal extension. To study the effects of O-glycosylation on the secretion and assembly of hCG, expression vectors containing either hCG..beta.. gene alone or together with the hCG..cap alpha.. gene were transfected into a mutant Chinese hamster ovary cell line, 1d1D, which exhibits a reversible defect in O-glycosylation. The results reveal that hCG..beta.. can be secreted normally in the absence of its O-linked oligosaccharides. hCG..beta.. devoid of O-linked carbohydrate can also combine efficiently with hCG..cap alpha.. and be secreted as an intact dimer. The authors conclude that in Chinese hamster ovary cells, the hCG..beta.. O-linked chains play no role in the assembly and secretion of hCG. The normal and O-linked oligosaccharide-deficient forms of hCG secreted by these cells should prove useful in examining the role of O-linked chains on the biological function of hCG.

  3. Cell-cycle specific expression of a small proline-rich protein in Chinese hamster ovary cells

    SciTech Connect

    Tesfaigzi, J.

    1994-11-01

    Squamous metaplasia of the bronchial epithelium is generally believed to be involved in the neoplastic progression toward squamous cell carcinomas. Thus, it is important to understand the mechanisms controlling this type of differentiation. The induction of two families of cDNAs encoding a small proline-rich protein (sPRP), sprI and sprII, was first identified in human keratinocytes exhibiting squamous differentiation. cDNAs similar to sprI have also been identified in cultured tracheal epithelial cells undergoing squamous differentiation. The first step during the squamous differentiation process is the inhibition of cell growth; it has also been noted that a sPRP mRNA in Chinese hamster ovary (CHO) cells is induced 10-fold just before the cultures reach confluence. Thus, sPRP may stop cell division in cells undergoing squamous differentation. In support of this possibility are the recent investigations correlating expression of sPRP with cell morphology. Specific immunoreactivity to sPRP, using affinity-purified antibodies, showed a strong immunostaining in cells with a round configuration, while less staining was observed in other cells. The major part of the CHO population showed no immunoreactivity. One interpretation of this observation is that the expression of sPRP may be cell-cyle regulated. The purpose of this investigation was to determine the phase of the cell cycle where induced synthesis of sPRP mRNA occurs.

  4. Cell growth stimulating effect of Ganoderma lucidum spores and their potential application for Chinese hamster ovary K1 cell cultivation.

    PubMed

    Li, Ding; Zhong, Qi; Liu, Tingting; Wang, Jufang

    2016-06-01

    In this work, water-soluble extracts of Ganoderma lucidum spores (Gls), a Chinese medicinal herb that possesses cell growth stimulating function, were found to be an effective growth factor for Chinese hamster ovary (CHO) cell cultivation. The Gls extract was prepared and supplemented to CHO K1 cell culture media with various serum levels. Our results obtained from both the static culture and the spinner-flask suspension culture showed that use of small-amount Gls extract effectively promoted cell growth and suppressed cell apoptosis induced by serum deprivation with normal cell cycle maintained in a low-serum medium. The low-serum medium containing 1 % (v/v) fetal bovine serum (FBS) and 0.01 % (w/v) Gls extract showed a comparable performance on both cell growth and fusion protein productivity with the conventional CHO culture medium containing 10 % (v/v) FBS and a commercial serum-free medium. This is the first study of the potential of Gls extracts for use as an alternative cell growth factor and nutrient for CHO cells. The findings have presented a new approach to economic cultivation of CHO cells for therapeutic protein production.

  5. Effect of Vitreoscilla hemoglobin expression on growth and specific tissue plasminogen activator productivity in recombinant Chinese hamster ovary cells

    SciTech Connect

    Pendse, G.J.; Bailey, J.E. . Dept. of Chemical Engineering)

    1994-12-01

    Previous studies suggest that secretion of cloned proteins synthesized by recombinant Chinese hamster ovary (CHO) cells can be adenosine triphosphate (ATP) limited. Other research indicates that the presence of cloned Vitreoscilla hemoglobin (VHb) enhances ATP production in oxygen-limited Escherichia coli. To evaluate the influence of VHb expression on recombinant CHO cell productivity, the vhb gene has been fused to the mouse mammary tumor virus (MMTV) promoter and cloned in a CHO cell line previously engineered to express human tissue plasminogen activator (tPA). Western blot analysis confirms dexamethasone-inducible VHb expression in all of the clones tested. Batch cultivation experiments with one VHb-expressing clone and the parental CHO-tPA cells show a reduced specific growth rate in the VHb-expressing cells. The VHb-expressing clone exhibits specific tPA production 40 to 100% greater than the parental CHO-tPA culture.

  6. Reduced cytotoxicity in PCB-exposed Chinese Hamster Ovary (CHO) cells pretreated with vitamin E.

    PubMed

    Murati, Teuta; Šimić, Branimir; Pleadin, Jelka; Vukmirović, Maja; Miletić, Marina; Durgo, Ksenija; Kniewald, Jasna; Kmetič, Ivana

    2017-01-01

    The aim of this study was to evaluate protective effects of vitamin E (50 -150 μM) in ovary cells upon cytotoxic effects induced by two structurally distinct PCB congeners - planar "dioxin-like" PCB 77 and non-planar di-ortho-substituted PCB 153 with an emphasis on identifying differences in the mechanism of vitamin E action depending on the structure of congeners. Application of three bioassays confirmed that PCBs decrease ovarian cell proliferation with slightly profound effects of PCB 77. PCB - induced ROS production and lipid peroxidation were significant for both congeners with also more noticeable effect for PCB 77. Vitamin E pre-incubation has improved viability of cells, reduced ROS formation and lipid peroxidation induced by PCBs' treatment. Preincubation with vitamin E was more effective when cells where treated with non-planar PCB 153. Altogether, vitamin E action was protective, congener specific and more effective when ovary cells were exposed to ortho-substituted PCB congener.

  7. Sister chromatid exchange response of human diploid fibroblasts and Chinese hamster ovary cells to dimethylnitrosamine and benzo(a)pyrene

    SciTech Connect

    Tomkins, D.J.; Kwok, S.E.; Douglas, G.R.; Biggs, D.

    1982-01-01

    In the search for relevant assays for mutagenicity testing, considerable attention has been given to the use of mammalian cells in vitro and the incorporation of metabolic activation in the protocol. Chinese hamster ovary (CHO) cells are commonly chosen as the target cells for cytogenetic tests because of their excellent growth characteristics and long lifespan in culture. However, there may be cellular factors affecting the uptake, metabolism, and repair of damage which are not the same in cell lines. The response of CHO cells and three human diploid fibroblast strains (1MR-90, WI-38, S-3299) to benzo(a)pyrene (BP) and dimethylnitrosamine (DMN) were compared using sister chromatid exchange (SCE) analysis as a measure of genetic damage. For both BP and DMN the human cells and the CHO cells showed dose-response slopes that were significantly different from zero, except CHO cells treated with BP for 1 hr and S-3299 cells treated with DMN. Whereas human and CHO cells showed similar dose-response to BP and the three human cell strains had similar dose-responses to BP and DMN, the dose-response of the human cells to DMN was statistically less significant than that of CHO cells. Reducing the duration of chemical treatment in CHO cells had no effect on the slope of the dose-response curves for BP or DMN. The observed differences between human and CHO cells may reflect differences in the fate of metabolic intermediates of DMN.

  8. Regulation of cell growth and apoptosis through lactate dehydrogenase C over-expression in Chinese hamster ovary cells.

    PubMed

    Fu, Tuo; Zhang, Cunchao; Jing, Yu; Jiang, Cheng; Li, Zhenhua; Wang, Shengyu; Ma, Kai; Zhang, Dapeng; Hou, Sheng; Dai, Jianxin; Kou, Geng; Wang, Hao

    2016-06-01

    Lactate has long been credited as a by-product, which jeopardizes cell growth and productivity when accumulated over a certain concentration during the manufacturing process of therapeutic recombinant proteins by Chinese hamster ovary (CHO) cells. A number of efforts to decrease the lactate concentration have been developed; however, the accumulation of lactate is still a critical issue by the late stage of fed-batch culture. Therefore, a lactate-tolerant cell line was developed through over-expression of lactate dehydrogenase C (LDH-C). In fed-batch culture, sodium lactate or sodium pyruvate was supplemented into the culture medium to simulate the environment of lactate accumulation, and LDH-C over-expression increased the highest viable cell density by over 30 and 50 %, respectively, on day 5, meanwhile the viability was also improved significantly since day 5 compared with that of the control. The percentages of cells suffering early and late apoptosis decreased by 3.2 to 12.5 and 2.0 to 4.3 %, respectively, from day 6 onwards in the fed-batch culture when 40 mM sodium pyruvate was added compared to the control. The results were confirmed by mitochondrial membrane potential assay. In addition, the expression of cleaved caspases 3 and 7 decreased in cells over-expressing LDH-C, suggesting the mitochondrial pathway was involved in the LDH-C regulated anti-apoptosis. In conclusion, a novel cell line with higher lactate tolerance, lowered lactate production, and alleviated apoptosis response was developed by over-expression of LDH-C, which may potentially represent an efficient and labor-saving approach in generating recombinant proteins.

  9. Increased expression of the integral membrane proteins EGFR and FGFR3 in anti-apoptotic Chinese hamster ovary cell lines.

    PubMed

    Ohsfeldt, Erika; Huang, Szu-Han; Baycin-Hizal, Deniz; Kristoffersen, Linda; Le, Thuy-My T; Li, Edwin; Hristova, Kalina; Betenbaugh, Michael J

    2012-01-01

    Membrane proteins such as receptor tyrosine kinases (RTKs) have a vital role in many cellular functions, making them potential targets for therapeutic research. In this study, we investigated the coexpression of the anti-apoptosis gene Bcl-x(L) with model membrane proteins as a means of increasing membrane protein expression in mammalian cells. Chinese hamster ovary (CHO) cells expressing heterologous Bcl-x(L) and wild-type CHO cells were transfected with either epidermal growth factor receptor or fibroblast growth factor receptor 3. The CHO-Bcl-x(L) cell lines showed increased expression of both RTK proteins as compared with the wild-type CHO cell lines in transient expression analysis, as detected by Western blot and flow cytometry after 15 days of antibiotic selection in stable expression pools. Increased expression was also seen in clonal isolates from the CHO-Bcl-x(L) cell lines, whereas the clonal cell line expression was minimal in wild-type CHO cell lines. Our results demonstrate that application of the anti-apoptosis gene Bcl-x(L) can increase expression of RTK proteins in CHO cells. This approach may be applied to improve stable expression of other membrane proteins in the future using mammalian cell lines with Bcl-x(L) or perhaps other anti-apoptotic genes.

  10. /sup 31/P NMR analysis of membrane phospholipid organization in viable, reversibly electropermeabilized Chinese hamster ovary cells

    SciTech Connect

    Lopez, A.; Rols, M.P.; Teissie, J.

    1988-02-23

    Chinese hamster ovary (CHO) cells were reversibly permeabilized by submitting them to short, high-intensity, square wave pulses (1.8 kV/cm, 100 ..mu..s). The cells remained in a permeable state without loss of viability for several hours at 4/sup 0/C. A new anisotropic peak with respect to control cells was observed on /sup 31/P NMR spectroscopic analysis of the phospholipid components. This peak is only present when the cells are permeable, and normal anisotropy is recovered after resealing. Taking into account the fusogenicity of electropermeabilized cells, comparative studies were performed on 5% poly(ethylene glycol) treated cells. The /sup 31/P NMR spectra of the phospholipids displayed the same anisotropic peak as in the case of the electropermeabilized cells. In the two cases, this anisotropic peak was located downfield from the main peak associated to the phospholipids when organized in bilayers. The localization of this anisotropic peak is very different from the one of a hexagonal phase. The authors proposed a reorganization of the polar head group region leading to a weakening of the hydration layer to account for these observations. This was also thought to explain the electric field induced fusogenicity of these cells.

  11. A Systematic Approach to Time-series Metabolite Profiling and RNA-seq Analysis of Chinese Hamster Ovary Cell Culture.

    PubMed

    Hsu, Han-Hsiu; Araki, Michihiro; Mochizuki, Masao; Hori, Yoshimi; Murata, Masahiro; Kahar, Prihardi; Yoshida, Takanobu; Hasunuma, Tomohisa; Kondo, Akihiko

    2017-03-02

    Chinese hamster ovary (CHO) cells are the primary host used for biopharmaceutical protein production. The engineering of CHO cells to produce higher amounts of biopharmaceuticals has been highly dependent on empirical approaches, but recent high-throughput "omics" methods are changing the situation in a rational manner. Omics data analyses using gene expression or metabolite profiling make it possible to identify key genes and metabolites in antibody production. Systematic omics approaches using different types of time-series data are expected to further enhance understanding of cellular behaviours and molecular networks for rational design of CHO cells. This study developed a systematic method for obtaining and analysing time-dependent intracellular and extracellular metabolite profiles, RNA-seq data (enzymatic mRNA levels) and cell counts from CHO cell cultures to capture an overall view of the CHO central metabolic pathway (CMP). We then calculated correlation coefficients among all the profiles and visualised the whole CMP by heatmap analysis and metabolic pathway mapping, to classify genes and metabolites together. This approach provides an efficient platform to identify key genes and metabolites in CHO cell culture.

  12. Caffeine-induced Release of Intracellular Ca2+ from Chinese Hamster Ovary Cells Expressing Skeletal Muscle Ryanodine Receptor

    PubMed Central

    Bhat, Manjunatha B.; Zhao, Jiying; Zang, Weijin; Balke, C. William; Takeshima, Hiroshi; Wier, W. Gil; Ma, Jianjie

    1997-01-01

    The ryanodine receptor (RyR)/Ca2+ release channel is an essential component of excitation–contraction coupling in striated muscle cells. To study the function and regulation of the Ca2+ release channel, we tested the effect of caffeine on the full-length and carboxyl-terminal portion of skeletal muscle RyR expressed in a Chinese hamster ovary (CHO) cell line. Caffeine induced openings of the full length RyR channels in a concentration-dependent manner, but it had no effect on the carboxyl-terminal RyR channels. CHO cells expressing the carboxyl-terminal RyR proteins displayed spontaneous changes of intracellular [Ca2+]. Unlike the native RyR channels in muscle cells, which display localized Ca2+ release events (i.e., “Ca2+ sparks” in cardiac muscle and “local release events” in skeletal muscle), CHO cells expressing the full length RyR proteins did not exhibit detectable spontaneous or caffeine-induced local Ca2+ release events. Our data suggest that the binding site for caffeine is likely to reside within the amino-terminal portion of RyR, and the localized Ca2+ release events observed in muscle cells may involve gating of a group of Ca2+ release channels and/or interaction of RyR with muscle-specific proteins. PMID:9382901

  13. Phosphatidylserine biosynthesis in cultured Chinese hamster ovary cells. III. Genetic evidence for utilization of phosphatidylcholine and phosphatidylethanolamine as precursors

    SciTech Connect

    Kuge, O.; Nishijima, M.; Akamatsu, Y.

    1986-05-05

    We reported that Chinese hamster ovary (CHO) cells contain two different serine-exchange enzymes (I and II) which catalyze the base-exchange reaction of phospholipid(s) with serine and that a phosphatidylserine-requiring mutant (strain PSA-3) of CHO cells is defective in serine-exchange enzyme I and lacks the ability to synthesize phosphatidylserine. In this study, we examined precursor phospholipids for phosphatidylserine biosynthesis in CHO cells. When mutant PSA-3 and parent (CHO-K1) cells were cultured with (/sup 32/P)phosphatidylcholine, phosphatidylserine in the parent accumulated radioactivity while that in the mutant was not labeled significantly. On the contrary, when cultured with (/sup 32/P)phosphatidylethanolamine, the mutant incorporated the label into phosphatidylserine more efficiently than the parent. Furthermore, we found that mutant PSA-3 grew normally in growth medium supplemented with 30 microM phosphatidylethanolamine as well as phosphatidylserine and that the biosynthesis of phosphatidylserine in the mutant was normal when cells were cultured in the presence of exogenous phosphatidylethanolamine. The simplest interpretation of these findings is that phosphatidylserine in CHO cells is biosynthesized through the following sequential reactions: phosphatidylcholine----phosphatidylserine----phosphatidylethanolamine--- - phosphatidylserine. The three reactions are catalyzed by serine-exchange enzyme I, phosphatidylserine decarboxylase, and serine-exchange enzyme II, respectively.

  14. A Systematic Approach to Time-series Metabolite Profiling and RNA-seq Analysis of Chinese Hamster Ovary Cell Culture

    PubMed Central

    Hsu, Han-Hsiu; Araki, Michihiro; Mochizuki, Masao; Hori, Yoshimi; Murata, Masahiro; Kahar, Prihardi; Yoshida, Takanobu; Hasunuma, Tomohisa; Kondo, Akihiko

    2017-01-01

    Chinese hamster ovary (CHO) cells are the primary host used for biopharmaceutical protein production. The engineering of CHO cells to produce higher amounts of biopharmaceuticals has been highly dependent on empirical approaches, but recent high-throughput “omics” methods are changing the situation in a rational manner. Omics data analyses using gene expression or metabolite profiling make it possible to identify key genes and metabolites in antibody production. Systematic omics approaches using different types of time-series data are expected to further enhance understanding of cellular behaviours and molecular networks for rational design of CHO cells. This study developed a systematic method for obtaining and analysing time-dependent intracellular and extracellular metabolite profiles, RNA-seq data (enzymatic mRNA levels) and cell counts from CHO cell cultures to capture an overall view of the CHO central metabolic pathway (CMP). We then calculated correlation coefficients among all the profiles and visualised the whole CMP by heatmap analysis and metabolic pathway mapping, to classify genes and metabolites together. This approach provides an efficient platform to identify key genes and metabolites in CHO cell culture. PMID:28252038

  15. Carbamates: A study on genotoxic, cytotoxic, and apoptotic effects induced in Chinese hamster ovary (CHO-K1) cells.

    PubMed

    Soloneski, Sonia; Kujawski, Maciej; Scuto, Anna; Larramendy, Marcelo L

    2015-08-01

    In vitro effects of the carbamates pirimicarb and zineb and their formulations Aficida® (50% pirimicarb) and Azzurro® (70% zineb), respectively, were evaluated in Chinese hamster ovary (CHO-K1) cells. Whereas the cytokinesis-blocked micronucleus cytome assay was employed to test for genotoxicity, MTT, neutral red (NR), and apoptosis evaluation were used as tests for estimating cell viability and succinic dehydrogenase activity, respectively. Concentrations tested were 10-300 μg/ml for pirimicarb and Aficida®, and 1-50 μg/ml for zineb and Azzurro®. All compounds were able to increase the frequency of micronuclei. A marked reduction in the nuclear division index was observed after treatment with 5 μg/ml of zineb and Azzurro® and 10 μg/ml of Azzurro®. Alterations in the cellular morphology not allowing the recognition of binucleated cells exposed to 300 μg/ml pirimicarb and Aficida® as well as 10-50 μg/ml zineb and Azzurro®. All four compounds induced inhibition of both cell viability and succinic dehydrogenase activity and trigger apoptosis in CHO-K1 cells, at least when exposed for 24 h. The data herein demonstrate the genotoxic and cytotoxic effects exerted by these carbamates and reveal the potential risk factor of these pesticides, still extensively used worldwide, for both human health and the environment.

  16. High-antibody-producing Chinese hamster ovary cells up-regulate intracellular protein transport and glutathione synthesis.

    PubMed

    Orellana, Camila A; Marcellin, Esteban; Schulz, Benjamin L; Nouwens, Amanda S; Gray, Peter P; Nielsen, Lars K

    2015-02-06

    Chinese hamster ovary (CHO) cells are the preferred production host for therapeutic monoclonal antibodies (mAb) due to their ability to perform post-translational modifications and their successful approval history. The completion of the genome sequence for CHO cells has reignited interest in using quantitative proteomics to identify markers of good production lines. Here we applied two different proteomic techniques, iTRAQ and SWATH, for the identification of expression differences between a high- and low-antibody-producing CHO cell lines derived from the same transfection. More than 2000 proteins were quantified with 70 of them classified as differentially expressed in both techniques. Two biological processes were identified as differentially regulated by both methods: up-regulation of glutathione biosynthesis and down-regulation of DNA replication. Metabolomic analysis confirmed that the high producing cell line displayed higher intracellular levels of glutathione. SWATH further identified up-regulation of actin filament processes and intracellular transport and down regulation of several growth-related processes. These processes may be important for conferring high mAb production and as such are promising candidates for targeted engineering of high-expression cell lines.

  17. Characterization of murine monoclonal antibodies that recognize defined epitopes of pertussis toxin and neutralize its toxic effect on Chinese hamster ovary cells.

    PubMed Central

    Walker, M J; Wehland, J; Timmis, K N; Raupach, B; Schmidt, M A

    1991-01-01

    Three murine monoclonal antibodies (MAb), E19, E205, and E251, raised against pertussis toxin reacted in Western blots (immunoblots) with the S1, S4, and S2-S3 subunits, respectively, and neutralized the Chinese hamster ovary cell-clustering activity of pertussis toxin. MAb E251 recognized a linear synthetic peptide corresponding to amino acids 107 to 120 of the S2 subunit, suggesting a role for this region in receptor binding. Images PMID:1718872

  18. Studies on cytotoxic and genotoxic effects of cadmium nitrate and lead nitrate in Chinese hamster ovary cells

    SciTech Connect

    Ruey H. Lin; Ching H. Lee; Wen K. Chen

    1994-12-31

    Cadmium nitrate decreased the viability of Chinese hamster ovary (CHO) cells in a concentration-dependent manner; 50% inhibition (IC{sub 50}) was achieved at 0.015 mM. In contrast, lead nitrate appeared to be less toxic. Neither cadmium nitrate nor lead nitrate significantly increased frequencies of binucleated CHO cells with micronuclei (MN). However, both cadmium nitrate and lead nitrate could augment sister chromatid exchanges (SCEs). Cadmium nitrate induced SCEs with a potency approximately equal to that of mitomycin C and more than 10 times higher than lead nitrate. Cadmium nitrate also increased chromosome aberrations (CAs), which included breaks, acentrics, interchanges, and dicentrics of chromosomes. In addition, cadmium nitrate induced a decrease in the mitotic index (MI), but lead nitrate increased it. In summary, it appears that both of these two heavy metal salts have cytogenetic toxicities with different degrees of effects on the cytotoxicity, MN, CAs, and SCEs and CHO cells. However, SCE was the most sensitive endpoint for indicating mutagenetic effects of cadmium and lead in the present study. 31 refs., 3 figs., 3 tabs.

  19. Molecular characterization of the AdeI mutant of Chinese hamster ovary cells: a cellular model of adenylosuccinate lyase deficiency.

    PubMed

    Vliet, Lydia K; Wilkinson, Terry G; Duval, Nathan; Vacano, Guido; Graham, Christine; Zikánová, Marie; Skopova, Vaclava; Baresova, Veronika; Hnízda, Aleš; Kmoch, Stanislav; Patterson, David

    2011-01-01

    Adenylosuccinate lyase (ADSL, E. C. 4.3.2.2) carries out two non-sequential steps in de novo AMP synthesis, the conversion of succinylaminoimidazole carboxamide ribotide (SAICAR) to aminoimidazolecarboxamide ribotide (AICAR) and the conversion of succinyl AMP (AMPS) to AMP. In humans, mutations in ADSL lead to an inborn error of metabolism originally characterized by developmental delay, often with autistic features. There is no effective treatment for ADSL deficiency. Hypotheses regarding the pathogenesis include toxicity of high levels of SAICAR, AMPS, or their metabolites, deficiency of the de novo purine biosynthetic pathway, or lack of a completely functional purine cycle in muscle and brain. One important approach to understand ADSL deficiency is to develop cell culture models that allow investigation of the properties of ADSL mutants and the consequences of ADSL deficiency at the cellular level. We previously reported the isolation and initial characterization of mutants of Chinese hamster ovary (CHO-K1) cells (AdeI) that lack detectable ADSL activity, accumulate SAICAR and AMPS, and require adenine for growth. Here we report the cDNA sequences of ADSL from CHO-K1 and AdeI cells and describe a mutation resulting in an alanine to valine amino acid substitution at position 291 (A291V) in AdeI ADSL. This substitution lies in the "signature sequence" of ADSL, inactivates the enzyme, and validates AdeI as a cellular model of ADSL deficiency.

  20. TOXICOLOGY STUDIES OF LEWISITE AND SULFUR MUSTARD AGENTS:GENETIC TOXICITY OF LEWISITE (L) IN CHINESE HAMSTER OVARY CELLS

    SciTech Connect

    Jostes,R.F. Jr.; Sasser, LB; Rausch, R.J.

    1989-05-31

    The cytotoxic clastogenic and mutagenic effects of the arsenic containing vesicant, Lewisite (L) [dichloro(2-chlorovinyl) arsine], have been investigated using Chinese hamster ovary cells. One hour exposures to Lewisite were cytotoxic in uM amounts. The cell survival response yields a D37 of 0.6 uM and an extrapolation number of 2.5. The mutagenic response at the hypoxantnine-guanine phosporibosyl transferase (HGPRT) locus was sporadic and not significantly greater than control values when cells were exposed over a range of 0.125 to2.0 uM. Sister chromatid exchange (SCE) induction, a measure of chromosomal rearrangement, was weakly positive over a range of 0.25 to 1.0 uM but the values were not significantly greater than the control response. Chromosomal aberrations were induced at 0.75 and 1.0 UMin one experiment and 0.5 and 0.75 uM in another experiment. The Induced values were significantly greater than the control values. Lewisite appears to be cytotoxic and clastogenic in our investigations but SCE and mutation at the HGPRT locus are not significantly greater than control values. Lewisita toxicity was in some ways similar to radiomimetic chemicals such as bleomycin.

  1. Novel Role of ER Stress and Autophagy in Microcystin-LR Induced Apoptosis in Chinese Hamster Ovary Cells

    PubMed Central

    Zhang, Shenshen; Liu, Chuanrui; Li, Yang; Imam, Mustapha U.; Huang, Hui; Liu, Haohao; Xin, Yongjuan; Zhang, Huizhen

    2016-01-01

    Microcystin-LR (MC-LR) is a ubiquitous peptide that exhibits strong reproductive toxicity, although the mechanistic basis for such toxicity remains largely unknown. The present study was conducted to investigate the mechanisms underlying the adverse effects of exposure to MC-LR in Chinese hamster ovary (CHO) cells. The results showed that MC-LR inhibited the in vitro proliferation of CHO cells significantly, with an IC50 of 10 μM. Moreover, MC-LR-treated CHO cells revealed strong induction of cell cycle arrest and apoptosis. Additionally, exposure of CHO cells to MC-LR resulted in excess reactive oxygen species production and intracellular calcium release, with resultant endoplasmic reticulum stress (ERs). There was also extensive accumulation of autophagic vacuoles with the highest concentration of MC-LR used (10 μM). Furthermore, the expression of ERs (GRP78, ATF-6, PERK, IRE1, CHOP) and autophagy (Beclin1 and LC3II) proteins was increased, with concomitantly reduced expression of LC3I suggesting that ERs and autophagy were induced in CHO cells by MC-LR treatment. Conversely, pretreatment of CHO cells with 4-Phenyl butyric acid, the ERs inhibitor reduced the MC-LR-induced apoptotic cell death and cellular autophagy as evidenced by the reduced expression of Beclin1 and LC3II. Similarly, MC-LR treatment in combination with an autophagy inhibitor (3-methyladenine) increased apoptotic cell death compared with MC-LR alone, and induced ERs via upregulating ERs proteins. The overall results indicated that activation of ERs and autophagy are both associated with MC-LR-induced apoptosis in CHO cells. ERs may be a trigger of autophagy in this process. PMID:27877136

  2. Molecular nature of spontaneous mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in Chinese hamster ovary cells.

    PubMed

    Xu, Z; Yu, Y; Schwartz, J L; Meltz, M L; Hsie, A W

    1995-01-01

    The hypoxanthine-guanine phosphoribosyltransferase (hprt) locus has been widely used as a selectable genetic marker for studies of mammalian cell mutagenesis. We report here the spontaneous mutation spectrum at the hprt locus in 64 independently isolated mutants of Chinese hamster ovary (CHO) cells. All nine hprt exons were simultaneously analyzed via multiplex polymerase chain reaction (PCR) for rapid detection of gene deletions or insertions. Structural point mutations were identified by direct sequence analysis of the PCR amplified cDNA. The molecular nature of RNA splicing errors and insertions was analyzed by solid-phase direct exon sequencing. Single base substitutions were found in 24 mutants (38%), of which 21 were missense and 3 were nonsense mutations. Transversions were about twice as frequent as transitions. Fifteen mutants (23%) had deletions involving either intragenic small fragments (2), single exons (9), or multiple exons (4). The majority of deletion breakpoints (71%) were located in regions surrounding exons 4, 5, and 6. RNA splicing mutations were observed in 15 mutants (23%) and affected exons 3-8; most (6/15) resulted in the loss of exon 7. Two insertion mutants, one with a 209 bp insert in exon 4 and the other with a 88 bp insert accompanied by a 24 bp deletion in exon 6, represent novel mutations reported for the first time in spontaneous mutants of the mammalian hprt gene.

  3. Improved transgene integration into the Chinese hamster ovary cell genome using the Cre-loxP system.

    PubMed

    Inao, Takanori; Kawabe, Yoshinori; Yamashiro, Takuro; Kameyama, Yujiro; Wang, Xue; Ito, Akira; Kamihira, Masamichi

    2015-07-01

    Genetic engineering of cellular genomes has provided useful tools for biomedical and pharmaceutical studies such as the generation of transgenic animals and producer cells of biopharmaceutical proteins. Gene integration using site-specific recombinases enables precise transgene insertion into predetermined genomic sites if the target site sequence is introduced into a specific chromosomal locus. We previously developed an accumulative site-specific gene integration system (AGIS) using Cre and mutated loxPs. The system enabled the repeated integration of multiple transgenes into a predetermined locus of a genome. In this study, we explored applicable mutated loxP pairs for AGIS to improve the integration efficiency. The integration efficiencies of 52 mutated loxP sequences, including novel sequences, were measured using an in vitro evaluation system. Among mutated loxP pairs that exhibited a high integration efficiency, the applicability of the selected pairs to AGIS was confirmed for transgene integration into the Chinese hamster ovary cell genome. The newly found mutated loxP pairs should be useful for Cre-mediated integration of transgenes and AGIS.

  4. Modulation of [(35)S]GTPgammaS binding to chinese hamster ovary cell membranes by D(2(short)) dopamine receptors.

    PubMed

    Terasmaa, A; Finnman, U B; Owman, C; Ferré, S; Fuxe, K; Rinken, A

    2000-02-18

    Rat dopamine D(2short) expressed in Chinese hamster ovary (CHO) cells were characterized by means of activation of [(35)S]-guanosine 5'-O-(gamma-thiotriphosphate) ([(35)S]GTPgammaS) binding and inhibition of [(3)H]raclopride binding. Among 18 dopaminergic ligands studied dopamine, NPA, apomorphine and quinpirole were full agonists in activation of [(35)S]GTPgammaS binding, while seven ligands were partial agonists with efficacies from 16 to 69% of the effect of dopamine and seven ligands were antagonists having no effect on the basal level of [(35)S]GTPgammaS binding, but inhibited dopamine-dependent activation in a dose-response manner. Despite the different efficacies, the potencies of all 18 ligands to modulate [(35)S]GTPgammaS binding revealed a good correlation with their potencies to inhibit [(3)H]raclopride binding in the CHO cell membranes. This indicates that the binding of the ligand to the receptor determines its potency, but has no direct correlation with its intrinsic activity.

  5. Induction of chromosomal aberrations by the fuel additive methylcyclopentadienyl-manganese tricarbonyl (MMT) in Chinese hamster ovary cells

    SciTech Connect

    Blakey, D.H.; Bayley, J.M.

    1995-11-01

    Methylcyclopentadienyl-manganese tricarbonyl (MMT) is a fuel additive used throughout Canada as replacement for lead-based antiknock compounds in gasoline and as an anti-smoking compound in other fuels. Because of the widespread use of MMT in Canadian gasoline, it is important to determine whether MMT is a safe alternative to alkyllead as a fuel additive. Although environmental exposure to MMT is unlikely because it is almost completely consumed during combustion and any MMT exhaust emissions would be degraded rapidly, human contact can occur occupationally through accidental exposure, or incidentally while refuelling gasoline-powered engines. In order to determine the intrinsic mutagenicity of MMT, an in vitro chromosomal aberration assay was performed using Chinese hamster ovary cells. In the presence of metabolic activation, MMT was a potent inducer of structural chromosomal aberrations. There was significant (p{le}0.0114), reproducible increase in chromosomal aberrations at concentrations as low as 0.02 {mu}l/ml (0.12 mM). Without metabolic activation, MMT failed to induce a significant increase in chromosomal aberrations following either a 3 hr (p = 0.412) or continuous (p = 0.178) exposure. In order to determine whether the intrinsic mutagenicity identified in vitro is expressed in vivo, a mouse bone marrow micronucleus assay will be performed. In addition, the mutagenicity of MMT combustion byproducts will be evaluated.

  6. Contribution of chlorination to the mutagenic activity of drinking water extracts in Salmonella and Chinese hamster ovary cells

    SciTech Connect

    Douglas, G.R.; Nestmann, E.R.; Lebel, G.

    1986-11-01

    The production of chlorinated by-products through chlorine disinfection of drinking water has been well documented. Natural organic precursors for these chemicals include fulvic and humic acids, the chlorination of which leads to the production of mutagenic compounds. Comparisons of extracts of raw versus treated waters have confirmed that clorination during water treatment produces mutagenic activity in the Salmonella (Ames) test. Present work on XAD-2 extracts of raw and chlorinated water from six municipalities in the Great Lakes region of Canada has involved a battery of mutagenicity assays for various genetic endpoints: the Salmonella test, the sister-chromatid exchange (SCE) and the micronucleus (MN) induction in Chinese hamster ovary (CHO) cells. All extracts of treated (chlorinated), but none of untreated, water were mutagenic in the Salmonella assay. On the other hand, extracts of both treated and untreated water samples showed activity in the SCE and MN assays, but no consistent pattern of response with regard to treatment (chlorination) was evident. These data show that chlorination contributes mutagens to drinking water and suggest that mammalian in vitro assays may be more sensitive for detecting mutagenicity in water samples than the Salmonella test.

  7. Multi-omic profiling -of EPO-producing Chinese hamster ovary cell panel reveals metabolic adaptation to heterologous protein production.

    PubMed

    Ley, Daniel; Seresht, Ali Kazemi; Engmark, Mikael; Magdenoska, Olivera; Nielsen, Kristian Fog; Kildegaard, Helene Faustrup; Andersen, Mikael Rørdam

    2015-11-01

    Chinese hamster ovary (CHO) cells are the preferred production host for many therapeutic proteins. The production of heterologous proteins in CHO cells imposes a burden on the host cell metabolism and impact cellular physiology on a global scale. In this work, a multi-omics approach was applied to study the production of erythropoietin (EPO) in a panel of CHO-K1 cells under growth-limited and unlimited conditions in batch and chemostat cultures. Physiological characterization of the EPO-producing cells included global transcriptome analysis, targeted metabolome analysis, including intracellular pools of glycolytic intermediates, NAD(P)H/NAD(P)(+) , adenine nucleotide phosphates (ANP), and extracellular concentrations of sugars, organic acids, and amino acids. Potential impact of EPO expression on the protein secretory pathway was assessed at multiple stages using quantitative PCR (qPCR), reverse transcription PCR (qRT-PCR), Western blots (WB), and global gene expression analysis to assess EPO gene copy numbers, EPO gene expression, intracellular EPO retention, and differentially expressed genes functionally related to secretory protein processing, respectively. We found no evidence supporting the existence of production bottlenecks in energy metabolism (i.e., glycolytic metabolites, NAD(P)H/NAD(P)(+) and ANPs) in batch culture or in the secretory protein production pathway (i.e., gene dosage, transcription and post-translational processing of EPO) in chemostat culture at specific productivities up to 5 pg/cell/day. Time-course analysis of high- and low-producing clones in chemostat culture revealed rapid adaptation of transcription levels of amino acid catabolic genes in favor of EPO production within nine generations. Interestingly, the adaptation was followed by an increase in specific EPO productivity.

  8. Chromosomal damage and repair in G{sub 1}-phase Chinese hamster ovary cells exposed to charged-particle beams

    SciTech Connect

    Goodwin, E.H.; Blakely, E.A.; Tobias, C.A.

    1994-06-01

    Chromosomal fragmentation was examined in G{sub 1}-phase Chinese hamster ovary cells using the premature chromosome condensation (PCC) technique. The yield and distribution of chromatin breaks, the lesions revealed by PCC, were measured in cells exposed to X rays or each of nine particle beams covering a range of LET from 0.56 to 2700 keV/{mu}m. The average number of breaks per cell was found to be linearly proportional to the fluence of high-LET neon ions (183 keV/{mu}m). Assuming a linear response for the other beams, the level of breakage per unit dose rose from a plateau at the lowest LET values to a peak in the 100-200 keV/{mu}m range and then declined continuously thereafter, eventually falling well below the low-LET plateau. The maximum breakage RBE was 1.5. The average number of breaks per particle traversal rose steadily from 0.006 to 11 breaks/cell as the LET increased from 0.56 to 2700 keV/{mu}m. The breaks were distributed randomly within the cell population after low-LET irradiation, but became progressively overdispersed with increasing LET. Rejoining of prematurely condensed chromosomes plus fragments was followed for up to 5 h for four particle beams having LET values between 0.56 and 183 keV/{mu}m. An LET-dependent trend toward higher levels of residual fragments was observed. 34 refs., 5 figs., 4 tabs.

  9. Induction and expression of mutations at multiple drug-resistance marker loci in Chinese hamster ovary cells

    SciTech Connect

    Adair, G.M.; Carver, J.H.

    1983-01-01

    We observed quantitative and qualitative differences in the mutability and mutagen-specificity of various drug-resistance marker loci in Chinese hamster ovary (THO) cells, which suggest that mammalian gene loci may differ in their relative mutability by a given mutagenic agent. We have used the CHO-AT3-2 multiple-marker mutagenesis assay system to examine the dose-dependent induction and kinetics of expression of mutations at four well-characterized, drug-resistance marker loci, after treatment with chemical agents which produce various types of DNA damage. The CHO-AT3-2 subline allows simultaneous quantitation and direct comparison of induced mutation frequencies at the hgprt, oua (Na/sup +//K/sup +/ ATPase), aprt, and tk loci. The agents tested in this study included ethyl methanesulfonate, methyl methanesulfonate, mitomycin C, ICR-191, benzo(a)pyrene, and dimethylnitrosamine. The expression kinetics and optimal expression times for each drug-resistance marker were determined in dose-response experiments in which cells from mutagen-treated populations were plated at 1-2-day intervals over a period of 10 days following mutagenesis. Comparison of induced mutation frequencies for each drug-resistance marker after mutagen treatments yielding equivalent cell survivals (equitoxic doses resulting in relative cell survivals of 0.37) revealed locus-specific differences in the relative mutagenicities of the agents tested. These results indicate that the apparent mutagenicity of a particular agent at a single genetic locus may not necessarily be an accurate indicator of that agent's mutagenic potential for the genome as a whole.

  10. Posttreatment with sodium arsenite alters the mutational spectrum induced by ultraviolet light irradiation in Chinese hamster ovary cells

    SciTech Connect

    Yang, Jia-Ling; Chen, Mei-Fang; Wu, Cheng-Wen; Lee, Te-Chang )

    1992-01-01

    Arsenic, a potent carcinogen, fails to induce gene mutations in mammalian cells. However, posttreatment of ultraviolet light (UV)-irradiated cells with sodium arsenite synergstically enhances the mutation frequency on the hypoxanthine (Guanine) phosphoribosyltransferase locus. To investigate the molecular mechanism of the comutagenic effects of sodium arsenite, the authors characterized the alternations of nucleotide sequences in 30 UV-induced and 39 sodium arsenite enhanced hprt mutants from CHinese hamster ovary K1 cells by direct sequencing of mRNA-PCR amplified cDNA. The majority of sequence alterations derived from UV irradiation (80%) and from sodium arsenite posttreatment (70%) were single base substitutions. UV irradiation induced all types of base substitutions. Among them, 57% were transversions. The frequency of transversion increased to 70% in sodium arsenite enhanced mutants. While base substitutions observed in UV-induced mutants were evenly distributed along with the whole coding region, exons 3 and 8 were most frequently mutated in sodium arsenite enhanced mutants. Sodium arsenite posttreatment did not alter the strand bias for mutation induction, i.e., 73% and 78%, of the mutations were located on the non-transcribed strand in UV-induced and sodium arsenite enhanced mutants, respectively. In contrast to UV-induced mutations, bases at the 5' position of TT and the 3' position of CT sequences were the most frequent mutation sites observed in sodium arsenite enhanced mutants. The authors hypothesize that sodium arsenite may interfere with the process of mutation fixation of TT and CT dimers during DNA replication. 50 refs., 2 figs., 6 tabs.

  11. Organization and genesis of dihydrofolate reductase amplicons in the genome of a methotrexate-resistant Chinese hamster ovary cell line.

    PubMed

    Ma, C; Looney, J E; Leu, T H; Hamlin, J L

    1988-06-01

    We have recently isolated overlapping recombinant cosmids that represent the equivalent of two complete dihydrofolate reductase (dhfr) amplicon types from the methotrexate-resistant Chinese hamster ovary (CHO) cell line CHOC 400. In the work described in this report, we used pulse-field gradient gel electrophoresis to analyze large SfiI restriction fragments arising from the amplified dhfr domains. The junction between the 260-kilobase type I amplicons (which are arranged in head-to-tail configurations in the genome) has been localized, allowing the construction of a linear map of the parental dhfr locus. We also show that the 220-kilobase type II amplicons are arranged as inverted repeat structures in the CHOC 400 genome and arose from the type I sequence relatively early in the amplification process. Our data indicate that there are a number of minor amplicon types in the CHOC 400 cell line that were not detected in previous studies; however, the type II amplicons represent ca. 75% of all the amplicons in the CHOC 400 genome. Both the type I and type II amplicons are shown to be composed entirely of sequences that were present in the parental dhfr locus. Studies of less resistant cell lines show that initial amplicons can be larger than those observed in CHOC 400. Once established, a given amplicon type appears to be relatively stable throughout subsequent amplification steps. We also present a modification of an in-gel renaturation method that gives a relatively complete picture of the size and variability of amplicons in the genome.

  12. Molecular structural analysis of HPRT mutations induced by thermal and epithermal neutrons in Chinese hamster ovary cells.

    PubMed

    Kinashi, Y; Sakurai, Y; Masunaga, S; Suzuki, M; Takagaki, M; Akaboshi, M; Ono, K

    2000-09-01

    Chinese hamster ovary (CHO) cells were exposed to thermal and epithermal neutrons, and the occurrence of mutations at the HPRT locus was investigated. The Kyoto University Research Reactor (KUR), which has been improved for use in neutron capture therapy, was the neutron source. Neutron energy spectra ranging from nearly pure thermal to epithermal can be chosen using the spectrum shifters and thermal neutron filters. To determine mutant frequency and cell survival, cells were irradiated with thermal and epithermal neutrons under three conditions: thermal neutron mode, mixed mode with thermal and epithermal neutrons, and epithermal neutron mode. The mutagenicity was different among the three irradiation modes, with the epithermal neutrons showing a mutation frequency about 5-fold that of the thermal neutrons and about 1.5-fold that of the mixed mode. In the thermal neutron and mixed mode, boron did not significantly increase the frequency of the mutants at the same dose. Therefore, the effect of boron as used in boron neutron capture therapy (BNCT) is quantitatively minimal in terms of mutation induction. Over 300 independent neutron-induced mutant clones were isolated from 12 experiments. The molecular structure of HPRT mutations was determined by analysis of all nine exons by multiplex polymerase chain reaction. In the thermal neutron and mixed modes, total and partial deletions were dominant and the fraction of total deletions was increased in the presence of boron. In the epithermal neutron mode, more than half of the mutations observed were total deletions. Our results suggest that there are clear differences between thermal and epithermal neutron beams in their mutagenicity and in the structural pattern of the mutants that they induce. Mapping of deletion breakpoints of 173 partial-deletion mutants showed that regions of introns 3-4, 7/8-9 and 9-0 are sensitive to the induction of mutants by neutron irradiation.

  13. Exploring the capabilities of fluorometric online monitoring on chinese hamster ovary cell cultivations producing a monoclonal antibody.

    PubMed

    Schwab, Karen; Amann, Thomas; Schmid, Jakob; Handrick, René; Hesse, Friedemann

    2016-11-01

    Online monitoring of Chinese hamster ovary fed-batch cell cultures via two-dimensional fluorescence spectroscopy (2DFS) was evaluated in this work. Particular attention was directed toward different process strategies regarding the use of nutrient-rich feed media and temperature shifts. These intentionally performed process manipulations broadened the variances in the obtained fluorescence spectra and this was suspected to hamper the generation of reliable soft sensors. Principal component analysis of the obtained fluorescence data showed that temperature shift and feeding strategy had a considerable impact on the fluorescence signals. Partial least square regression models were calculated for the prediction of glucose, lactate, monoclonal antibody (mAb), and viable cell concentrations (VCC). It was aimed to integrate all 2DFS datasets in the respective calibration models regardless of the process-strategy-dependent diversity. Contrary to the expectations, it was feasible to calibrate soft sensors for the online prediction of glucose (7 latent variables (LVs), Rcal2 = 0.97, rout mean squared error of prediction (RMSEP) = 1.1 g L(-1) ), lactate (5 LV; Rcal2 = 0.96; RMSEP = 0.5 g L(-1) ) and mAb concentrations (4 LV; Rcal2 = 0.99; RMSEP = 11.4 mg L(-1) ). Feeding and temperature shifts had the highest impact on the VCC model (3 LV; Rcal2 = 0.94; RMSEP 3.8 × 10(5) mL(-1) ), nevertheless the prediction of VCC from the fed-batch 2DFS data was feasible. The results strongly indicate that variances in the datasets due to the process strategy can be tolerated to some extent by the respective soft sensors. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1592-1600, 2016.

  14. Generation of high-producing cell lines by overexpression of cell division cycle 25 homolog A in Chinese hamster ovary cells.

    PubMed

    Lee, Kyoung Ho; Tsutsui, Tomomi; Honda, Kohsuke; Asano, Ryutaro; Kumagai, Izumi; Ohtake, Hisao; Omasa, Takeshi

    2013-12-01

    To improve the efficiency of conventional gene amplification systems, the effect of cell cycle modification during the gene amplification process on IgG production was investigated in Chinese hamster ovary (CHO) cells. The full-length cDNA of CHO cell division cycle 25 homolog A (Cdc25A) was introduced into CHO DG44 cells and the effects of CDC25A overexpression on the cell cycle, transgene copy number and IgG productivity were examined. Both wild-type and mutated CDC25A-overexpressing CHO cells showed a rapid increase in transgene copy number compared with mock cells during the gene amplification process, in both cell pools and individual clones. High-producing clones were obtained with high frequency in CDC25A-overexpressing cell pools. The specific production rate of the isolated clone CHO SD-S23 was up to 2.9-fold higher than that of mock cells in the presence of 250 nM methotrexate (MTX). Cell cycle analysis revealed that the G2 to M phase transition rate was increased ∼1.5-fold in CDC25A-overexpressing CHO cells under MTX treatment. Our results show the improvement of conventional gene amplification systems via cell cycle engineering at an early stage of cell line development.

  15. Formycin B-resistant mutants of Chinese hamster ovary cells: novel genetic and biochemical phenotype affecting adenosine kinase.

    PubMed Central

    Mehta, K D; Gupta, R S

    1983-01-01

    Stable mutants which are approximately three- and eightfold resistant to the pyrazolopyrimidine nucleosides formycin A and formycin B (FomR) have been selected in a single step from mutagenized Chinese hamster ovary cells. In cell extracts, the two FomR mutants which were examined were both found to contain no measurable activity of the enzyme adenosine kinase (AK). However, cross-resistance studies with other adenosine analogs such as toyocamycin and tubercidin show that these mutants are distinct from toyocamycin or tubercidin resistant (Toyr) mutants which also contain no measurable AK activity in cell extracts. Studies on the uptake and incorporation of [3H]adenosine and [3H]tubercidin by various mutants and parental cell lines show that unlike the Toyr mutants, which are severely deficient in the phosphorylation of these compounds, the FomR mutants possess nearly normal capacity to phosphorylate these compounds and incorporate them into cellular macromolecules. These results suggest that the FomR mutants contain normal levels of AK activity in vivo. In cell hybrids formed between FomR X FomS cells and FomR X Toyr cells, the formycin-resistant phenotype of both of the FomR mutants behaved codominantly. However, the extracts from these hybrid cells contained either congruent to 50% (FomR X FomS) or no measurable (FomR X Toyr) AK activity, indicating that the lesion in these mutants neither suppresses the wild-type AK activity nor complements the AK deficiency of the Toyr mutants. The presence of AK activity in the FomR mutants in vivo, but not in their cell extracts, along with the codominant behavior of the mutants in hybrids, indicates that the lesions in the FomR mutant are of a novel nature. It is suggested that the genetic lesion in these mutants affects AK activity indirectly and that it may involve an essential cellular function which exists in a complex form with AK. Some implications of these results regarding the mechanism of action of formycin B are

  16. Short hairpin RNA targeted to dihydrofolate reductase enhances the immunoglobulin G expression in gene-amplified stable Chinese hamster ovary cells.

    PubMed

    Wu, Suh-Chin; Hong, Willy W L; Liu, Jin-Hwang

    2008-09-08

    The dihydrofolate reductase (dhfr)/methotrexate (MTX) selection is a common method to conduct gene amplification in stable clones of Chinese hamster ovary (CHO) cells. We previously reported the use of a short hairpin RNA (shRNA) vector targeted to the dhfr gene resulted in improving the intracellular antigen expression in gene-amplified stable CHO cells [Hong, W.W., Wu, S.C., 2007. A novel RNA silencing vector to improve antigen expression and stability in Chinese hamster ovary cells. Vaccine 25 (20), 4103-4111]. Here we investigated the use of the dhfr-targeted shRNA vector for immunoglobulin G (IgG) expression in gene-amplified stable CHO cells. With the use of the dhfr-targeted shRNA vector, the gene-amplified CHO/dhFr(-) cells were found to increase IgG expression at 1.0 microM MTX by more than 100% and to improve the genomic stability of IgG expression in MTX-free cultures by approximately 30%. The use of the dhfr-targeted shRNA vector can enhance the IgG expression in the gene-amplified stable CHO cells and uphold the IgG expression in MTX-free cultures. Utilizing the dhfr-targeted shRNA vector may provide an alternative way to maneuver CHO cell factories for IgG production in cultures.

  17. Expression of vascular endothelial growth factor does not promote transformation but confers a growth advantage in vivo to Chinese hamster ovary cells.

    PubMed Central

    Ferrara, N; Winer, J; Burton, T; Rowland, A; Siegel, M; Phillips, H S; Terrell, T; Keller, G A; Levinson, A D

    1993-01-01

    Vascular endothelial growth factor (VEGF) is a mitogen with a specificity for endothelial cells in vitro and an angiogenic inducer in vivo. We tested the hypothesis that VEGF may confer on expressing cells a growth advantage in vivo. Dihydrofolatereductase--Chinese hamster ovary cells were transfected with expression vectors which direct the constitutive synthesis of VEGF. Neither the expression nor the exogenous administration of VEGF stimulated anchorage-dependent or anchorage-independent growth of Chinese hamster ovary cells in vitro. However, VEGF-expressing clones, unlike control cells, demonstrated an ability to proliferate in nude mice. Histologic examination revealed that the proliferative lesions were compact, well vascularized, and nonedematous. Ultrastructural analysis revealed that capillaries within the lesions were of the continuous type. These findings indicate that the expression of VEGF may confer on cells the ability to grow in vivo in the absence of transformation by purely paracrine mechanisms. Since VEGF is a widely distributed protein, this property may have relevance for a variety of physiological and pathological proliferative processes. Images PMID:8423215

  18. Folk medicine Terminalia catappa and its major tannin component, punicalagin, are effective against bleomycin-induced genotoxicity in Chinese hamster ovary cells.

    PubMed

    Chen, P S; Li, J H; Liu, T Y; Lin, T C

    2000-05-01

    Terminalia catappa L. is a popular folk medicine for preventing hepatoma and treating hepatitis in Taiwan. In this paper, we examined the protective effects of T. catappa leaf water extract (TCE) and its major tannin component, punicalagin, on bleomycin-induced genotoxicity in cultured Chinese hamster ovary cells. Pre-treatment with TCE or punicalagin prevented bleomycin-induced hgprt gene mutations and DNA strand breaks. TCE and punicalagin suppressed the generation of bleomycin-induced intracellular free radicals, identified as superoxides and hydrogen peroxides. The effectiveness of TCE and punicalagin against bleomycin-induced genotoxicity could be, at least in part, due to their antioxidative potentials.

  19. Hydrogen peroxide cytotoxicity under conditions of normal or reduced catalase activity in H2O2-sensitive and -resistant Chinese hamster ovary (CHO) cell variants.

    PubMed

    Cantoni, O; Guidarelli, A; Sestili, P; Mannello, F; Gazzanelli, G; Cattabeni, F

    1994-09-01

    H2O2-sensitive and -resistant sublines of Chinese Hamster Ovary (CHO) cells were tested for their sensitivity to the growth inhibitory effect elicited by increasing concentrations of the oxidant under conditions of normal or reduced catalase activity. Experimental results have demonstrated that, under conditions of reduced catalase activity, the cytotoxic action of H2O2 was differentially regulated in resistant and sensitive cells. Indeed, the parental cell line and cells resistant to low concentrations of H2O2 (V 250 cells) depended on catalase to a lower extent than did highly resistant cells (V 850 cells). It is interesting to note that V 250 cells had more catalase, on a per million cell basis, than V 850 cells. We conclude that acquired resistance to oxidative stress is not entirely dependent on catalase and that the contribution of catalase depends on the degree of resistance to the oxidant.

  20. Effects of asbestos fibers on cell division, cell survival, and formation of thioguanine-resistant mutants in Chinese hamster ovary cells

    SciTech Connect

    Kenne, K.; Ljungquist, S.; Ringertz, N.R.

    1986-04-01

    The ability of crocidolite fibers to induce point mutations and mitotic abnormalities in Chinese hamster ovary (CHO) cells was examined in cell cultures. The purpose has been to study the possibilities for establishing in vitro test methods to quantify genetic damage induced by asbestos and other mineral fibers. Results obtained with the CHO/hypoxanthine guanine phosphoribosyl transferase system indicated that crocidolite fibers per se do not significantly increase the number of thioguanine-resistant mutants. Crocidolite fibers also failed to potentiate the mutagenicity of benzo(a)pyrene. Time-lapse cinematography and microscopy showed that asbestos (crocidolite) fibers were markedly cytotoxic. Among surviving cells some underwent abnormal cell divisions which resulted in multi- and micronucleate cells. Many cells that contained a few asbestos fibers, however, underwent mitosis and successfully formed two mononucleate daughter cells capable of further divisions. Individual, fiber-containing cells were examined by time-lapse television recordings for 4-5 days. During this time period some cells underwent six divisions and generated an almost normal number of daughter cells. Cells which contained fibers that were longer or equivalent to the diameter of the mitotic cell (20 ..mu..m), showed different forms of mitotic abnormalities. The frequency of multinucleate cells was drastically increased following exposure to asbestos fibers. Only rarely, however, did these cells divide to produce viable daughter cells capable of continued cell multiplication. The frequency of multinucleate cells was dependent on the dose of exposure to asbestos fibers and could possible be used as an index of the degree of mitotic disturbances induced by mineral fibers.

  1. In Vitro Chromosome Aberrations Study in Chinese Hamster Ovary (CHO) Cells

    DTIC Science & Technology

    2016-06-07

    24, 1996 Experimental Start Date: Experimental Termination Date: Study Coŕpletion Date: 10.0 Test System: Exponentially growing CHO-K1 cells...by a decline in cell growth , or by achieving the highest possible concentration (i.e., 100.0%). If possible, the high dose was selected to give at...8.0) was detected in the toxicity test cultures at doses selected for clastogenic testing. 13.2 Toxicity Test: Exponentially growing cells seeded

  2. Functional nucleotide excision repair is required for the preferential removal of N-ethylpurines from the transcribed strand of the dihydrofolate reductase gene of Chinese hamster ovary cells.

    PubMed Central

    Sitaram, A; Plitas, G; Wang, W; Scicchitano, D A

    1997-01-01

    Transcription-coupled repair of DNA adducts is an essential factor that must be considered when one is elucidating biological endpoints resulting from exposure to genotoxic agents. Alkylating agents comprise one group of chemical compounds which modify DNA by reacting with oxygen and nitrogen atoms in the bases of the double helix. To discern the role of transcription-coupled DNA repair of N-ethylpurines present in discrete genetic domains, Chinese hamster ovary cells were exposed to N-ethyl-N-nitrosourea, and the clearance of the damage from the dihydrofolate reductase gene was investigated. The results indicate that N-ethylpurines were removed from the dihydrofolate reductase gene of nucleotide excision repair-proficient Chinese hamster ovary cells; furthermore, when repair rates in the individual strands were determined, a statistically significant bias in the removal of ethyl-induced, alkali-labile sites was observed, with clearance occurring 30% faster from the transcribed strand than from its nontranscribed counterpart at early times after exposure. In contrast, removal of N-ethylpurines was observed in the dihydrofolate reductase locus in cells that lacked nucleotide excision repair, but both strands were repaired at the same rate, indicating that transcription-coupled clearance of these lesions requires the presence of active nucleotide excision repair. PMID:9001209

  3. Splicing mutants and their second-site suppressors at the dihydrofolate reductase locus in Chinese hamster ovary cells.

    PubMed

    Carothers, A M; Urlaub, G; Grunberger, D; Chasin, L A

    1993-08-01

    Point mutants induced with a variety of mutagens at the dihydrofolate reductase (dhfr) locus in Chinese hamster ovary (CHO) cells were screened for aberrantly spliced dhfr mRNA by RNase protection and/or reverse transcriptase coupled with cDNA amplification by the polymerase chain reaction (PCR). Of 115 mutants screened, 28 were found to be affected in splicing. All exhibited less than 1% correct splicing, probably because the selection procedure was stringent. All 26 unique mutations were located within the consensus splice sequences; changes were found at 9 of 10 possible sites in this 25-kb six-exon gene. Mutations at the sites flanking the first and last exons resulted in the efficient recruitment of a cryptic site within each exon. In contrast, mutations bordering internal exons caused predominantly exon skipping. In many cases, multiple exons were skipped, suggesting the clustering of adjacent exons prior to actual splicing. Six mutations fell outside the well-conserved GU and AG dinucleotides. All but one were donor site single-base substitutions that decreased the agreement with the consensus and resulted in little or no correct splicing. Starting with five of these donor site mutants, we isolated 31 DHFR+ revertants. Most revertants carried a single-base substitution at a site other than that of the original mutation, and most had only partially regained the ability to splice correctly. The second-site suppression occurred through a variety of mechanisms: (i) a second change within the consensus sequence that produced a better agreement with the consensus; (ii) a change close to but beyond the consensus boundaries, as far as 8 bases upstream in the exon or 28 bases downstream in the intron; (iii) mutations in an apparent pseudo 5' site in the intron, 84 and 88 bases downstream of a donor site; and (iv) mutations that improved the upstream acceptor site of the affected exon. Taken together, these second-site suppressor mutations extend the definition of a

  4. uv excision-repair gene transfer in Chinese hamster ovary (CHO) cells

    SciTech Connect

    MacInnes, M.A.; Bingham, J.M.; Strniste, G.F.; Thompson, L.H.

    1983-01-01

    uvc-sensitive mutants of CHO cells provide a model system for molecular studies of DNA repair. We present our recent results which show that these mutants are competent recipients for plasmid marker gene transfer and incorporation of a putative CHO repair gene. The applicability and advantages of this system for interspecies human repair gene identification are discussed.

  5. Biosynthesis and turnover of the mannose 6-phosphate receptor in cultured Chinese hamster ovary cells.

    PubMed

    Sahagian, G G; Neufeld, E F

    1983-06-10

    The natural history of the mannose 6-phosphate receptor was examined by radiolabeling cells in monolayers or in suspension; the receptor was isolated by immuno- or affinity precipitation followed by polyacrylamide gel electrophoresis. The receptor was found to contain asparagine-linked oligosaccharide chains and phosphorylated serine residues. Newly made receptor was sensitive to endo-beta-N-acetylglucosaminidase H (endo-H) and was slowly converted to a mature endo-H resistant form; phosphate was found on the mature receptor only. The receptor had an apparent molecular weight of 215,000 at all times, as determined under reducing and denaturing conditions; unreduced receptor had a greater electrophoretic mobility, suggesting the presence of intrachain disulfide linkages. The synthesis of immunoreactive receptor occurred with a lag of 50 min and of functional receptor with a lag of 70 min, indicating a requirement for some post-translational event(s) for acquisition of immunoreactivity and binding activity. Maturation of asparagine-linked oligosaccharides was not the requisite modification, since endo-H sensitive or deglycosylated receptor bound to both antibody and to insoluble phosphomannan; however, much less immunoreactive and functional receptor was detected in the presence of tunicamycin. Immunoprecipitable [3H]leucine-labeled receptor was degraded with a t1/2 of 16 h and 6 h for cells in monolayers and suspension, respectively, whereas 32P was lost with a corresponding t1/2 of 2.3 and 4 h. A pool of cell surface mannose 6-phosphate receptor was identified by separation on Percoll gradients as well as by iodination of cells with 125I; receptor in this pool was resistant to endo-H and had a t1/2 similar to that of the total [3H]leucine-labeled receptor, even in the presence of a saturating concentration of ligand. During endocytosis, ligand (beta-galactosidase) and 125I-receptor separated, the ligand accumulating within lysosomes. These results are consistent

  6. Matrix attachment region combinations increase transgene expression in transfected Chinese hamster ovary cells.

    PubMed

    Zhao, Chun-Peng; Guo, Xiao; Chen, Si-Jia; Li, Chang-Zheng; Yang, Yun; Zhang, Jun-He; Chen, Shao-Nan; Jia, Yan-Long; Wang, Tian-Yun

    2017-02-20

    Matrix attachment regions (MARs) are cis-acting DNA elements that can increase transgene expression levels in a CHO cell expression system. To investigate the effects of MAR combinations on transgene expression and the underlying regulatory mechanisms, we generated constructs in which the enhanced green fluorescent protein (eGFP) gene flanked by different combinations of human β-interferon and β-globin MAR (iMAR and gMAR, respectively), which was driven by the cytomegalovirus (CMV) or simian virus (SV) 40 promoter. These were transfected into CHO-K1 cells, which were screened with geneticin; eGFP expression was detected by flow cytometry. The presence of MAR elements increased transfection efficiency and transient and stably expression of eGFP expression under both promoters; the level was higher when the two MARs differed (i.e., iMAR and gMAR) under the CMV but not the SV40 promoter. For the latter, two gMARs showed the highest activity. We also found that MARs increased the ratio of stably transfected positive colonies. These results indicate that combining the CMV promoter with two different MAR elements or the SV40 promoter with two gMARs is effective for inducing high expression level and stability of transgenes.

  7. Matrix attachment region combinations increase transgene expression in transfected Chinese hamster ovary cells

    PubMed Central

    Zhao, Chun-Peng; Guo, Xiao; Chen, Si-Jia; Li, Chang-Zheng; Yang, Yun; Zhang, Jun-He; Chen, Shao-Nan; Jia, Yan-Long; Wang, Tian-Yun

    2017-01-01

    Matrix attachment regions (MARs) are cis-acting DNA elements that can increase transgene expression levels in a CHO cell expression system. To investigate the effects of MAR combinations on transgene expression and the underlying regulatory mechanisms, we generated constructs in which the enhanced green fluorescent protein (eGFP) gene flanked by different combinations of human β-interferon and β-globin MAR (iMAR and gMAR, respectively), which was driven by the cytomegalovirus (CMV) or simian virus (SV) 40 promoter. These were transfected into CHO-K1 cells, which were screened with geneticin; eGFP expression was detected by flow cytometry. The presence of MAR elements increased transfection efficiency and transient and stably expression of eGFP expression under both promoters; the level was higher when the two MARs differed (i.e., iMAR and gMAR) under the CMV but not the SV40 promoter. For the latter, two gMARs showed the highest activity. We also found that MARs increased the ratio of stably transfected positive colonies. These results indicate that combining the CMV promoter with two different MAR elements or the SV40 promoter with two gMARs is effective for inducing high expression level and stability of transgenes. PMID:28216629

  8. The Use of Transcription Terminators to Generate Transgenic Lines of Chinese Hamster Ovary Cells (CHO) with Stable and High Level of Reporter Gene Expression

    PubMed Central

    Gasanov, N. B.; Toshchakov, S. V.; Georgiev, P. G.; Maksimenko, O. G.

    2015-01-01

    Mammalian cell lines are widely used to produce recombinant proteins. Stable transgenic cell lines usually contain many insertions of the expression vector in one genomic region. Transcription through transgene can be one of the reasons for target gene repression after prolonged cultivation of cell lines. In the present work, we used the known transcription terminators from the SV40 virus, as well as the human β- and γ-globin genes, to prevent transcription through transgene. The transcription terminators were shown to increase and stabilize the expression of the EGFP reporter gene in transgenic lines of Chinese hamster ovary (CHO) cells. Hence, transcription terminators can be used to create stable mammalian cells with a high and stable level of recombinant protein production. PMID:26483962

  9. Cholesterol oxidation switches the internalization pathway of endothelin receptor type A from caveolae to clathrin-coated pits in Chinese hamster ovary cells.

    PubMed

    Okamoto, Y; Ninomiya, H; Miwa, S; Masaki, T

    2000-03-03

    We investigated the mechanism of endothelin receptor type A (ETA) internalization in Chinese hamster ovary cells using two assays; flow cytometric quantification of cell surface myc-ETA and in situ localization of Cy5-labeled ET-1. In both assays, agonist-dependent internalization of myc-ETA was inhibited by nystatin and filipin, both of which disrupt internalization via caveolae, whereas it was barely affected by chlorpromazine and hypertonic sucrose, both of which disrupt internalization via clathrin-coated pits. In addition to myc-ETA, ET-1 caused intracellular translocation of caveolin-1 and this translocation was also blocked by nystatin but not by chlorpromazine. These results strongly argue that ETA is internalized via caveolae but not clathrin-coated pits. Treatment of the cells with cholesterol oxidase reduced cellular cholesterol and caused intracellular translocation of caveolin-1 but did not affect cell surface localization of myc-ETA. In cholesterol oxidase-treated cells, however, both chlorpromazine and hypertonic sucrose effectively blocked ET-1-induced myc-ETA internalization and nystatin was less effective than in untreated cells. Accordingly, expression of a dominant negative form of beta-arrestin blocked myc-ETA internalization in cholesterol oxidase-treated cells but not in untreated cells. These results suggest that, in Chinese hamster ovary cells, 1) agonist-occupied ETA can be internalized either via caveolae or clathrin-coated pits; 2) of the two, the former is the default pathway; and 3) the oxidative state of cell surface cholesterol is one of the factors involved in the pathway selection.

  10. The impact of homologous recombination repair deficiency on depleted uranium clastogenicity in Chinese hamster ovary cells: XRCC3 protects cells from chromosome aberrations, but increases chromosome fragmentation.

    PubMed

    Holmes, Amie L; Joyce, Kellie; Xie, Hong; Falank, Carolyne; Hinz, John M; Wise, John Pierce

    2014-04-01

    Depleted uranium (DU) is extensively used in both industry and military applications. The potential for civilian and military personnel exposure to DU is rising, but there are limited data on the potential health hazards of DU exposure. Previous laboratory research indicates DU is a potential carcinogen, but epidemiological studies remain inconclusive. DU is genotoxic, inducing DNA double strand breaks, chromosome damage and mutations, but the mechanisms of genotoxicity or repair pathways involved in protecting cells against DU-induced damage remain unknown. The purpose of this study was to investigate the effects of homologous recombination repair deficiency on DU-induced genotoxicity using RAD51D and XRCC3-deficient Chinese hamster ovary (CHO) cell lines. Cells deficient in XRCC3 (irs1SF) exhibited similar cytotoxicity after DU exposure compared to wild-type (AA8) and XRCC3-complemented (1SFwt8) cells, but DU induced more break-type and fusion-type lesions in XRCC3-deficient cells compared to wild-type and XRCC3-complemented cells. Surprisingly, loss of RAD51D did not affect DU-induced cytotoxicity or genotoxicity. DU induced selective X-chromosome fragmentation irrespective of RAD51D status, but loss of XRCC3 nearly eliminated fragmentation observed after DU exposure in wild-type and XRCC3-complemented cells. Thus, XRCC3, but not RAD51D, protects cells from DU-induced breaks and fusions and also plays a role in DU-induced chromosome fragmentation.

  11. Perturbation of N-linked oligosaccharide structure results in an altered incorporation of (/sup 3/H)palmitate into specific proteins in Chinese hamster ovary cells

    SciTech Connect

    Wellner, R.B.; Ghosh, P.C.; Roecklein, B.; Wu, H.C.

    1987-09-25

    Increased (/sup 3/H)palmitate incorporation into specific cellular proteins has been reported to occur in Chinese hamster ovary and yeast mutant cells. In this paper we report studies concerning the relationship between N-linked oligosaccharide structure and (/sup 3/H)palmitate incorporation into proteins of Chinese hamster ovary (CHO) cells. We have compared the incorporation of (/sup 3/H)palmitate into proteins of wild-type and four different mutant CHO cell lines defective in various steps of N-linked protein glycosylation. Sodium dodecyl sulfate-gel electrophoretic analysis showed that three of the mutants exhibited increased (/sup 3/H)palmitate incorporation into several CHO cellular proteins (approximately 30,000-38,000 molecular weight) as compared to the wild-type cells. One of the affected mutants which accumulates the Man5Gn2Asn intermediate structure was examined in detail. In agreement with earlier reports, virtually all of the (/sup 3/H) palmitate-labeled proteins of both wild-type and mutant cell lines are membrane-bound. Pretreatment of the mutant cell line with tunicamycin blocked the increased (/sup 3/H)palmitate incorporation into the two specific proteins (both of approximately 30,000 molecular weight) observed in untreated cells; the decreased incorporation of (/sup 3/H)palmitate into the 30,000 molecular weight species was accompanied by a concomitant increase in the incorporation of (/sup 3/H)palmitate into two proteins of approximately 20,000 molecular weight. Pretreatment of wild-type cells with tunicamycin also caused increased (/sup 3/H)palmitate incorporation into the 20,000 molecular weight species.

  12. Improved gene amplification by cell-cycle engineering combined with the Cre-loxP system in Chinese hamster ovary cells.

    PubMed

    Matsuyama, Rima; Tsutsui, Tomomi; Lee, Kyoung Ho; Onitsuka, Masayoshi; Omasa, Takeshi

    2015-12-01

    The dihydrofolate reductase gene amplification system is widely used in Chinese hamster ovary (CHO) cells for the industrial production of therapeutic proteins. To enhance the efficiency of conventional gene amplification systems, we previously presented a novel method using cell-cycle checkpoint engineering. Here, we constructed high-producing and stable cells by the conditional expression of mutant cell division cycle 25 homolog B (CDC25B) using the Cre-loxP system. A bispecific antibody-producing CHO DG44-derived cell line was transfected with floxed mutant CDC25B. After inducing gene amplification in the presence of 250 nM methotrexate, mutant CDC25B sequence was removed by Cre recombinase protein expression. Overexpression of the floxed mutant CDC25B significantly enhanced the efficiency of transgene amplification and productivity. Moreover, the specific production rate of the isolated clone CHO Cre-1 and Cre-2 were approximately 11-fold and 15-fold higher than that of mock-transfected clone CHO Mock-S. Chromosomal aneuploidy was increased by mutant CDC25B overexpression, but Cre-1 and Cre-2 did not show any changes in chromosome number during long-term cultivation, as is the case with CHO Mock-S. Our results suggest that high-producing and stable cells can be constructed by conditionally controlling a cell-cycle checkpoint integrated in conventional gene amplification systems.

  13. Enhanced efflux of (/sup 3/H)vinblastine from Chinese hamster ovary cells transfected with a full-length complementary DNA clone for the mdr1 gene

    SciTech Connect

    Hammond, J.R.; Johnstone, R.M.; Gros, P.

    1989-07-15

    Multidrug-resistant Chinese hamster ovary cell clones stably transfected with, and overexpressing, the mouse mdr1 complementary DNA clone along with drug-sensitive Chinese hamster ovary control cells were characterized for their capacities to accumulate and retain (/sup 3/H)vinblastine. Multidrug-resistant mdr1 transfectants show a 3-4-fold decrease in (/sup 3/H)vinblastine accumulation, compared to their drug-sensitive counterparts. After ATP depletion, this difference in (/sup 3/H)vinblastine accumulation between mdr1 transfectants and control cells effectively disappears. This ATP-dependent decreased drug accumulation is paralleled in mdr1 transfectants by an enhanced capacity of these cells to extrude the drug in an ATP-dependent manner. In medium containing glucose and glutamine, the mdr1 transfectants release preloaded drug at a rate five times that of control, drug-sensitive cells. In ATP-depleted control and mdr1-transfected cells, there is little difference in the rate or extent of (/sup 3/H)vinblastine release. The observation that the mdr1 transfectants show a decreased (/sup 3/H)vinblastine accumulation and an increased vinblastine release, both of which are abolished when cellular ATP levels are reduced, provides a direct demonstration that the product of the transfected mdr1 gene is responsible for a mechanism controlling cellular drug levels in an ATP-dependent manner. However, attempts to establish competition for (/sup 3/H)vinblastine transport by vincristine, daunomycin, and actinomycin D were only partly successful in mdr1 transfectants.

  14. Phytosphingosine and C2-phytoceramide induce cell death and inhibit carbachol-stimulated phospholipase D activation in Chinese hamster ovary cells expressing the Caenorhabditis elegans muscarinic acetylcholine receptor.

    PubMed

    Lee, J S; Min, D S; Park, C; Park, C S; Cho, N J

    2001-06-15

    Sphingolipid metabolites, such as sphingosine and ceramide, are known to play important roles in cell proliferation, differentiation and apoptosis, but the physiological roles of phytosphingosine (PHS) and phytoceramide (PHC) are poorly understood. In this study we investigated the effects of PHS, C2-PHC (N-acetylPHS) and C6-PHC (N-hexanoylPHS) on cell growth and intracellular signalling enzymes. Treatment of Chinese hamster ovary (CHO) cells with PHS, C2-PHC or C6-PHC resulted in cell death in a time- and dose-dependent manner. C2-PHC induced internucleosomal DNA fragmentation, whereas PHS or C6-PHC had little if any effect on DNA fragmentation under the same experimental conditions. Both PHS and C2-PHC inhibited carbachol-induced activation of phospholipase D (PLD), but not of phospholipase C (PLC), in CHO cells expressing the Caenorhabditis elegans muscarinic acetylcholine receptor (mAChR). On the other hand, no significant effect of C6-PHC on PLD or PLC was observed. Our results show that PHS and C2-PHC exert strong cytotoxic effects on CHO cells and modulate the mAChR-mediated signal transduction pathway.

  15. 7-Hydroxystaurosporine (UCN-01) causes redistribution of proliferating cell nuclear antigen and abrogates cisplatin-induced S-phase arrest in Chinese hamster ovary cells.

    PubMed

    Bunch, R T; Eastman, A

    1997-07-01

    A variety of agents, such as caffeine, have been shown to abrogate the DNA damage-dependent G2 checkpoint and enhance cytotoxicity. However, these agents are too toxic for clinical use. We have reported that the potent protein kinase inhibitor 7-hydroxystaurosporine (UCN-01) at nontoxic doses abrogates the G2 arrest caused by the DNA-damaging agent cisplatin. Here, using Chinese hamster ovary cells, we show that cisplatin causes predominantly an S-phase arrest; UCN-01 abrogates this S-phase arrest, causing progression of cells to G2 and, subsequently, apoptotic cell death. In searching for an explanation for this accelerate DNA synthesis, we discovered that UCN-01 caused translocation of proliferating cell nuclear antigen (PCNA) to the detergent-insoluble, DNA-bound fraction. PCNA acts as a sliding clamp for DNA polymerase delta. Sequestering of PCNA by p21waf1/cip1 is required for p53-dependent G1 arrest in damaged cells. However, the S-phase arrest occurs independently of p53 and p21waf1/cip1. Our results suggest that PCNA is also a component of this S-phase checkpoint, despite the fact that CHO cells are defective for p53, and no increase in p21waf1/cip1 was observed. The mechanism by which PCNA is sequestered in the absence of p21waf1/cip1 and the mechanism by which UCN-01 disrupts this sequestration remain to be elucidated.

  16. Efficient selection of high-producing subclones during gene amplification of recombinant Chinese hamster ovary cells by flow cytometry and cell sorting.

    PubMed

    Borth, N; Zeyda, M; Kunert, R; Katinger, H

    The screening procedure for high-producing cell lines is extremely time- and labor-intensive and costly, and is at present guided by an empirical approach based on individual experience. Flow cytometry and cell sorting, with its ability to analyze and separate single cells, an ideal method in the selection of such rare cells. The isolation of recombinant cell lines is especially difficult due to repeated gene amplification, which introduces high mutational variation into the population. We have established and evaluated a modification of a previous method that traps secreted product on the surface of the secreting cell, thus allowing direct analysis of single cell specific production rates. This method was used to select for high-producing subclones of a recombinant Chinese hamster ovary (CHO) cell line producing a human antibody against HIV-1 by repeated rounds of gene amplification and cell sorting. This cell line has been amplified in previous investigations, so that the amount of work and testing required by traditional methods can be compared with the protocol described herein. Forty-five 96-well plates were necessary to obtain a high-producing subclone by limited dilution methods, whereas only five plates were required when cell sorting was used. The specific production rate of the best clone obtained by sorting, however, was five times that of the clone obtained by traditional methods. In contrast to the clones obtained by limited dilution, which consisted of several populations of low- and high-producing cells even at high methotrexate concentrations (6.4 microM), the clones isolated by sorting were already homogeneous at 0.8 microM methotrexate.

  17. Toxicology Studies on Lewisite and Sulfur Mustard Agents: Genetic Toxicity of Sulfur Mustard (HD) in Chinese Hamster Ovary Cells Final Report

    SciTech Connect

    Jostes, Jr., R. F.; Sasser, L. B.; Rausch, R. J.

    1989-05-01

    The cytotoxic, clastogenic and mutagenic effects of sulfur nustard in Chinese hamster ovary cells are described in this reoort. The cytotoxicity data indicate that micromolar amounts of HC are highly toxic in microrolar amounts. Chromosone aberration frequencies increased in a dose-dependent manner over a dose range of 0. 5 to 1.0 {micro}m and SCE increased in a dose-dependent fashion in the dose range of 0.0625 to 0.25 {micro}M. Mutation induction at the HGPRT locus was sporadic, but the majority of the exoosures resulted in mutation frequencies which were 1.2 to 4.3 fold higher than the spontaneous frequencies.

  18. Development, qualification, validation and application of the neutral red uptake assay in Chinese Hamster Ovary (CHO) cells using a VITROCELL® VC10® smoke exposure system.

    PubMed

    Fields, Wanda; Fowler, Kathy; Hargreaves, Victoria; Reeve, Lesley; Bombick, Betsy

    2017-04-01

    Cytotoxicity assessment of combustible tobacco products by neutral red uptake (NRU) has historically used total particulate matter (TPM) or solvent captured gas vapor phase (GVP), rather than fresh whole smoke. Here, the development, validation and application of the NRU assay in Chinese Hamster Ovary (CHO) cells, following exposure to fresh whole smoke generated with the VITROCELL® VC10® system is described. Whole smoke exposure is particularly important as both particulate and vapor phases of tobacco smoke show cytotoxicity in vitro. The VITROCELL® VC10® system provides exposure at the air liquid interface (ALI) to mimic in vivo conditions for assessing the toxicological impact of smoke in vitro. Instrument and assay validations are crucial for comparative analyses.

  19. Ability of four potential topoisomerase II inhibitors to enhance the cytotoxicity of cis-diamminedichloroplatinum (II) in Chinese hamster ovary cells and in an epipodophyllotoxin-resistant subline.

    PubMed

    Eder, J P; Teicher, B A; Holden, S A; Senator, L; Cathcart, K N; Schnipper, L E

    1990-01-01

    Four drugs known to interact with topoisomerase II were assessed for their ability to enhance the cytotoxicity of cis-diamminedichloroplatinum(II) (CDDP) in Chinese hamster ovary (CHO) cell lines sensitive and resistant to VM-26. The combination treatments were analyzed by isobologram methodology. On 24 h exposure, there was no significant difference in the cytotoxicity of novobiocin or ciprofloxacin toward either cell line. The resistant cells were approximately 9-fold more resistant to 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) and approximately 170-fold more resistant to etoposide after a 24-h exposure. The combination of novobiocin and cisplatin produced greater than additive cell kill over the entire dose range of cisplatin tested in both cell lines. m-AMSA and CDDP produced cell kill that fell within the envelope of additivity. Etoposide and CDDP resulted in cytotoxicity that was slightly greater than additive at low CDDP concentrations and additive at the highest concentration of CDDP tested in the parental cell line and was slightly greater than additive in the resistant cell line. Ciprofloxacin and CDDP, like novobiocin, resulted in greater than additive cell kill in both cell lines. The enhancement of CDDP cytotoxicity by novobiocin that was seen in exponentially growing cells was lost in stationary-phase cultures. In these studies, novobiocin and, to a lesser degree, ciprofloxacin produced greater than additive cell kill in combination with CDDP in parental and epipodophyllotoxin-resistant CHO cells.

  20. Engineering the cellular protein secretory pathway for enhancement of recombinant tissue plasminogen activator expression in Chinese hamster ovary cells: effects of CERT and XBP1s genes.

    PubMed

    Rahimpour, Azam; Vaziri, Behrouz; Moazzami, Reza; Nematollahi, Leila; Barkhordari, Farzaneh; Kokabee, Leila; Adeli, Ahmad; Mahboudi, Fereidoun

    2013-08-01

    Cell line development is the most critical and also the most time-consuming step in the production of recombinant therapeutic proteins. In this regard, a variety of vector and cell engineering strategies have been developed for generating high-producing mammalian cells; however, the cell line engineering approach seems to show various results on different recombinant protein producer cells. In order to improve the secretory capacity of a recombinant tissue plasminogen activator (t-PA)-producing Chinese hamster ovary (CHO) cell line, we developed cell line engineering approaches based on the ceramide transfer protein (CERT) and X-box binding protein 1 (XBP1) genes. For this purpose, CERT S132A, a mutant form of CERT that is resistant to phosphorylation, and XBP1s were overexpressed in a recombinant t-PA-producing CHO cell line. Overexpression of CERT S132A increased the specific productivity of t-PA-producing CHO cells up to 35%. In contrast, the heterologous expression of XBP1s did not affect the t-PA expression rate. Our results suggest that CERTS132A- based secretion engineering could be an effective strategy for enhancing recombinant t- PA production in CHO cells.

  1. Analysis of dynamic changes in the proteome of a Bcl-XL overexpressing Chinese hamster ovary cell culture during exponential and stationary phases.

    PubMed

    Carlage, Tyler; Kshirsagar, Rashmi; Zang, Li; Janakiraman, Vijay; Hincapie, Marina; Lyubarskaya, Yelena; Weiskopf, Andy; Hancock, William S

    2012-01-01

    Mammalian cell cultures used for biopharmaceutical production undergo various dynamic biological changes over time, including the transition of cells from an exponential growth phase to a stationary phase during cell culture. To better understand the dynamic aspects of cell culture, a quantitative proteomics approach was used to identify dynamic trends in protein expression over the course of a Chinese hamster ovary (CHO) cell culture for the production of a recombinant monoclonal antibody and overexpressing the antiapoptotic gene Bcl-xl. Samples were analyzed using a method incorporating iTRAQ labeling, two-dimensional LC/MS, and linear regression calculations to identify significant dynamic trends in protein abundance. Using this approach, 59 proteins were identified with significant temporal changes in expression. Pathway analysis tools were used to identify a putative network of proteins associated with cell growth and apoptosis. Among the differentially expressed proteins were molecular chaperones and isomerases, such as GRP78 and PDI, and reported cell growth markers MCM2 and MCM5. In addition, two proteins with growth-regulating properties, transglutaminase-2 and clusterin, were identified. These proteins are associated with tumor proliferation and apoptosis and were observed to be expressed at relatively high levels during stationary phase, which was confirmed by western blotting. The proteomic methodology described here provides a dynamic view of protein expression throughout a CHO fed-batch cell culture, which may be useful for further elucidating the biological processes driving mammalian cell culture performance.

  2. Down-regulation of lactate dehydrogenase-A by siRNAs for reduced lactic acid formation of Chinese hamster ovary cells producing thrombopoietin.

    PubMed

    Kim, Sung Hyun; Lee, Gyun Min

    2007-02-01

    Lactate, one of the major waste products in mammalian cell culture, can inhibit cell growth and affect cellular metabolism at high concentrations. To reduce lactate formation, lactate dehydrogenase-A (LDH-A), an enzyme catalyzing the conversion of glucose-derived pyruvate to lactate, was down-regulated by an expression vector of small interfering RNAs (siRNA) in recombinant Chinese hamster ovary (rCHO) cells producing human thrombopoietin (hTPO). Three clones expressing low levels of LDH-A, determined by reverse transcription-PCR and an enzyme activity test, were established in addition to a negative control cell line. LDH-A activities in the three clones were decreased by 75-89%, compared with that of the control CHO cell line, demonstrating that the effect of siRNA is more significant than that of other traditional methods such as homologous recombination (30%) and antisense mRNA (29%). The specific glucose consumption rates of the three clones were reduced to 54-87% when compared to the control cell line. Similarly, the specific lactate production rates were reduced to 45-79% of the control cell line level. In addition, reduction of LDH-A did not impair either cell proliferation or hTPO productivity. Taken together, these results show that the lactate formation rate in rCHO cell culture can be efficiently reduced through the down-regulation of LDH via siRNA.

  3. Detection of DNA single-strand breaks induced by procarcinogens in Chinese hamster ovary cells cocultured with rat hepatocytes

    SciTech Connect

    Yang, K.H.; Shin, C.G.; Choe, S.Y.; Kim, D.H.

    1984-01-01

    DNA single-strand breaks induced by procarcinogens were detected in Chinese hamster overy (CHO) cell cocultured with adult rat hepatocytes. Freshly isolated adult rat hepatocytes were added to the CHO cell culture prelabeled with (/sup 3/H) thymidine. After allowing the hepatocytes to attach on or near the CHO cells, aflatoxin B/sub 1/ or benzo(a)pyrene was added to the culture and incubated for the desired time. DNA single-strand breaks in CHO cells were measured by the alkaline elution technique. Aflatoxin B/sub 1/ induced some DNA single-strand breaks in CHO cells cultured alone, but in coculture system with hepatocytes the number of DNA single-strand breaks increased greatly. The magnitude of the increase was related to the dose and the time of exposure to aflatoxin B/sub 1/. Addition of proteinase-K to the cell lysates increased the elution of DNA compared to that of samples without proteinase-K. Benzo(a)pyrene did not induce any DNA single-strand breaks in CHO cells in the absence of liver cells, but a significant number of single-strand breaks were detected in the coculture system.

  4. Analysis of solvent control and 1-nitrosopyrene-induced chinese hamster ovary cell mutants by southern and northern blots and the polymerase chain reaction

    SciTech Connect

    Newton, R.K.; Mittelstaedt, R.A.; Heflich, R.H. )

    1992-01-01

    1-Nitrosopyrene, a metabolite of the tumorigenic environmental pollutant 1-nitropyrene, is a potent mutagen at the hprt locus in Chinese hamster ovary (CHO) cells. A single DNA adduct, N-(deoxyguanosin-8-yl)-l-aminopyrene, is produced in CHO cells treated with 1-nitrosopyrene, and this adduct is found in rats and mice exposed to 1-nitropyrene. In this study, the structure of the hprt gene and the structure and amount of hprt mRNA were analyzed in 43 CHO cell mutants (16 isolated from solvent control cultures and 27 isolated from 1-nitrosopyrene-treated cultures). PstI- and BamHI-digested DNA from the mutants were subjected to Southern blot analysis using a hamster hprt cDNA probe. None of the 1-nitrosopyrene-induced mutants and only one of the control mutants displayed hybridization patterns that were different from the parent CHO cells. Northern blot analysis revealed that two control mutants had truncated hprt mRNAs, while 56% of the control mutants and 78% of the induced mutants had reduced levels of hprt mRNA. Using polymerase chain reaction amplification of cDNA synthesized from RNA, the hprt protein-coding region could be amplified from 23 of the 1-nitrosopyrene-induced mutants and 11 of the control mutants. The amplification products from 3 of the control mutants and 5 of the induced mutants were smaller than that found with RNA from parental CHO cells. These results indicate that the mutagenic DNA damage produced by 1-nitrosopyrene in CHO cells does not cause major structural alterations in the hprt gene and suggest that 1-nitrosopyrene acts as a point mutagen. A large number of both control and 1-nitrosopyrene-induced mutants exhibited a marked reduction in hprt mRNA concentration or possessed truncated mRNA hprt protein coding sequences. These alterations may contribute to the 6-thioguanine-resistant phenotype.

  5. Chinese Hamster Ovary (CHO) Host Cell Engineering to Increase Sialylation of Recombinant Therapeutic Proteins by Modulating Sialyltransferase Expression

    PubMed Central

    Lin, Nan; Mascarenhas, Joaquina; Sealover, Natalie R.; George, Henry J.; Brooks, Jeanne; Kayser, Kevin J.; Gau, Brian; Yasa, Isil; Azadi, Parastoo; Archer-Hartmann, Stephanie

    2015-01-01

    N-Glycans of human proteins possess both α2,6- and α2,3-linked terminal sialic acid (SA). Recombinant glycoproteins produced in Chinese hamster overy (CHO) only have α2,3-linkage due to the absence of α2,6-sialyltransferase (St6gal1) expression. The Chinese hamster ST6GAL1 was successfully overexpressed using a plasmid expression vector in three recombinant immunoglobulin G (IgG)-producing CHO cell lines. The stably transfected cell lines were enriched for ST6GAL1 overexpression using FITC-Sambucus nigra (SNA) lectin that preferentially binds α2,6-linked SA. The presence of α2,6-linked SA was confirmed using a novel LTQ Linear Ion Trap Mass Spectrometry (LTQ MS) method including MSn fragmentation in the enriched ST6GAL1 Clone 27. Furthermore, the total SA (mol/mol) in IgG produced by the enriched ST6GAL1 Clone 27 increased by 2-fold compared to the control. For host cell engineering, the CHOZN® GS host cell line was transfected and enriched for ST6GAL1 overexpression. Single-cell clones were derived from the enriched population and selected based on FITC-SNA staining and St6gal1 expression. Two clones (“ST6GAL1 OE Clone 31 and 32”) were confirmed for the presence of α2,6-linked SA in total host cell protein extracts. ST6GAL1 OE Clone 32 was subsequently used to express SAFC human IgG1. The recombinant IgG expressed in this host cell line was confirmed to have α2,6-linked SA and increased total SA content. In conclusion, overexpression of St6gal1 is sufficient to produce recombinant proteins with increased sialylation and more human-like glycoprofiles without combinatorial engineering of other sialylation pathway genes. This work represents our ongoing effort of glycoengineering in CHO host cell lines for the development of “bio-better” protein therapeutics and cell culture vaccine production. PMID:25641927

  6. Chinese hamster ovary (CHO) host cell engineering to increase sialylation of recombinant therapeutic proteins by modulating sialyltransferase expression.

    PubMed

    Lin, Nan; Mascarenhas, Joaquina; Sealover, Natalie R; George, Henry J; Brooks, Jeanne; Kayser, Kevin J; Gau, Brian; Yasa, Isil; Azadi, Parastoo; Archer-Hartmann, Stephanie

    2015-01-01

    N-Glycans of human proteins possess both α2,6- and α2,3-linked terminal sialic acid (SA). Recombinant glycoproteins produced in Chinese hamster overy (CHO) only have α2,3-linkage due to the absence of α2,6-sialyltransferase (St6gal1) expression. The Chinese hamster ST6GAL1 was successfully overexpressed using a plasmid expression vector in three recombinant immunoglobulin G (IgG)-producing CHO cell lines. The stably transfected cell lines were enriched for ST6GAL1 overexpression using FITC-Sambucus nigra (SNA) lectin that preferentially binds α2,6-linked SA. The presence of α2,6-linked SA was confirmed using a novel LTQ Linear Ion Trap Mass Spectrometry (LTQ MS) method including MSn fragmentation in the enriched ST6GAL1 Clone 27. Furthermore, the total SA (mol/mol) in IgG produced by the enriched ST6GAL1 Clone 27 increased by 2-fold compared to the control. For host cell engineering, the CHOZN(®) GS host cell line was transfected and enriched for ST6GAL1 overexpression. Single-cell clones were derived from the enriched population and selected based on FITC-SNA staining and St6gal1 expression. Two clones ("ST6GAL1 OE Clone 31 and 32") were confirmed for the presence of α2,6-linked SA in total host cell protein extracts. ST6GAL1 OE Clone 32 was subsequently used to express SAFC human IgG1. The recombinant IgG expressed in this host cell line was confirmed to have α2,6-linked SA and increased total SA content. In conclusion, overexpression of St6gal1 is sufficient to produce recombinant proteins with increased sialylation and more human-like glycoprofiles without combinatorial engineering of other sialylation pathway genes. This work represents our ongoing effort of glycoengineering in CHO host cell lines for the development of "bio-better" protein therapeutics and cell culture vaccine production.

  7. A novel regulatory element (E77) isolated from CHO‐K1 genomic DNA enhances stable gene expression in Chinese hamster ovary cells

    PubMed Central

    Kang, Shin‐Young; Kim, Yeon‐Gu; Kang, Seunghee; Lee, Hong Weon

    2016-01-01

    Abstract Vectors flanked by regulatory DNA elements have been used to generate stable cell lines with high productivity and transgene stability; however, regulatory elements in Chinese hamster ovary (CHO) cells, which are the most widely used mammalian cells in biopharmaceutical production, are still poorly understood. We isolated a novel gene regulatory element from CHO‐K1 cells, designated E77, which was found to enhance the stable expression of a transgene. A genomic library was constructed by combining CHO‐K1 genomic DNA fragments with a CMV promoter‐driven GFP expression vector, and the E77 element was isolated by screening. The incorporation of the E77 regulatory element resulted in the generation of an increased number of clones with high expression, thereby enhancing the expression level of the transgene in the stable transfectant cell pool. Interestingly, the E77 element was found to consist of two distinct fragments derived from different locations in the CHO genome shotgun sequence. High and stable transgene expression was obtained in transfected CHO cells by combining these fragments. Additionally, the function of E77 was found to be dependent on its site of insertion and specific orientation in the vector construct. Our findings demonstrate that stable gene expression mediated by the CMV promoter in CHO cells may be improved by the isolated novel gene regulatory element E77 identified in the present study. PMID:26762773

  8. A novel regulatory element (E77) isolated from CHO-K1 genomic DNA enhances stable gene expression in Chinese hamster ovary cells.

    PubMed

    Kang, Shin-Young; Kim, Yeon-Gu; Kang, Seunghee; Lee, Hong Weon; Lee, Eun Gyo

    2016-05-01

    Vectors flanked by regulatory DNA elements have been used to generate stable cell lines with high productivity and transgene stability; however, regulatory elements in Chinese hamster ovary (CHO) cells, which are the most widely used mammalian cells in biopharmaceutical production, are still poorly understood. We isolated a novel gene regulatory element from CHO-K1 cells, designated E77, which was found to enhance the stable expression of a transgene. A genomic library was constructed by combining CHO-K1 genomic DNA fragments with a CMV promoter-driven GFP expression vector, and the E77 element was isolated by screening. The incorporation of the E77 regulatory element resulted in the generation of an increased number of clones with high expression, thereby enhancing the expression level of the transgene in the stable transfectant cell pool. Interestingly, the E77 element was found to consist of two distinct fragments derived from different locations in the CHO genome shotgun sequence. High and stable transgene expression was obtained in transfected CHO cells by combining these fragments. Additionally, the function of E77 was found to be dependent on its site of insertion and specific orientation in the vector construct. Our findings demonstrate that stable gene expression mediated by the CMV promoter in CHO cells may be improved by the isolated novel gene regulatory element E77 identified in the present study.

  9. Evaluation of two public genome references for Chinese hamster ovary cells in the context of RNA-seq based gene expression analysis.

    PubMed

    Chen, Chun; Le, Huong; Goudar, Chetan T

    2017-03-15

    RNA-Seq is a powerful transcriptomics tool for mammalian cell culture process development. Successful RNA-Seq data analysis requires a high quality reference for read mapping and gene expression quantification. Currently, there are two public genome references for Chinese hamster ovary (CHO) cells, the predominant mammalian cell line in the biopharmaceutical industry. In this study, we compared these two references by analyzing 60 RNA-Seq samples from a variety of CHO cell culture conditions. Among the 20,891 common genes in both references, we observed that 31.5% have more than 7.1% quantification differences, implying gene definition differences in the two references. We propose a framework to quantify this difference using two metrics, Consistency and Stringency, which account for the average quantification difference between the two references over all samples, and the sample-specific effect on the quantification result, respectively. These two metrics can be used to identify potential genes for future gene model improvement and to understand the reliability of differentially expressed genes identified by RNA-Seq data analysis. Before a more comprehensive genome reference for CHO cells emerges, the strategy proposed in this study can enable a more robust transcriptome analysis for CHO cell RNA-Seq data. This article is protected by copyright. All rights reserved.

  10. Relationship between Fluorescence Intensity of GFP and the Expression Level of Prestin in a Prestin-Expressing Chinese Hamster Ovary Cell Line

    NASA Astrophysics Data System (ADS)

    Iida, Koji; Nagaoka, Tomoyuki; Tsumoto, Kouhei; Ikeda, Katsuhisa; Kumagai, Izumi; Kobayashi, Toshimitsu; Wada, Hiroshi

    Outer hair cells (OHCs) in mammals can elongate and contract at frequencies up to 100kHz in response to changes in their membrane potential. The origin of this unique motility is the motor protein prestin, which is densely packed in the lateral membrane of the OHCs. In a previous work, we constructed a prestin-expressing cell line using Chinese hamster ovary (CHO) cells to obtain a stable supply of prestin. When we research prestin using constructed cells, it is necessary to estimate the expression level of prestin in the cells easily and non-invasively. As the prestin gene and a green fluorescent protein (GFP) gene were introduced into constructed cells using the same vector, the expression level of prestin and fluorescence intensity of GFP are possibly correlated. Since this correlation is not clear, however, in this study, we therefore investigated whether the expression level of prestin evaluated by patch-clamp recording and the fluorescence intensity of GFP obtained from fluorescence images are correlated or not. As a result, it was demonstrated that they were correlated. The expression level of prestin can therefore be evaluated by measuring the fluorescence intensity of GFP.

  11. Molecular characterization of X-ray-induced mutations at the HPRT locus in plateau-phase Chinese hamster ovary cells.

    PubMed

    Morgan, T L; Fleck, E W; Poston, K A; Denovan, B A; Newman, C N; Rossiter, B J; Miller, J H

    1990-10-01

    CHO-K1 cells were irradiated in plateau phase to determine the effect of dose, dose fractionation, and delayed replating on the type, location and frequency of mutations induced by 250 kVp X-rays at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus. Independent HPRT-deficient cell lines were isolated from each group for Southern blot analysis using a hamster HPRT cDNA probe. When compared with irradiation with 4 Gy and immediate replating, dose fractionation (2 Gy + 24 h + 2 Gy) the entire gene. Since an increase in survival was noted under these conditions, these data suggest that repair of sublethal and potentially lethal damage acts equally on all premutagenic lesions, regardless of type or location. Differences in the mutation spectrum were noted when cells were irradiated at 2 Gy and replated immediately. The location of the deletion breakpoints was determined in 15 mutants showing partial loss of the HPRT locus. In 12 of these cell lines one or both of the breakpoints appeared to be located near the center of the gene, indicating a nonrandom distribution of mutations. These results indicate that damage induced by ionizing radiation results in a nonrandom distribution of genetic damage, suggesting that certain regions of the genome may be acutely sensitive to the mutagenic effects of ionizing radiation.

  12. Use of an antikinetochore antibody and DNA probes to measure aneuploidy induction in interphase human lymphocytes and Chinese hamster ovary cells

    SciTech Connect

    Eastmond, D.A.; Tucker, J.D.; Pinkel, D.

    1988-12-05

    Aneuploidy in germ cells is associated with birth defects, spontaneous abortions, and infertility, whereas in somatic cells aneuploidy may lead to cell death and carcinogenesis. The nonrandom numerical chromosomal changes that are often observed in tumors or transformed cells suggest that aneuploidy induction by chemicals may be involved in carcinogenesis. The identification of aneuploidy inducing agents (aneuploidogens) and studies into the mechanisms by which aneuploidy may be involved in carcinogenesis are currently limited in that standard cytogenetic techniques are time consuming, require highly skilled personnel and are prone to technical artifacts. Recent developments in immunology and molecular biology have resulted in new techniques which may allow simple and rapid identification of aneuploidogens. We report the development of two new approaches to determine the aneuploidy-inducing potential of chemicals. The first approach involves the induction of micronuclei in human lymphocytes and Chinese hamster ovary (CHO) cells and the use of an antikinetochore antibody to determine whether micronuclei contain centromeres---a condition indicating potential aneuploidy. The second approach involves the use of in situ hybridization with fluorescently labeled chromosome-specific DNA probes and the subsequent counting of the number of copies of that chromosome in the interphase nuclei of human lymphocytes. 8 refs., 1 fig., 1 tab.

  13. Pichia pastoris secretes recombinant proteins less efficiently than Chinese hamster ovary cells but allows higher space-time yields for less complex proteins.

    PubMed

    Maccani, Andreas; Landes, Nils; Stadlmayr, Gerhard; Maresch, Daniel; Leitner, Christian; Maurer, Michael; Gasser, Brigitte; Ernst, Wolfgang; Kunert, Renate; Mattanovich, Diethard

    2014-04-01

    Chinese hamster ovary (CHO) cells are currently the workhorse of the biopharmaceutical industry. However, yeasts such as Pichia pastoris are about to enter this field. To compare their capability for recombinant protein secretion, P. pastoris strains and CHO cell lines producing human serum albumin (HSA) and the 3D6 single chain Fv-Fc anti-HIV-1 antibody (3D6scFv-Fc) were cultivated in comparable fed batch processes. In P. pastoris, the mean biomass-specific secretion rate (qp ) was 40-fold lower for 3D6scFv-Fc compared to HSA. On the contrary, qp was similar for both proteins in CHO cells. When comparing both organisms, the mean qp of the CHO cell lines was 1011-fold higher for 3D6scFv-Fc and 26-fold higher for HSA. Due to the low qp of the 3D6scFv-Fc producing strain, the space-time yield (STY) was 9.6-fold lower for P. pastoris. In contrast, the STY of the HSA producer was 9.2-fold higher compared to CHO cells because of the shorter process time and higher biomass density. The results indicate that the protein secretion machinery of P. pastoris is much less efficient and the secretion rate strongly depends on the complexity of the recombinant protein. However, process efficiency of the yeast system allows higher STYs for less complex proteins.

  14. Mapping of a locus correcting lack of phosphoribosylaminoimidazole carboxylase activity in Chinese hamster ovary cell Ade-D mutants to human chromosome 4.

    PubMed

    Barton, J W; Hart, I M; Patterson, D

    1991-02-01

    The human phosphoribosylaminoimidazole (AIR) carboxylase locus has been until this report one of the genes encoding purine biosynthetic enzymes that had not been assigned to an individual human chromosome. Characterization of Chinese hamster ovary (CHO) cell mutant Ade-D showed that the cell line was unable to produce IMP and accumulated AIR. CHO Ade-D cells were fused with normal human lymphocytes utilizing inactivated Sendai virus and the resulting hybrid cell lines were selected for purine prototrophy. Cytogenetic analysis showed a 100% concordance value for chromosome 4. Two of the isolated subclones contained only the long arm of chromosome 4 translocated onto a CHO chromosome, providing evidence for a regional assignment of the Ade-D gene to the long arm of chromosome 4. Two of the subclones containing chromosome 4 were subjected to the BrdU visible light segregation. All of the isolated purine auxotrophic cell lines showed a loss of the q arm of chromosome 4. The localization of the Ade-D locus to the long arm of chromosome 4 may reveal further clustering of the mammalian purine genes since the Ade-A locus has previously been regionally assigned to 4pter-q21.

  15. Three-dimensional optical force field on a Chinese hamster ovary cell in a fiber-optical dual-beam trap

    NASA Astrophysics Data System (ADS)

    Wei, Ming-Tzo; Yang, Kun-Ta; Karmenyan, Artahses; Chiou, Arthur

    2006-04-01

    We used a fiber-optical dual-beam trap (single-mode fiber, λ = 532nm, trapping power ~ 22mW, the distance between the two fiber end-faces = 125μm) to capture a Chinese hamster ovary (CHO) cell with a diameter of approximately 15μm and tracked its three-dimensional Brownian motion via a pair of orthogonal quadrant photodiodes. By analyzing the Brownian motion of the trapped CHO cell, we determined the force constants of the optical force field on the CHO cell to be kx=6.75 pN/μm, ky=5.53 pN/μm, kz=1.96 pN/μm, and kx=2.91 pN/μm, ky=2.7 pN/μm, kz=0.79 pN/μm, respectively, before and after the CHO cell was treated with latrunculin, a toxic drug known to disrupt the cytoskeleton of the cell.

  16. Monitoring utilizations of amino acids and vitamins in culture media and Chinese hamster ovary cells by liquid chromatography tandem mass spectrometry.

    PubMed

    Qiu, Jinshu; Chan, Pik Kay; Bondarenko, Pavel V

    2016-01-05

    Monitoring amino acids and vitamins is important for understanding human health, food nutrition and the culture of mammalian cells used to produce therapeutic proteins in biotechnology. A method including ion pairing reversed-phase liquid chromatography with tandem mass spectrometry was developed and optimized to quantify 21 amino acids and 9 water-soluble vitamins in Chinese hamster ovary (CHO) cells and culture media. By optimizing the chromatographic separation, scan time, monitoring time window, and sample preparation procedure, and using isotopically labeled (13)C, (15)N and (2)H internal standards, low limits of quantitation (≤0.054 mg/L), good precision (<10%) and good accuracy (100±10%) were achieved for nearly all the 30 compounds. Applying this method to CHO cell extracts, statistically significant differences in the metabolite levels were measured between two cell lines originated from the same host, indicating differences in genetic makeup or metabolic activities and nutrient supply levels in the culture media. In a fed-batch process of manufacturing scale bioreactors, two distinguished trends for changes in amino acid concentrations were identified in response to feeding. Ten essential amino acids showed a zigzag pattern with maxima at the feeding days, and 9 non-essential amino acids displayed a smoothly changing profile as they were mainly products of cellular metabolism. Five of 9 vitamins accumulated continuously during the culture period, suggesting that they were fed in access. The method serves as an effective tool for the development and optimization of mammalian cell cultures.

  17. Identification of shed proteins from Chinese hamster ovary cells: Application of statistical confidence using human and mouse protein databases

    SciTech Connect

    Ahram, Mamoun; Strittmatter, Eric F.; Monroe, Matthew E.; Adkins, Joshua N.; Hunter, Joel C.; Miller, John H.; Springer, David L.

    2005-05-01

    The shedding process releases ligands, receptors, and other proteins from the surface of the cell and is a mechanism whereby cells communicate. Even though altered regulation of this process has been implicated in several diseases, global approaches to evaluate shed proteins have not been developed. A goal of this study was to identify global changes in shed proteins in media taken from cells exposed to low-doses of radiation in an effort to develop a fundamental understanding of the bystander response. CHO cells were chosen for this study because they have been widely used for radiation studies and since they have been reported to respond to radiation by releasing factors into the media that cause genomic instability and cytotoxicity in unexposed cells, i.e., a bystander effect. Media samples taken for irradiated cells were evaluated using a combination of tandem- and FTICR-mass spectrometry analysis. Since the hamster genome has not been sequenced, mass spectrometry data was searched against the mouse and human proteins databases. Nearly 150 proteins that were identified by tandem mass spectrometry were confirmed by FTICR. When both types of mass spectrometry data were evaluated with a new confidence scoring tool, which is based on discriminant analyses, about 500 protein were identified. Approximately 20% of these identifications were either integral membrane proteins or membrane associated proteins, suggesting that they were derived from the cell surface, hence were likely shed. However, estimates of quantitative changes, based on two independent mass spectrometry approaches, did not identify any protein abundance changes attributable to the bystander effect. Results from this study demonstrate the feasibility of global evaluation of shed proteins using mass spectrometry in conjunction with cross-species protein databases and that significant improvement in peptide/protein identifications is provided by the confidence scoring tool.

  18. Effects of agonist efficacy on desensitization of phosphoinositide hydrolysis mediated by m1 and m3 muscarinic receptors expressed in Chinese hamster ovary cells

    SciTech Connect

    Hu, J.; Wang, S.Z.; el-Fakahany, E.E. )

    1991-06-01

    Muscarinic receptor agonist-induced desensitization of phosphoinositide (PI) hydrolysis and loss of receptors were studied in Chinese hamster ovary (CHO) cells transfected with the m1 and m3 muscarinic receptor genes. Long-term exposure to the full agonist carbamylcholine (CBC) resulted in a time-dependent attenuation of the maximal PI response and a decrease in agonist potency. This desensitization was accompanied by a parallel loss of maximal ligand binding without an alteration of the binding affinity. The time course of both receptor desensitization and down-regulation was similar in m1 and m3 CHO cells. The PI response to the partial agonist McN-A-343 (McN) in m1 cells was more sensitive to desensitization by CBC than the response to the latter agonist, and this desensitization was faster than receptor down-regulation. Desensitization of the PI response to McN was reflected as a decrease in the maximal response without a marked change in potency. McN induced slow desensitization of the PI response to CBC but a much faster desensitization of its own response. Our data provide evidence that although muscarinic agonist-induced desensitization of PI hydrolysis in CHO cells is due mainly to loss of receptors, there are other important factors which play a role in this process, e.g., receptor-effector uncoupling. The relative contribution of these different mechanisms depends on the efficacy of the agonists used for the receptor desensitization and activation steps.

  19. Optimization of cultivation conditions in spin tubes for Chinese hamster ovary cells producing erythropoietin and the comparison of glycosylation patterns in different cultivation vessels.

    PubMed

    Strnad, Jure; Brinc, Matjaz; Spudić, Vatroslav; Jelnikar, Nadja; Mirnik, Lidija; Carman, Barbara; Kravanja, Zdravko

    2010-01-01

    This article describes the optimization of cultivation factor settings, that is the shaking rate and working volume in 50 mL spin tubes for a Chinese hamster ovary cell line expressing recombinant human alpha-erythropoietin, using a response D-optimal surface method. The main objectives of the research were, firstly, to determine a setting in which the product titer and product quality attributes in spin tubes are equivalent to those in 250 mL shake flasks in a seven day batch and, secondly, to find a setting in which the product titer is maximal. The model for product titer prediction as a function of shaking rate and working volume in the defined design space was successfully applied to the optimization of cultivation conditions in spin tubes for the tested cell line. Subsequently, validation experiments were carried out simultaneously in spin tubes, shake flasks and bench scale bioreactors to compare cell culture performance parameters such as growth, productivity and product quality attributes in the form of isoform profiles and glycan antennarity structures. The results of the experiments showed that similar cell culture performance and product quality could be achieved in spin tubes when compared to shake flasks. Additionally, bioreactor titers could be reproduced in spin tubes at high shaking rates and low working volumes, but with differing product quality. Cultivation at lower shaking rates in spin tubes and shake flasks produced a glycoprotein with a product quality slightly comparable to that from bioreactors, but with titers being only two thirds.

  20. A novel function for selenium in biological system: selenite as a highly effective iron carrier for Chinese hamster ovary cell growth and monoclonal antibody production.

    PubMed

    Zhang, Jinyou; Robinson, David; Salmon, Peter

    2006-12-20

    As the market for biopharmaceuticals especially monoclonal antibodies (MAbs) rapidly grows, their manufacturing methods are coming under increasing regulatory scrutiny, particularly due to concerns about the potential introduction of adventitious agents from animal-sourced components in the media used for their production in mammalian cell culture. Chinese hamster ovary (CHO) cells are by far the most commonly used production vehicles for these recombinant glycoproteins. In developing animal-component free media for CHO and other mammalian cell lines, the iron-transporter function of serum or human/bovine transferrin is usually replaced by certain organic or inorganic chelators capable of delivering iron for cell respiration and metabolism, but few of them are sufficiently effective. Selenium is a well-known essential trace element (TE) for cell growth and development, and its positive role in biological system includes detoxification of free radicals by activating glutathione peroxidase. In cell culture, selenium in the form of selenite can help cells to detoxify the medium thus protect them from oxidative damage. In this presentation, we describe the discovery and application of a novel function of selenite, that is, as a highly effective carrier to deliver iron for cell growth and function. In our in-house-developed animal protein-free (APF) medium for CHO cells, using an iron-selenite compound to replace the well-established tropolone delivery system for iron led to comparable or better cell growth and antibody production. A high cell density of >10 x 10(6) viable cells/mL and excellent antibody titer of approximately 3 g/L were achieved in 14-day fed-batch cultures in shake flasks, followed by successful scale-up to stirred bioreactors. The preparation of the commercially unavailable iron-selenite compound from respective ions, and its effectiveness in cell-culture performance, were dependent on reaction time, substrates, and other conditions.

  1. Effects of radiofrequency radiation and simultaneous exposure with mitomycin C on the frequency of sister chromatid exchanges in Chinese hamster ovary cells

    SciTech Connect

    Ciaravino, V.; Meltz, M.L.; Erwin, D.N.

    1987-01-01

    Chinese hamster ovary (CHO) cells were exposed for 2 hr with and without mitomycin C (MMC) to pulsed wave radiofrequency radiation (RFR) at 2450 MHz. The repetition rate of 25,000 pulses per sec (pps), and exposure geometry used, resulted in a specific absorption rate (SAR) of 33.8 W/kg. The following exposure regimens were used: 1) a 37 C water bath control; 2) a water bath temperature control (TC) in which the continuously monitored medium temperature closely followed teh temperature rise in the RFR-exposed flasks; and 3) the RFR-exposed cells in a water bath set at 37 C prior to exposure. RFR exposure resulted in a maximum cell culture medium temperature of 39.2 C. In the absence of MMC, there was no significant increase in sister chromatid exchange (SCE) in the RFR-exposed or TC groups over that of teh 37 C control. When a simultaneous treatment of RFR and MMC occurred there was no statistical difference in SCE frequency from that caused by chemical treatment alone.

  2. Effects of radiofrequency radiation and simultaneous exposure with mitomycin C on the frequency of sister chromatid exchanges in Chinese hamster ovary cells

    SciTech Connect

    Ciaravino, V.; Meltz, M.L.; Erwin, D.N.

    1987-01-01

    Chinese hamster ovary (CHO) cells were exposed for 2 hr with and without mitomycin C (MMC) (1 X 10(-8)M) to pulsed wave radiofrequency radiation (RFR) at 2450 MHz. The repetition rate of 25,000 pulses per sec (pps), pulse width of 10 microseconds, and exposure geometry used, resulted in a specific absorption rate (SAR) of 33.8 W/kg. The following exposure regimens were used: a 37 degrees C water bath control; a water bath temperature control (TC) in which the continuously monitored medium temperature closely followed the temperature rise in the RFR-exposed flasks; and the RFR-exposed cells in a water bath set at 37 degrees C prior to exposure. RFR exposure resulted in a maximum cell culture medium temperature of 39.2 degrees C. In the absence of MMC, there was no significant increase in sister chromatid exchange (SCE) in the RFR-exposed or TC groups over that of the 37 degrees C control. When a simultaneous treatment of RFR and MMC occurred there was no statistical difference in SCE frequency from that caused by chemical treatment alone.

  3. Sp1 involvement in the 4beta-phorbol 12-myristate 13-acetate (TPA)-mediated increase in resistance to methotrexate in Chinese hamster ovary cells.

    PubMed

    Noé, V; Alemany, C; Nicolás, M; Ciudad, C J

    2001-06-01

    4beta-Phorbol 12-myristate 13-acetate (TPA) increases the number of colonies resistant to methotrexate (MTX), mainly by amplification of the dihydrofolate reductase (dhfr) locus. We showed previously that inhibition of protein kinase C (PKC) prevents this resistance. Here, we studied the molecular changes involved in the development of TPA-mediated MTX resistance in Chinese hamster ovary (CHO) cells. TPA incubation increased the expression and activity of DHFR. Because Sp1 controls the dhfr promoter, we determined the effect of TPA on the expression of Sp1 and its binding to DNA. TPA incubation increased Sp1 binding and the levels of Sp1 protein. The latter effect was due to an increase in Sp1 mRNA. Dephosphorylation of nuclear extracts from control or TPA-treated cells reduced the binding of Sp1. Stable transfectants of PKCalpha showed increased Sp1 binding, and when treated with MTX, developed a greater number of resistant colonies than control cells. Seventy-five percent of the isolated colonies showed increased copy number for the dhfr gene. Transient expression of PKCalpha increased DHFR activity. Over-expression of Sp1 increased resistance to MTX, and inhibition of Sp1 binding by mithramycin decreased this resistance. We conclude that one mechanism by which TPA enhances MTX resistance, mainly by gene amplification, is through an increase in Sp1 expression which leads to DHFR activation.

  4. Transient production of recombinant proteins by Chinese hamster ovary cells using polyethyleneimine/DNA complexes in combination with microtubule disrupting anti-mitotic agents.

    PubMed

    Tait, Andrew S; Brown, Catherine J; Galbraith, Douglas J; Hines, Michael J; Hoare, Mike; Birch, John R; James, David C

    2004-12-20

    We have developed a simple and robust transient expression system utilizing the 25 kDa branched cationic polymer polyethylenimine (PEI) as a vehicle to deliver plasmid DNA into suspension-adapted Chinese hamster ovary cells synchronized in G2/M phase of the cell cycle by anti-mitotic microtubule disrupting agents. The PEI-mediated transfection process was optimized with respect to PEI nitrogen to DNA phosphate molar ratio and the plasmid DNA mass to cell ratio using a reporter construct encoding firefly luciferase. Optimal production of luciferase was observed at a PEI N to DNA P ratio of 10:1 and 5 mug DNA 10(6) cells(-1). To manipulate transgene expression at mitosis, we arrested cells in G2/M phase of the cell cycle using the microtubule depolymerizing agent nocodazole. Using secreted human alkaline phosphatase (SEAP) and enhanced green fluorescent protein (eGFP) as reporters we showed that continued inclusion of nocodazole in cell culture medium significantly increased both transfection efficiency and reporter protein production. In the presence of nocodazole, greater than 90% of cells were eGFP positive 24 h post-transfection and qSEAP was increased almost fivefold, doubling total SEAP production. Under optimal conditions for PEI-mediated transfection, transient production of a recombinant chimeric IgG4 encoded on a single vector was enhanced twofold by nocodazole, a final yield of approximately 5 microg mL(-1) achieved at an initial viable cell density of 1 x 10(6) cells mL(-1). The glycosylation of the recombinant antibody at Asn297 was not significantly affected by nocodazole during transient production by this method.

  5. Chemometrics and in-line near infrared spectroscopic monitoring of a biopharmaceutical Chinese hamster ovary cell culture: prediction of multiple cultivation variables.

    PubMed

    Clavaud, Matthieu; Roggo, Yves; Von Daeniken, Ralph; Liebler, André; Schwabe, Jan-Oliver

    2013-07-15

    In the present study near infrared (NIR) spectroscopy was used to monitor the cultivation of mammalian Chinese hamster ovary (CHO) cells producing a monoclonal antibody in a fed-batch cell culture process. A temperature shift was applied during the cultivation. The cells were incubated at 37 °C and 33 °C. The Fourier transform near infrared (FT-NIR) multiplex process analyzer spectroscopy was investigated to monitor cultivation variables of the CHO cell culture from 10 independent batches using two channels of the FT-NIR. The measurements were performed on production scale bioreactors of 12,500 L. The cell cultures were analyzed with the spectrometer coupled to a transflection sterilizable fiber optic probe inserted into the bioreactors. Multivariate data analysis (MVDA) employing unsupervised principal component analysis (PCA) and partial least squares regression methods (PLS) were applied. PCA demonstrated that 96% of the observed variability was explained by the process trajectory and the inter-batch variability. PCA was found to be a significant tool in identifying batch homogeneity between lots and in detecting abnormal fermentation runs. Seven different cell culture parameters such as osmolality, glucose concentration, product titer, packed cell volume (PCV), integrated viable packed cell volume (ivPCV), viable cell density (VCD), and integrated viable cell count (iVCC) were monitored inline and predicted by NIR. NIR spectra and reference analytics data were computed using control charts to evaluate the monitoring abilities. Control charts of each media component were under control by NIR spectroscopy. The PLS calibration plots offered accurate predictive capabilities for each media. This paper underlines the capability for inline prediction of multiple cultivation variables during bioprocess monitoring.

  6. Sensitization to the cytotoxicity of melphalan by ethacrynic acid and hyperthermia in drug-sensitive and multidrug-resistant Chinese hamster ovary cells.

    PubMed

    Turcotte, S; Averill-Bates, D A

    2001-09-01

    The ability of physical and pharmacological modulators to increase the cytotoxicity of melphalan was investigated in Chinese hamster ovary cells using a clonogenic cell survival assay. Hyperthermia has potential for use in cancer treatment, particularly as an adjuvant to chemotherapy or radiotherapy. Ethacrynic acid is a glutathione S-transferase inhibitor and also undergoes conjugation with glutathione. Interactions between hyperthermia (41-43 degrees C), ethacrynic acid and melphalan were evaluated in multidrug-resistant (CH(R)C5) cells with overexpression of P-glycoprotein (33.69-fold), and in drug-sensitive (AuxB1) cells. GST alpha was expressed at a higher level (3.65-fold) in CH(R)C5 cells than in sensitive cells, whereas levels of isoforms pi and mu were the same. GST pi was the most highly expressed isoform in the two cell populations. Ethacrynic acid was cytotoxic at elevated temperatures, while it caused little or no cytotoxicity at 37 degrees C. This effect occurred in drug-resistant and drug-sensitive cells, and attributes thermosensitizing properties to ethacrynic acid. Ethacrynic acid (20 microM) alone did not alter the cytotoxicity of melphalan at 37 degrees C. Hyperthermia potentiated drug cytotoxicity in cells, both with and without ethacrynic acid treatment. Ethacrynic acid could be useful in cancer treatment by acting as a thermosensitizer when combined with heat and by enhancing the cytotoxicity of melphalan at elevated temperatures. A major advantage arising from the use of regional hyperthermia is the ability to target drug cytotoxicity to the tumor volume. A useful finding is that ethacrynic acid, heat and/or melphalan are also effective against multidrug-resistant cells with overexpression of P-glycoprotein.

  7. Development of a standard protocol for in vitro cytogenetic testing with Chinese hamster ovary cells: comparison of results for 22 compounds in two laboratories

    SciTech Connect

    Galloway, S.M.; Bloom, A.D.; Resnick, M.; Margolin, B.H.; Nakamura, F.; Archer, P.; Zeiger, E.

    1985-01-01

    A major problem of cytogenetics testing in mammalian cells is lack of agreement of results among laboratories. The objective of this study was to develop a sensitive in vitro test protocol that was applicable to large-scale chemical screening and yielded comparable results in two laboratories. The authors used sister chromatid exchange (SEC) and chromosome aberration (CAb) tests in Chinese hamster ovary (CHO) cells. Five clastogens were tested in the first two-laboratory comparison: mitomycin-C, triethylenemelamine, N-methyl-N'-nitro-N-nitrosoguanidine, cyclophosphamide, and benzo(..cap alpha..)pyrene. There was quite good agreement between laboratories. Seventeen compounds were then tested blind in the two laboratories. As testing proceeded, some discrepancies occurred between the laboratories, and the protocol was modified in attempts to improve the resolution of marginal responses and make dose selection more consistent. This protocol gave comparable results in the two laboratories in many cases and by testing up to a maximum dose, limited by solubility and/or toxicity, should detect a high proportion of clastogens and SCE inducers.

  8. Ammonia affects the glycosylation patterns of recombinant mouse placental lactogen-I by chinese hamster ovary cells in a pH-dependent manner

    SciTech Connect

    Borys, M.C.; Linzer, D.I.H.; Papoutsakis, E.T. )

    1994-03-15

    The N-linked glycosylation of the recombinant protein mouse placental lactogen-I (mPL-I) expressed by Chinese hamster ovary (CHO) cells under nongrowth conditions was inhibited by increasing levels of ammonium chloride in a serum-free, protein expression medium. The effect of ammonia on glycosylation was dependent on the extracellular pH (pH[sub e]). In media containing 0 and 9 mM ammonium chloride, the percentage of the most heavily glycosylated forms of secreted mPL-I decreased from ca. 90% to ca. 25% at pH[sub e] 8.0, and from ca. 90% to ca. 65% at pH[sub e] 7.6, respectively. However, at pH[sub e] 7.2, the most heavily glycosylated forms of secreted mPL-I decreased from ca. 90% to ca. 80% in media containing 0 and 9 mM ammonium chloride, respectively. Inhibition of mPL-I glycosylation was found to correlate with the calculated concentration of the ammonia species. Control experiments showed that the ammonia species. Control experiments showed that the ammonia effect on mPL-I glycosylation could not be attributed to increased chloride concentration or osmolarity, or to extracellular events after secretion of the recombinant protein into the supernatant. Ammonium chloride, inhibited the expression rate of mPL-I by CHO cells at low pH[sub e].

  9. Alpha 2-adrenergic receptor stimulation of phospholipase A2 and of adenylate cyclase in transfected Chinese hamster ovary cells is mediated by different mechanisms

    SciTech Connect

    Jones, S.B.; Halenda, S.P.; Bylund, D.B. )

    1991-02-01

    The effect of alpha 2-adrenergic receptor activation on adenylate cyclase activity in Chinese hamster ovary cells stably transfected with the alpha 2A-adrenergic receptor gene is biphasic. At lower concentrations of epinephrine forskolin-stimulated cyclic AMP production is inhibited, but at higher concentrations the inhibition is reversed. Both of these effects are blocked by the alpha 2 antagonist yohimbine but not by the alpha 1 antagonist prazosin. Pretreatment with pertussis toxin attenuates inhibition at lower concentrations of epinephrine and greatly potentiates forskolin-stimulated cyclic AMP production at higher concentrations of epinephrine. alpha 2-Adrenergic receptor stimulation also causes arachidonic acid mobilization, presumably via phospholipase A2. This effect is blocked by yohimbine, quinacrine, removal of extracellular Ca2+, and pretreatment with pertussis toxin. Quinacrine and removal of extracellular Ca2+, in contrast, have no effect on the enhanced forskolin-stimulated cyclic AMP production. Thus, it appears that the alpha 2-adrenergic receptor in these cells can simultaneously activate distinct signal transduction systems; inhibition of adenylate cyclase and stimulation of phospholipase A2, both via G1, and potentiation of cyclic AMP production by a different (pertussis toxin-insensitive) mechanism.

  10. Digital mRNA profiling of N-glycosylation gene expression in recombinant Chinese hamster ovary cells treated with sodium butyrate.

    PubMed

    Lee, Sang Min; Kim, Yeon-Gu; Lee, Eun Gyo; Lee, Gyun Min

    2014-02-10

    To understand the effects of sodium butyrate (NaBu) on protein glycosylation, recombinant Chinese hamster ovary (rCHO) cells producing Fc-fusion glycoprotein were subjected to 3mM NaBu. The addition of NaBu to the cultures reduced the relative proportion of acidic isoforms and sialic acid content of the glycoprotein. Fifty-two N-glycosylation-related gene expressions were also assessed by the NanoString nCounter system, which can provide a direct digital readout using custom-designed color-coded probes. Among them, ten genes (ugp, slc35a2, ganc, man1a, man1c, mgat5a, st3gal5, glb1, neu1, and neu3) were up-regulated and three genes (b4galt2, st3gal3, and neu2) were down-regulated significantly. Altered expression patterns in st3gal3, neu1, and neu3, which have roles in the sialic acid biosynthesis pathway, correlated with reduced sialic acid content of the glycoprotein by NaBu. Taken together, the results obtained in this study provide a better understanding of the detrimental effect of NaBu on N-glycosylation in rCHO cells.

  11. High-frequency structural gene deletion as the basis for functional hemizygosity of the adenine phosphoribosyltransferase locus in Chinese hamster ovary cells.

    PubMed

    Adair, G M; Stallings, R L; Nairn, R S; Siciliano, M J

    1983-10-01

    The CHO-AT3-2 Chinese hamster ovary cell line is functionally hemizygous for the adenine phosphoribosyltransferase (APRT; EC 2.4.2.7) locus. Class 1 APRT +/- heterozygotes, such as CHO-AT3-2, can be isolated at high spontaneous frequencies from wild-type CHO cell populations. Simon et al. [Simon, A. E., Taylor, M. W., Bradley, W. E. C. & Thompson, L. (1982) Mol. Cell. Biol. 2, 1126-1133] have proposed that a high-frequency event that inactivates one APRT allele might be responsible for both the spontaneous generation of class 1 APRT +/- heterozygotes and the high-frequency occurrence of APRT- mutants in class 2 APRT +/- heterozygote populations. This event appears to occur at only one of the two APRT alleles. To investigate the nature of this high-frequency event, and to determine the genetic basis for functional hemizygosity of the APRT locus in CHO-AT3-2 cells, we have mapped the APRT locus by using CHO-AT3-2-mouse somatic cell hybrids. Our data confirm that CHO-AT3-2 cells have a single functional APRT allele, which is located on the Z7 chromosome. Karyotypic analysis of CHO-AT3-2 revealed an interstitial deletion on the long arm of the Z4 chromosome, in the very region where the other APRT allele should be located. To determine whether the Z4q interstitial deletion had resulted in physical loss of the APRT gene, DNA from CHO-AT3-2-mouse cell hybrids that had either lost or retained the Z4q- chromosome was analyzed for the presence of CHO APRT coding sequences. Our data suggest that allele-specific high-frequency structural gene deletion events involving the long arm of chromosome Z4 are responsible for the spontaneous generation of functional hemizygosity at the APRT locus in CHO cells.

  12. ATF6β-based fine-tuning of the unfolded protein response enhances therapeutic antibody productivity of Chinese Hamster Ovary cells.

    PubMed

    Pieper, Lisa A; Strotbek, Michaela; Wenger, Till; Olayioye, Monilola A; Hausser, Angelika

    2017-02-06

    The dynamics of protein folding and secretion are key issues in improving the productivity and robustness of Chinese Hamster Ovary (CHO) producer cells. High recombinant protein secretion in CHO producer clones triggers the activation of the unfolded protein response (UPR), an intracellular response to the accumulation of unfolded and misfolded proteins in the endoplasmic reticulum (ER). We previously reported that the human microRNA (miRNA) miR-1287 enhances productivity in IgG expressing CHO cells (CHO-IgG). Here, through next generation sequencing (NGS) we identified the activating transcription factor 6 beta (ATF6β), a repressor of the pro-survival and UPR promoting factor ATF6α, as a direct target gene of miR-1287 in CHO-IgG cells. We show that the transient depletion of ATF6β resulted in enhanced specific productivity comparable to that of miR-1287 expressing CHO-IgG cells. Strikingly, stable ATF6β knockdown in CHO-IgG cells significantly improved antibody titer and viable cell density under fed-batch conditions. This was associated with the elevated expression of the UPR genes glucose regulated protein 78 (GRP78), homocysteine inducible ER protein with ubiquitin like domain 1 (Herpud1) and CCAAT/enhancer-binding protein homologous protein (CHOP). We hence demonstrate that ATF6β-based cell line engineering is a promising strategy to improve the productivity of CHO producer cells by activating an optimally balanced UPR program. This article is protected by copyright. All rights reserved.

  13. Chinese hamster ovary cells cultured in low concentrations of fetal bovine serum: cloning efficiency, growth in suspension, and selection of drug-resistant mutant phenotypes

    SciTech Connect

    Carver, J.H.; Salazar, E.P.; Knize, M.G.

    1983-09-01

    Reducing serum concentrations in media provides a potential cost advantage. To determine whether such media could be used for applied mutagenesis assays, cloning efficiency and growth parameters in suspension of Chinese hamster ovary cells cultured were measured in reduced serum with or without additives (1 ..mu..g/ml insulin, 3 x 10/sup -7/ M linoleic acid, 1 x 10/sup -8/ M H/sub 2/SeO/sub 3/) or bovine serum albumin (BSA, 1% wt/vol). With the additives and less than or equal to 0.5% fetal bovine serum (FBS), Ham's F12 medium (without hypoxanthine and thymidine) was more optimal than alpha Eagle's minimum essential medium (MEM) (without ribosides and deoxyribosides) for low density cloning and high density suspension growth. The spontaneous frequency of azaadenine-resistant phenotypes (mutant at the aprt locus) in 1% FBS plus BSA was significantly lower than the frequency observed in 2% FBS plus BSA or 10% DFBS. Frequencies of spontaneous mutants resistant to thioguanine (hgprt locus) or fluorodeoxy-uridine (tk locus) were similar with 10% DFBS, 1% FBS plus BSA, or 2% FBS plus BSA. Compared to alpha MEM with 10% DFBS, frequencies of drug-resistant mutants induced by ethyl methanesulfonate or mitomycin C (MMC) were not significantly lower in alpha MEM with 2% FBS plus BSA; observed mutant frequencies induced by dimethylnitrosamine or benzo(a)pyrene seemed to be decreased at lower survival levels.

  14. Overexpression of phospholipase D prevents actinomycin D-induced apoptosis through potentiation of phosphoinositide 3-kinase signalling pathways in Chinese-hamster ovary cells.

    PubMed Central

    Yamada, Momoko; Banno, Yoshiko; Takuwa, Yoh; Koda, Masahiro; Hara, Akira; Nozawa, Yoshinori

    2004-01-01

    To examine the roles of PLD (phospholipase D) in the regulation of the apoptotic process, PLD1 and PLD2 were stably overexpressed in S1P3-CHO cells [CHO (Chinese-hamster ovary) cells expressing the S1P (sphingosine 1-phosphate) receptor S1P3]. Treatment of S1P3-CHO cells with ActD (actinomycin D) induced apoptosis, as shown by the occurrence of nuclear fragmentation and the caspase-dependent proteolytic cleavage of PARP [poly(ADP-ribose) polymerase] and protein kinase Cd. Overexpression of either PLD1 or PLD2 protected S1P3-CHO cells from ActD-induced apoptosis, as demonstrated by an increased number of viable cells and inhibition of PARP and protein kinase Cd cleavage. However, in the early phase of apoptosis, ActD induced an increase in PLD activity and activation of key factors in the cell-survival signalling pathways, such as PI3K (phosphoinositide 3-kinase), Akt, p70S6K (p70 S6 kinase) and ERK (extracellular-signal-regulated kinase). Furthermore, the ActD-induced activation of these survival signalling enzymes was potentiated by overexpression of either PLD1 or PLD2. The PI3K inhibitor LY294002 inhibited the ActD-induced activation of Akt and p70S6K, and completely abolished the effects of PLD1 or PLD2, whereas inhibition of ERK activity by the MEK inhibitor U0126 had a milder effect. The ActD-induced activation of p70S6K and ERKs was blocked by 1-butanol, but not by t-butanol; similar to S1P, exogenous PLD suppressed the ActD-induced events in the apoptosis signalling pathways. These results show that, in S1P3-CHO cells, increased expression of PLDs prevents ActD-induced apoptosis by enhanced activation of the PI3K signalling pathways. PMID:14640974

  15. Effect of Temperature Downshift on the Transcriptomic Responses of Chinese Hamster Ovary Cells Using Recombinant Human Tissue Plasminogen Activator Production Culture.

    PubMed

    Bedoya-López, Andrea; Estrada, Karel; Sanchez-Flores, Alejandro; Ramírez, Octavio T; Altamirano, Claudia; Segovia, Lorenzo; Miranda-Ríos, Juan; Trujillo-Roldán, Mauricio A; Valdez-Cruz, Norma A

    2016-01-01

    Recombinant proteins are widely used as biopharmaceuticals, but their production by mammalian cell culture is expensive. Hence, improvement of bioprocess productivity is greatly needed. A temperature downshift (TDS) from 37°C to 28-34°C is an effective strategy to expand the productive life period of cells and increase their productivity (qp). Here, TDS in Chinese hamster ovary (CHO) cell cultures, initially grown at 37°C and switched to 30°C during the exponential growth phase, resulted in a 1.6-fold increase in the qp of recombinant human tissue plasminogen activator (rh-tPA). The transcriptomic response using next-generation sequencing (NGS) was assessed to characterize the cellular behavior associated with TDS. A total of 416 (q > 0.8) and 3,472 (q > 0.9) differentially expressed transcripts, with more than a 1.6-fold change at 24 and 48 h post TDS, respectively, were observed in cultures with TDS compared to those at constant 37°C. In agreement with the extended cell survival resulting from TDS, transcripts related to cell growth arrest that controlled cell proliferation without the activation of the DNA damage response, were differentially expressed. Most upregulated genes were related to energy metabolism in mitochondria, mitochondrial biogenesis, central metabolism, and avoidance of apoptotic cell death. The gene coding for rh-tPA was not differentially expressed, but fluctuations were detected in the transcripts encoding proteins involved in the secretory machinery, particularly in glycosylation. Through NGS the dynamic processes caused by TDS were assessed in this biological system.

  16. Effect of Temperature Downshift on the Transcriptomic Responses of Chinese Hamster Ovary Cells Using Recombinant Human Tissue Plasminogen Activator Production Culture

    PubMed Central

    Bedoya-López, Andrea; Estrada, Karel; Sanchez-Flores, Alejandro; Ramírez, Octavio T.; Altamirano, Claudia; Segovia, Lorenzo; Miranda-Ríos, Juan; Trujillo-Roldán, Mauricio A.; Valdez-Cruz, Norma A.

    2016-01-01

    Recombinant proteins are widely used as biopharmaceuticals, but their production by mammalian cell culture is expensive. Hence, improvement of bioprocess productivity is greatly needed. A temperature downshift (TDS) from 37°C to 28–34°C is an effective strategy to expand the productive life period of cells and increase their productivity (qp). Here, TDS in Chinese hamster ovary (CHO) cell cultures, initially grown at 37°C and switched to 30°C during the exponential growth phase, resulted in a 1.6-fold increase in the qp of recombinant human tissue plasminogen activator (rh-tPA). The transcriptomic response using next-generation sequencing (NGS) was assessed to characterize the cellular behavior associated with TDS. A total of 416 (q > 0.8) and 3,472 (q > 0.9) differentially expressed transcripts, with more than a 1.6-fold change at 24 and 48 h post TDS, respectively, were observed in cultures with TDS compared to those at constant 37°C. In agreement with the extended cell survival resulting from TDS, transcripts related to cell growth arrest that controlled cell proliferation without the activation of the DNA damage response, were differentially expressed. Most upregulated genes were related to energy metabolism in mitochondria, mitochondrial biogenesis, central metabolism, and avoidance of apoptotic cell death. The gene coding for rh-tPA was not differentially expressed, but fluctuations were detected in the transcripts encoding proteins involved in the secretory machinery, particularly in glycosylation. Through NGS the dynamic processes caused by TDS were assessed in this biological system. PMID:26991106

  17. Genotoxic and cytotoxic effects of 60Co gamma-rays and 90Sr/90Y beta-rays on Chinese hamster ovary cells (CHO-K1).

    PubMed

    Murakami, Daniella; Suzuki, Miriam Fussae; da Silva Dias, Mauro; Okazaki, Kayo

    2004-07-01

    Among various types of ionizing radiation, the beta emitter radionuclides are involved in many sectors of human activity, such as nuclear medicine, nuclear industries and biomedicine, with a consequently increased risk of accidental, occupational or therapeutic exposure. Despite their recognized importance, there is little information about the effect of beta particles at the cellular level when compared to other types of ionizing radiation. Thus, the objective of the present study was to evaluate the genotoxic and cytotoxic effects of (90)Sr/(90)Y-a pure, highly energetic beta source-on Chinese hamster ovary (CHO) cells and to compare them with data obtained with (60)Co. CHO cells irradiated with different doses of (60)Co (0.34 Gy min(-1)) and (90)Sr/(90)Y (0.23 Gy min(-1)) were processed for analysis of clonogenic death, induction of micronuclei (MN) and interphase death. The survival curves obtained for both types of radiation were fitted by the exponential quadratic model and were found to be similar. Also, the cytogenetic results showed similar frequencies of radio-induced MN between gamma and beta radiations and the MN distribution pattern among cells did not follow the expected Poisson probability pattern. The relative variance values were significantly higher in cells irradiated with (90)Sr/(90)Y than with (60)Co in all exposure doses. The irradiated cells showed more necrotic cells 72 h and 96 h after exposure to beta than to gamma radiation. In general, the (90)Sr/(90)Y beta-radiation was more damaging than (60)Co gamma-rays. The data obtained also demonstrated the need to use several parameters for a better estimate of cellular sensitivity to the action of genotoxic agents, which would be important in terms of radiobiology, oncology and therapeutics.

  18. Optical forced oscillation for the study of lectin-glycoprotein interaction at the cellular membrane of a Chinese hamster ovary cell

    NASA Astrophysics Data System (ADS)

    Liu, Shang-Ling; Karmenyan, Artashes; Wei, Ming-Tzo; Huang, Chun-Chieh; Lin, Chi-Hung; Chiou, Arthur

    2007-03-01

    We report the application of a set of twin optical tweezers to trap and oscillate a ConA (lectin)- coated polystyrene particle and to measure its interaction with glycoprotein receptors at the cellular plasma membrane of a Chinese hamster ovary (CHO) cell. The particle was trapped between two quadratic potential wells defined by a set of twin optical tweezers and was forced to oscillate by chopping on and off one of the trapping beams. We tracked the oscillatory motion of the particle via a quadrant photodiode and measured with a lock-in amplifier the amplitude of the oscillation as a function of frequency at the fundamental component of the driving frequency over a frequency range from 10Hz to 600Hz. By analyzing the amplitude as a function of frequency for a free particle suspended in buffer solution without the presence of the CHO cell and compared with the corresponding data when the particle was interacting with the CHO cell, we deduced the transverse force constant associated with the optical trap and that associated with the interaction by treating both the optical trap and the interaction as linear springs. The force constants were determined to be approximately 2.15pN/μm for the trap and 2.53pN/μm for the lectin-glycoprotein interaction. When the CHO cell was treated with lantrunculin A, a drug that is known to destroy the cytoskeleton of the cell, the oscillation amplitude increased with time, indicating the softening of the cellular membrane, until a steady state with a smaller force constant was reached. The steady state value of the force constant depended on the drug concentration.

  19. Phosphatidylserine biosynthesis in cultured Chinese hamster ovary cells. I. Inhibition of de novo phosphatidylserine biosynthesis by exogenous phosphatidylserine and its efficient incorporation

    SciTech Connect

    Nishijima, M.; Kuge, O.; Akamatsu, Y.

    1986-05-05

    The effect of phosphatidylserine exogenously added to the medium on de novo biosynthesis of phosphatidylserine was investigated in cultured Chinese hamster ovary cells. When cells were cultured for several generations in medium supplemented with phosphatidylserine and /sup 32/Pi, the incorporation of /sup 32/Pi into cellular phosphatidylserine was remarkably inhibited, the degree of inhibition being dependent upon the concentration of added phosphatidylserine. /sup 32/Pi uptake into cellular phosphatidylethanolamine was also partly reduced by the addition of exogenous phosphatidylserine, consistent with the idea that phosphatidylethanolamine is biosynthesized via decarboxylation of phosphatidylserine. However, incorporation of /sup 32/Pi into phosphatidylcholine, sphingomyelin, and phosphatidylinositol was not significantly affected. In contrast, the addition of either phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, or phosphatidylinositol to the medium did not inhibit endogenous biosynthesis of the corresponding phospholipid. Radiochemical and chemical analyses of the cellular phospholipid composition revealed that phosphatidylserine in cells grown with 80 microM phosphatidylserine was almost entirely derived from the added phospholipid. Phosphatidylserine uptake was also directly determined by using (/sup 3/H)serine-labeled phospholipid. Pulse and pulse-chase experiments with L-(U-/sup 14/C) serine showed that when cells were cultured with 80 microM phosphatidylserine, the rate of synthesis of phosphatidylserine was reduced 3-5-fold. Enzyme assaying of extracts prepared from cells grown with and without phosphatidylserine indicated that the inhibition of de novo phosphatidylserine biosynthesis by the added phosphatidylserine appeared not to be caused by a reduction in the level of the enzyme involved in the base-exchange reaction between phospholipids and serine.

  20. Folate-dependent enzymes in cultured Chinese hamster ovary cells: impaired mitochondrial serine hydroxymethyltransferase activity in two additional glycine-auxotroph complementation classes

    SciTech Connect

    Taylor, R.T.; Hanna, M.L.

    1982-09-01

    Two glycine-requiring Chinese hamster ovary (CHO) auxotrophs (GLYB and AUXB2) representative of the Gly/sup -/ mutant classes B and C are shown to have defects in folate metabolism. These defects result in 10-fold lower rates of whole cell L-(U-/sup 14/C)serine-to-(/sup 14/C)glycine conversion relative to the parental CHO lines (2 vs 20 nmol/h/10/sup 6/ cells). This restriction in serine hydroxymethyltransferase (SHMT) activity is localized in the mitochondria. Intact mitochondria from GLYB and AUXB2 convert labeled serine to glycine at 1-4% the rate and with only 1-3% of the total capacity of parental CHO mitochondria. Yet, GLYB and AUXB2 contain parental cell amounts of cytosolic and mitochondrial SHMT, the latter displaying normal substrate K/sub m/ values. The whole cell and mitochondrial impairments in glycine formation are corrected in GLYB (but not AUXB2) by a prior growth with 100 ..mu..M dl-folinate. They are also partially restored in spontaneous or chemically induced Gly/sup +/ revertants of GLYB and AUXB2. Subcellular fractionation experiments suggest that a low content (one-fifth parental) of mitochondrial folylpolyglutamates contributes to the auxotrophy of GLYB. These studies demonstrate that mitochondrial SHMT is potentially functional in the Gly/sup -/ mutant classes B (GLYB) and C (AUXB2). The impaired SHMT activity in vivo and in isolated mitochondria may result from a deficiency in mitochondrial recycling of 5,10-methylenetetrahydrofolate back to tetrahydrofolate.

  1. Prolonged activation of phospholipase D in Chinese hamster ovary cells expressing platelet-activating-factor receptor lacking cytoplasmic C-terminal tail.

    PubMed Central

    Liu, B; Nakashima, S; Adachi, T; Ito, Y; Takano, T; Shimizu, T; Nozawa, Y

    1997-01-01

    The mechanism and role of phospholipase D (PLD) activation by platelet-activating factor (PAF) were examined with Chinese hamster ovary cells stably expressing wild-type PAF receptor (WT-H cells) and truncated PAF receptor lacking the C-terminal cytoplasmic tail (D-H cells). Treatment of D-H cells with PAF resulted in the rapid formation of Ins(1,4,5)P3, which was followed by a sustained phase for more than 10 min. In these cells, PAF-induced PLD activation lasted for more than 20 min. In contrast, PLD activation in WT-H cells was transient. PAF stimulation caused the biphasic formation of 1,2-diacylglycerol (DG) in both types of cell. The first phase was rapid and transient, coinciding with the Ins(1,4,5)P3 peak. The second sustained phase of DG formation was attenuated by butanol, which produces phosphatidylbutanol at the expense of phosphatidic acid (PA) by transphosphatidylation activity of PLD, and by propranolol, a selective inhibitor for PA phosphohydrolase catalysing the conversion of PA into DG. The DG level returned nearly to basal at 20 min after PAF stimulation in WT-H cells, whereas in D-H cells the elevated DG level was sustained for more than 20 min. The profile of translocation of protein kinase Calpha (PKCalpha) to membrane was similar to that of DG formation. In WT-H cells, PKCalpha was transiently associated with membranes and then returned to the cytosol. However, in D-H cells PKCalpha was rapidly translocated to and remained in membranes for more than 20 min. Butanol suppressed this sustained translocation of PKCalpha. Furthermore the mRNA levels of c-fos and c-jun by PAF in WT-H cells were much lower than those in D-H cells. Propranolol and butanol at concentrations that inhibited the formation of DG suppressed the PAF-induced mRNA expression of c-fos and c-jun. Taken together, the prolonged PLD activation in D-H cells confirmed a primary role for phospholipase C/PKC in PLD activation by PAF. Furthermore the results obtained here suggest that

  2. Understanding of altered N-glycosylation-related gene expression in recombinant Chinese hamster ovary cells subjected to elevated ammonium concentration by digital mRNA counting.

    PubMed

    Ha, Tae Kwang; Kim, Yeon-Gu; Lee, Gyun Min

    2015-08-01

    To understand the effects of ammonium on N-glycosylation, recombinant Chinese hamster ovary (rCHO) cells that produce the Fc-fusion protein were cultivated in serum-free suspension cultures with 10 mM ammonium addition. The addition of ammonium to the cultures reduced the relative proportion of acidic isoforms and sialic acid content of an Fc-fusion protein. Fifty two N-glycosylation-related gene expressions were assessed by the NanoString nCounter system, which provides a digital readout using custom-designed color-coded probes. Among these queried genes, thirteen genes (gale, nans, gpi, man2a1, b4galt5, b4galt7, st3gal2, st3gal5, glb1, hexa, hexb, neu1, and neu3) were up-regulated over 1.5 times in the culture with ammonium addition after 5 days of culture; however, none of the 54 genes were significantly different after 3 days of culture. In particular, the expression level of neu1 (sialidase-1) and neu3 (sialidase-3), which play a role in reduction of sialylation, increased over 2 times. Likewise, the protein expression levels of sialidase-1 and sialidase-3 determined by Western blot analysis were also increased significantly in the culture with ammonium addition. Transient transfection of neu-1 or neu3-targeted siRNAs significantly improved the sialic acid content of the Fc-fusion protein in the culture with ammonium addition, indicating that the decreased sialic acid content was in part due to the increased expression level of sialidase. Taken together, the results obtained in this study provide a better understanding of the detrimental effect of ammonium on N-glycosylation, especially sialylation, in rCHO cells.

  3. The effect of ammonia on the O-linked glycosylation of granulocyte colony-stimulating factor produced by chinese hamster ovary cells

    SciTech Connect

    Andersen, D.C.; Goochee, C.F.

    1995-07-05

    Ammonium ion concentrations ranging from 0 to 10 mM are shown to significantly reduce the sialylation of granulocyte colony-stimulating factor (G-CSF) produced by recombinant Chinese hamster ovary cells. Specifically, the degree of completion of the final reaction in the O-linked glycosylation pathway, the addition of sialic acid in an {alpha}(2,6) linkage to N-acetylgalactosamine, is reduced by NH{sub 4}{sup +} concentrations of as low as 2 mM. The effect of ammonia on sialylation is rapid, sustained, and does not affect the secretion rate of G-CSF. Additionally, the effect can be mimicked using the weak base chloroquine, suggesting that the effect is related to the weak base characteristics of ammonia. In support of this hypothesis, experiments using brefeldin A suggest that the addition of sialic acid in an {alpha}(2,6) linkage to N-acetylgalactosamine occurs in the trans-Golgi compartment prior to the trans-Golgi network, which would be expected under normal conditions to have a slightly acidic pH in the range from 6.5 to 6.75. Ammonium ion concentrations of 10mM would be expected to reduce significantly the differences in pH between acidic intracellular compartments and the cytoplasm. The pH-activity profile for the CHO O-linked {alpha}(2,6)sialytransferase using monosialylated G-CSF as a substrate reveals a twofold decrease in enzymatic activity across the pH range from 6.75 to 7.0. Mathematical modeling of this sialylation reaction supports the hypothesis that this twofold decrease in sialyltransferase activity resulting from an ammonia-induced increase in trans-Golgi pH could produce the observed decrease in G-CSF sialylation.

  4. Nuclear Hormone Receptor Activity of Polybrominated Diphenyl Ethers and Their Hydroxylated and Methoxylated Metabolites in Transactivation Assays Using Chinese Hamster Ovary Cells

    PubMed Central

    Kojima, Hiroyuki; Takeuchi, Shinji; Uramaru, Naoto; Sugihara, Kazumi; Yoshida, Takahiko; Kitamura, Shigeyuki

    2009-01-01

    Background An increasing number of studies are reporting the existence of polybrominated diphenyl ethers (PBDEs) and their hydroxylated (HO) and methoxylated (MeO) metabolites in the environment and in tissues from wildlife and humans. Objective Our aim was to characterize and compare the agonistic and antagonistic activities of principle PBDE congeners and their HO and MeO metabolites against human nuclear hormone receptors. Methods We tested the hormone receptor activities of estrogen receptor α (ERα), ERβ, androgen receptor (AR), glucocorticoid receptor (GR), thyroid hormone receptor α1 (TRα1), and TRβ1 against PBDE congeners BDEs 15, 28, 47, 85, 99, 100, 153, and 209, four para-HO-PBDEs, and four para-MeO-PBDEs by highly sensitive reporter gene assays using Chinese hamster ovary cells. Results Of the 16 compounds tested, 6 and 2 showed agonistic activities in the ERα and ERβ assays, respectively, and 6 and 6 showed antagonistic activities in these assays. 4′-HO-BDE-17 showed the most potent estrogenic activity via ERα/β, and 4′-HO-BDE-49 showed the most potent anti estrogenic activity via ERα/β. In the AR assay, 13 compounds showed antagonistic activity, with 4′-HO-BDE-17 in particular inhibiting AR-mediated transcriptional activity at low concentrations in the order of 10−8 M. In the GR assay, seven compounds, including two HO-PBDEs and two MeO-PBDEs, showed weak antagonistic activity. In the TRα1 and TRβ1 assays, only 4-HO-BDE-90 showed weak antagonistic activity. Conclusions Taken together, these results suggest that PBDEs and their metabolites might have multiple endocrine-disrupting effects via nuclear hormone receptors, and para-HO-PBDEs, in particular, possess more potent receptor activities compared with those of the parent PBDEs and corresponding para-MeO-PBDEs. PMID:19672399

  5. Molecular polygamy: The promiscuity of l-phenylalanyl-tRNA-synthetase triggers misincorporation of meta- and ortho-tyrosine in monoclonal antibodies expressed by Chinese hamster ovary cells.

    PubMed

    Popp, Oliver; Larraillet, Vincent; Kettenberger, Hubert; Gorr, Ingo H; Hilger, Maximiliane; Lipsmeier, Florian; Zeck, Anne; Beaucamp, Nicola

    2015-06-01

    In-depth analytical characterization of biotherapeutics originating from different production batches is mandatory to ensure product safety and consistent molecule efficacy. Previously, we have shown unintended incorporation of tyrosine (Tyr) and leucine/isoleucine (Leu/Ile) at phenylalanine (Phe) positions in a recombinant produced monoclonal antibody (mAb) using an orthogonal MASCOT/SIEVE based approach for mass spectrometry data analysis. The misincorporation could be avoided by sufficient supply of phenylalanine throughout the process. Several non-annotated signals in the primarily chromatographic peptide separation step for apparently single Phe→Tyr sequence variants (SVs) suggest a role for isobar tyrosine isoforms. Meta- and ortho-Tyr are spontaneously generated during aerobic fed-batch production processes using Chinese hamster ovary (CHO) cell lines. Process induced meta- and ortho-Tyr but not proteinogenic para-Tyr are incorporated at Phe locations in Phe-starved CHO cultures expressing a recombinant mAb. Furthermore, meta- and ortho-Tyr are preferably misincorporated over Leu. Structural modeling of the l-phenylalanyl-tRNA-synthetase (PheRS) substrate activation site indicates a possible fit of non-cognate ortho-Tyr and meta-Tyr substrates. Dose-dependent misincorporations of Tyr isoforms support the hypothesis that meta- and ortho-Tyr are competing, alternative substrates for PheRS in CHO processes. Finally, easily accessible at-line surrogate markers for Phe→Tyr SV formation in biotherapeutic production were defined by the calculation of critical ratios for meta-Tyr/Phe and ortho-Tyr/Phe to support early prediction of SV probability, and finally, to allow for immediate process controlled Phe→Tyr SV prevention.

  6. Screening for estrogen and androgen receptor activities in 200 pesticides by in vitro reporter gene assays using Chinese hamster ovary cells.

    PubMed Central

    Kojima, Hiroyuki; Katsura, Eiji; Takeuchi, Shinji; Niiyama, Kazuhito; Kobayashi, Kunihiko

    2004-01-01

    We tested 200 pesticides, including some of their isomers and metabolites, for agonism and antagonism to two human estrogen receptor (hER) subtypes, hERalpha and hERbeta, and a human androgen receptor (hAR) by highly sensitive transactivation assays using Chinese hamster ovary cells. The test compounds were classified into nine groups: organochlorines, diphenyl ethers, organophosphorus pesticides, pyrethroids, carbamates, acid amides, triazines, ureas, and others. These pesticides were tested at concentrations < 10-5 M. Of the 200 pesticides tested, 47 and 33 showed hER- and hERbeta-mediated estrogenic activities, respectively. Among them, 29 pesticides had both hERalpha and hERbeta agonistic activities, and the effects of the organochlorine insecticides beta-benzene hexachloride (BHC) and delta-BHC and the carbamate insecticide methiocarb were predominantly hERbeta rather than hERalpha agonistic. Weak antagonistic effects toward hERalpha and hERbeta were shown in five and two pesticides, respectively. On the other hand, none of tested pesticides showed hAR-mediated androgenic activity, but 66 of 200 pesticides exhibited inhibitory activity against the transcriptional activity induced by 5alpha-dihydrotestosterone. In particular, the antiandrogenic activities of two diphenyl ether herbicides, chlornitrofen and chlomethoxyfen, were higher than those of vinclozolin and p,p -dichlorodiphenyl dichloroethylene, known AR antagonists. The results of our ER and AR assays show that 34 pesticides possessed both estrogenic and antiandrogenic activities, indicating pleiotropic effects on hER and hAR. We also discussed chemical structures related to these activities. Taken together, our findings suggest that a variety of pesticides have estrogenic and/or antiandrogenic potential via ER and/or AR, and that numerous other manmade chemicals may also possess such estrogenic and antiandrogenic activities. PMID:15064155

  7. A quantitative assay of mutation induction at the hypoxanthine-guanine phosphoribosyl transferase locus in Chinese hamster ovary cells (CHO/HGPRT system): development and definition of the system.

    PubMed

    O'Neill, J P; Brimer, P A; Machanoff, R; Hirsch, G P; Hsie, A W

    1977-10-01

    An assay is described for the measurement of mutation induction at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary (CHO) cells utilizing resistance to 6-thioguanine (TG). Optimal selection conditions are defined for such parameters as phenotypic expression time prior to selection, and TG concentration and cell density which permits maximum mutant recovery. The nature of the TG-resistant mutants is characterized by several physiological and biochemical methods. The data demonstrate that more than 98% of the mutant clones isolated by this selection procedure contain altered HGPRTase activity. The CHO/HGPRT system thus shows the specificity necessary for a specific gene locus mutational assay.

  8. Clastogenicity, photo-clastogenicity or pseudo-photo-clastogenicity: Genotoxic effects of zinc oxide in the dark, in pre-irradiated or simultaneously irradiated Chinese hamster ovary cells.

    PubMed

    Dufour, Eric K; Kumaravel, Tirukalikundram; Nohynek, Gerhard J; Kirkland, David; Toutain, Hervé

    2006-09-05

    Zinc oxide (ZnO), a widely used ingredient in dermatological preparations and sunscreens, is clastogenic in vitro, but not in vivo. Given that ZnO has an approximately four-fold greater clastogenic potency in the presence of UV light when compared with that in the dark, it has been suggested to be photo-clastogenic. In order to clarify whether this increased potency is a genuine photo-genotoxic effect, we investigated the clastogenicity of ZnO (mean particle size, 100 nm) in Chinese hamster ovary (CHO) cells in the dark (D), in pre-irradiated (PI, i.e. UV irradiation of cells followed by treatment with ZnO) and in simultaneously irradiated (SI, i.e. ZnO treatment concurrent with UV irradiation) CHO cells at UV doses of 350 and 700 mJ/cm(2). The cytotoxicity of ZnO to CHO cells under the different irradiation conditions was as follows: SI>PI>D. In the dark, ZnO produced a concentration-related increase in chromosome aberrations (CA). In PI or SI CHO cells, ZnO was clastogenic at significantly lower concentrations (approximately two- to four-fold) when compared with effective concentrations in the dark, indicating an increased susceptibility of CHO cells to ZnO-mediated clastogenic effects due to UV irradiation per se. The incidence of CA in SI or PI cells was generally higher than that in the dark. At similar ZnO concentrations, SI conditions generally produced higher CA incidence than PI conditions. However, when ZnO concentrations producing similar cytotoxicity were compared, CA incidences under PI or SI conditions were nearly identical. The modest increase in the clastogenic potency of ZnO following UV irradiation contrasts with the results observed with genuine photo-clastogenic agents, such as 8-MOP, which may produce an increase in clastogenic potency of >15,000-fold under SI conditions. Our results provide evidence that, under conditions of in vitro photo-clastogenicity tests, UV irradiation of the cellular test system per se may produce a slight increase in

  9. Comparison of five different in vitro assays for assessment of sodium metavanadate cytotoxicity in Chinese hamster ovary cells (CHO-K1 line).

    PubMed

    Zwolak, Iwona

    2015-08-01

    This investigation was undertaken to compare five different in vitro cytotoxicity assays for their power in revealing vanadium-mediated toxicity in Chinese hamster ovary (CHO)-K1 cells. The cells were exposed to sodium metavanadate (NaVO(3)) in the range of 10-1000 µM for 24 h and thereafter the cytotoxic effects of NaVO(3) were measured by colorimetric in vitro assays: the neutral red (NR) test, the 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide inner salt (XTT) assay, the resazurin assay, the sulforhodamine B (SR-B) assay, and by microscopic assessment of cell viability using the trypan blue (TB) staining method. Among the assays used, the NR test was the most sensitive, since it revealed metavanadate cytotoxicity at the lowest NaVO(3) dose (=50 µM). Also, NaVO(3) cytotoxicity expressed as inhibitory concentration (IC) showed the lowest values for the NR test. Three other tests XTT, resazurin, and SR-B assays showed intermediate sensitivity revealing the cytotoxicity of NaVO(3) at 100 µM. The corresponding IC10 and IC50 values calculated for the XTT, resazurin, and SR-B tests were similar. The TB staining method was the least sensitive, since it recorded metavanadate cytotoxicity at the highest NaVO(3) concentration tested (=600 µM). Based on the cytotoxicity end points measured with the above assays, it can be concluded that lysosomal/Golgi apparatus damage (measured by NR assay) may be the primary effect of NaVO(3) on CHO-K1 cells. The disintegration of mitochondria (assessed with the XTT and resazurin assays) probably follows lysosomal impairment. Plasma membrane permeability (staining with TB) occurs at a late stage of NaVO(3)-induced cytotoxicity on CHO-K1 cells. The results obtained in this research work show that the NR test can be recommended as a very sensitive assay for the assessment of NaVO(3) cytotoxicity in the CHO-K1 cell culture model. Considering the convenience of assay performance along with adequate sensitivity

  10. Cloning and Expression of Major Surface Antigen 1 Gene of Toxoplasma gondii RH Strain Using the Expression Vector pVAX1 in Chinese Hamster Ovary Cells

    PubMed Central

    Abdizadeh, Rahman; Maraghi, Sharif; Ghadiri, Ata A.; Tavalla, Mehdi; Shojaee, Saeedeh

    2015-01-01

    Background: Toxoplasmosis is an opportunistic protozoan infection with a high prevalence in a broad range of hosts infecting up to one-third of the world human population. Toxoplasmosis leads to serious medical problems in immunocompromised individuals and fetuses and also induces abortion and mortality in domestic animals. Therefore, there is a huge demand for the development of an effective vaccine. Surface Antigen 1 (SAG1) is one of the important immunodominant surface antigens of Toxoplasma gondii, which interacts with host cells and primarily involved in adhesion, invasion and stimulation of host immune response. Surface antigen 1 is considered as the leading candidate for development of an effective vaccine against toxoplasmosis. Objectives: The purpose of this study was to clone the major surface antigen1 gene (SAG1) from the genotype 1 of T. gondii, RH strain into the eukaryotic expression vector pVAX1 in order to use for a DNA vaccine. Materials and Methods: Genomic DNA was extracted from tachyzoite of the parasite using the QIAamp DNA mini kit. After designing the specific primers, SAG1 gene was amplified by Polymerase Chain Reaction (PCR). The purified PCR products were then cloned into a pPrime plasmid vector. The aforementioned product was subcloned into the pVAX1 eukaryotic expression vector. The recombinant pVAX1-SAG1 was then transfected into Chinese Hamster Ovary (CHO) cells and expression of SAG1 antigen was evaluated using Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), Immunofluorescence Assay (IFA) and Western Blotting (WB). Results: The cloning and subcloning products (pPrime-SAG1 and pVAX1-SAG1 plasmid vectors) of SAG1 gene were verified and confirmed by enzyme digestion and sequencing. A 30 kDa recombinant protein was expressed in CHO cells as shown by IFA and WB methods. Conclusions: The pVAX1 expression vector and CHO cells are a suitable system for high-level recombinant protein production for SAG1 gene from T. gondii parasites

  11. Isolation and characterization of Chinese hamster ovary (CHO) cells deficient in acyl coenzyme A: cholesterol acyltransferase (ACAT) activity

    SciTech Connect

    Cadigan, K.M.; Heider, J.G.; Chang, T.Y.

    1986-05-01

    The specific ACAT inhibitor compound 58-035 has been used to mimic the phenotype of an ACAT deficient mutant in 25-RA cells. 25-RA is a CHO cell line resistant to 25-hydroxycholesterol and contains five times more cholesterol ester than wild-type (WT) cells. 25-RA cells preincubated with 58-035 are 100 to 500 times more resistant to amphotericin B killing than untreated 25-RA. 100 x 10/sup 6/ mutagenized 25-RA cells underwent three rounds of amphotericin B killing and two rounds of 25-hydroxycholesterol killing (to remove WT revertants which are amphotericin B resistant). Thus far, three biochemically distinct mutants have been isolated containing 33% (AC27), 25% (AC90), and 10% (AC232) of the parental ACAT activity as measured by an /sup 3/H-oleate pulse in intact cells. When parental and mutant cell extracts are reconstituted into cholesterol containing liposomes the differences in ACAT activity remain. They have also found that 25-RA cells can survive in cholesterol free medium containing TMD, an inhibitor of cholesterol biosynthesis, presumably because of adequate supply of endogenous cholesterol from hydrolysis of its stored cholesterol ester. In contrast, under the same conditions, mutant AC232 is effectively killed ( greater than or equal to 99%) by cholesterol starvation, thus providing a potential selection procedure for isolating revertants of ACAT mutants.

  12. PDADMAC flocculation of Chinese hamster ovary cells: enabling a centrifuge-less harvest process for monoclonal antibodies.

    PubMed

    McNerney, Thomas; Thomas, Anne; Senczuk, Anna; Petty, Krista; Zhao, Xiaoyang; Piper, Rob; Carvalho, Juliane; Hammond, Matthew; Sawant, Satin; Bussiere, Jeanine

    2015-01-01

    High titer (>10 g/L) monoclonal antibody (mAb) cell culture processes are typically achieved by maintaining high viable cell densities over longer culture durations. A corresponding increase in the solids and sub-micron cellular debris particle levels are also observed. This higher burden of solids (≥15%) and sub-micron particles typically exceeds the capabilities of a continuous centrifuge to effectively remove the solids without a substantial loss of product and/or the capacity of the harvest filtration train (depth filter followed by membrane filter) used to clarify the centrate. We discuss here the use of a novel and simple two-polymer flocculation method used to harvest mAb from high cell mass cell culture processes. The addition of the polycationic polymer, poly diallyldimethylammonium chloride (PDADMAC) to the cell culture broth flocculates negatively-charged cells and cellular debris via an ionic interaction mechanism. Incorporation of a non-ionic polymer such as polyethylene glycol (PEG) into the PDADMAC flocculation results in larger flocculated particles with faster settling rate compared to PDADMAC-only flocculation. PDADMAC also flocculates the negatively-charged sub-micron particles to produce a feed stream with a significantly higher harvest filter train throughput compared to a typical centrifuged harvest feed stream. Cell culture process variability such as lactate production, cellular debris and cellular densities were investigated to determine the effect on flocculation. Since PDADMAC is cytotoxic, purification process clearance and toxicity assessment were performed.

  13. PDADMAC flocculation of Chinese hamster ovary cells: Enabling a centrifuge-less harvest process for monoclonal antibodies

    PubMed Central

    McNerney, Thomas; Thomas, Anne; Senczuk, Anna; Petty, Krista; Zhao, Xiaoyang; Piper, Rob; Carvalho, Juliane; Hammond, Matthew; Sawant, Satin; Bussiere, Jeanine

    2015-01-01

    High titer (>10 g/L) monoclonal antibody (mAb) cell culture processes are typically achieved by maintaining high viable cell densities over longer culture durations. A corresponding increase in the solids and sub-micron cellular debris particle levels are also observed. This higher burden of solids (≥15%) and sub-micron particles typically exceeds the capabilities of a continuous centrifuge to effectively remove the solids without a substantial loss of product and/or the capacity of the harvest filtration train (depth filter followed by membrane filter) used to clarify the centrate. We discuss here the use of a novel and simple two-polymer flocculation method used to harvest mAb from high cell mass cell culture processes. The addition of the polycationic polymer, poly diallyldimethylammonium chloride (PDADMAC) to the cell culture broth flocculates negatively-charged cells and cellular debris via an ionic interaction mechanism. Incorporation of a non-ionic polymer such as polyethylene glycol (PEG) into the PDADMAC flocculation results in larger flocculated particles with faster settling rate compared to PDADMAC-only flocculation. PDADMAC also flocculates the negatively-charged sub-micron particles to produce a feed stream with a significantly higher harvest filter train throughput compared to a typical centrifuged harvest feed stream. Cell culture process variability such as lactate production, cellular debris and cellular densities were investigated to determine the effect on flocculation. Since PDADMAC is cytotoxic, purification process clearance and toxicity assessment were performed. PMID:25706650

  14. Effect of pH, temperature, and salt on the stability of Escherichia coli- and Chinese hamster ovary cell-derived IgG1 Fc.

    PubMed

    Li, Cynthia H; Narhi, Linda O; Wen, Jie; Dimitrova, Mariana; Wen, Zai-qing; Li, Jenny; Pollastrini, Joseph; Nguyen, Xichdao; Tsuruda, Trace; Jiang, Yijia

    2012-12-18

    The circulation half-life of a potential therapeutic can be increased by fusing the molecule of interest (an active peptide, the extracellular domain of a receptor, an enzyme, etc.) to the Fc fragment of a monoclonal antibody. For the fusion protein to be a successful therapeutic, it must be stable to process and long-term storage conditions, as well as to physiological conditions. The stability of the Fc used is critical for obtaining a successful therapeutic protein. The effects of pH, temperature, and salt on the stabilities of Escherichia coli- and Chinese hamster ovary cell (CHO)-derived IgG1 Fc high-order structure were probed using a variety of biophysical techniques. Fc molecules derived from both E. coli and CHO were compared. The IgG1 Fc molecules from both sources (glycosylated and aglycosylated) are folded at neutral pH and behave similarly upon heat- and low pH-induced unfolding. The unfolding of both IgG1 Fc molecules occurs via a multistep unfolding process, with the tertiary structure and C(H)2 domain unfolding first, followed by changes in the secondary structure and C(H)3 domain. The acid-induced unfolding of IgG1 Fc molecules is only partially reversible, with the formation of high-molecular weight species. The CHO-derived Fc protein (glycosylated) is more compact (smaller hydrodynamic radius) than the E. coli-derived protein (aglycosylated) at neutral pH. Unfolding is dependent on pH and salt concentration. The glycosylated C(H)2 domain melts at a temperature 4-5 °C higher than that of the aglycosylated domain, and the low-pH-induced unfolding of the glycosylated Fc molecule occurs at a pH ~0.5 pH unit lower than that of the aglycosylated protein. The difference observed between E. coli- and CHO-derived Fc molecules primarily involves the C(H)2 domain, where the glycosylation of the Fc resides.

  15. Carboxypeptidase D is the only enzyme responsible for antibody C-terminal lysine cleavage in Chinese hamster ovary (CHO) cells.

    PubMed

    Hu, Zhilan; Zhang, Henry; Haley, Benjamin; Macchi, Frank; Yang, Feng; Misaghi, Shahram; Elich, Joseph; Yang, Renee; Tang, Yun; Joly, John C; Snedecor, Bradley R; Shen, Amy

    2016-10-01

    Heterogeneity of C-terminal lysine levels often observed in therapeutic monoclonal antibodies is believed to result from the proteolysis by endogenous carboxypeptidase(s) during cell culture production. Identifying the responsible carboxypeptidase(s) for C-terminal lysine cleavage in CHO cells would provide valuable insights for antibody production cell culture processes development and optimization. In this study, five carboxypeptidases, CpD, CpM, CpN, CpB, and CpE, were studied for message RNA (mRNA) expression by qRT-PCR analysis in two most commonly used blank hosts (DUXB-11 derived DHFR-deficient DP12 host and DHFR-positive CHOK1 host), used for therapeutic antibody production, as well an antibody-expressing cell line derived from each host. Our results showed that CpD had the highest mRNA expression. When CpD mRNA levels were reduced by RNAi (RNA interference) technology, C-terminal lysine levels increased, whereas there was no obvious change in C-terminal lysine levels when a different carboxypeptidase mRNA level was knocked down suggesting that carboxypeptidase D is the main contributor for C-terminal lysine processing. Most importantly, when CpD expression was knocked out by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology, C-terminal lysine cleavage was completely abolished in CpD knockout cells based on mass spectrometry analysis, demonstrating that CpD is the only endogenous carboxypeptidase that cleaves antibody heavy chain C-terminal lysine in CHO cells. Hence, our work showed for the first time that the cleavage of antibody heavy chain C-terminal lysine is solely mediated by the carboxypeptidase D in CHO cells and our finding provides one solution to eliminating C-terminal lysine heterogeneity for therapeutic antibody production by knocking out CpD gene expression. Biotechnol. Bioeng. 2016;113: 2100-2106. © 2016 Wiley Periodicals, Inc.

  16. In Vivo Crystallization of Human IgG in the Endoplasmic Reticulum of Engineered Chinese Hamster Ovary (CHO) Cells

    PubMed Central

    Hasegawa, Haruki; Wendling, John; He, Feng; Trilisky, Egor; Stevenson, Riki; Franey, Heather; Kinderman, Francis; Li, Gary; Piedmonte, Deirdre Murphy; Osslund, Timothy; Shen, Min; Ketchem, Randal R.

    2011-01-01

    Protein synthesis and secretion are essential to cellular life. Although secretory activities may vary in different cell types, what determines the maximum secretory capacity is inherently difficult to study. Increasing protein synthesis until reaching the limit of secretory capacity is one strategy to address this key issue. Under highly optimized growth conditions, recombinant CHO cells engineered to produce a model human IgG clone started housing rod-shaped crystals in the endoplasmic reticulum (ER) lumen. The intra-ER crystal growth was accompanied by cell enlargement and multinucleation and continued until crystals outgrew cell size to breach membrane integrity. The intra-ER crystals were composed of correctly folded, endoglycosidase H-sensitive IgG. Crystallizing propensity was due to the intrinsic physicochemical properties of the model IgG, and the crystallization was reproduced in vitro by exposing a high concentration of IgG to a near neutral pH. The striking cellular phenotype implicated the efficiency of IgG protein synthesis and oxidative folding exceeded the capacity of ER export machinery. As a result, export-ready IgG accumulated progressively in the ER lumen until a threshold concentration was reached to nucleate crystals. Using an in vivo system that reports accumulation of correctly folded IgG, we showed that the ER-to-Golgi transport steps became rate-limiting in cells with high secretory activity. PMID:21464137

  17. Characterization of alanine to valine sequence variants in the Fc region of nivolumab biosimilar produced in Chinese hamster ovary cells

    PubMed Central

    Li, Yantao; Fu, Tuo; Liu, Tao; Guo, Huaizu; Guo, Qingcheng; Xu, Jin; Zhang, Dapeng; Qian, Weizhu; Dai, Jianxin; Li, Bohua; Guo, Yajun; Hou, Sheng; Wang, Hao

    2016-01-01

    ABSTRACT Nivolumab is a therapeutic fully human IgG4 antibody to programmed death 1 (PD-1). In this study, a nivolumab biosimilar, which was produced in our laboratory, was analyzed and characterized. Sequence variants that contain undesired amino acid sequences may cause concern during biosimilar bioprocess development. We found that low levels of sequence variants were detected in the heavy chain of the nivolumab biosimilar by ultra performance liquid chromatography (UPLC) and tandem mass spectrometry. It was further identified with UPLC-MS/MS by IdeS or trypsin digestion. The sequence variant was confirmed through addition of synthetic mutant peptide. Subsequently, the mixing base signal of normal and mutant sequence was detected through DNA sequencing. The relative levels of mutant A424V in the Fc region of the heavy chain have been detected and demonstrated to be 12.25% and 13.54%, via base peak intensity (BPI) and UV chromatography of the tryptic peptide mapping, respectively. A424V variant was also quantified by real-time PCR (RT-PCR) at the DNA and RNA level, which was 19.2% and 16.8%, respectively. The relative content of the mutant was consistent at the DNA, RNA and protein level, indicating that the A424V mutation may have little influence at transcriptional or translational levels. These results demonstrate that orthogonal state-of-the-art techniques such as LC- UV- MS and RT-PCR should be implemented to characterize recombinant proteins and cell lines for development of biosimilars. Our study suggests that it is important to establish an integrated and effective analytical method to monitor and characterize sequence variants during antibody drug development, especially for antibody biosimilar products. PMID:27050807

  18. Cell-permeable ceramides preferentially inhibit coated vesicle formation and exocytosis in Chinese hamster ovary compared with Madin-Darby canine kidney cells by preventing the membrane association of ADP-ribosylation factor.

    PubMed Central

    Abousalham, Abdelkarim; Hobman, Tom C; Dewald, Jay; Garbutt, Michael; Brindley, David N

    2002-01-01

    Differential effects of acetyl(C2-) ceramide (N-acetylsphingosine) were studied on coated vesicle formation from Golgi-enriched membranes of Chinese hamster ovary (CHO) and Madin-Darby canine kidney (MDCK) cells. C2-ceramide blocked the translocation of ADP-ribosylation factor-1 (ARF-1) and protein kinase C-alpha (PKC-alpha) to the membranes from CHO cells, but not those of MDCK cells. Consequently, C2-ceramide blocked the stimulation of phospholipase D1 (PLD1) by the cytosol and guanosine 5'-[gamma-thio]triphosphate (GTP[S]) in membranes from CHO cells. Basal specific activity of PLD1 and the concentration of ARF-1 were 3-4 times higher in Golgi-enriched membranes from MDCK cells compared with CHO cells. Moreover, PLD1 activity in MDCK cells was stimulated less by cytosol and GTP[S]. PLD2 was not detectable in the Golgi-enriched membranes. Incubation of intact CHO cells or their Golgi-enriched membranes with C2-ceramide also inhibited COP1 vesicle formation by membranes from CHO, but not MDCK, cells. Specificity was demonstrated, since dihydro-C2-ceramide had no significant effect on ARF-1 translocation, PLD1 activation or vesicle formation in membranes from both cell types. C2-ceramide also decreased the secretion of virus-like particles to a greater extent in CHO compared with MDCK cells, whereas dihydro-C2-ceramide had no significant effect. The results demonstrate a biological effect of C2-ceramide in CHO cells by decreasing ARF-1 and PKC-alpha binding to Golgi-enriched membranes, thereby preventing COP1 vesicle formation. PMID:11802796

  19. Induction of tolerance to lipopolysaccharide and mycobacterial components in Chinese hamster ovary/CD14 cells is not affected by overexpression of Toll-like receptors 2 or 4.

    PubMed

    Medvedev, A E; Henneke, P; Schromm, A; Lien, E; Ingalls, R; Fenton, M J; Golenbock, D T; Vogel, S N

    2001-08-15

    Down-regulation of cell surface expression of Toll-like receptor (TLR) 4 following LPS stimulation has been suggested to underlie endotoxin tolerance. In this study, we examined whether overexpression of TLR2 or TLR4 would affect the ability of cells to become tolerant to LPS or the mycobacterial components, arabinose-capped lipoarabinomannan (LAM) and soluble tuberculosis factor (STF). To this end, Chinese hamster ovary/CD14 cells stably transfected with a NF-kappaB-dependent reporter construct, endothelial leukocyte adhesion molecule CD25 (the 3E10 clone), were engineered to overexpress either human TLR2 or TLR4. Transfected TLRs exhibited proper signaling functions, as evidenced by increased LPS responsiveness of 3E10/TLR4 cells and acquisition of sensitivity to TLR2-specific ligands upon transfection of TLR2 into TLR2-negative 3E10 cells. Pretreatment of cells with LPS, LAM, or STF did not modulate TLR2 or TLR4 cell surface expression. Following LPS exposure, 3E10, 3E10/TLR2, and 3E10/TLR4 cells exhibited comparable decreases in LPS-mediated NF-kappaB activation and mitogen-activated protein (MAP) kinase phosphorylation. Likewise, LPS pretreatment profoundly inhibited LPS-induced NF-kappaB translocation in Chinese hamster ovary cells that concomitantly overexpressed human TLR4 and myeloid differentiation protein-2 (MD-2), but failed to modulate TLR4 or MD-2 cell surface expression. Pretreatment of 3E10/TLR2 cells with LAM or STF decreased their NF-kappaB responses induced by subsequent stimulation with these substances or LPS. Conversely, prior exposure of 3E10/TLR2 cells to LPS led to hyporesponsiveness to LPS, LAM, and STF, indicating that LPS and mycobacterial products induce cross-tolerance. Thus, tolerance to LPS and mycobacterial components cannot be attributed solely to a decrease in TLR/MD-2 expression levels, suggesting inhibition of expression or function of other signaling intermediates.

  20. Comparison of protein patterns of xrs-5, a radiosensitive Chinese hamster ovary cell line, and CHO-K1, its radioresistant parent, using two-dimensional gel-electrophoresis

    SciTech Connect

    Kramer, J.M. . Dept. of Zoology)

    1991-01-01

    X-ray sensitive strains of Chinese hamster ovary cell lines have been used to analyze radiation repair mechanisms. One cell line, xrs-5, has been shown to be very sensitive to ionizing radiation and radical forming chemical mutagens. This sensitivity is thought to be a result a mutation in the DNA double strand break (DSB) repair mechanism, and its characterization has been a goal of several repair mechanism studies. Using two-dimensional gel electrophoresis, we have detected a protein (MW approximately 55KD) in the DNA/Nuclear Matrix (nucleoid) cell fraction of CHO-Kl cells that is absent in the nucleoid fraction of xrs-5. This protein is present, however, in both CHO-Kl and xrs-5 whole cell protein maps. To determine whether the 55KD protein is responsible for the radiosensitive and defective DSB repair phenotype of xrs-5 cells, studies are now underway to analyze revertants of xrs-5 that are proficient in DSB repair. Furthermore, an effort to sequence the protein in question is planned. 23 refs., 2 figs.

  1. The induction/loss of the oxidant-resistant phenotype of Chinese hamster ovary (CHO) cell variants does not correlate with sensitivity to DNA single strand breakage by hydrogen peroxide.

    PubMed

    Sestili, P; Cattabeni, F; Cantoni, O

    1994-11-01

    Hydrogen peroxide resistant variants of Chinese hamster ovary (CHO) cells characterized by different levels of resistance to growth inhibition induced by the oxidant displayed a decreased susceptibility to the induction of DNA single strand breakage by hydrogen peroxide. Resistance to DNA damage, however, was maximal in cells resistant to killing by low concentrations of H2O, and did not increase further in cells characterized by a much higher resistance to the toxic action of the oxidant. Different sensitivities to the induction of DNA single strand breakage observed in wild type and resistant sublines were related to a decreased susceptibility/differential depletion of H2O2, rather than being dependent on different velocities in DNA repair processes. Growth of resistant cells in the absence of H2O2 resulted in a rapid loss of resistance to induction of DNA strand scission by H2O2. Cells retained resistance to the growth-inhibitory effect of the oxidant under conditions where resistance to the production of DNA single strand breaks was lost. Experiments aimed at elucidating the molecular basis for resistance to DNA damage induction by H2O2 have demonstrated that this effect is dependent upon the catalase activity of the specific sublines as well as on their different total protein content.

  2. Cytotoxic and mutagenic properties of shale oil byproducts II. Comparison of mutagenic effects at five genetic markers induced by retort process water plus near ultraviolet light in Chinese hamster ovary cells.

    PubMed

    Chen, D J; Strniste, G F

    1982-01-01

    A chinese hamster ovary (CHO) cell line heterozygous at the adenine phosphoribosyl transferase (APRT) locus was used for selection of induced mutants resistant to 8-azaadenine (8AA), 6-thioguanine (6TG), ouabain (OUA), emetine (EMT) and diphtheria toxin (DIP). The expression times necessary for optimizing the number of mutants recovered at the different loci have been determined using the know direct acting mutagen, far ultraviolet light (FUV), and a complex aqueous organic mixture (shale oil process water) activated with near ultraviolet light (NUV). Our results indicate that optimal expression times following treatment with either mutagen was between 2 and 8 days (depending on the genetic marker examined). For CHO cells treated with shale oil process water and subsequently exposed to NUV a linear dose response for mutant induction was observed for all five genetic loci. At 10% surviving fraction of cells, between 35- and 130-fold increases above background mutation frequencies were observed for the various markers examined. Among the five genetic loci tested, OUAR was the most sensitive marker tested.

  3. Stable expression of human H1-histamine-receptor cDNA in Chinese hamster ovary cells. Pharmacological characterisation of the protein, tissue distribution of messenger RNA and chromosomal localisation of the gene.

    PubMed

    Moguilevsky, N; Varsalona, F; Noyer, M; Gillard, M; Guillaume, J P; Garcia, L; Szpirer, C; Szpirer, J; Bollen, A

    1994-09-01

    A cDNA clone for the histamine H1 receptor was isolated from a human lung cDNA library; it encoded a protein of 487 amino acids which showed characteristic features of G-protein-coupled receptors. The percentages of identity of the deduced amino acid sequence with bovine, rat and guinea pig H1 histamine receptors were 82.6%, 79.4% and 73.3%, respectively, whereas these percentages decreased to 74.6%, 66% and 56.7% for the amino acid sequence of the third intracellular loop. The human H1-receptor cDNA was transfected into Chinese hamster ovary cells (CHO) via an eukaryotic expression vector; the receptor protein present on cell membranes specifically bound [3H]mepyramine with a Kd of 3.7 nM. The binding was displaced by H1-histamine-receptor antagonists and histamine. Northern blot analysis indicated the presence of two histamine H1 receptor mRNAs of 3.5 kb and 4.1 kb in various human tissues and an additional mRNA of 4.8 kb restricted to the human brain. Finally, by means of somatic cell hybrids segregating either human or rat chromosomes, the gene for histamine H1 receptor was found to reside on human chromosome 3 and rat chromosome 4.

  4. The impact of cell adaptation to serum-free conditions on the glycosylation profile of a monoclonal antibody produced by Chinese hamster ovary cells.

    PubMed

    Costa, Ana Rita; Withers, Joanne; Rodrigues, Maria Elisa; McLoughlin, Niaobh; Henriques, Mariana; Oliveira, Rosário; Rudd, Pauline M; Azeredo, Joana

    2013-06-25

    N-glycosylation is one of the most crucial parameters affecting the biological activity of therapeutic monoclonal antibodies (mAbs), and should therefore be closely monitored and controlled to guarantee a consistent and high-quality product in biopharmaceutical processes. In the present work, the effect of the time-consuming step of gradual cell adaptation to serum-free conditions on the glycosylation profile of a mAb produced by CHO-K1 cells was evaluated. High-performance liquid chromatography analysis revealed important changes in mAb glycosylation patterns in all steps of serum reduction. These changes could be grouped in two distinct phases of the process of adaptation: middle (2.5 to 0.15% serum) and final (0.075 and 0% serum). For intermediate levels of serum, a desirable increase of galactosylation and decrease of fucosylation, but an undesirable increase in sialylation were observed; while the inverse was obtained at the final stages of adaptation. These divergences may be related to the reduction of serum supplementation, to variations in the levels of cell density and viability achieved at these stages, and to the natural shift of the cell growth mode during adaptation from adherent to suspended. The divergent glycan profiles obtained in this study demonstrate a strong influence of the adaptation process on mAb glycosylation, suggesting that control and monitoring of product quality should be implemented at the early stages of process development.

  5. Cocytotoxicity/comutagenicity of arsenic in a Chinese hamster ovary triple auxotroph

    SciTech Connect

    Taylor, R.T.; Stewart, S.A.; Hanna, M.L.

    1984-06-04

    Among four forms of As that are measurable in human tissues (arsenite, arsenate, monomethylarsonate, and dimethylarsinate), non-cytotoxic concentrations of arsenite specifically enhance cell killing by various mutagenic agents in a Chinese hamster ovary auxotroph that requires glycine + adenosine + thymidine (CHO AUXB1). Arsenite is cocytotoxic at low concentrations of 2 to 12 ..mu..M. It is also the most growth rate inhibitory and cytotoxic of these As compounds, when each is incubated alone in AUXB1 cell cultures. None of these four As compounds are mutagenic per se, using an assay that we have developed to measure reversion to prototrophy at the FPGS gene locus. But arsenite (10 ..mu..M) specifically enhances induced reversion by the direct acting chemical mutagens cis-Pt(NH/sub 3/)/sub 2/Cl/sub 2/, methylglyoxal, and glycidal with which it is also cocytotoxic. Its comutagenicity with other agents is being tested. The foregoing experiments represent the first systematic comparison in mammalian cells of As growth inhibition, cytotoxicity, and mutagenicity versus its chemical form. Moreover, they provide the first evidence for the specific cocytotoxicity/comutagenicity of trivalent As in a mammalian cell line. Our findings support the suggestion that one role of As in its association with cancer could be to serve as a cocarcinogen. By functioning as a comutagen, perhaps through the inhibition of DNA repair, trivalent As may increase the initiation of tumor formation by enhancing the mutagenic activities of a large collection of primary environmental carcinogens. 55 references, 11 figures.

  6. The C. elegans VIG-1 and FRM-1 modulate carbachol-stimulated ERK1/2 activation in chinese hamster ovary cells expressing the muscarinic acetylcholine receptor GAR-3.

    PubMed

    Shin, Youngmi; Cho, Nam Jeong

    2014-04-01

    Many neurotransmitter receptors are known to interact with a variety of intracellular proteins that modulate signaling processes. In an effort to understand the molecular mechanism by which acetylcholine (ACh) signaling is modulated, we searched for proteins that interact with GAR-3, the Caenorhabditis elegans homolog of muscarinic ACh receptors. We isolated two proteins, VIG-1 and FRM-1, in a yeast two-hybrid screen of a C. elegans cDNA library using the third intracellular (i3) loop of GAR-3 as bait. To test whether these proteins regulate ACh signaling, we utilized Chinese hamster ovary (CHO) cells stably expressing GAR-3 (GAR-3/CHO cells). Previously we have shown that the cholinergic agonist carbachol stimulates extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation in an atropine-sensitive manner in this cell line. When VIG-1 was transiently expressed in GAR-3/CHO cells, carbachol-stimulated ERK1/2 activation was substantially reduced. In contrast, transient expression of FRM-1 significantly enhanced carbachol-stimulated ERK1/2 activation. Neither VIG-1 nor FRM-1 expression appeared to alter the affinity between GAR-3 and carbachol. In support of this notion, expression of these proteins did not affect GAR-3-mediated phospholipase C activation. To verify the modulation of ERK1/2 activity by VIG-1 and FRM-1, we used an i3 loop deletion mutant of GAR-3 (termed GAR-3Δi3). Carbachol treatment evoked robust ERK1/2 activation in CHO cells stably expressing the deletion mutant (GAR-3Δi3/CHO cells). However, transient expression of either VIG-1 or FRM-1 had little effect on carbachol-stimulated ERK1/2 activation in GAR-3Δi3/CHO cells. Taken together, these results indicate that VIG-1 and FRM-1 regulate GAR-3-mediated ERK1/2 activation by interacting with the i3 loop of GAR-3.

  7. Poly(N-isopropylacrylamide)-coated thermo-responsive nanoparticles for controlled delivery of sulfonated Zn-phthalocyanine in Chinese hamster ovary cells in vitro and zebra fish in vivo

    NASA Astrophysics Data System (ADS)

    He, Jia; Chen, Ji-Yao; Wang, Pu; Wang, Pei-Nan; Guo, Jia; Yang, Wu-Li; Wang, Chang-Chun; Peng, Qian

    2007-10-01

    Poly(N-isopropylacrylamide) (PNIPAM)-coated Fe3O4@SiO2@CdTe multifunctional nanoparticles with photoluminescent (PL), thermosensitive and magnetic properties, were investigated as carriers to deliver water-soluble, fluorescent sulfonated Zn-phthalocyanine (ZnPcS), a photosensitizing drug for photodynamic therapy of cancer, in Chinese hamster ovary (CHO) cells in vitro and zebra fish in vivo. PNIPAM is a well-known thermo-responsive polymer with a volume phase transition temperature. This property allows it to be swollen in water at temperatures lower than 32-34 °C to take up ZnPcS and shrunken to expel the drug at higher temperatures. Since the PL band of CdTe quantum dots (QDs) as indicators for the nanoparticles is at 585 nm and the emission band of ZnPcS is at 680 nm, it is possible to study the temperature-dependent release of ZnPcS from the nanoparticles by fluorescence measurements. ZnPcS was embedded in the PNIPAM of the nanoparticles at 25 °C in phosphate buffered saline (PBS) solution and released at 37 °C, measured with a spectrophotometer. When CHO cells had been incubated with the ZnPcS-loaded nanoparticles at 27 °C, a similar intracellular localization pattern of CdTe QDs and ZnPcS was seen by multichannel measurements in confocal laser scanning microscopy (CLSM), but a diffuse pattern of only ZnPcS fluorescence was detected in the cytoplasm of the cells at 37 °C, indicating a release of ZnPcS from the nanoparticles. Similar results were also found in the intestinal tract of zebra fish in vivo after intake of the nanoparticles. Since the nanoparticles contain magnetic (Fe3O4) material, the nanoparticles could also be manipulated to change their location in the intestinal tract of the zebra fish with an external magnetic field gradient of 300 G mm-1. The results presented suggest that such multifunctional nanoparticles may have combined potential for temperature-dependent drug delivery, QD photodetection and magnetic manipulation in diagnosis and

  8. [Evaluation of the critical process parameters for the cultivation of recombinant Chinese hamster ovary cells in serum-free fed-batch mode].

    PubMed

    Liu, Xingmao; Liu, Hong; Ye, Lingling; Li, Shichong; Wu, Benchuan; Wang, Qiwei; Chen, Zhaolie

    2011-02-01

    Taking a suspension adapted recombinant CHO cell line, 11G-S expressing human Pro-urokinase (Pro-UK) as the object of study, the impacts of different feeding nutrients, the start time of feeding and cell inoculation density on the growth and Pro-UK production of 11G-S cells in serum-free fed-batch culture were evaluated in 100 mL shacking flasks. The results indicated that amino acids, serum-free supplements and inorganic salts played important role in cell growth, cell viability and protein expression. And the effects of cells fed-batch culture was much better with the initial cell inoculation density at 3 x 10(5)-4 x 10(5) cells/mL and the start time of feeding set at 72 h, a maximum viable cells density of 7.8 x 10(6) cells/mL with a peak Pro-UK activity at 8570 IU/mL was achieved during 12 d fed-batch culture. Further, the mu of the 11G-S cells at the middle phase of the fed-batch culture, and both the q(glu) and q(gln) of the 11G-S cells at the middle and later phases of the fed-batch culture was higher than that of the 11G-S cells at the same phase of the batch culture, respectively.

  9. Dissociation of centrosome replication events from cycles of DNA synthesis and mitotic division in hydroxyurea-arrested Chinese hamster ovary cells

    PubMed Central

    1995-01-01

    Relatively little is known about the mechanisms used by somatic cells to regulate the replication of the centrosome complex. Centrosome doubling was studied in CHO cells by electron microscopy and immunofluorescence microscopy using human autoimmune anticentrosome antiserum, and by Northern blotting using the cDNA encoding portion of the centrosome autoantigen pericentriolar material (PCM)-1. Centrosome doubling could be dissociated from cycles of DNA synthesis and mitotic division by arresting cells at the G1/S boundary of the cell cycle using either hydroxyurea or aphidicolin. Immunofluorescence micros-copy using SPJ human autoimmune anticentrosome antiserum demonstrated that arrested cells were able to undergo numerous rounds of centrosome replication in the absence of cycles of DNA synthesis and mitosis. Northern blot analysis demonstrated that the synthesis and degradation of the mRNA encoding PCM-1 occurred in a cell cycle-dependent fashion in CHO cells with peak levels of PCM-1 mRNA being present in G1 and S phase cells before mRNA amounts dropped to undetectable levels in G2 and M phases. Conversely, cells arrested at the G1/S boundary of the cell cycle maintained PCM-1 mRNA at artificially elevated levels, providing a possible molecular mechanism for explaining the multiple rounds of centrosome replication that occurred in CHO cells during prolonged hydroxyurea-induced arrest. The capacity to replicate centrosomes could be abolished in hydroxyurea-arrested CHO cells by culturing the cells in dialyzed serum. However, the ability to replicate centrosomes and to synthesize PCM-1 mRNA could be re- initiated by adding EGF to the dialyzed serum. This experimental system should be useful for investigating the positive and negative molecular mechanisms used by somatic cells to regulate the replication of centrosomes and for studying and the methods used by somatic cells for coordinating centrosome duplication with other cell cycle progression events. PMID:7790366

  10. Understanding of decreased sialylation of Fc-fusion protein in hyperosmotic recombinant Chinese hamster ovary cell culture: N-glycosylation gene expression and N-linked glycan antennary profile.

    PubMed

    Lee, Jong Hyun; Jeong, Yeong Ran; Kim, Yeon-Gu; Lee, Gyun Min

    2017-03-07

    To understand the effects of hyperosmolality on protein glycosylation, recombinant Chinese hamster ovary (rCHO) cells producing the Fc-fusion protein were cultivated in hyperosmolar medium resulting from adding NaCl (415 mOsm/kg). The hyperosmotic culture showed increased specific Fc-fusion protein productivity (qFc ) but a decreased proportion of acidic isoforms and sialic acid content of the Fc-fusion protein. The intracellular and extracellular sialidase activities in the hyperosmotic cultures were similar to those in the control culture (314 mOsm/kg), indicating that reduced sialylation of Fc-fusion protein at hyperosmolality was not due to elevated sialidase activity. Expression of 52 N-glycosylation-related genes was assessed by the NanoString nCounter system, which provides a direct digital readout using custom-designed color-coded probes. After three days of hyperosmotic culture, nine genes (ugp, slc35a3, slc35d2, gcs1, manea, mgat2, mgat5b, b4galt3, and b4galt4) were differentially expressed over 1.5-fold of the control, and all these genes were down-regulated. N-linked glycan analysis by anion exchange and hydrophilic interaction HPLC showed that the proportion of highly sialylated (di-, tri-, tetra-) and tetra-antennary N-linked glycans was significantly decreased upon hyperosmotic culture. Addition of betaine, an osmoprotectant, to the hyperosmotic culture significantly increased the proportion of highly sialylated and tetra-antennary N-linked glycans (P ≤ 0.05), while it increased the expression of the N-glycan branching/antennary genes (mgat2 and mgat4b). Thus, decreased expression of the genes with roles in the N-glycan biosynthesis pathway correlated with reduced sialic acid content of Fc-fusion protein caused by hyperosmolar conditions. Taken together, the results obtained in this study provide a better understanding of the detrimental effects of hyperosmolality on N-glycosylation, especially sialylation, in rCHO cells. This article is protected

  11. Hamster thecal cells express muscle characteristics

    SciTech Connect

    Self, D.A.; Schroeder, P.C.; Gown, A.M.

    1988-08-01

    Contraction of the follicular wall about the time of ovulation appears to be a coordinated event; however, the cells that mediate it remain poorly studied. We examined the theca externa cells in the wall of hamster follicles for the presence of a functional actomyosin system, both in developing follicles and in culture. We used a monoclonal antibody (HHF35) that recognizes the alpha and gamma isoelectric variants of actin normally found in muscle, but not the beta variant associated with non-muscle sources, to evaluate large preovulatory follicles for actin content and composition. Antibody staining of sectioned ovaries showed intense circumferential reactivity in the outermost wall of developing follicles. Immunoblots from two-dimensional gels of theca externa lysates demonstrated the presence of the two muscle-specific isozymes of actin. Immunofluorescence of cultured follicular cells pulse-labeled with (3H) thymidine (for autoradiographic detection of DNA replication) revealed the presence, in many dividing cells, of actin filaments aligned primarily along the longitudinal axis of the cells. In cultures exposed to the calcium ionophore A23187 (10(-4) M) for varying periods (5 min to 1 h), contraction of many individual muscle-actin-positive cells was observed. Immunofluorescence of these cells, fixed immediately after ionophore-induced contraction, revealed compaction of the actin filaments. Our findings demonstrate that the cells of the theca externa contain muscle actins from an early stage and that these cells are capable of contraction even while proliferating in subconfluent cultures. They suggest that follicular growth may include a naturally occurring developmental sequence in which a contractile cell type proliferates in the differentiated state.

  12. Role of protein synthesis in the repair of sublethal x-ray damage in a mutant Chinese hamster ovary cell line

    SciTech Connect

    Yezzi, M.J.

    1985-04-01

    A temperature-sensitive mutant for protein synthesis, CHO-TSH1, has been compared to the wild-type cell, CHO-sC1, in single- and split-radiation-dose schemes. When the exponentially growing TS mutant and the wild-type cells were treated at 40/sub 0/C for up to 2 hrs prior to graded doses of x rays, the survival curves were identical and were the same as those obtained without heat treatment. If the cultures were incubated at 40/sup 0/C for 2 hrs before a first dose and maintained at 40/sup 0/C during a 2 hr dose fractionation interval, repair of radiation damage was reduced in the mutant compared to the wild type. These observations implied that a pool of proteins was involved in the repair of sublethal x-ray damage. However, if repair was measured by the alkaline-unwinding technique under the same time and temperature schemes, no difference in the kientics of DNA strand rejoining was observed. Misrepair processes may permit restoration of DNA strand integrity but not allow functional repair. The effect of diminished repair under conditions of inhibition of protein synthesis was found to be cell-cycle dependent in survival studies with synchronized mutant cell populations. Repair was found to be almost completely eliminated if the temperature sequence described above was applied in the middle of the DNA synthetic phase. Treatment of cell populations in the middle of G/sub 1/-phase yielded repair inhibition comparable to that observed with the asynchronous cells. Splitdose experiments were done using pre-incubation with cycloheximide to chemically inhibit protein synthesis. WT cells and TS cells were treated with cycloheximide at 35/sup 0/C for 2 hrs before a first dose and during a 2 hr dose fractionation interval. 23 figs., 7 tabs.

  13. Laminin on Toxoplasma gondii mediates parasite binding to the beta 1 integrin receptor alpha 6 beta 1 on human foreskin fibroblasts and Chinese hamster ovary cells.

    PubMed

    Furtado, G C; Cao, Y; Joiner, K A

    1992-11-01

    We investigated the role of parasite-bound laminin and the host cell beta 1 integrin receptors for this extracellular matrix protein in Toxoplasma gondii binding to fibroblasts. Laminin but not fibronectin was detected on extracellular tachyzoites by immunofluorescence and immunoblotting. Binding of parasites to CHO cells was inhibited by polyclonal antibodies to laminin and by a monoclonal antibody directed against the globular carboxyl-terminal portion of the long arm of laminin (at or near the suggested ligand-binding sites for alpha 3 beta 1 and alpha 6 beta 1), but not by a monoclonal antibody directed against the lateral short arms of laminin near the cross region of the molecule. Antibodies to the alpha 6 but not the alpha 2, alpha 3, or alpha 5 chains of the beta 1 family of integrins blocked parasite attachment to human foreskin fibroblasts and CHO cells. Attachment of T. gondii to cells via laminin on the parasite surface and laminin receptors on the mammalian cell is consistent with the capacity of the parasite to invade almost all nucleated cells.

  14. Comparison between pulsed-field and constant-field gel electrophoresis for measurement of DNA double-strand breaks in irradiated Chinese hamster ovary cells.

    PubMed

    Wlodek, D; Banáth, J; Olive, P L

    1991-11-01

    Pulsed-field gel electrophoresis (PFGE) is one of the most sensitive methods for detecting DNA double-strand breaks in mammalian cells. However, it has been observed that constant-field gel electrophoresis (CFGE), when optimized, can detect breaks with equal efficiency. The migration of DNA from the well and the separation of DNA molecules according to size appear to be different processes; only the latter requires the application of PFGE. CFGE is very sensitive and can detect DNA damage produced by less than 5 Gy of radiation. Low voltage (ca. 0.6 V/cm) during electrophoresis appears to be essential for the migration of the largest fraction of DNA from the agarose plug containing the cells; the electrophoresis run time, cell density in the plug, agarose concentration, nature of detergent and extent of radiolabelling are less important. It is concluded that CFGE is equally sensitive but more rapid and economical than PFGE for the measurement of DNA damage.

  15. Analysis of possible genotoxicity of the herbicide flurochloridone and its commercial formulations: Endo III and Fpg alkaline comet assays in Chinese hamster ovary (CHO-K1) cells.

    PubMed

    Soloneski, Sonia; Nikoloff, Noelia; Larramendy, Marcelo L

    2016-02-01

    Cytotoxic and genotoxic effects of flurochloridone (FLC) and its formulations Twin Pack Gold(®) and Rainbow(®) were evaluated in CHO-K1 cells. Using the alkaline single-cell gel electrophoresis (SCGE) assay, we observed that FLC (15 μg/ml), Twin Pack Gold(®) or Rainbow(®) induced primary DNA damage, increasing the frequency of damaged nucleoids. Vitamin E pretreatment did not modify the effect. Decreased cell viability was observed only in Twin Pack Gold(®)-treated cultures and was significantly ameliorated by vitamin E. Post-treatment of herbicide-damaged CHO-K1 cells with the enzymes Endo III or Fpg did not increase FLC-, Twin Pack Gold(®)-, or Rainbow(®)-induced DNA damage. These results demonstrate that neither FLC nor FLC-based formulations induce DNA damage through hydroxyl radical or lipid alkoxyl radical production, and that the induced DNA lesions were not related to oxidative damage at the purine/pyrimidine level. Our observations strongly suggest that the cytotoxic effects observed after Twin Pack Gold(®) exposure are due to the excipients contained within the technical formulation rather than FLC itself.

  16. Increased intracellular calcium activates serum and glucocorticoid-inducible kinase 1 (SGK1) through a calmodulin-calcium calmodulin dependent kinase kinase pathway in Chinese hamster ovary cells.

    PubMed

    Imai, Seiji; Okayama, Naotsuka; Shimizu, Manabu; Itoh, Makoto

    2003-04-04

    SGK1 is one of the protein-serine/threonine kinases that is activated by insulin in a PI3K-dependent manner. Although SGK1 mediates a variety of biological activities, the mechanisms regulating its activity remain unclear. In this study, we examined the potential roles of calcium signaling in the activation of SGK1. Treatment of CHO-IR cells with a cell-permeable calcium chelator, BAPTA-AM, abolished the insulin-induced activation of SGK1. Increasing intracellular calcium concentration by treating cells with thapsigargin or ionomycin induced a 6-8 fold increase in SGK1 activation. This was not affected by a PI3K inhibitor, wortmannin, but was completely inhibited by the calmodulin inhibitors, W 7 and W 5. Co-transfection of CHO cells with FLAG-SGK1 and CaMKK revealed the direct association of CaMKK with SGK1. These results suggest a calcium-triggered signaling cascade in which an increase in intracellular calcium concentration directly stimulates SGK1 through CaMKK.

  17. Effect of Brazilian propolis (AF-08) on genotoxicity, cytotoxicity and clonogenic death of Chinese hamster ovary (CHO-K1) cells irradiated with (60)Co gamma-radiation.

    PubMed

    Santos, Geyza Spigoti; Tsutsumi, Shigetoshi; Vieira, Daniel Perez; Bartolini, Paolo; Okazaki, Kayo

    2014-03-01

    The present study was conducted in order to evaluate the effect of Brazilian propolis (AF-08; 5, 10, 15, 30, 50, 100, and 200μg/mL) in protecting CHO-K1 cells against genotoxic and cytotoxic damage and clonogenic death induced by (60)Co gamma-radiation (1.0, 2.0, 4.0, and 6.0Gy). For this purpose, three interlinked endpoints were analyzed: induction of DNA damage by use of the micronucleus (MN) test (genotoxic damage), cell viability by means of the MTS assay, and differential staining (cytotoxic damage) and clonogenic death via the colony-formation test (cytotoxic damage). The MN test revealed that propolis alone (5-100μg/mL) was not genotoxic up to 100μg/mL and that 30μg/mL of propolis reduced the radiation-induced DNA damage (∼56% reduction, p<0.05), exhibiting a radio-protective effect on irradiated CHO-K1 cells. On the other hand, analysis of cytotoxicity showed that a concentration of 50μg/mL presented a significant proliferative effect (p<0.001) when associated with radiation, decreasing the percentage of necrotic cells (p<0.01). No mediated cytotoxic effect was found, but the concentration of 200μg/mL was toxic when analyzed at 24 and 48h via the differential staining technique, but not at 72h after irradiation, analyzed with the MTS assay. Differential staining also showed that necrosis was the main death modality in irradiated cells and that apoptosis was induced only at the toxic concentration of propolis (200μg/mL). Concerning the clonogenic capacity, a concentration of 50μg/mL also exhibited a significant stimulating effect on cell proliferation (p<0.001), in agreement with the data from differential staining. Taken together, these data suggest that the use of propolis AF-08 for the prevention of the adverse effects of ionizing radiation is promising. Nevertheless, additional investigations are necessary for a better understanding of potential applications of propolis to improve human health.

  18. Construction of the plasmid, expression by Chinese hamster ovary cell, purification and characterization of the first three short consensus repeat modules of human complement receptor type 1.

    PubMed

    Yamaguchi, Atsushi; Takagawa, Hiroaki; Iwakaji, Hirofumi; Miyagawa, Shuji; Wang, Pi-Chao; Ishii, Noriyuki

    2009-04-01

    Short consensus repeat (SCR1-3), the first three SCR modules from N-terminus of type 1 complement receptor (CR1), is expected to accelerate dissociation of complement components and suppress complement activity by binding the main component of complement C4b. In order to clarify the three-dimensional structure, which triggers the activity of SCR1-3 on complement, we constructed an over-expression system in CHO DG44 cells which facilitated mass production of SCR1-3. The mass production was achieved by a two-stage culture system and optimum culture conditions using ASF104N medium and MTX-, NaBu-containing alpha-MEM/10% FBS medium, respectively. The constructed gene of SCR1-3 was confirmed by restriction enzyme digestion and DNA sequence analysis, and the expressed protein by CHO DG44 cells was confirmed by western blotting. The expressed SCR1-3 was proved containing N-linked sugar chain, an important factor to the proper expression of protein, by the cleavage with glycosidase of N-linked oligosaccharide (PNGase F). The suppression effect of the yield protein on complement-mediated inflammation was investigated by haemolytic assay and necrosis assay of stromal cells. Both assays showed that SCR1-3 possessed complement control activity. However, residing sugar chain on SCR1-3 did not show significant difference in the complement control activity.

  19. Inhibition of the ubiquitin ligase activity improves the production of biologically active fusion protein HSA-HGF in Chinese hamster ovary cells.

    PubMed

    Xu, Dongsheng; Wan, Aini; Zhang, Jingjing; Peng, Lin; Chen, Yun; He, Yang; Yang, Jianfeng; Jin, Jian

    2016-10-18

    Hepatocyte growth factor (HGF) is a potent multi-functional protein that stimulates proliferation, survival, motility, scattering and differentiation during growth and development, and has been considered to be a potential therapeutic agent for the treatment of a number of intractable diseases. The aim of this study was to enhance the expression of recombinant fusion protein HSA-HGF (R494E) in CHO cells by inhibiting the intracellular ubiquitin ligase activity. The high stable expression sub-clones with different signal peptides were selected by western blot (WB) analysis and used for suspension culture. We found that the expression of fusion protein HSA-HGF (R494E) on day 3 achieved 50 mg/L during the 8 day culture process, a large number of fusion proteins were intracellular degradated by ubiquitination pathway during day 4 to day 8. Furthermore, ubiquitin ligase inhibitor, thalidomide, was added in culture process, and resulted in efficient and stable secretion of HSA-HGF (R494E) in CHO cells. According to biological activity assays, HSA-HGF (R494E) possessed various biological activities similar to native HGF. In conclusion, innhibition of intracellular ubiquitin ligase activity was successfully improve the expression of biologically active fusion protein HSA-HGF (R494E) in CHO cells. Our data may be beneficial to enhance the production of other therapeutic proteins in fed-batch culture.

  20. Non-stationary 13C metabolic flux analysis of Chinese hamster ovary cells in batch culture using extracellular labeling highlights metabolic reversibility and compartmentation

    PubMed Central

    2014-01-01

    Background Mapping the intracellular fluxes for established mammalian cell lines becomes increasingly important for scientific and economic reasons. However, this is being hampered by the high complexity of metabolic networks, particularly concerning compartmentation. Results Intracellular fluxes of the CHO-K1 cell line central carbon metabolism were successfully determined for a complex network using non-stationary 13C metabolic flux analysis. Mass isotopomers of extracellular metabolites were determined using [U-13C6] glucose as labeled substrate. Metabolic compartmentation and extracellular transport reversibility proved essential to successfully reproduce the dynamics of the labeling patterns. Alanine and pyruvate reversibility changed dynamically even if their net production fluxes remained constant. Cataplerotic fluxes of cytosolic phosphoenolpyruvate carboxykinase and mitochondrial malic enzyme and pyruvate carboxylase were successfully determined. Glycolytic pyruvate channeling to lactate was modeled by including a separate pyruvate pool. In the exponential growth phase, alanine, glycine and glutamate were excreted, and glutamine, aspartate, asparagine and serine were taken up; however, all these amino acids except asparagine were exchanged reversibly with the media. High fluxes were determined in the pentose phosphate pathway and the TCA cycle. The latter was fueled mainly by glucose but also by amino acid catabolism. Conclusions The CHO-K1 central metabolism in controlled batch culture proves to be robust. It has the main purpose to ensure fast growth on a mixture of substrates and also to mitigate oxidative stress. It achieves this by using compartmentation to control NADPH and NADH availability and by simultaneous synthesis and catabolism of amino acids. PMID:24773761

  1. Evaluation of Heavy Chain C-terminal Deletions on Productivity and Product Quality of Monoclonal Antibodies in Chinese Hamster Ovary (CHO) Cells.

    PubMed

    Hu, Zhilan; Tang, Danming; Misaghi, Shahram; Jiang, Guoying; Yu, Christopher; Yim, Mandy; Shaw, David; Snedecor, Brad; Laird, Michael; Shen, Amy

    2017-02-11

    Monoclonal antibodies (mAbs) have been well established as potent therapeutic agents and are used to treat many different diseases. During cell culture production, antibody charge variants can be generated by cleavage of heavy chain (HC) C-terminal lysine and proline amidation. Differences in levels of charge variants during manufacturing process changes make it challenging to demonstrate process comparability. In order to reduce heterogeneity and achieve consistent product quality, we generated and expressed antibodies with deletion of either HC C-terminal lysine (-K) or lysine and glycine (-GK). Interestingly, clones that express antibodies lacking HC C-terminal lysine (-K) had considerably lower specific productivities compared to clones that expressed either wild type antibodies (WT) or antibodies lacking HC glycine and lysine (-GK). While no measurable differences in antibody HC and LC mRNA levels, glycosylation and secretion were observed, our analysis suggests that the lower specific productivity of clones expressing antibody lacking HC C-terminal lysine was due to slower antibody HC synthesis and faster antibody degradation. This article is protected by copyright. All rights reserved.

  2. The use of primary rat hepatocytes to achieve metabolic activation of promutagens in the Chinese hamster ovary/hypoxantine-guanine phosphoribosyl transferase mutational assay

    SciTech Connect

    Bermudez, E.; Couch, D.B.; Tillery, D.

    1982-01-01

    A method is described in which primary rat hepatocytes have been cocultured with chinese hamster ovary (CHO) cells to provide metabolic activation of promutgens in the Chinese hamster ovary/hypoxanthine-guanine phosphoribosyl transferase (CHO/HGPRT) mutational assay. Single cell hepatocyte suspensions were prepared from male Fisher-344 rats using the in situ collagenase perfusion technique. Hepatocytes were allowed to attach for 1.5 hours in tissue culture dishes containing an approximately equal number of CHO cells in log growth. The cocultures were exposed to promutagens for up to 20 hours in serum-free medium. The survival and 6-thioguanine-resistant fraction of treated CHO cells were then determined as in the standard CHO/HGPRT assay. Aflatoxin B/sub 1/ (AFB/sub 1/) 7,12-dimethylbenz(a)anthracene (DMBA) and benzo(a)pyrene (B(a)P) were found to produce increases in the mutant fractions of treated CHO cells as a function of concentration. The time required for optimum expression of the mutant phenotype following exposure to DMBA and AFB/sub 1/ was approximately 8 days. Primary cell-mediated mutagenesis may be useful in elucidating methobolic pathways important in the production and detoxification of genotoxic products in vivo.

  3. Cell killing and mutation induction on Chinese hamster cells by photoradiations

    SciTech Connect

    Lam, C.K.C.

    1982-11-01

    Applying radiation directly on cells, far-uv is more effective than black light, and black light is more effective than white light in inducing proliferative death and in inducing resistance to 6-thioguanine (6-TG), ouabain and diptheria toxin (DT). Gold light has no killing and mutagenic effects on CHO (Chinese hamster ovary) cells. Use of filters showed that a small percentage of shorter wavelengths in the far-uv region is responsible for most of the killing and mutagenic effects in the unfiltered broad spectra of black and white light.

  4. Effects of 13 T Static Magnetic Fields (SMF) in the Cell Cycle Distribution and Cell Viability in Immortalized Hamster Cells and Human Primary Fibroblasts Cells

    NASA Astrophysics Data System (ADS)

    Zhao, Guoping; Chen, Shaopeng; Zhao, Ye; Zhu, Lingyan; Huang, Pei; Bao, Lingzhi; Wang, Jun; Wang, Lei; Wu, Lijun; Wu, Yuejin; Xu, An

    2010-02-01

    Magnetic resonance image (MRI) systems with a much higher magnetic flux density were developed and applied for potential use in medical diagnostic. Recently, much attention has been paid to the biological effects of static, strong magnetic fields (SMF). With the 13 T SMF facility in the Institute of Plasma Physics, Chinese Academy of Sciences, the present study focused on the cellular effects of the SMF with 13 T on the cell viability and the cell cycle distribution in immortalized hamster cells, such as human-hamster hybrid (AL) cells, Chinese hamster ovary (CHO) cells, DNA double-strand break repair deficient mutant (XRS-5) cells, and human primary skin fibroblasts (AG1522) cells. It was found that the exposure of 13 T SMF had less effect on the colony formation in either nonsynchronized or synchronized AL cells. Moreover, as compared to non-exposed groups, there were slight differences in the cell cycle distribution no matter in either synchronized or nonsynchronized immortalized hamster cells after exposure to 13 T SMF. However, it should be noted that the percentage of exposed AG1522 cells at G0/G1 phase was decreased by 10% as compared to the controls. Our data indicated that although 13 T SMF had minimal effects in immortalized hamster cells, the cell cycle distribution was slightly modified by SMF in human primary fibroblasts.

  5. A rapid method for simultaneous evaluation of free light chain content and aggregate content in culture media of Chinese hamster ovary cells expressing monoclonal antibodies for cell line screening.

    PubMed

    Ishii, Yoichi; Tsukahara, Masayoshi; Wakamatsu, Kaori

    2016-04-01

    The goal of developing a monoclonal antibody (mAb) production process is high productivity and high quality. Because the productivity and quality of mAbs depend on cell line properties, the selection of cell lines suitable for large-scale production is an important stage in process development for mAb production. The light chain (LC) is important for antibody folding and assembly in the endoplasmic reticulum; cell lines that secrete a large amount of LCs in the medium secrete high-quality antibodies with high productivity. LC contents in culture media have been estimated by western blotting, reverse-phase high-performance liquid chromatography, and enzyme-linked immunosorbent assay. However, these analyses require fine tuning of experimental conditions for each antibody analyzed. Here we report a rapid and simple high-sensitivity size-exclusion chromatography (HS-SEC) method to evaluate the contents of low-molecular weight species (LMWS, mainly consisting of LC monomers and dimers) and high-molecular weight species (HMWS, aggregates) in the media for cell line screening. Because LMWS and HMWS are important indicators of productivity and quality, respectively, for cell line screening, HS-SEC will be useful in the first step of cell line selection needed for large-scale production.

  6. Transformation of Hamster Embryo Cells and Tumor Induction in Newborn Hamsters by Simian Adenovirus SV11

    PubMed Central

    Casto, Bruce C.

    1969-01-01

    Simian adenovirus, SV11, readily transformed hamster embryo cell cultures in vitro and produced tumors in vivo when inoculated into newborn hamsters. Foci consisting of small, loosely attached, rounded cells could be seen as early as 7 days postinoculation. Many of these cells contained several nuclei or the nucleus was multilobed. The cells grew without extensive cell to cell contact or formed small chains or clusters when passaged in vitro. This pattern of cell morphology and growth has not been reported with other simian or human adenovirus-transformed cells. Linearity of foci formation with virus dilution was observed when the virus multiplicity was less than 3 plaque-forming units (PFU)/cell. The PFU to focus-forming units ratio for SV11 was found to be 2 × 104 to 4 × 104, which is approximately 5- to 10-fold and 50- to 100-fold lower than those reported for simian adenovirus, SA7, and human adenovirus type 12, respectively. Cells transformed by SV11: (i) produced tumors when inoculated into young hamsters, (ii) contained tumor antigen which reacts with serum obtained from hamsters bearing SV11 passaged tumors, and (iii) could be propagated in vitro through an indefinite number of generations. Images PMID:5786181

  7. Chinese hamster ovary-sphingomyelin synthase2 biospecific extraction and liquid chromatography with tandem mass spectrometry analysis for the prediction of bioactive components of Rhizoma Polygoni Cuspidati.

    PubMed

    Xue, Ying; Dong, Jibin; Liang, Jianying

    2016-03-01

    A novel strategy for predicting bioactive components in traditional Chinese medicines using Chinese hamster ovary-sphingomyelin synthase2 (CHO-SMS2 ) cell biospecific extraction and high-performance liquid chromatography with diode array detection and tandem mass spectrometry analysis was proposed. The hypothesis is that when cells are incubated with the extract of traditional Chinese medicines, the potential bioactive components in the traditional Chinese medicines should selectively combine with the cells, while the cell-combining components would be detectable in the extract of denatured cells. The identities of the cell-combining components could be determined by liquid chromatography with tandem mass spectrometry. Using the proposed approach, the potential bioactive components of Rhizoma Polygoni Cuspidati, a commonly used traditional Chinese medicine for atherosclerosis, were detected and identified. Eight compounds in the extract of Rhizoma Polygoni Cuspidati were detected as the components selectively combined with CHO-SMS2 cells, which is a stable cell line that highly expresses sphingomyelin synthases, it was found that piceid, resveratrol, emodin-8-β-d-glucoside, physcion-8-β-d-glucoside, emodin, physcion, 3,5,4'-trihydroxystilbene-3-O-(6"-galloyl)-glucoside, and emodin-1-O-glucoside combined specifically with CHO-SMS2 cells. The results indicate that the proposed approach may be applied to predict the bioactive candidates in traditional Chinese medicines.

  8. Mutation and repair induced by the carcinogen 2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP) in the dihydrofolate reductase gene of Chinese hamster ovary cells and conformational modeling of the dG-C8-PhIP adduct in DNA.

    PubMed

    Carothers, A M; Yuan, W; Hingerty, B E; Broyde, S; Grunberger, D; Snyderwine, E G

    1994-01-01

    Three experiments using 20 microM 2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP) were performed to induce mutations in the dihydrofolate reductase (DHFR) gene of a hemizygous Chinese hamster ovary (CHO) cell line (UA21). Metabolized forms of this chemical primarily bind at the C-8 position of guanine in DNA. In total, 21 independent induced mutants were isolated and 20 were characterized. DNA sequencing showed that the preferred mutation type found in 75% of the induced DHFR- clones was G.C-->T.A single and tandem double transversions. In addition to base substitutions, one mutant carried a-1 frameshift and another one had lost the entire locus by deletion. The induced changes affected purine targets on the nontranscribed strand of the gene in nearly all of the mutants sequenced (18/19). At the time that the first two experiments were performed, the initial adduct levels were quantitated in treated cells at the mutagenic dose by 32P-postlabeling. While the induced frequency of mutation was relatively low (approximately 5 x 10(-6), the adduct levels after a 1-h exposure of UA21 cells to 20 microM N-OH-PhIP were relatively high (13 adducts x 10(-6) nucleotides). This latter method was then employed to learn if the induced mutation frequency correlated with rapid overall genome repair of PhIP-DNA adducts. Total adduct levels, determined using DNA samples from treated cells collected after intervals of time, were reduced by about 50% after 6 h, and about 70% after 24 h. Since overall genome repair in CHO cells is relatively slow compared with preferential gene repair, the removal of dG-C8-PhIP adducts was apparently efficient. In order to better understand the mutational and repair results, we performed computational modeling to determine the lowest energy structure for the major dG-C8-PhIP adduct in a repetitively mutated duplex sequence opposite dA. Results of this analysis indicate that the PhIP-modified base resembles previous structural

  9. Stem cells, progenitor cells, and lineage decisions in the ovary.

    PubMed

    Hummitzsch, Katja; Anderson, Richard A; Wilhelm, Dagmar; Wu, Ji; Telfer, Evelyn E; Russell, Darryl L; Robertson, Sarah A; Rodgers, Raymond J

    2015-02-01

    Exploring stem cells in the mammalian ovary has unleashed a Pandora's box of new insights and questions. Recent evidence supports the existence of stem cells of a number of the different cell types within the ovary. The evidence for a stem cell model producing mural granulosa cells and cumulus cells is strong, despite a limited number of reports. The recent identification of a precursor granulosa cell, the gonadal ridge epithelial-like cell, is exciting and novel. The identification of female germline (oogonial) stem cells is still very new and is currently limited to just a few species. Their origins and physiological roles, if any, are unknown, and their potential to produce oocytes and contribute to follicle formation in vivo lacks robust evidence. The precursor of thecal cells remains elusive, and more compelling data are needed. Similarly, claims of very small embryonic-like cells are also preliminary. Surface epithelial cells originating from gonadal ridge epithelial-like cells and from the mesonephric epithelium at the hilum of the ovary have also been proposed. Another important issue is the role of the stroma in guiding the formation of the ovary, ovigerous cords, follicles, and surface epithelium. Immune cells may also play key roles in developmental patterning, given their critical roles in corpora lutea formation and regression. Thus, while the cellular biology of the ovary is extremely important for its major endocrine and fertility roles, there is much still to be discovered. This review draws together the current evidence and perspectives on this topic.

  10. Stem Cells, Progenitor Cells, and Lineage Decisions in the Ovary

    PubMed Central

    Hummitzsch, Katja; Anderson, Richard A.; Wilhelm, Dagmar; Wu, Ji; Telfer, Evelyn E.; Russell, Darryl L.; Robertson, Sarah A.

    2015-01-01

    Exploring stem cells in the mammalian ovary has unleashed a Pandora's box of new insights and questions. Recent evidence supports the existence of stem cells of a number of the different cell types within the ovary. The evidence for a stem cell model producing mural granulosa cells and cumulus cells is strong, despite a limited number of reports. The recent identification of a precursor granulosa cell, the gonadal ridge epithelial-like cell, is exciting and novel. The identification of female germline (oogonial) stem cells is still very new and is currently limited to just a few species. Their origins and physiological roles, if any, are unknown, and their potential to produce oocytes and contribute to follicle formation in vivo lacks robust evidence. The precursor of thecal cells remains elusive, and more compelling data are needed. Similarly, claims of very small embryonic-like cells are also preliminary. Surface epithelial cells originating from gonadal ridge epithelial-like cells and from the mesonephric epithelium at the hilum of the ovary have also been proposed. Another important issue is the role of the stroma in guiding the formation of the ovary, ovigerous cords, follicles, and surface epithelium. Immune cells may also play key roles in developmental patterning, given their critical roles in corpora lutea formation and regression. Thus, while the cellular biology of the ovary is extremely important for its major endocrine and fertility roles, there is much still to be discovered. This review draws together the current evidence and perspectives on this topic. PMID:25541635

  11. Isolation and identification of normal killer cells from Syrian hamsters

    SciTech Connect

    Matveeva, V.A.; Klyuchareva, T.E.

    1986-09-01

    This paper gives data on isolation of normal killer cells from the blood and various tissues of Syrian hamsters in a Percoll density gradient and their identification on the basis of morphologic criteria and cytotoxic activity (CTA). CTA of the isolated cells was studied in the cytotoxic test with target cells of a human MOLT-4 thymoma cell labeled with /sup 51/Cr. Isolation of large granular lymphocytes from blood, spleen, and bone marrow of Syrian hamsters in Percoll density gradient is shown in the results of five experiments used for cells of each type.

  12. The insecticide 1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane (DDT) alters the membrane raft location of the TSH receptor stably expressed in Chinese hamster ovary cells

    SciTech Connect

    De Gregorio, Francesca; Pellegrino, Mario; Picchietti, Simona; Belardinelli, Maria C.; Taddei, Anna Rita; Fausto, Anna Maria; Rossi, Mario; Maggio, Roberto; Giorgi, Franco

    2011-06-01

    DDT is a highly lipophilic molecule known to deplete membrane rafts of their phosphoglycolipid and cholesterol contents. However, we have recently shown that DDT can also alter the thyroid homeostasis by inhibiting TSH receptor (TSHr) internalization. The present study was undertaken to verify whether DDT goitrogenic effects are due to the insecticide acting directly on TSHr or via alteration of the membrane rafts hosting the receptor itself. Our results demonstrate that, in CHO-TSHr transfected cells, TSHr is activated in the presence of TSH, while it is inhibited following DDT exposure. DDT can also reduce the endocytic vesicular traffic, alter the extension of multi-branched microvilli along their plasma membranes and induce TSHr shedding in vesicular forms. To verify whether TSHr displacement might depend on DDT altering the raft constitution of CHO-TSHr cell membranes the extent of TSHr and lipid raft co-localization was examined by confocal microscopy. Evidence shows that receptor/raft co-localization increased significantly upon exposure to TSH, while receptors and lipid rafts become dislodged on opposite cell poles in DDT-exposed CHO-TSHr cells. As a control, under similar culturing conditions, diphenylethylene, which is known to be a lipophilic substance that is structurally related to DDT, did not affect the extent of TSHr and lipid raft co-localization in CHO-TSHr cells treated with TSH. These findings corroborate and extend our view that, in CHO cells, the DDT disrupting action on TSHr is primarily due to the insecticide acting on membranes to deplete their raft cholesterol content, and that the resulting inhibition on TSHr internalization is due to receptor dislodgement from altered raft microdomains of the plasma membrane. - Highlights: >DDT is a pesticide with a severe environmental impact >Epidemiologic correlation exists between exposition to DDT and thyroid dysfunction >DDT is a lipophilic molecule that has been shown to inhibit TSH receptor

  13. Suppression of hamster lymphocyte reactivity to simian virus 40 tumor surface antigens by spleen cells from pregnant hamsters

    SciTech Connect

    Weppner, W.A.; Adkinson, L.R.; Coggin, J.H.Jr

    1980-09-01

    SV40-transformed tumor cells in hamsters have been found to have cell surface antigens cross-reactive with antigens temporally expressed on fetal tissues. Using a lymphocyte transformation assay, spleen cells from pregnant hamsters were found to be incapable of responding to preparations of either hamster fetal tissue or SV40-transformed cells. However, a suppressor component can be demonstrated in spleen cell populations of both primi-and multiparous hamsters during pregnancy that is capable of reducing the response of lymphocytes sensitized against SV40 tumor-associated antigens. The degree of suppression is proportional to the ratio of responder cells to spleen cells from pregnant animals. These results suggest there is a subpopulation of spleen cells involved in immunoregulation during pregnancy that has the ability to suppress the reactivity of lymphocytes sensitized against SV40-associated oncofetal antigens.

  14. Isolation of Undifferentiated Female Germline Cells from Adult Drosophila Ovaries.

    PubMed

    Lim, Robyn Su May; Osato, Motomi; Kai, Toshie

    2015-08-03

    This unit describes a method for isolating undifferentiated, stem cell-like germline cells from adult Drosophila ovaries. Here, we demonstrate that this population of cells can be effectively purified from hand-dissected ovaries in considerably large quantities. Tumor ovaries with expanded populations of undifferentiated germline cells are first removed from fly abdomens and dissociated into a cell suspension with the aid of protease treatment. The target cells, which express Vasa-green fluorescent protein (GFP) fusion protein under the control of the germline-specific vasa promoter, are specifically selected from the suspension via fluorescence-activated cell sorting (FACS). These protocols can be adapted to isolate other cell types from fly ovaries, such as somatic follicle cells or escort cells, by driving GFP expression in the respective target cells.

  15. Molecular characterization of mutation and comparison of mutation profiles in the hprt gene of Chinese hamster ovary cells treated with benzo[a]pyrene trans-7,8-diol-anti-9,10-epoxide, 1-nitrobenzol[a]pyrene trans-7,8-diol-anti-9,10-epoxide, and 3-nitrobenzol[a]pyrene trans-7,8-diol-anti-9,10-epoxide

    SciTech Connect

    Zhan, D.J.; Heflich, R.H.; Fu, P.P.

    1996-12-31

    Both 1- and 3-nitrobenzol[a] pyrene (nitro-BaP) are environmental contaminants, potent mutagens in Salmonella, and moderate mutagens in Chinese hamster ovary (CHO) cells. The mutagenicity of their oxidized metabolites, trans-7,8-dihydroxy-anti-9, 10-epoxy-7,8,9,10-tetrahydro-1-nitrobenzol[a]pyrene (1-nitro-BaP-DE) and trans-7,8-dihydroxy-anti-9, 10-epoxy-7,8,9,10-tetrahydro-3-nitrobenzo[a]pyrene (3-nitro-BaP-DE), together with trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzol[a]pyren (BaP-DE), was determined in CHO-K1 cells, and the resulting mutations at the hprt locus were characterized by polymerase chain reaction (PCR) amplification of reverse-transcribed hprt mRNA, followed by DNA sequence analysis. The mutant frequencies, in mutants/10{sup 6} clonable cells, at 30 and 100 ng/ml, were BaP-DE, 248 and 456; 1-nitro-BaP-DE, 68 and 260; 3-nitro-BaP-DE, 81 and 232, respectively. In general, the three diolepoxides exhibited similar mutational spectra: (1) 64% (23/36 sequenced mutants) of BaP-DE, 53% (19/36) of 1-nitro-BaP-DE, and 64% (23/36) of 3-nitro-BaP-DE mutants resulted from simple base pair substitution, with the predominant mutation being G{r_arrow}T transversion: (2) 90%, 100%, and 100% of mutations at G:C had the mutated dG on the nontranscribed DNA strand; and (3) about one quarter of the mutants produced by each mutagen had one or more PCR products with partial or complete exon deletions. 61 refs., 1 fig., 7 tabs.

  16. Absence of interaction between X-rays and UV light in inducing ouabain- and thioguanine-resistant mutants in Chinese hamster cells.

    PubMed

    Cleaver, J E

    1978-11-01

    Chinese hamster ovary cells were irradiated with X-rays at times from 0 to 17 h before being irradiated with ultraviolet (UV) light. No synergism was observed between the two radiations for the production of mutants resistant to either ouabain or 6-thioguanine. These experiments were designed to test whether X-rays induced an error-prone repair system that would increase the frequency of mutations produced by UV light, but no such system was detected.

  17. Test for Chemical Induction of Chromosome Aberrations in Cultured Chinese Hamster (CHO) Cells With and Without Metabolic Activation. Test Article. Diethylene triamine trinitrate (DETN)

    DTIC Science & Technology

    2010-02-25

    chromatid interchanges between chromosomes leading to four-armed configurations. This could be asymmetrical with formation of a dicentric and an acentric...fragment which may be misaligned and a shortened monocentric chromosome , and where there is no sister chromatid union. Dicentric - an asymmetrical...Test for Chemical Induction of Chromosome Aberrations in Cultured Chinese Hamster Ovary (CHO) Cells With and Without Metabolic Activation Test

  18. A theoretical estimate for nucleotide sugar demand towards Chinese Hamster Ovary cellular glycosylation

    PubMed Central

    del Val, Ioscani Jimenez; Polizzi, Karen M.; Kontoravdi, Cleo

    2016-01-01

    Glycosylation greatly influences the safety and efficacy of many of the highest-selling recombinant therapeutic proteins (rTPs). In order to define optimal cell culture feeding strategies that control rTP glycosylation, it is necessary to know how nucleotide sugars (NSs) are consumed towards host cell and rTP glycosylation. Here, we present a theoretical framework that integrates the reported glycoproteome of CHO cells, the number of N-linked and O-GalNAc glycosylation sites on individual host cell proteins (HCPs), and the carbohydrate content of CHO glycosphingolipids to estimate the demand of NSs towards CHO cell glycosylation. We have identified the most abundant N-linked and O-GalNAc CHO glycoproteins, obtained the weighted frequency of N-linked and O-GalNAc glycosites across the CHO cell proteome, and have derived stoichiometric coefficients for NS consumption towards CHO cell glycosylation. By combining the obtained stoichiometric coefficients with previously reported data for specific growth and productivity of CHO cells, we observe that the demand of NSs towards glycosylation is significant and, thus, is required to better understand the burden of glycosylation on cellular metabolism. The estimated demand of NSs towards CHO cell glycosylation can be used to rationally design feeding strategies that ensure optimal and consistent rTP glycosylation. PMID:27345611

  19. Characterization of a Chinese hamster-human hybrid cell line with increased system L amino acid transport activity.

    PubMed Central

    Lobaton, C D; Moreno, A; Oxender, D L

    1984-01-01

    We have studied leucine transport in several Chinese hamster-human hybrid cell lines obtained by fusion of a temperature-sensitive line of Chinese hamster ovary cells, ts025C1, and normal human leukocytes. A hybrid cell line exhibiting a twofold increase in L-leucine uptake over that in the parental cell line was found. This hybrid cell line, 158CnpT-1, was temperature resistant, whereas the parental Chinese hamster ovary mutant, ts025C1, contained a temperature-sensitive leucyl-tRNA synthetase mutation. An examination of the different amino acid transport systems in this hybrid cell line revealed a specific increase of system L activity with no significant changes in systems A and ASC. The Vmax for L-leucine uptake exhibited by the hybrid 158CnpT-1 was twice that in the CHO parental mutant, ts025C1. Cytogenetic analysis showed that the hybrid 158CnpT-1 contains four complete human chromosomes (numbers 4, 5, 10, and 21) and three interspecific chromosomal translocations in a total complement of 34 chromosomes. Biochemical and cytogenetic analysis of segregant clones obtained from hybrid 158CnpT-1 showed that the primary temperature resistance and high system L transport phenotypes can be segregated from this hybrid independently. The loss of the primary temperature resistance was associated with the loss of the human chromosome 5, as previously reported by other laboratories, whereas the loss of the high leucine transport phenotype, which is associated with a lesser degree of temperature resistance, was correlated with the loss of human chromosome 20. Images PMID:6717430

  20. Characterization of a Chinese hamster-human hybrid cell line with increased system L amino acid transport activity.

    PubMed

    Lobaton, C D; Moreno, A; Oxender, D L

    1984-03-01

    We have studied leucine transport in several Chinese hamster-human hybrid cell lines obtained by fusion of a temperature-sensitive line of Chinese hamster ovary cells, ts025C1, and normal human leukocytes. A hybrid cell line exhibiting a twofold increase in L-leucine uptake over that in the parental cell line was found. This hybrid cell line, 158CnpT-1, was temperature resistant, whereas the parental Chinese hamster ovary mutant, ts025C1, contained a temperature-sensitive leucyl-tRNA synthetase mutation. An examination of the different amino acid transport systems in this hybrid cell line revealed a specific increase of system L activity with no significant changes in systems A and ASC. The Vmax for L-leucine uptake exhibited by the hybrid 158CnpT-1 was twice that in the CHO parental mutant, ts025C1. Cytogenetic analysis showed that the hybrid 158CnpT-1 contains four complete human chromosomes (numbers 4, 5, 10, and 21) and three interspecific chromosomal translocations in a total complement of 34 chromosomes. Biochemical and cytogenetic analysis of segregant clones obtained from hybrid 158CnpT-1 showed that the primary temperature resistance and high system L transport phenotypes can be segregated from this hybrid independently. The loss of the primary temperature resistance was associated with the loss of the human chromosome 5, as previously reported by other laboratories, whereas the loss of the high leucine transport phenotype, which is associated with a lesser degree of temperature resistance, was correlated with the loss of human chromosome 20.

  1. Centrosomal amplification and aneuploidy induced by the antiretroviral drug AZT in hamster and human cells

    PubMed Central

    Borojerdi, Jennifer P.; Ming, Jessica; Cooch, Catherine; Ward, Yvona; Semino-Mora, Cristina; Yu, Mia; Braun, Hannan M.; Taylor, Barbara J.; Poirier, Miriam C.; Olivero, Ofelia A.

    2009-01-01

    The centrosome directs chromosomal migration by a complex process of tubulin-chromatin binding. In this contribution centrosomal abnormalities, including centrosomal amplification, were explored in Chinese Hamster Ovary (CHO) and Normal Human Mammary Epithelial (NHMEC) cells exposed to the antiretroviral drug zidovudine (3’-azido-3’-deoxythymidine, AZT). Centrosomal amplification/fragmentation was observed in both cell types and kinetochore positive micronuclei were found in AZT-exposed CHO cells in correlation with dose. Normal human mammary epithelial cell (NMHEC), strain M99005, previously identified as a strain that incorporates high levels of AZT into DNA (High incorporator, HI), showed greater centrosomal amplification when compared with a second strain, NHMEC M98040, which did not incorporate AZT into DNA (Low incorporator, LI). Additionally, an abnormal tubulin distribution was observed in AZT-exposed HI cells bearing multiple centrosomes. Immunofluorescent staining of human cells with Aurora A, a kinase involved in the maturation of the centrosome, confirmed the induction of centrosomal amplification and revealed multipolar mitotic figures. Flow cytometric studies revealed that cells bearing abnormal numbers of centrosomes and abnormal tubulin distribution had similar S-phase percentages suggesting that cells bearing unbalanced chromosomal segregation could divide. Therefore, AZT induces genomic instability and clastogenicity as well as alterations in proteins involved in centrosomal activation, all of which may contribute to the carcinogenic properties of this compound. PMID:19427513

  2. Photoperiod-dependent modulation of anti-Müllerian hormone in female Siberian hamsters, Phodopus sungorus.

    PubMed

    Kabithe, Esther W; Place, Ned J

    2008-03-01

    Fertility and fecundity decline with advancing age in female mammals, but reproductive aging was decelerated in Siberian hamsters (Phodopus sungorus) raised in a short-day (SD) photoperiod. Litter success was significantly improved in older hamsters when reared in SD and the number of primordial follicles was twice that of females held in long days (LD). Because anti-Müllerian hormone (AMH) appears to inhibit the recruitment of primordial follicles in mice, we sought to determine whether the expression patterns of AMH differ in the ovaries and serum of hamsters raised in SD versus LD. Ovaries of SD female hamsters are characterized by a paucity of follicular development beyond the secondary stage and are endowed with an abundance of large eosinophilic cells, which may derive from granulosa cells of oocyte-depleted follicles. In ovaries from 10-week-old SD hamsters, we found that the so-called 'hypertrophied granulosa cells' were immunoreactive for AMH, as were granulosa cells within healthy-appearing primary and secondary follicles. Conversely, ovaries from age-matched LD animals lack the highly eosinophilic cells present in SD ovaries. Therefore, AMH staining in LD was limited to primary and secondary follicles that are comparable in number to those found in SD ovaries. The substantially greater AMH expression in SD ovaries probably reflects the abundance of hypertrophied granulosa cells in SD ovaries and their relative absence in LD ovaries. The modulation of ovarian AMH by day length is a strong mechanistic candidate for the preservation of primordial follicles in female hamsters raised in a SD photoperiod.

  3. Repair-deficient xeroderma pigmentosum cells made UV light resistant by fusion with X-ray-inactivated Chinese hamster cells

    SciTech Connect

    Karentz, D.; Cleaver, J.E.

    1986-10-01

    Xeroderma pigmentosum (XP) is an autosomal recessive human disease, characterized by an extreme sensitivity to sunlight, caused by the inability of cells to repair UV light-induced damage to DNA. Cell fusion was used to transfer fragments of Chinese hamster ovary (CHO) chromosomes into XP cells. The hybrid cells exhibited UV resistance and DNA repair characteristics comparable to those expressed by CHO cells, and their DNA had greater homology with CHO DNA than did the DNA from XP cells. Control experiments consisted of fusion of irradiated and unirradiated XP cells and repeated exposure of unfused XP cells to UV doses used for hybrid selection. These treatments did not result in an increase in UV resistance, repair capability, or homology with CHO DNA. The hybrid cell lines do not, therefore, appear to be XP revertants. The establishment of these stable hybrid cell lines is an initial step toward identifying and cloning CHO DNA repair genes that complement the XP defect in human cells. The method should also be applicable to cloning genes for other diseases, such as ataxia-telangiectasia and Fanconi's anemia.

  4. Potential role of mast cells in hamster cheek pouch carcinogenesis.

    PubMed

    Aromando, Romina F; Pérez, Miguel A; Heber, Elisa M; Trivillin, Verónica A; Tomasi, Víctor H; Schwint, Amanda E; Itoiz, María E

    2008-11-01

    During the process of activation, mast cells release products stored in their granules. Tryptase, a protease released from mast cell granules after activation, induces tumor cell proliferation through the activation of PAR-2 (protease activated receptor 2) on the plasma membrane of carcinoma cells. Chemical cancerization (DMBA) of the hamster cheek pouch is the most accepted model of oral cancer. However, there are no reports on the activation of mast cells during experimental carcinogenesis or on the correlation between mast cell activation and cell proliferation. The aim of the present study was to evaluate the potential effect of mast cells on the proliferation of epithelial cells at different times during the cancerization process. Paraffin serial sections of cancerized, tumor-bearing pouches were stained with Alcian Blue-Safranin to identify the different degrees of mast cell activation. Immunohistochemistry was performed to identify BrdU-positive cells to study tumor cell proliferation. Mast cells were counted and grouped into two categories: inactive mast cells AB-S+++ (red) and active mast cells AB+++S- (blue). Mast cell counts were performed in tumor stroma, base of the tumor (connective tissue immediately below the exophytic tumor), connective and muscle tissue underlying the cancerized epithelium (pouch wall) and adventitious tissue underlying the pouch wall. There was a significant increase in the number of mast cells at the base of tumors (p<0.001) compared to the number of mast cells in the wall of the pouch and in tumor stroma. In normal non-cancerized pouches, inactive mast cells were prevalent both in the wall (AB:S=1:2.15; p<0.001) and in the adventitious tissue (AB:S=1:1.6; p<0.004) of the hamster cheek pouch. At most of the experimental times examined, the ratio of active/inactive mast cells (AB/S) in the wall approximated unity and even reverted. The ratio of mast cells was AB:S 1:1.05 at the base of the tumor and 1:0.24 in tumor stroma (p<0

  5. Neuroendocrine cells are present in the domestic fowl ovary

    PubMed Central

    Hofmann, Pablo G; Báez Saldaña, Armida; Fortoul Van Der Goes, Teresa; González del Pliego, Margarita; Gutiérrez Ospina, Gabriel

    2013-01-01

    Neuroendocrine cells are present in virtually all organs of the vertebrate body; however, it is yet uncertain whether they exist in the ovaries. Previous reports of ovarian neurons and neuron-like cells in mammals and birds might have resulted from misidentification. The aim of the present work was to determine the identity of neuron-like cells in immature ovaries of the domestic fowl. Cells immunoreactive to neurofilaments, synaptophysin, and chromogranin-A, with small, dense-core secretory granules, were consistently observed throughout the sub-cortical ovarian medulla and cortical interfollicular stroma. These cells also displayed immunoreactivity for tyrosine, tryptophan and dopamine β-hydroxylases, as well as to aromatic L-DOPA decarboxylase, implying their ability to synthesize both catecholamines and indolamines. Our results support the argument that the ovarian cells previously reported as neuron-like in birds, are neuroendocrine cells. PMID:23083425

  6. Molecular characterisation of camptothecin-induced mutations at the hprt locus in Chinese hamster cells.

    PubMed

    Balestrieri, E; Zanier, R; Degrassi, F

    2001-05-09

    The capacity of the topoisomerase I inhibitor camptothecin (CPT) to induce single locus mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene and the DNA changes underlying induced mutations were analysed in Chinese hamster ovary cells. Camptothecin treatments increased hprt mutations up to 50-fold over the spontaneous levels at highly cytotoxic doses. Genomic DNA was isolated from 6-thioguanine resistant clones and subjected to multiplex PCR to screen for gross alterations in the gene structure. The molecular analysis revealed that deletion mutants represented 80% of the analysed clones, including total hprt deletion, multiple and single exon deletions. Furthermore, a fraction of the analysed clones showed deletions of more than one exon that were characterised by the absence of non-contiguous exons. These data show that single locus mutations induced by camptothecin are characterised by large deletions or complex rearrangements rather than single base substitutions and suggest that the recombinational repair of camptothecin-induced strand breaks at replication fork may be involved in the generations of these alterations at the chromatin structure level.

  7. Molecular characterization of mutation and comparison of mutation profiles in the hprt gene of Chinese hamster ovary cells treated with benzo[a]pyrene trans-7,8-diol-anti-9,10-epoxide, 1-nitrobenzo[a]pyrene trans-7,8-diol-anti-9,10-epoxide, and 3-nitrobenzo[a]pyrene trans-7,8- diol-anti-9,10-epoxide.

    PubMed

    Zhan, D J; Heflich, R H; Fu, P P

    1996-01-01

    Both 1- and 3-nitrobenzo[a]pyrene (nitro-BaP) are environmental contaminants, potent mutagens in Salmonella, and moderate mutagens in Chinese hamster ovary (CHO) cells. The mutagenicity of their oxidized metabolites,trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-epoxy -7,8,9,10-tetrahydro-1-nitrobenzo[a]pyrene (1-nitro-BaP-DE) and trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydro-3-nitrobenzo[a]- pyrene (3-nitro-BaPDE), together with trans-7,8-dihydroxy-anti-9, 10-ep- oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BaP-DE), was determined in CHO-K1 cells, and the resulting mutations at the hprt locus were characterized by polymerase chain reaction (PCR) amplification of reverse-transcribed hprt mRNA, followed by DNA sequence analysis. The mutant frequencies, in mutants/10(6) clonable cells, at 30 and 100 ng/ml, were BaP-DE, 248 and 456; 1-nitro-BaP-DE, 68 and 260; 3-nitro-BaP-DE, 81 and 232, respectively. In general, the three diolepoxides exhibited similar mutational spectra: 1) 64% (23/36 sequenced mutants) of BaP-DE, 53% (19/36) of 1-nitro-BaP-DE, and 64% (23/36) of 3-nitro-BaP-DE mutants resulted from simple base pair substitution, with the predominant mutation being G-->T transversion; 2) 90%, 100%, and 100% of mutations at G:C had the mutated dG on the nontranscribed DNA strand; and 3) about one quarter of the mutants produced by each mutagen had one or more PCR products with partial or complete exon deletions. The mutagens induced few frameshifts or complex mutations. Among the differences in mutational specificity for the three diolepoxides, the proportion of substituted dGs with 3' purines was significant (P < 0.05) for BaP-DE (16/19, 84%) and 3-nitro-BaP-DE (17/20, 85%), but not significant for 1-nitro-BaP-DE-induced mutants (11/17, 65%, P > 0.05). Also, high proportions of BaP-DE and 3-nitro-BaP-DE base pair substitutions at G:C occurred in DNA sequence contexts of 5'-GG-3', 5'-GGA-3', and 5'-TGGA-3', while the proportions of 1-nitro-BaP-DE mutants in these

  8. Neoplastic transformation of hamster embyro cells by heavy ions.

    PubMed

    Han, Z; Suzuki, H; Suzuki, F; Suzuki, M; Furusawa, Y; Kato, T; Ikenaga, M

    1998-01-01

    We have studied the induction of morphological transformation of Syrian hamster embryo cells by low doses of heavy ions with different linear energy transfer (LET), ranging from 13 to 400 keV/micrometer. Exponentially growing cells were irradiated with 12C or 28Si ion beams generated by the Heavy Ion Medical Accelerator in Chiba (HIMAC), inoculated to culture dishes, and transformed colonies were identified when the cells were densely stacked and showed a crisscross pattern. Over the LET range examined, the frequency of transformation induced by the heavy ions increased sharply at very low doses no greater than 5 cGy. The relative biological effectiveness (RBE) of the heavy ions relative to 250 kVp X-rays showed an initial increase with LET, reaching a maximum value of about 7 at 100 keV/micrometer, and then decreased with the further increase in LET. Thus, we confirmed that high LET heavy ions are significantly more effective than X-rays for the induction of in vitro cell transformation.

  9. Propagation of Asian isolates of canine distemper virus (CDV) in hamster cell lines

    PubMed Central

    Sultan, Serageldeen; Lan, Nguyen Thi; Ueda, Toshiki; Yamaguchi, Ryoji; Maeda, Ken; Kai, Kazushige

    2009-01-01

    Backgrounds The aim of this study was to confirm the propagation of various canine distemper viruses (CDV) in hamster cell lines of HmLu and BHK, since only a little is known about the possibility of propagation of CDV in rodent cells irrespective of their epidemiological importance. Methods The growth of CDV in hamster cell lines was monitored by titration using Vero.dogSLAMtag (Vero-DST) cells that had been proven to be susceptible to almost all field isolates of CDV, with the preparations of cell-free and cell-associated virus from the cultures infected with recent Asian isolates of CDV (13 strains) and by observing the development of cytopathic effect (CPE) in infected cultures of hamster cell lines. Results Eleven of 13 strains grew in HmLu cells, and 12 of 13 strains grew in BHK cells with apparent CPE of cell fusion in the late stage of infection. Two strains and a strain of Asia 1 group could not grow in HmLu cells and BHK cells, respectively. Conclusion The present study demonstrates at the first time that hamster cell lines can propagate the majority of Asian field isolates of CDV. The usage of two hamster cell lines suggested to be useful to characterize the field isolates biologically. PMID:19835588

  10. Langerhans cell function dictates induction of contact hypersensitivity or unresponsiveness to DNFB in Syrian hamsters

    SciTech Connect

    Streilein, J.W.; Bergstresser, P.R.

    1981-09-01

    The relationship between distribution and function of Langerhans cells within the epidermis and the capacity of cutaneous surfaces to promote the induction of contact hypersensitivity to DNFB have been examined in inbred Syrian hamsters. In a manner very similar to previous findings in mice, the results indicate that hamster cutaneous surfaces deficient in normally functioning Langerhans cells, naturally (cheek pouch epithelium) or artificially (after perturbation with ultraviolet light), are inefficient at promoting DNFB sensitization. Instead, DNFB applied to these regions of skin results in the induction of a state of specific unresponsiveness. Viable lymphoid cells from unresponsive hamsters can transfer the unresponsiveness to naive hamsters suggesting that active suppression is at least partly responsible, probably mediated by T lymphocytes.

  11. Honeybee product therapeutic as stem cells homing for ovary failure

    PubMed Central

    Safitri, Erma; Widiyatno, Thomas V.; Prasetyo, R. Heru

    2016-01-01

    Aim: Complexity of the method of isolation, cultivation in vitro and the expensive cost of transplantation process of stem cells, it would require an innovation to homing and differentiation of stem cells and increase folliculogenesis. The stem cells homing was achieved through the provision of food or beverages derived from natural materials like honeybee product. Through honeybee product, there will be homing of stem cells and accompany with the sources from the body itself will take place in regeneration of the ovary. Materials and Methods: Female rats model of degenerative ovary was obtained through food fasting but still have drinking water for 5 days. It caused malnutrition and damage of the ovarian tissue. The administration of 50% honeybee product (T1) was performed for 10 consecutive days, while the positive control group (T0+) was fasted and not given honeybee product and the negative control (T0−) not fasted and without honeybee product. Observations were taken for homing of stem cells, raised of folliculogenesis, differentiation of stem cells, and regeneration of the ovarian tissue using routine H&E staining. Results: Homing of stem cells shown the vascular endothelial growth factor and granulocyte colony-stimulating factor expression; enhancement of folliculogenesis was indicated by an increase of follicle dee Graaf count; enhancement of differentiation of stem cells was indicated by growth differentiation factor-9 expression; and regeneration of ovarian tissue indicated by intact ovarian tissue with growing follicles. Conclusion: Honeybee product can be induced endogenous stem cells in regeneration of ovary failure due to malnutrition. PMID:27956789

  12. Characterization of a Mutant Diphtheria Toxin that is Defective in Binding to Cell Membrane Receptors on Vero Cells

    DTIC Science & Technology

    1982-08-13

    Uniformed Services University of the Health Sciences, Bethesda, MD and have been maintained by S. Hansen since 1966. Chinese hamster ovary ( CHO ...et proprietes immunisantes. Ann. lnst. Pasteur~. 1-22 163. Ray, B.J. and Wu. H.C. (1981) Internalization of r1C1n in Chinese hamster ovary cells . Mol...cytotoxicity of toxin for Chinese hamster V79 cells and the inhibitory effects were reversed by methyl-a-mannoslde and N-acetyl glucosamine

  13. Regulation of germ cell meiosis in the fetal ovary.

    PubMed

    Spiller, Cassy M; Bowles, Josephine; Koopman, Peter

    2012-01-01

    Fertility depends on correct regulation of meiosis, the special form of cell division that gives rise to haploid gametes. In female mammals, germ cells enter meiosis during fetal ovarian development, while germ cells in males avoid entering meiosis until puberty. Decades of research have shown that meiotic entry, and germ cell sex determination, are not initiated intrinsically within the germ cells. Instead, meiosis is induced by signals produced by the surrounding somatic cells. More recently, retinoic acid (RA), the active derivative of vitamin A, has been implicated in meiotic induction during fetal XX and postnatal XY germ cell development. Evidence for an intricate system of RA synthesis and degradation in the fetal ovary and testis has emerged, explaining past observations of infertility in vitamin A-deficient rodents. Here we review how meiosis is triggered in fetal ovarian germ cells, paying special attention to the role of RA in this process.

  14. Hematologic Assessment in Pet Rats, Mice, Hamsters, and Gerbils: Blood Sample Collection and Blood Cell Identification.

    PubMed

    Lindstrom, Nicole M; Moore, David M; Zimmerman, Kurt; Smith, Stephen A

    2015-09-01

    Hamsters, gerbils, rats, and mice are presented to veterinary clinics and hospitals for prophylactic care and treatment of clinical signs of disease. Physical examination, history, and husbandry practice information can be supplemented greatly by assessment of hematologic parameters. As a resource for veterinarians and their technicians, this article describes the methods for collection of blood, identification of blood cells, and interpretation of the hemogram in mice, rats, gerbils, and hamsters.

  15. Hematologic assessment in pet rats, mice, hamsters, and gerbils: blood sample collection and blood cell identification.

    PubMed

    Lindstrom, Nicole M; Moore, David M; Zimmerman, Kurt; Smith, Stephen A

    2015-01-01

    Hamsters, gerbils, rats, and mice are presented to veterinary clinics and hospitals for prophylactic care and treatment of clinical signs of disease. Physical examination, history, and husbandry practice information can be supplemented greatly by assessment of hematologic parameters. As a resource for veterinarians and their technicians, this article describes the methods for collection of blood, identification of blood cells, and interpretation of the hemogram in mice, rats, gerbils, and hamsters.

  16. Effect of Gonadectomy on the Androgen-Dependent Behavior of Ganglion Cell-Like Cells in Djungarian Hamsters (Phodopus sungorus).

    PubMed

    Nakahira, Rei; Yoshida, Rumika; Michishita, Masaki; Ohkusu-Tsukada, Kozo; Takahashi, Kimimasa

    2016-02-01

    Ganglion cell-like (GL) cells reside in the dermis of the ventral skin of mature male Djungarian hamsters (Phodopus sugorus) and express androgen receptor (AR). To assess whether GL cells have androgen-dependent behavior, we evaluated the histologic changes of GL cells after gonadectomy. Five male and 5 female hamsters were gonadectomized at the age of 4 wk and necropsied 14 wk later. The number, distribution, and proliferative activity of GL cells in the thoracoabdominal and dorsal skins were evaluated histologically and compared with those of corresponding intact animals. GL cells were more numerous, were distributed throughout the skin more widely, and had higher proliferative activity in the intact male hamsters than in their gonadectomized counterparts. Similar trends regarding these 3 parameters were seen in ovariectomized compared with intact female hamsters and between intact male and intact female hamsters. These results suggest that the GL cells of Djungarian hamsters demonstrate sex-associated differences in their distribution and proliferative activity and that androgen may be involved in the development of these cells.

  17. Vincristine sulfate-induced cell transformation, mitotic inhibition and aneuploidy in cultured Syrian hamster embryo cells

    SciTech Connect

    Tsutsui, T.; Suzuki, N.; Maizumi, H.; Barrett, J.C.

    1986-01-01

    Vincristine, a naturally occurring Vinca alkaloid and widely used anti-neoplastic agent, was examined for its ability to induce cell transformation, inhibition of growth and mitosis, and genetic effects in Syrian hamster embryo cells in culture. Treatment of the cells with doses of less than or equal to 1 ng/ml vincristine sulfate (VCR) had no effect on cell growth, while exposure to greater than or equal to 3 ng/ml reduced the growth rate and treatment with 30 ng/ml resulted in no detectable increase in cell number. At this latter dose the mitotic index of the cells increased significantly suggesting that VCR delayed completion of mitosis. Exposure of the cells to VCR at doses of 1-10 ng/ml for 48 h resulted in morphological transformation of the cells in a doserelated fashion. The vincristine-treated transformed colonies were morphologically indistinguishable from colonies transformed by benzo(a)pyrene or other chemical carcinogens. Morphological transformation was induced by VCR at non-toxic and slightly toxic doses as measured by a reduction in colony-forming ability of the treated cells. Over the dose range which resulted in cell transformation, VCR failed to induce either detectable gene mutations at two genetic loci, unscheduled DNA synthesis, or chromosome aberrations in the Syrian hamster embryo cells. However, a significant dose-dependent increase in aneuploid cells with a near-diploid chromosome number was induced by VCR. Both chromosome losses and gains were induced which is consistent with a non-disjunctional mechanism. These results further support our hypothesis that aneuploidy is one possible mechanism for induction of this early step in the neoplastic transformation of Syrian hamster embryo cells. Furthermore, these findings indicate that VCR may have some carcinogenic potential if exposure to rapidly dividing cells occurs.

  18. Retinoic acid derived from the fetal ovary initiates meiosis in mouse germ cells.

    PubMed

    Mu, Xinyi; Wen, Jing; Guo, Meng; Wang, Jianwei; Li, Ge; Wang, Zhengpin; Wang, Yijing; Teng, Zhen; Cui, Yan; Xia, Guoliang

    2013-03-01

    Meiotic initiation of germ cells at 13.5 dpc (days post-coitus) indicates female sex determination in mice. Recent studies reveal that mesonephroi-derived retinoic acid (RA) is the key signal for induction of meiosis. However, whether the mesonephroi is dispensable for meiosis is unclear and the role of the ovary in this meiotic process remains to be clarified. This study provides data that RA derived from fetal ovaries is sufficient to induce germ cell meiosis in a fetal ovary culture system. When fetal ovaries were collected from 11.5 to 13.5 dpc fetuses, isolated and cultured in vitro, germ cells enter meiosis in the absence of mesonephroi. To exclude RA sourcing from mesonephroi, 11.5 dpc urogenital ridges (UGRs; mesonephroi and ovary complexes) were treated with diethylaminobenzaldehyde (DEAB) to block retinaldehyde dehydrogenase (RALDH) activity in the mesonephros and the ovary. Meiosis occurred when DEAB was withdrawn and the mesonephros was removed 2 days later. Furthermore, RALDH1, rather than RALDH2, serves as the major RA synthetase in UGRs from 12.5 to 15.5 dpc. DEAB treatment to the ovary alone was able to block germ cell meiotic entry. We also found that exogenously supplied RA dose-dependently reduced germ cell numbers in ovaries by accelerating the entry into meiosis. These results suggest that ovary-derived RA is responsible for meiosis initiation.

  19. Expression of a functional human type I interferon receptor in hamster cells: application of functional yeast artificial chromosome (YAC) screening.

    PubMed

    Soh, J; Mariano, T M; Lim, J K; Izotova, L; Mirochnitchenko, O; Schwartz, B; Langer, J A; Pestka, S

    1994-07-08

    The previously cloned human interferon alpha/beta (Hu-IFN-alpha/beta; Type I interferon) receptor cDNA appears to be only one component of a receptor complex since expression of the cDNA in mouse cells confers sensitivity only to Hu-IFN-alpha B2, but a monoclonal antibody against this cloned receptor subunit inhibits biological activities of Hu-IFN-alpha A, Hu-IFN-alpha B2, Hu-IFN-omega, and Hu-IFN-beta. Here we report that a yeast artificial chromosome (YAC) containing a segment of human chromosome 21 introduced into Chinese hamster ovary (CHO) cells confers upon these cells a greatly enhanced response to Hu-IFN-alpha A and Hu-IFN-alpha B2 as well as an increased response to Hu-IFN-omega, Hu-IFN-alpha A/D(Bgl), andd Hu-IFN-beta. These responses were measured by induction of class I MHC antigens and by protection against encephalomyocarditis virus and vesicular stomatitis virus. Furthermore, these cells exhibit specific high affinity binding of Hu-IFN-alpha A and Hu-IFN-alpha B2, Hu-IFN-beta, and Hu-IFN-omega. The results indicate that all the genes necessary to reconstitute a biologically active Type I human IFN receptor complex are located within the human DNA insert of this YAC clone.

  20. Sex, stem cells and tumors in the Drosophila ovary.

    PubMed

    Salz, Helen K

    2013-01-01

    The Drosophila Sex-lethal (Sxl) gene encodes a female-specific RNA binding protein that in somatic cells globally regulates all aspects of female-specific development and behavior. Sxl also has a critical, but less well understood, role in female germ cells. Germ cells without Sxl protein can adopt a stem cell fate when housed in a normal ovary, but fail to successfully execute the self-renewal differentiation fate switch. The failure to differentiate is accompanied by the inappropriate expression of a set of male specific markers, continued proliferation, and formation of a tumor. The findings in Chau et al., (2012) identify the germline stem cell maintenance factor nanos as one of its target genes, and suggest that Sxl enables the switch from germline stem cell to committed daughter cell by posttranscriptional downregulation of nanos expression. These studies provide the basis for a new model in which Sxl directly couples sexual identity with the self-renewal differentiation decision and raises several interesting questions about the genesis of the tumor phenotype.

  1. [An initial investigation on the in vitro culture system of primordial germ cells in golden hamsters].

    PubMed

    Li, Hong; Zhang, Hao; Liang, Ying; Zhan, Li; Wu, Desheng

    2006-06-01

    To establish the in vitro culture system of primordial germ cells (PGCs) of golden hamsters, PGCs of hamster were isolated from genital ridge of embryos at 10. 5th dpc (day post coitum), obtained by enzyme-mechanical method, and cultured on feeder cells. Then the PGCs were identified by alkaline phosphatase (ALP) activity staining. In order to induce the PGCs to differentiate in vitro, we removed the differential inhibition factors in the conditioned medium and observed the formation of embryoids and differentiated cells from three layers. The result showed that the pluripotent primordial germ cells could be successfully obtained from the golden hamsters at 10. 5th dpc by the enzyme-mechanical method and that PGCs were identified by both their strong positive reaction in ALP activity staining test and their differentiation into three-layer derived cells in vitro. The result suggests that the establishment of in vitro PGCs culture system of golden hamsters will provide new cell source for biomedical engineering.

  2. A hybrid approach identifies metabolic signatures of high-producers for chinese hamster ovary clone selection and process optimization.

    PubMed

    Popp, Oliver; Müller, Dirk; Didzus, Katharina; Paul, Wolfgang; Lipsmeier, Florian; Kirchner, Florian; Niklas, Jens; Mauch, Klaus; Beaucamp, Nicola

    2016-09-01

    In-depth characterization of high-producer cell lines and bioprocesses is vital to ensure robust and consistent production of recombinant therapeutic proteins in high quantity and quality for clinical applications. This requires applying appropriate methods during bioprocess development to enable meaningful characterization of CHO clones and processes. Here, we present a novel hybrid approach for supporting comprehensive characterization of metabolic clone performance. The approach combines metabolite profiling with multivariate data analysis and fluxomics to enable a data-driven mechanistic analysis of key metabolic traits associated with desired cell phenotypes. We applied the methodology to quantify and compare metabolic performance in a set of 10 recombinant CHO-K1 producer clones and a host cell line. The comprehensive characterization enabled us to derive an extended set of clone performance criteria that not only captured growth and product formation, but also incorporated information on intracellular clone physiology and on metabolic changes during the process. These criteria served to establish a quantitative clone ranking and allowed us to identify metabolic differences between high-producing CHO-K1 clones yielding comparably high product titers. Through multivariate data analysis of the combined metabolite and flux data we uncovered common metabolic traits characteristic of high-producer clones in the screening setup. This included high intracellular rates of glutamine synthesis, low cysteine uptake, reduced excretion of aspartate and glutamate, and low intracellular degradation rates of branched-chain amino acids and of histidine. Finally, the above approach was integrated into a workflow that enables standardized high-content selection of CHO producer clones in a high-throughput fashion. In conclusion, the combination of quantitative metabolite profiling, multivariate data analysis, and mechanistic network model simulations can identify metabolic

  3. Restriction of human adenovirus replication in Chinese hamster cell lines and their hybrids with human cells.

    PubMed

    Radna, R L; Foellmer, B; Feldman, L A; Francke, U; Ozer, H L

    1987-11-01

    We have found that the replication of human adenovirus (Ad2) is restricted in multiple Chinese hamster cell lines including CHO and V79. The major site of restriction involves differential accumulation of late viral proteins as demonstrated by immunofluorescence assay and polyacrylamide gel electrophoresis with and without prior immunoprecipitation. Synthesis of fiber and penton base are markedly reduced, whereas others, such as the 100K polypeptide, are synthesized efficiently. This pattern of restriction is similar to that previously reported for Ad2 infection of several monkey cell lines; however, the restriction is more marked in the Chinese hamster cell lines. The restriction is most likely due to a deficient cellular function since stable cell hybrids between V79 or CHO and human cells are permissive for virus replication. By analysis of a series of hybrids with reduced numbers of human chromosomes, fiber synthesis was correlated with the presence of the short arm of human chromosome 3. More hybrids showed restoration of fiber synthesis than production of progeny virus, suggesting that more than one unlinked function is required for the latter.

  4. Serotonergic modulation of hippocampal pyramidal cells in euthermic, cold-acclimated, and hibernating hamsters

    NASA Technical Reports Server (NTRS)

    Horrigan, D. J.; Horwitz, B. A.; Horowitz, J. M.

    1997-01-01

    Serotonergic fibers project to the hippocampus, a brain area previously shown to have distinctive changes in electroencephalograph (EEG) activity during entrance into and arousal from hibernation. The EEG activity is generated by pyramidal cells in both hibernating and nonhibernating species. Using the brain slice preparation, we characterized serotonergic responses of these CA1 pyramidal cells in euthermic, cold-acclimated, and hibernating Syrian hamsters. Stimulation of Shaffer-collateral/commissural fibers evoked fast synaptic excitation of CA1 pyramidal cells, a response monitored by recording population spikes (the synchronous generation of action potentials). Neuromodulation by serotonin (5-HT) decreased population spike amplitude by 54% in cold-acclimated animals, 80% in hibernating hamsters, and 63% in euthermic animals. The depression was significantly greater in slices from hibernators than from cold-acclimated animals. In slices from euthermic animals, changes in extracellular K+ concentration between 2.5 and 5.0 mM did not significantly alter serotonergic responses. The 5-HT1A agonist 8-hydroxy-2(di-n-propylamino)tetralin mimicked serotonergic inhibition in euthermic hamsters. Results show that 5-HT is a robust neuromodulator not only in euthermic animals but also in cold-acclimated and hibernating hamsters.

  5. Morphological cell transformation of Syrian hamster embryo (SHE) cells by the cyanotoxin, cylindrospermopsin.

    PubMed

    Maire, M-A; Bazin, E; Fessard, V; Rast, C; Humpage, A R; Vasseur, P

    2010-06-15

    Cylindrospermopsin (CYN) is a cyanotoxin which has been implicated in human intoxication and animal mortality. Genotoxic activity of this hepatotoxin is known but its carcinogenic activity remains to be elucidated. In this work, CYN was assessed for its cell-transforming activity using the Syrian hamster embryo (SHE) cell transformation assay. This in vitro assay is used to evaluate the carcinogenic potential of chemical, physical and biological agents in SHE cells, which are primary, normal, diploid, genetically stable and capable of metabolic activation. We demonstrated that CYN induced a significant increase in morphological cell transformation in SHE cells following a 7-day continuous treatment in the range of non-cytotoxic concentrations 1 x 10(-7)-1 x 10(-2) ng/mL.

  6. Misoprostol-induced radioprotection of Syrian hamster embryo cells in utero from cell death and oncogenic transformation

    SciTech Connect

    Miller, R.C.; LaNasa, P.; Hanson, W.R.

    1994-07-01

    Misoprostol, a PGE analog, is an effective radioprotector of murine intestine and hematopoietic and hair cell renewal systems. The radioprotective nature of misoprostol was extended to examine its ability to influence clonogenic cell survival and induction of oncogenic transformation in Syrian hamster embryo cells exposed to X rays in utero and assayed in vitro. Hamsters in their 12th day of pregnancy were injected subcutaneously with misoprostal, and 2 h later the pregnant hamsters were exposed to graded doses of X rays. Immediately after irradiation, hamsters were euthanized and embryonic tissue was explanted into culture dishes containing complete growth medium. After a 2-week incubation period, clongenic cell survival and morphologically transformed foci were determined. Survival of misoprostol-treated SHE cells was increased and yielded a dose reduction factor of 1.5 compared to SHE cells treated with X rays alone. In contrast, radiation-induced oncogenic transformation of misoprostol-treated cells was reduced by a factor of 20 compared to cells treated with X rays alone. These studies suggest that misoprostol not only protects normal tissues in vivo from acute radiation injury, but also protects cells, to a large extent, from injury leading to transforming events. 26 refs., 6 figs., 2 tabs.

  7. Antigenic modulation of metastatic breast and ovary carcinoma cells by intracavitary injection of IFN-alpha.

    PubMed Central

    Giacomini, P.; Mottolese, M.; Fraioli, R.; Benevolo, M.; Venturo, I.; Natali, P. G.

    1992-01-01

    Antigenic modulation of major histocompatibility and tumour associated antigens was observed in neoplastic cells obtained from patients with pleural and abdominal effusions of breast and ovary carcinomas following a single intracavitary dose of 18 x 10(6) U recombinant IFN-alpha. This regimen resulted in antigenic modulation in seven out of 11 tested cases, suggesting a potential, although limited, responsiveness of at least a fraction of breast and ovary carcinoma cells to in situ biomodification with IFN-alpha. PMID:1503908

  8. New insights into melatonin/CRH signaling in hamster Leydig cells.

    PubMed

    Rossi, Soledad P; Matzkin, María E; Terradas, Claudio; Ponzio, Roberto; Puigdomenech, Elisa; Levalle, Oscar; Calandra, Ricardo S; Frungieri, Mónica B

    2012-08-01

    We have previously described that melatonin inhibits androgen production in hamster testes via melatonin subtype 1a (mel1a) receptors and the local corticotrophin-releasing hormone (CRH) system. This study attempted to determine the initial events of the melatonin/CRH signaling pathway. In Leydig cells from reproductively active Syrian hamsters, Western blotting, reverse transcription quantitative polymerase chain reaction (RT-qPCR) and a colorimetric assay demonstrated that melatonin and CRH activate tyrosine phosphatases and subsequently reduce the phosphorylation levels of extracellular signal-regulated kinase (erk) and c-jun N-terminal kinase (jnk), down-regulate the expression of c-jun, c-fos and steroidogenic acute regulatory (StAR), and inhibit the production of testosterone. These effects were prevented by a highly selective CRH antagonist, thus indicating that melatonin does not exert a direct role. Specific mitogen-activated protein kinase kinase (MEK) and jnk blockers inhibited expression of c-jun, c-fos, StAR and the production of testosterone, confirming that these are events triggered downstream of erk and jnk. In Leydig cells from photoperiodically regressed adult hamsters, CRH inhibited the production of androstane-3α,17β-diol (3α-diol), the main androgen produced, through the same signaling pathway. Testicular melatonin concentration was 3-4-fold higher in reproductively inactive hamsters than that detected in active animals. Since melatonin, CRH, and their receptors are present not only in hamster testes but also in testicular biopsies of infertile men, we can conjecture about the relevance of this previously uncharacterized pathway in human fertility disorders. In summary, our study identifies crucial intracellular events triggered by melatonin/CRH in the testis that lead to a down-regulation of the steroidogenic process.

  9. Effects of microwave exposure on the hamster immune system. I. Natural killer cell activity

    SciTech Connect

    Yang, H.K.; Cain, C.A.; Lockwood, J.; Tompkins, W.A.

    1983-01-01

    Hamsters were exposed to repeated or single doses of microwave energy and monitored for changes in core body temperature, circulating leukocyte profiles, serum corticosteroid levels, and natural killer (NK) cell activity in various tissues. NK cytotoxicity was measured in a /sup 51/Cr-release assay employing baby hamster kidney (BHK) targets or BHK infected with herpes simplex virus. Repeated exposure of hamsters at 15 mW/cm2 for 60 min/day had no significant effect on natural levels of spleen-cell NK activity against BHK targets. Similarly, repeated exposure at 15 mW/cm2 over a 5-day period had no demonstrable effect on the induction of spleen NK activity by vaccinia virus immunization, that is, comparable levels of NK were induced in untreated and microwave-treated animals. In contrast, treatment of hamsters with a single 60-min microwave exposure at 25 mW/cm2 caused a significant suppression in induced spleen NK activity. A similar but less marked decrease in spleen NK activity was observed in sham-exposed animals. Moreover, the sham effects on NK activity were not predictable and appeared to represent large individual animal variations in the response to stress factors. Depressed spleen NK activity was evident as early as 4 h postmicrowave treatment and returned to normal levels by 8 h. Hamsters exposed at 25 mW/cm2 showed an elevated temperature of 3.0-3.5 degrees C that returned to normal within 60 min after termination of microwave exposure. These animals also showed a marked lymphopenia and neutrophilia by 1 h posttreatment that returned to normal by 8-10 h. Serum glucocorticosteroids were elevated between 1 aNd 8 h after microwave treatment. Sham-exposed animals did not demonstrate significant changes in core body temperature, peripheral blood leukocyte (PBL) profile, or glucocorticosteroid levels as compared to minimum-handling controls.

  10. Night temperature and source–sink effects on overall growth, cell number and cell size in bell pepper ovaries

    PubMed Central

    Darnell, Rebecca L.; Cruz-Huerta, Nicacio; Williamson, Jeffrey G.

    2012-01-01

    Background and Aims Ovary swelling, and resultant fruit malformation, in bell pepper flowers is favoured by low night temperature or a high source–sink ratio. However, the interaction between night temperature and source–sink ratio on ovary swelling and the contribution of cell size and cell number to ovary swelling are unknown. The present research examined the interactive effects of night temperature and source–sink ratio on ovary size, cell number and cell size at anthesis in bell pepper flowers. Methods Bell pepper plants were grown in growth chambers at night temperatures of either 20 °C (HNT) or 12 °C (LNT). Within each temperature treatment, plants bore either 0 (non-fruiting) or two developing fruits per plant. Ovary fresh weight, cell size and cell number were measured. Key Results Ovary fresh weights in non-fruiting plants grown at LNT were the largest, while fresh weights were smallest in plants grown at HNT with fruits. In general, mesocarp cell size in ovaries was largest in non-fruiting plants grown at either LNT or HNT and smallest in fruiting plants at HNT. Mesocarp cell number was greater in non-fruiting plants under LNT than in the rest of the night temperature/fruiting treatments. These responses were more marked in ovaries sampled after 18 d of treatment compared with those sampled after 40 d of treatment. Conclusions Ovary fresh weight of flowers at anthesis increased 65 % in non-fruiting plants grown under LNT compared with fruiting plants grown under HNT. This increase was due primarily to increases in mesocarp cell number and size. These results indicate that the combined effects of LNT and high source–sink ratio on ovary swelling are additive. Furthermore, the combined effects of LNT and low source–sink ratio or HNT and high source–sink ratio can partially overcome the detrimental effects of LNT and high source–sink ratio. PMID:22933415

  11. The Role of the MHV Receptor and Related Glycoproteins in Murine Hepatitis Virus Infection of Murine Cell Lines

    DTIC Science & Technology

    1995-04-13

    Chinese hamster ovary ( CHO ) cells (Asanaka and Lai, 1993). The fusion event can be controlled at multiple levels. As mentioned above, some receptor...receptor can mediate virus infection, since transfection of the receptor cDNA clone into MHV - resistant hamster or human cell lines confers MHV...expressed in MHV - resistant cells such as baby hamster kidney cells (BHK-21) or human RD cell lines, the cells became susceptible to infection by

  12. Primary cell cultures from sea urchin ovaries: a new experimental tool.

    PubMed

    Mercurio, Silvia; Di Benedetto, Cristiano; Sugni, Michela; Candia Carnevali, M Daniela

    2014-02-01

    In the present work, primary cell cultures from ovaries of the edible sea urchin Paracentrotus lividus were developed in order to provide a simple and versatile experimental tool for researches in echinoderm reproductive biology. Ovary cell phenotypes were identified and characterized by different microscopic techniques. Although cell cultures could be produced from ovaries at all stages of maturation, the cells appeared healthier and viable, displaying a higher survival rate, when ovaries at early stages of gametogenesis were used. In terms of culture medium, ovarian cells were successfully cultured in modified Leibovitz-15 medium, whereas poor results were obtained in minimum essential medium Eagle and medium 199. Different substrates were tested, but ovarian cells completely adhered only on poly-L-lysine. To improve in vitro conditions and stimulate cell proliferation, different serum-supplements were tested. Fetal calf serum and an originally developed pluteus extract were detrimental to cell survival, apparently accelerating processes of cell death. In contrast, cells cultured with sea urchin egg extract appeared larger and healthier, displaying an increased longevity that allowed maintaining them for up to 1 month. Overall, our study provides new experimental bases and procedures for producing successfully long-term primary cell cultures from sea urchin ovaries offering a good potential to study echinoid oogenesis in a controlled system and to investigate different aspects of echinoderm endocrinology and reproductive biology.

  13. Generation of exogenous germ cells in the ovaries of sterile NANOS3-null beef cattle

    PubMed Central

    Ideta, Atsushi; Yamashita, Shiro; Seki-Soma, Marie; Yamaguchi, Ryosaku; Chiba, Shiori; Komaki, Haruna; Ito, Tetsuya; Konishi, Masato; Aoyagi, Yoshito; Sendai, Yutaka

    2016-01-01

    Blastocyst complementation (BC) systems have enabled in vivo generation of organs from allogeneic pluripotent cells, compensating for an empty germ cell niche in gene knockout (KO) animals. Here, we succeeded in producing chimeric beef cattle (Wagyu) by transferring allogenic germ cells into ovaries using somatic cell nuclear transfer and BC technology. The KO of NANOS3 (NANOS3−/−) in Wagyu bovine ovaries produced a complete loss of germ cells. Holstein blastomeres (NANOS3+/+) were injected into NANOS3−/− Wagyu embryos. Subsequently, exogenous germ cells (NANOS3+/+) were identified in the NANOS3−/− ovary. These results clearly indicate that allogeneic germ cells can be generated in recipient germ cell-free gonads using cloning and BC technologies. PMID:27117862

  14. Generation of exogenous germ cells in the ovaries of sterile NANOS3-null beef cattle.

    PubMed

    Ideta, Atsushi; Yamashita, Shiro; Seki-Soma, Marie; Yamaguchi, Ryosaku; Chiba, Shiori; Komaki, Haruna; Ito, Tetsuya; Konishi, Masato; Aoyagi, Yoshito; Sendai, Yutaka

    2016-04-27

    Blastocyst complementation (BC) systems have enabled in vivo generation of organs from allogeneic pluripotent cells, compensating for an empty germ cell niche in gene knockout (KO) animals. Here, we succeeded in producing chimeric beef cattle (Wagyu) by transferring allogenic germ cells into ovaries using somatic cell nuclear transfer and BC technology. The KO of NANOS3 (NANOS3(-/-)) in Wagyu bovine ovaries produced a complete loss of germ cells. Holstein blastomeres (NANOS3(+/+)) were injected into NANOS3(-/-) Wagyu embryos. Subsequently, exogenous germ cells (NANOS3(+/+)) were identified in the NANOS3(-/-) ovary. These results clearly indicate that allogeneic germ cells can be generated in recipient germ cell-free gonads using cloning and BC technologies.

  15. Ciliated cells in vitamin A-deprived cultured hamster tracheal epithelium do divide

    SciTech Connect

    Rutten, A.A.; Beems, R.B.; Wilmer, J.W.; Feron, V.J.

    1988-09-01

    The pseudostratified tracheal epithelium, composed of a heterogeneous phenotypically varying cell population, was studied with respect to the in vitro cell proliferative activity of differentiated epithelial cells. Ciliated tracheal epithelial cells so far have been considered to be terminally differentiated, nonproliferating cells. Tracheal organ cultures obtained from vitamin A-deprived Syrian Golden hamsters were cultured in a vitamin A-deficient, serum-free, hormone-supplemented medium. In vitamin A-deprived tracheal epithelium treated with physiologically active all-trans retinol and low cigarette-smoke condensate concentrations it is possible to stimulate the cell proliferation of both basal and columnar cells. Therefore, the probability of finding proliferating columnar cells was increased compared with the in vivo and the vitamin A-deprived situation in which cell proliferative activity is relatively low. In the presence of cigarette-smoke condensate in a noncytotoxic concentration, basal, small mucous granule, ciliated, and indifferent tracheal epithelial cells incorporated (methyl-3H)-thymidine into the DNA during the S phase. The finding that ciliated cells were labeled was supported by serial sections showing the same labeled ciliated cell in two section planes separated by 2 to 3 micron, without labeled epithelial cells next to the ciliated cell. Furthermore, a ciliated tracheal epithelial cell incorporating (methyl-/sup 3/H)thymidine into DNA was also seen in tracheal cultures of vitamin A-deprived hamsters treated with all-trans retinol in a physiologic concentration.

  16. Regulation of neuroendocrine cells and neuron factors in the ovary by zinc oxide nanoparticles.

    PubMed

    Liu, Xin-Qi; Zhang, Hong-Fu; Zhang, Wei-Dong; Zhang, Peng-Fei; Hao, Ya-Nan; Song, Ran; Li, Lan; Feng, Yan-Ni; Hao, Zhi-Hui; Shen, Wei; Min, Ling-Jiang; Yang, Hong-Di; Zhao, Yong

    2016-08-10

    The pubertal period is an important window during the development of the female reproductive system. Development of the pubertal ovary, which supplies the oocytes intended for fertilization, requires growth factors, hormones, and neuronal factors. It has been reported that zinc oxide nanoparticles (ZnO NPs) cause cytotoxicity of neuron cells. However, there have been no reports of the effects of ZnO NPs on neuronal factors and neuroendocrine cells in the ovary (in vivo). For the first time, this in vivo study investigated the effects of ZnO NPs on gene and protein expression of neuronal factors and the population of neuroendocrine cells in ovaries. Intact NPs were detected in ovarian tissue and although ZnO NPs did not alter body weight, they reduced the ovary organ index. Compared to the control or ZnSO4 treatments, ZnO NPs treatments differentially regulated neuronal factor protein and gene expression, and the population of neuroendocrine cells. ZnO NPs changed the contents of essential elements in the ovary; however, they did not alter levels of the steroid hormones estrogen and progesterone. These data together suggest that intact ZnO NPs might pose a toxic effect on neuron development in the ovary and eventually negatively affect ovarian developmental at puberty.

  17. Infertility in the hyperplasic ovary of freshwater planarians: the role of programmed cell death.

    PubMed

    Harrath, Abdel Halim; Semlali, Abdelhabib; Mansour, Lamjed; Ahmed, Mukhtar; Sirotkin, Alexander V; Al Omar, Suliman Y; Arfah, Maha; Al Anazi, Mohamed S; Alhazza, Ibrahim M; Nyengaard, Jens R; Alwasel, Saleh

    2014-11-01

    Ex-fissiparous planarians produce infertile cocoons or, in very rare cases, cocoons with very low fertility. Here, we describe the features of programmed cell death (PCD) occurring in the hyperplasic ovary of the ex-fissiparous freshwater planarian Dugesia arabica that may explain this infertility. Based on TEM results, we demonstrate a novel extensive co-clustering of cytoplasmic organelles, such as lysosomes and microtubules, and their fusion with autophagosomes during the early stage of oocyte cell death occurring through an autophagic pattern. During a later stage of cell death, the generation of apoptotic vesicles in the cytoplasm can be observed. The immunohistochemical labeling supports the ultrastructural results because it has been shown that the proapoptotic protein bax was more highly expressed in the hyperplasic ovary than in the normal one, whereas the anti-apoptotic protein bcl2 was slightly more highly expressed in the normal ovary compared to the hyperplasic one. TUNEL analysis of the hyperplasic ovary confirmed that the nuclei of the majority of differentiating oocytes were TUNEL-positive, whereas the nuclei of oogonia and young oocytes were TUNEL-negative; in the normal ovary, oocytes are TUNEL-negative. Considering all of these data, we suggest that the cell death mechanism of differentiating oocytes in the hyperplasic ovary of freshwater planarians is one of the most important factors that cause ex-fissiparous planarian infertility. We propose that autophagy precedes apoptosis during oogenesis, whereas apoptotic features can be observed later.

  18. Effects of dinitrotoluenes on morphological cell transformation and intercellular communication in Syrian hamster embryo cells.

    PubMed

    Holen, I; Mikalsen, S O; Sanner, T

    1990-01-01

    The effects of four isomers of dinitrotoluene (DNT) and technical DNT (a mixture of DNT isomers and other compounds, with 2,4-DNT as the major constituent) were studied in two short-term in vitro assays. None of the isomers or technical DNT induced an increase in morphological transformation of Syrian hamster embryo (SHE) cells. Four DNT metabolites (2,4-diaminotoluene, 2-amino-4-nitrotoluene, 2-amino-6-nitrotoluene, and 2,4-dinitobenzoic acid), representing different stages in reduction or oxidation of DNT isomers, were also negative for induction of morphological transformation. The DNT isomers were tested in an intercellular communication assay based on dye transfer. 2,4-DNT, 2,6-DNT, and technical DNT inhibited intercellular communication in the SHE cell line BPNi at toxic concentrations. This may be reminiscent of in vivo data showing promoting activity of these compound. 2,3-DNT and 3,4-DNT did not inhibit communication.

  19. Unlike in Drosophila Meroistic Ovaries, hippo represses notch in Blattella germanica Panoistic ovaries, triggering the mitosis-endocycle switch in the follicular cells.

    PubMed

    Irles, Paula; Piulachs, Maria-Dolors

    2014-01-01

    During insect oogenesis, the follicular epithelium undergoes both cell proliferation and apoptosis, thus modulating ovarian follicle growth. The Hippo pathway is key in these processes, and has been thoroughly studied in the meroistic ovaries of Drosophila melanogaster. However, nothing is known about the role of the Hippo pathway in primitive panoistic ovaries. This work examines the mRNA expression levels of the main components of the Hippo pathway in the panoistic ovary of the basal insect species Blattella germanica, and demonstrates the function of Hippo through RNAi. In Hippo-depleted specimens, the follicular cells of the basal ovarian follicles proliferate without arresting cytokinesis; the epithelium therefore becomes bilayered, impairing ovarian follicle growth. This phenotype is accompanied by long stalks between the ovarian follicles. In D. melanogaster loss of function of Notch determines that the stalk is not developed. With this in mind, we tested whether Hippo and Notch pathways are related in B. germanica. In Notch (only)-depleted females, no stalks were formed between the ovarian follicles. Simultaneous depletion of Hippo and Notch rescued partially the stalk to wild-type. Unlike in the meroistic ovaries of D. melanogaster, in panoistic ovaries the Hippo pathway appears to regulate follicular cell proliferation by acting as a repressor of Notch, triggering the switch from mitosis to the endocycle in the follicular cells. The phylogenetically basal position of B. germanica suggests that this might be the ancestral function of Hippo in insect ovaries.

  20. The evolution of chromosomal instability in Chinese hamster cells: a changing picture?

    NASA Technical Reports Server (NTRS)

    Ponnaiya, B.; Limoli, C. L.; Corcoran, J.; Kaplan, M. I.; Hartmann, A.; Morgan, W. F.

    1998-01-01

    PURPOSE: To investigate the kinetics of chromosomal instability induced in clones of Chinese hamster cells following X-irradiation. MATERIALS AND METHODS: X-irradiated clones of GM10115, human-hamster hybrid cells containing a single human chromosome 4 (HC4), have been previously established. These clones were defined as unstable if they contained > or = three subpopulations of cells with unique rearrangements of HC4 as detected by FISH. Stable and unstable clones were analysed by FISH and Giemsa staining at various times post-irradiation. RESULTS: While most of the stable clones continued to show chromosomal stability of HC4 over time, one became marginally unstable at approximately 45 population doublings post-irradiation. Clones exhibiting chromosomal instability had one of several fates. Many of the unstable clones were showed similar levels of instability over time. However, one unstable clone became stable with time in culture, while another became even more unstable over time. Cytogenetic analyses of all clones after Giemsa staining indicated that in some clones the hamster chromosomes were rearranged independent of HC4, demonstrating increased frequencies of chromatid breaks and dicentric chromosomes. The majority of the unstable clones also had higher yields of chromatid gaps. CONCLUSIONS: These data demonstrate the dynamic nature of chromosomal instability as measured by two different cytogenetic assays.

  1. Polycystic ovary syndrome and the peripheral blood white cell count.

    PubMed

    Herlihy, A C; Kelly, R E; Hogan, J L; O'Connor, N; Farah, N; Turner, M J

    2011-01-01

    This retrospective cross-sectional study examined if the white cell count (WCC) is increased in women with polycystic ovary syndrome (PCOS) and if so, is it due to PCOS or to the associated obesity? Body mass index (BMI) was calculated and body composition was measured using bioelectrical impedance analysis. Of the 113 women studied, 36 had PCOS and 77 did not. The mean WCC was higher in the PCOS group compared with the non-PCOS group (8.9 × 10(9)/l vs 7.4 × 10(9)/l p = 0.002). This increase was due to a higher neutrophil count (5.6 × 10(9)/l vs 4.3 × 10(9)/l; p = 0.003). There was a leucocytosis (WCC >11 × 10(9)/l) present in 19% of the PCOS group compared with 1% in the non-PCOS group (p < 0.001). The neutrophil count was abnormally high (>7.7 × 10(9)/l) in 14% of the PCOS group compared with 4% in the non-PCOS group (p < 0.001). On regression analysis, however, the only independent variable which explained both the increased WCC and the increased neutrophil count was PCOS. We found that PCOS is associated with an increased WCC due to increased neutrophils, which supports the evidence that PCOS is associated with low-grade inflammation. The increase appears to be due to the underlying PCOS, and not to the increased adiposity associated with PCOS.

  2. Interspecies complementation analysis of xeroderma pigmentosum and UV-sensitive Chinese hamster cells

    SciTech Connect

    Stefanini, M.; Keijzer, W.; Westerveld, A.; Bootsma, D.

    1985-12-01

    Complementation analysis was performed 24 h after fusion of UV-sensitive CHO cells (CHO 12 RO) with XP cells of complementation groups A, B, C, D, F and G. The parental cells are characterized by low levels of unscheduled DNA synthesis (UDS). In all combinations, the UDS levels observed in heterokaryons were higher than those in parental mutant cells, clearly indicating cooperation of human and Chinese hamster repair functions. In heterokaryons of CHO 12 RO with XP-A and XP-C cells, the UDS values reached about the normal human level, whereas in heterokaryons with XP-B, XP-D and XP-F, UDS was restored at a level approaching that in wild-type CHO cells. The results obtained after fusion of CHO cells with two representative cell strains from the XP-G group, XP 2 BI and XP 3 BR, were inconsistent. Fusion with XP 3 BR cells yielded UDS levels ranging from wild-type Chinese hamster to normal human, whereas fusion with XP 2 BI cells resulted in a slight increase in UDS which even after 48 h remained below the level found in wild-type CHO cells. The occurrence of complementation in these interspecies heterokaryons indicates that the genetic defect in the CHO 12 RO cells is different from the defects in the XP complementation groups tested.

  3. Prolactin (PRL) induction of cyclooxygenase 2 (COX2) expression and prostaglandin (PG) production in hamster Leydig cells.

    PubMed

    Matzkin, María Eugenia; Ambao, Verónica; Carino, Mónica Herminia; Rossi, Soledad Paola; González, Lorena; Turyn, Daniel; Campo, Stella; Calandra, Ricardo Saúl; Frungieri, Mónica Beatriz

    2012-01-02

    Serum prolactin (PRL) variations play a crucial role in the photoperiodic-induced testicular regression-recrudescence transition in hamsters. We have previously shown that cyclooxygenase 2 (COX2), a key enzyme in the biosynthesis of prostaglandins (PGs), is expressed mostly in Leydig cells of reproductively active hamsters with considerable circulating and pituitary levels of PRL. In this study, we describe a stimulatory effect of PRL on COX2/PGs in hamster Leydig cells, which is mediated by IL-1β and prevented by P38-MAPK and JAK2 inhibitors. Furthermore, by preparative isoelectric focusing (IEF), we isolated PRL charge analogues from pituitaries of active [isoelectric points (pI): 5.16, 4.61, and 4.34] and regressed (pI: 5.44) hamsters. More acidic PRL charge analogues strongly induced COX2 expression, while less acidic ones had no effect. Our studies suggest that PRL induces COX2/PGs in hamster Leydig cells through IL-1β and activation of P38-MAPK and JAK2. PRL microheterogeneity detected in active/inactive hamsters may be responsible for the photoperiodic variations of COX2 expression in Leydig cells.

  4. Presence of SNAP-23 and syntaxin 4 in mouse and hamster peritoneal mast cells.

    PubMed

    Salinas, Eva; Rodríguez, Gonzalo; Quintanar, J Luis

    2007-01-01

    Mast cells (MCs) play a crucial role in inflammatory reactions. Their presence and number in the peritoneal cavity is important to overcome and enhance resistance to peritoneal infection. When MCs are activated they release a variety of biological mediators from their granules, such as histamine, that contribute to the appropriate and rapid local immune response. Granular content is released using a process of compound exocytosis, also termed degranulation. SNAP-23 and syntaxin 4 are plasma membrane proteins involved in degranulation of rat MCs. Their presence, however, has not been studied in MCs of other rodent species. The aim of the present study was to investigate using immunocytochemistry whether SNAP-23 and syntaxin 4 are present in peritoneal MCs of the mouse and hamster. In addition, the diameter, percentage and histamine content of these cells were also analyzed. Our results demonstrate that SNAP-23 and syntaxin 4 are present in the mouse and hamster peritoneal MCs, suggesting that proteins involved in the secretory process in MCs are conserved among species. Likewise, we conclude that peritoneal MCs of mouse and hamster are heterogeneous in size, percentage and histamine content.

  5. Lgr5 marks stem/progenitor cells in ovary and tubal epithelia.

    PubMed

    Ng, Annie; Tan, Shawna; Singh, Gurmit; Rizk, Pamela; Swathi, Yada; Tan, Tuan Zea; Huang, Ruby Yun-Ju; Leushacke, Marc; Barker, Nick

    2014-08-01

    The ovary surface epithelium (OSE) undergoes ovulatory tear and remodelling throughout life. Resident stem cells drive such tissue homeostasis in many adult epithelia, but their existence in the ovary has not been definitively proven. Lgr5 marks stem cells in multiple epithelia. Here we use reporter mice and single-molecule fluorescent in situ hybridization to document candidate Lgr5(+) stem cells in the mouse ovary and associated structures. Lgr5 is broadly expressed during ovary organogenesis, but becomes limited to the OSE in neonate life. In adults, Lgr5 expression is predominantly restricted to proliferative regions of the OSE and mesovarian-fimbria junctional epithelia. Using in vivo lineage tracing, we identify embryonic and neonate Lgr5(+) populations as stem/progenitor cells contributing to the development of the OSE cell lineage, as well as epithelia of the mesovarian ligament and oviduct/fimbria. Adult Lgr5(+) populations maintain OSE homeostasis and ovulatory regenerative repair in vivo. Thus, Lgr5 marks stem/progenitor cells of the ovary and tubal epithelia.

  6. Chromosomal mutations and chromosome loss measured in a new human-hamster hybrid cell line, ALC: studies with colcemid, ultraviolet irradiation, and 137Cs gamma-rays

    NASA Technical Reports Server (NTRS)

    Kraemer, S. M.; Waldren, C. A.; Chatterjee, A. (Principal Investigator)

    1997-01-01

    Small mutations, megabase deletions, and aneuploidy are involved in carcinogenesis and genetic defects, so it is important to be able to quantify these mutations and understand mechanisms of their creation. We have previously quantified a spectrum of mutations, including megabase deletions, in human chromosome 11, the sole human chromosome in a hamster-human hybrid cell line AL. S1- mutants have lost expression of a human cell surface antigen, S1, which is encoded by the M1C1 gene at 11p13 so that mutants can be detected via a complement-mediated cytotoxicity assay in which S1+ cells are killed and S1- cells survive. But loss of genes located on the tip of the short arm of 11 (11p15.5) is lethal to the AL hybrid, so that mutants that have lost the entire chromosome 11 die and escape detection. To circumvent this, we fused AL with Chinese hamster ovary (CHO) cells to produce a new hybrid, ALC, in which the requirement for maintaining 11p15.5 is relieved, allowing us to detect mutations events involving loss of 11p15.5. We evaluated the usefulness of this hybrid by conducting mutagenesis studies with colcemid, 137Cs gamma-radiation and UV 254 nm light. Colcemid induced 1000 more S1- mutants per unit dose in ALC than in AL; the increase for UV 254 nm light was only two-fold; and the increase for 137Cs gamma-rays was 12-fold. The increase in S1- mutant fraction in ALC cells treated with colcemid and 137Cs gamma-rays were largely due to chromosome loss and 11p deletions often containing a breakpoint within the centromeric region.

  7. Sequencing, Annotation and Analysis of the Syrian Hamster (Mesocricetus auratus) Transcriptome

    PubMed Central

    Tchitchek, Nicolas; Safronetz, David; Rasmussen, Angela L.; Martens, Craig; Virtaneva, Kimmo; Porcella, Stephen F.; Feldmann, Heinz

    2014-01-01

    Background The Syrian hamster (golden hamster, Mesocricetus auratus) is gaining importance as a new experimental animal model for multiple pathogens, including emerging zoonotic diseases such as Ebola. Nevertheless there are currently no publicly available transcriptome reference sequences or genome for this species. Results A cDNA library derived from mRNA and snRNA isolated and pooled from the brains, lungs, spleens, kidneys, livers, and hearts of three adult female Syrian hamsters was sequenced. Sequence reads were assembled into 62,482 contigs and 111,796 reads remained unassembled (singletons). This combined contig/singleton dataset, designated as the Syrian hamster transcriptome, represents a total of 60,117,204 nucleotides. Our Mesocricetus auratus Syrian hamster transcriptome mapped to 11,648 mouse transcripts representing 9,562 distinct genes, and mapped to a similar number of transcripts and genes in the rat. We identified 214 quasi-complete transcripts based on mouse annotations. Canonical pathways involved in a broad spectrum of fundamental biological processes were significantly represented in the library. The Syrian hamster transcriptome was aligned to the current release of the Chinese hamster ovary (CHO) cell transcriptome and genome to improve the genomic annotation of this species. Finally, our Syrian hamster transcriptome was aligned against 14 other rodents, primate and laurasiatheria species to gain insights about the genetic relatedness and placement of this species. Conclusions This Syrian hamster transcriptome dataset significantly improves our knowledge of the Syrian hamster's transcriptome, especially towards its future use in infectious disease research. Moreover, this library is an important resource for the wider scientific community to help improve genome annotation of the Syrian hamster and other closely related species. Furthermore, these data provide the basis for development of expression microarrays that can be used in functional

  8. Assignment of the human MARS gene, encoding methioninyl-tRNA synthetase, to chromosome 12 using human X Chinese hamster cell hybrids.

    PubMed

    Cirullo, R E; Wasmuth, J J

    1984-05-01

    We have isolated interspecific somatic cell hybrids between a temperature-sensitive Chinese hamster ovary (CHO) cell methioninyl -tRNA synthetase mutant and human peripheral leukocytes. The hybrids were selected at 39 degrees C which requires the retention and expression of the human gene, MARS , which complements the defective CHO gene. In vitro heat-inactivation experiments on the methioninyl -tRNA synthetase activity in cell-free extracts from heat-resistant hybrids indicate that the human form of this enzyme and, therefore, the human MARS gene is present in hybrid cells. Cytogenetic analysis of three independent temperature-resistant hybrids revealed the presence of a single human chromosome, number 12. Two other independent hybrids examined contained human chromosome 12 as well as a second human chromosome. Electrophoretic analysis of extracts from hybrid cell lines for a human chromosome 12 marker isozyme, LDH-B, showed a pattern of heterotetrameric bands consistent with the presence of the human form of this enzyme in these cells. The correlation between the presence of the human form of methioninyl -tRNA synthetase and human chromosome 12 in temperature-resistant hybrids indicates that the human MARS locus is located on this chromosome.

  9. Gibberellin reactivates and maintains ovary-wall cell division causing fruit set in parthenocarpic Citrus species.

    PubMed

    Mesejo, Carlos; Yuste, Roberto; Reig, Carmina; Martínez-Fuentes, Amparo; Iglesias, Domingo J; Muñoz-Fambuena, Natalia; Bermejo, Almudena; Germanà, M Antonietta; Primo-Millo, Eduardo; Agustí, Manuel

    2016-06-01

    Citrus is a wide genus in which most of the cultivated species and cultivars are natural parthenocarpic mutants or hybrids (i.e. orange, mandarin, tangerine, grapefruit). The autonomous increase in GA1 ovary concentration during anthesis was suggested as being the stimulus responsible for parthenocarpy in Citrus regardless of the species. To determine the exact GA-role in parthenocarpic fruit set, the following hypothesis was tested: GA triggers and maintains cell division in ovary walls causing fruit set. Obligate and facultative parthenocarpic Citrus species were used as a model system because obligate parthenocarpic Citrus sp (i.e. Citrus unshiu) have higher GA levels and better natural parthenocarpic fruit set compared to other facultative parthenocarpic Citrus (i.e. Citrus clementina). The autonomous activation of GA synthesis in C. unshiu ovary preceded cell division and CYCA1.1 up-regulation (a G2-stage cell cycle regulator) at anthesis setting a high proportion of fruits, whereas C. clementina lacked this GA-biosynthesis and CYCA1.1 up-regulation failing in fruit set. In situ hybridization experiments revealed a tissue-specific expression of GA20ox2 only in the dividing tissues of the pericarp. Furthermore, CYCA1.1 expression correlated endogenous GA1 content with GA3 treatment, which stimulated cell division and ovary growth, mostly in C. clementina. Instead, paclobutrazol (GA biosynthesis inhibitor) negated cell division and reduced fruit set. Results suggest that in parthenocarpic citrus the specific GA synthesis in the ovary walls at anthesis triggers cell division and, thus, the necessary ovary growth rate to set fruit.

  10. Marker genes identify three somatic cell types in the fetal mouse ovary.

    PubMed

    Rastetter, Raphael H; Bernard, Pascal; Palmer, James S; Chassot, Anne-Amandine; Chen, Huijun; Western, Patrick S; Ramsay, Robert G; Chaboissier, Marie-Christine; Wilhelm, Dagmar

    2014-10-15

    The two main functions of the ovary are the production of oocytes, which allows the continuation of the species, and secretion of female sex hormones, which control many aspects of female development and physiology. Normal development of the ovaries during embryogenesis is critical for their function and the health of the individual in later life. Although the adult ovary has been investigated in great detail, we are only starting to understand the cellular and molecular biology of early ovarian development. Here we show that the adult stem cell marker Lgr5 is expressed in the cortical region of the fetal ovary and this expression is mutually exclusive to FOXL2. Strikingly, a third somatic cell population can be identified, marked by the expression of NR2F2, which is expressed in LGR5- and FOXL2 double-negative ovarian somatic cells. Together, these three marker genes label distinct ovarian somatic cell types. Using lineage tracing in mice, we show that Lgr5-positive cells give rise to adult cortical granulosa cells, which form the follicles of the definitive reserve. Moreover, LGR5 is required for correct timing of germ cell differentiation as evidenced by a delay of entry into meiosis in Lgr5 loss-of-function mutants, demonstrating a key role for LGR5 in the differentiation of pre-granulosa cells, which ensure the differentiation of oogonia, the formation of the definitive follicle reserve, and long-term female fertility.

  11. Testosterone induction of prostaglandin-endoperoxide synthase 2 expression and prostaglandin F(2alpha) production in hamster Leydig cells.

    PubMed

    Matzkin, María E; Gonzalez-Calvar, Silvia I; Mayerhofer, Artur; Calandra, Ricardo S; Frungieri, Mónica B

    2009-07-01

    We have previously observed expression of prostaglandin-endoperoxide synthase 2 (PTGS2), the key enzyme in the biosynthesis of prostaglandins (PGs), in reproductively active Syrian hamster Leydig cells, and reported an inhibitory role of PGF(2alpha) on hamster testicular steroidogenesis. In this study, we further investigated PTGS2 expression in hamster Leydig cells during sexual development and photoperiodic gonadal regression. Since PTGS2 is mostly expressed in pubertal and reproductively active adult hamsters with high circulating levels of LH and androgens, we studied the role of these hormones in the regulation/maintenance of testicular PTGS2/PGF(2alpha). In active hamster Leydig cells, LH/hCG and testosterone induced PTGS2 and PGF(2alpha) production, and their actions were abolished by the antiandrogen bicalutamide (Bi). These results indicate that LH does not exert a direct effect on PG synthesis. Testosterone also stimulated phosphorylation of the mitogen-activated protein kinase isoforms 3/1 (MAPK3/1) within minutes and hours, but the testosterone metabolite dihydrotestosterone had no effect on PTGS2 and MAPK3/1. Because Bi and U0126, an inhibitor of the MAP kinase kinases 1 and 2 (MAP2K1/2), abolished testosterone actions on MAPK3/1 and PTGS2, our studies suggest that testosterone directly induces PTGS2/PGF(2alpha) in hamster Leydig cells via androgen receptors and a non-classical mechanism that involves MAPK3/1 activation. Since PGF(2alpha) inhibits testosterone production, it might imply the existence of a regulatory loop that is setting a brake on steroidogenesis. Thus, the androgen environment might be crucial for the regulation of testicular PG production at least during sexual development and photoperiodic variations in hamsters.

  12. Targeted delivery of antigen to hamster nasal lymphoid tissue with M-cell-directed lectins.

    PubMed Central

    Giannasca, P J; Boden, J A; Monath, T P

    1997-01-01

    The nasal cavity of a rodent is lined by an epithelium organized into distinct regional domains responsible for specific physiological functions. Aggregates of nasal lymphoid tissue (NALT) located at the base of the nasal cavity are believed to be sites of induction of mucosal immune responses to airborne antigens. The epithelium overlying NALT contains M cells which are specialized for the transcytosis of immunogens, as demonstrated in other mucosal tissues. We hypothesized that NALT M cells are characterized by distinct glycoconjugate receptors which influence antigen uptake and immune responses to transcytosed antigens. To identify glycoconjugates that may distinguish NALT M cells from other cells of the respiratory epithelium (RE), we performed lectin histochemistry on sections of the hamster nasal cavity with a panel of lectins. Many classes of glycoconjugates were found on epithelial cells in this region. While most lectins bound to sites on both the RE and M cells, probes capable of recognizing alpha-linked galactose were found to label the follicle-associated epithelium (FAE) almost exclusively. By morphological criteria, the FAE contains >90% M cells. To determine if apical glycoconjugates on M cells were accessible from the nasal cavity, an M-cell-selective lectin and a control lectin in parallel were administered intranasally to hamsters. The M-cell-selective lectin was found to specifically target the FAE, while the control lectin did not. Lectin bound to M cells in vivo was efficiently endocytosed, consistent with the role of M cells in antigen transport. Intranasal immunization with lectin-test antigen conjugates without adjuvant stimulated induction of specific serum immunoglobulin G, whereas antigen alone or admixed with lectin did not. The selective recognition of NALT M cells by a lectin in vivo provides a model for microbial adhesin-host cell receptor interactions on M cells and the targeted delivery of immunogens to NALT following intranasal

  13. Isolation and characterization of Chinese hamster cell mutants resistant to the cytotoxic effects of chromate.

    PubMed

    Campbell, C E; Gravel, R A; Worton, R G

    1981-09-01

    Stable mutants resistant to the toxic anion chromate have been isolated from a variety of Chinese hamster cell lines. The mechanism of chromate toxicity is not known, but it must involve internalization via the sulfate transport pathway. All mutant lines had a defective sulfate transport system, showing a 10-fold reduction in the rate of uptake of radioactive sulfate into the cell. The chromate resistance phenotype in CHO cell mutants behave recessively in somatic cell hybrids; in other cell lines the Chr(r) phenotype was partially expressed (codominant) in cell hybrids. Complementation analysis in cell hybrids between 18 different mutant pairs failed to reveal any complementation, indicating that chromate selects mutants primarily, if not exclusively, at a single gene locus.

  14. Reduction of Cullin-2 in somatic cells disrupts differentiation of germline stem cells in the Drosophila ovary.

    PubMed

    Ayyub, Champakali; Banerjee, Kushal Kr; Joti, Prakash

    2015-09-15

    Signaling from a niche consisting of somatic cells is essential for maintenance of germline stem cells (GSCs) in the ovary of Drosophila. Decapentaplegic (Dpp), a type of bone morphogenetic protein (BMP) signal, emanating from the niche, is the most important signal for this process. Cullin proteins constitute the core of a multiprotein E3-ligase important for their functions viz. degradation or modification of proteins necessary for different cellular processes. We have found that a Cullin protein called Cullin-2 (Cul-2) expresses in both somatic and germline cells of the Drosophila ovary. Reduction of Cul-2 in somatic cells causes upregulation of Dpp signal and produces accumulation of extra GSC-like cells inside germarium, the anteriormost structure of the ovary. Our results suggest that Cullin-2 protein present in the somatic cells is involved in a non cell-autonomous regulation of the extent of Dpp signaling and thus controls the differentiation of GSCs to cystoblasts (CBs).

  15. Evolution of Chromosomal Instability in Chinese Hamster Cells: aChanging Picture?

    SciTech Connect

    Ponnaiya, Brian; Limoli, Charles L.; Corcoran, James; Kaplan,Mark I.; Hartmann, Andreas; Morgan, William F.

    1998-07-14

    Purpose: To investigate the kinetics of chromosomalinstability induced in clones of Chinese hamster cells followingX-irradiation. Materials and methods: X-irradiated clones of GM10115,human hamster hybrid cells containing a single human chromosome 4 (HC4),have been previously established. These clones were defined as unstableif they contained three sub-populations of cells with uniquerearrangements of HC4 as detected by FISH. Stable and unstable cloneswere analysed by FISH and Giemsa staining at various timespost-irradiation. Results: While most of the stable clones continued toshow chromosomal stability of HC4 over time, one became marginallyunstable at approximately 45 population doublings postirradiation. Clonesexhibiting chromosomal instability had one of several fates. Many of theunstable clones were showed similar levels of instability over time.However, one unstable clone became stable with time in culture, whileanother became even more unstable over time. Cytogenetic analyses of allclones after Giemsa staining indicated that in some clones the hamsterchromosomes were rearranged independent of HC4, demonstrating increasedfrequencies of chromatid breaks and dicentric chromosomes. The majorityof the unstable clones also had higher yields of chromatid gaps.Conclusions: These data demonstrate the dynamic nature of chromosomalinstability as measured by two different cytogenetic assays.

  16. Restoration of Mitochondrial Gene Expression Using a Cloned Human Gene in Chinese Hamster Lung Cell Mutant

    PubMed Central

    Sherif, Zaki A; Broome, Carolyn W

    2015-01-01

    Background Gal−32 is a Chinese hamster lung cell nuclear mutant that is unable to grow in galactose due to a defect in mitochondrial protein synthesis. Since the product of the Gal−32 gene was unknown, it was imperative to use phenotypic complementation to clone a human gene that corrected the Gal−32 mutation. Results Recessive Gal−32 cells were co-transformed with pSV2-neo plasmid DNA and recombinant DNA from a human genomic library containing the dominant human Gal+ gene and a chloramphenicol-resistance (camr) gene present in the pSV13 vector. Primary transformants were selected by growth in galactose and the neomycin analog G418. In order to rescue the human Gal+ gene, a genomic library was constructed with primary transformant DNA and the pCV108 cosmid vector. The camr gene was used to identify clones with the nearby human sequences. DNA from two camr, Alu-hybridizing clones was able to transform the recessive Gal−32 cells to the Gal+ phenotype and to restore mitochondrial protein synthesis. Conclusion These data demonstrate the isolation of two pCV108-transformant recombinant clones containing a human gene that complements the Chinese hamster Gal−32 mutation and restores galactose metabolism. PMID:26052559

  17. A method for electrophysiological characterization of hamster retinal ganglion cells using a high-density CMOS microelectrode array

    PubMed Central

    Jones, Ian L.; Russell, Thomas L.; Farrow, Karl; Fiscella, Michele; Franke, Felix; Müller, Jan; Jäckel, David; Hierlemann, Andreas

    2015-01-01

    Knowledge of neuronal cell types in the mammalian retina is important for the understanding of human retinal disease and the advancement of sight-restoring technology, such as retinal prosthetic devices. A somewhat less utilized animal model for retinal research is the hamster, which has a visual system that is characterized by an area centralis and a wide visual field with a broad binocular component. The hamster retina is optimally suited for recording on the microelectrode array (MEA), because it intrinsically lies flat on the MEA surface and yields robust, large-amplitude signals. However, information in the literature about hamster retinal ganglion cell functional types is scarce. The goal of our work is to develop a method featuring a high-density (HD) complementary metal-oxide-semiconductor (CMOS) MEA technology along with a sequence of standardized visual stimuli in order to categorize ganglion cells in isolated Syrian Hamster (Mesocricetus auratus) retina. Since the HD-MEA is capable of recording at a higher spatial resolution than most MEA systems (17.5 μm electrode pitch), we were able to record from a large proportion of RGCs within a selected region. Secondly, we chose our stimuli so that they could be run during the experiment without intervention or computation steps. The visual stimulus set was designed to activate the receptive fields of most ganglion cells in parallel and to incorporate various visual features to which different cell types respond uniquely. Based on the ganglion cell responses, basic cell properties were determined: direction selectivity, speed tuning, width tuning, transience, and latency. These properties were clustered to identify ganglion cell types in the hamster retina. Ultimately, we recorded up to a cell density of 2780 cells/mm2 at 2 mm (42°) from the optic nerve head. Using five parameters extracted from the responses to visual stimuli, we obtained seven ganglion cell types. PMID:26528115

  18. A method for electrophysiological characterization of hamster retinal ganglion cells using a high-density CMOS microelectrode array.

    PubMed

    Jones, Ian L; Russell, Thomas L; Farrow, Karl; Fiscella, Michele; Franke, Felix; Müller, Jan; Jäckel, David; Hierlemann, Andreas

    2015-01-01

    Knowledge of neuronal cell types in the mammalian retina is important for the understanding of human retinal disease and the advancement of sight-restoring technology, such as retinal prosthetic devices. A somewhat less utilized animal model for retinal research is the hamster, which has a visual system that is characterized by an area centralis and a wide visual field with a broad binocular component. The hamster retina is optimally suited for recording on the microelectrode array (MEA), because it intrinsically lies flat on the MEA surface and yields robust, large-amplitude signals. However, information in the literature about hamster retinal ganglion cell functional types is scarce. The goal of our work is to develop a method featuring a high-density (HD) complementary metal-oxide-semiconductor (CMOS) MEA technology along with a sequence of standardized visual stimuli in order to categorize ganglion cells in isolated Syrian Hamster (Mesocricetus auratus) retina. Since the HD-MEA is capable of recording at a higher spatial resolution than most MEA systems (17.5 μm electrode pitch), we were able to record from a large proportion of RGCs within a selected region. Secondly, we chose our stimuli so that they could be run during the experiment without intervention or computation steps. The visual stimulus set was designed to activate the receptive fields of most ganglion cells in parallel and to incorporate various visual features to which different cell types respond uniquely. Based on the ganglion cell responses, basic cell properties were determined: direction selectivity, speed tuning, width tuning, transience, and latency. These properties were clustered to identify ganglion cell types in the hamster retina. Ultimately, we recorded up to a cell density of 2780 cells/mm(2) at 2 mm (42°) from the optic nerve head. Using five parameters extracted from the responses to visual stimuli, we obtained seven ganglion cell types.

  19. Resistance to DNA denaturation in irradiated Chinese hamster V79 fibroblasts is linked to cell shape

    SciTech Connect

    Olive, P.L.; Vanderbyl, S.; MacPhail, S.H. )

    1991-04-01

    Exponentially growing Chinese hamster V79-171b lung fibroblasts seeded at high density on plastic (approximately 7 x 10(3) cells/cm2) flatten, elongate, and produce significant amounts of extracellular fibronectin. When lysed in weak alkali/high salt, the rate of DNA denaturation following exposure to ionizing radiation is exponential. Conversely, cells plated at low density (approximately 7 x 10(2) cells/cm2) on plastic are more rounded 24 h later, produce little extracellular fibronectin, and display unusual DNA denaturation kinetics after X-irradiation. DNA in these cells resists denaturation, as though constraints to DNA unwinding have developed. Cell doubling time and distribution of cells in the growth cycle are identical for both high and low density cultures as is cell survival in response to radiation damage. The connection between DNA conformation and cell shape was examined further in low density cultures grown in conditioned medium. Under these conditions, cells at low density were able to elongate, and DNA denaturation of low density cultures was identical to that of high density cultures. Conversely, cytochalasin D, which interferes with actin polymerization causing cells to round up and release fibronectin, allowed development of constraints in high density cultures. These results suggest that DNA conformation is sensitive to changes in cell shape which result when cells are grown in different environments. However, these changes in DNA conformation detected by the DNA unwinding assay do not appear to play a direct role in radiation-induced cell killing.

  20. Phagocytosis and solubilization of fixed cells by metastatic hamster embryo fibroblasts, Nil2C2

    SciTech Connect

    Sakiyama, H.; Nishino, Y.; Nishimura, K.; Noda, Y.; Otsu, H.

    1984-05-01

    When Nil2C2, a metastatic clone derived from hamster embryo fibroblasts (Nil), was inoculated over (/sup 3/H)leucine-labeled fixed cells, Nil2C2 cells solubilized and phagocytosed fixed cells, and the radioactivity was released into the culture medium as trichloroacetic acid-soluble fragments. The solubilization of fixed cells was dependent on both the time of incubation of living cells with fixed cells and the number of living cells inoculated. Nil2C2 cells were shown by autoradiographic and electron microscopic studies to peel off fixed cells and ingest them as large fragments. The solubilization of fixed cells was significantly decreased when plasminogen was depleted from the culture medium. Protease inhibitors such as leupeptin, epsilon-aminocaproic acid, and soybean trypsin inhibitor partially inhibited the proteolysis and phagocytosis of Nil2C2 cells. Mouse peritoneal macrophages activated by Salmonella typhimurium solubilized fixed cells after the addition of 12-O-tetradecanoylphorbol-13-acetate. However, they did not phagocytose fixed cells as large fragments.

  1. Nesprin-2 epsilon: A novel nesprin isoform expressed in human ovary and Ntera-2 cells

    SciTech Connect

    Lam, Le Thanh; Boehm, Sabrina V.; Roberts, Roland G.; Morris, Glenn E.

    2011-08-26

    Highlights: {yields} A novel epsilon isoform of nesprin-2 has been discovered. {yields} This 120 kDa protein was predicted by bioinformatic analysis, but has not previously been observed. {yields} It is the main isoform expressed in a teratocarcinoma cell line and is also found in ovary. {yields} Like other nesprins, it is located at the nuclear envelope. {yields} We suggest it may have a role in very early development or in some ovary-specific function. -- Abstract: The nuclear envelope-associated cytoskeletal protein, nesprin-2, is encoded by a large gene containing several internal promoters that produce shorter isoforms. In a study of Ntera-2 teratocarcinoma cells, a novel isoform, nesprin-2-epsilon, was found to be the major mRNA and protein product of the nesprin-2 gene. Its existence was predicted by bioinformatic analysis, but this is the first direct demonstration of both the mRNA and the 120 kDa protein which is located at the nuclear envelope. In a panel of 21 adult and foetal human tissues, the nesprin-2-epsilon mRNA was strongly expressed in ovary but was a minor isoform elsewhere. The expression pattern suggests a possible link with very early development and a likely physiological role in ovary.

  2. Inhibitory effect of vitamin D-binding protein-derived macrophage activating factor on DMBA-induced hamster cheek pouch carcinogenesis and its derived carcinoma cell line

    PubMed Central

    TOYOHARA, YUKIYO; HASHITANI, SUSUMU; KISHIMOTO, HIROMITSU; NOGUCHI, KAZUMA; YAMAMOTO, NOBUTO; URADE, MASAHIRO

    2011-01-01

    This study investigated the inhibitory effect of vitamin D-binding protein-derived macrophage-activating factor (GcMAF) on carcinogenesis and tumor growth, using a 9,10-dimethyl-1,2-benzanthracene (DMBA)-induced hamster cheek pouch carcinogenesis model, as well as the cytocidal effect of activated macrophages against HCPC-1, a cell line established from DMBA-induced cheek pouch carcinoma. DMBA application induced squamous cell carcinoma in all 15 hamsters of the control group at approximately 10 weeks, and all 15 hamsters died of tumor burden within 20 weeks. By contrast, 2 out of the 14 hamsters with GcMAF administration did not develop tumors and the remaining 12 hamsters showed a significant delay of tumor development for approximately 3.5 weeks. The growth of tumors formed was significantly suppressed and none of the hamsters died within the 20 weeks during which they were observed. When GcMAF administration was stopped at the 13th week of the experiment in 4 out of the 14 hamsters in the GcMAF-treated group, tumor growth was promoted, but none of the mice died within the 20-week period. On the other hand, when GcMAF administration was commenced after the 13th week in 5 out of the 15 hamsters in the control group, tumor growth was slightly suppressed and all 15 hamsters died of tumor burden. However, the mean survival time was significantly extended. GcMAF treatment activated peritoneal macrophages in vitro and in vivo, and these activated macrophages exhibited a marked cytocidal effect on HCPC-1 cells. Furthermore, the cytocidal effect of activated macrophages was enhanced by the addition of tumor-bearing hamster serum. These findings indicated that GcMAF possesses an inhibitory effect on tumor development and growth in a DMBA-induced hamster cheek pouch carcinogenesis model. PMID:22848250

  3. Inhibitory effect of vitamin D-binding protein-derived macrophage activating factor on DMBA-induced hamster cheek pouch carcinogenesis and its derived carcinoma cell line.

    PubMed

    Toyohara, Yukiyo; Hashitani, Susumu; Kishimoto, Hiromitsu; Noguchi, Kazuma; Yamamoto, Nobuto; Urade, Masahiro

    2011-07-01

    This study investigated the inhibitory effect of vitamin D-binding protein-derived macrophage-activating factor (GcMAF) on carcinogenesis and tumor growth, using a 9,10-dimethyl-1,2-benzanthracene (DMBA)-induced hamster cheek pouch carcinogenesis model, as well as the cytocidal effect of activated macrophages against HCPC-1, a cell line established from DMBA-induced cheek pouch carcinoma. DMBA application induced squamous cell carcinoma in all 15 hamsters of the control group at approximately 10 weeks, and all 15 hamsters died of tumor burden within 20 weeks. By contrast, 2 out of the 14 hamsters with GcMAF administration did not develop tumors and the remaining 12 hamsters showed a significant delay of tumor development for approximately 3.5 weeks. The growth of tumors formed was significantly suppressed and none of the hamsters died within the 20 weeks during which they were observed. When GcMAF administration was stopped at the 13th week of the experiment in 4 out of the 14 hamsters in the GcMAF-treated group, tumor growth was promoted, but none of the mice died within the 20-week period. On the other hand, when GcMAF administration was commenced after the 13th week in 5 out of the 15 hamsters in the control group, tumor growth was slightly suppressed and all 15 hamsters died of tumor burden. However, the mean survival time was significantly extended. GcMAF treatment activated peritoneal macrophages in vitro and in vivo, and these activated macrophages exhibited a marked cytocidal effect on HCPC-1 cells. Furthermore, the cytocidal effect of activated macrophages was enhanced by the addition of tumor-bearing hamster serum. These findings indicated that GcMAF possesses an inhibitory effect on tumor development and growth in a DMBA-induced hamster cheek pouch carcinogenesis model.

  4. Origin of germ cells and formation of new primary follicles in adult human ovaries

    PubMed Central

    Bukovsky, Antonin; Caudle, Michael R; Svetlikova, Marta; Upadhyaya, Nirmala B

    2004-01-01

    Recent reports indicate that functional mouse oocytes and sperm can be derived in vitro from somatic cell lines. We hypothesize that in adult human ovaries, mesenchymal cells in the tunica albuginea (TA) are bipotent progenitors with a commitment for both primitive granulosa and germ cells. We investigated ovaries of twelve adult women (mean age 32.8 ± 4.1 SD, range 27–38 years) by single, double, and triple color immunohistochemistry. We show that cytokeratin (CK)+ mesenchymal cells in ovarian TA differentiate into surface epithelium (SE) cells by a mesenchymal-epithelial transition. Segments of SE directly associated with ovarian cortex are overgrown by TA, forming solid epithelial cords, which fragment into small (20 micron) epithelial nests descending into the lower ovarian cortex, before assembling with zona pellucida (ZP)+ oocytes. Germ cells can originate from SE cells which cover the TA. Small (10 micron) germ-like cells showing PS1 meiotically expressed oocyte carbohydrate protein are derived from SE cells via asymmetric division. They show nuclear MAPK immunoexpression, subsequently divide symmetrically, and enter adjacent cortical vessels. During vascular transport, the putative germ cells increase to oocyte size, and are picked-up by epithelial nests associated with the vessels. During follicle formation, extensions of granulosa cells enter the oocyte cytoplasm, forming a single paranuclear CK+ Balbiani body supplying all the mitochondria of the oocyte. In the ovarian medulla, occasional vessels show an accumulation of ZP+ oocytes (25–30 microns) or their remnants, suggesting that some oocytes degenerate. In contrast to males, adult human female gonads do not preserve germline type stem cells. This study expands our previous observations on the formation of germ cells in adult human ovaries. Differentiation of primitive granulosa and germ cells from the bipotent mesenchymal cell precursors of TA in adult human ovaries represents a most

  5. Bystander effect induced by UVC radiation in Chinese hamster V79 cells.

    PubMed

    Wu, Shengwen; Jin, Cuihong; Lu, Xiaobo; Yang, Jinghua; Liu, Qiufang; Qi, Ming; Lu, Shuai; Zhang, Lifeng; Cai, Yuan

    2014-01-01

    In past decades, researches on radiation-induced bystander effect mainly focused on ionizing radiation such as α-particle, β-particle, X-ray and γ-ray. But few researches have been conducted on the ability of ultraviolet (UV) radiation-induced bystander effect, and knowledge of UVC-induced bystander effect is far limited. Here, we adopted medium transfer experiment to detect whether UVC could cause bystander effect in Chinese hamster V79 cells. We determined the cell viability, apoptosis rate, chromosome aberration and ultrastructure changes, respectively. Our results showed that: (1) the viability of UVC-irradiated V79 cells declined significantly with the dosage of UVC; (2) similar to the irradiated cells, the main death type of bystander cells cultured in irradiation conditioned medium (ICMs) was also apoptosis; (3) soluble factors secreted by UVC-irradiated cells could induce bystander effect in V79 cells; (4) cells treated with 4 h ICM collected from 90 mJ cm(-2) UVC-irradiated cells displayed the strongest response. Our data revealed that UVC could cause bystander effect through the medium soluble factors excreted from irradiated cells and this bystander effect was a novel quantitative and kinetic response. These findings might provide a foundation to further explore the exact soluble bystander factors and detailed mechanism underlying UVC-induced bystander effect.

  6. Caffeine-enhanced survival of radiation-sensitive, repair-deficient Chinese hamster cells

    SciTech Connect

    Utsumi, H.; Elkind, M.M.

    1983-11-01

    A clone of V79 Chinese hamster cells (V79-AL162/S-10) with unique properties has been isolated after a challenge of parental cells (V79-AL162) with 1 mM ouabain. Compared with parental cells, or with other clones isolated after the ouabain challenge, these cells form smaller colonies, are more sensitive to both x rays and fission-spectrum neutrons, and respond atypically to a postirradiation treatment with caffeine. Their enhanced response to x rays results mainly from a large reduction in the shoulder of their survival curve, probably because in late S phase, the most resistant phase in the cell cycle, the survival curve of these cells has a reduced shoulder width. Caffeine, and to a lesser extent theophylline, added to the colony-forming medium immediately after exposure appreciably increases the width of the shoulder of these sensitive cells, whereas caffeine has the opposite effect on the response of normal V79 cells. Thus the unique response of the V79-AL162/S-10 cells to a radiation posttreatment with caffeine (increased survival) results from a net increase in their ability to repair damage that is otherwise lethal; caffeine treatment ordinarly prevents normal V79 cells from repairing damage that is only potentially lethal.

  7. The Regenerative Effect of Bone Marrow-Derived Stem Cells in Spermatogenesis of Infertile Hamster

    PubMed Central

    Vahdati, Akbar; Fathi, Alireza; Hajihoseini, Mehrdokht; Aliborzi, Ghaem; Hosseini, Ebrahim

    2017-01-01

    BACKGROUND Infertility is a serious social problem in advanced nations, with male factor in half of all cases of infertility. This study was conducted to determine the regenerative effect of bone marrow-derived stem cells in spermatogenesis of infertile hamster. METHODS Twelve adult male hamsters were equally divided into azoospermic and control groups. Busulfan was intraperitoneally used for induction of azoospermia, while the right testis was treated with bone marrow-derived stem cells (106 BM-SCs), labeled with sterile trypan blue, 35 days after busulfan injection. The left testis served as positive control for azoospermia. Sixty days after cell transplantation, the animals were euthanized and both testes were removed and evaluated histologically. RESULTS BM-SCs were spindle-shaped, adherent to the culture flasks and had positive expression of CD29 and CD73 and negative expression of CD45. Alcian blue staining confirmed differentiation of BM-SCs into chondrocytes. Karyotyping denoted to stability of chromosomes. Treatment with busulfan in seminiferous tubules resulted into distruption of spermatogenesis. After two months in busulfan treatment group, seminiferous tubular atrophy and germinal epitheliums degenerations were noticed with no spermatozoa in epididymis. After treatment of busulfan group with BM-SCs, spermatogonia, primary spermatocytes, spermatids and sperms were present in seminiferous tubules. CONCLUSION As cell transplantation in seminiferous tubules resulted into a rapid repair of pathological changes, BM-SCs can be recommended an effective treatment measure in azoospermia. It seems that more studies are necessary to confirm the use of this technique in treatment of azoospermia and infertility in human. PMID:28289609

  8. Induction of the bystander effect in Chinese hamster V79 cells by actinomycin D.

    PubMed

    Jin, Cuihong; Wu, Shengwen; Lu, Xiaobo; Liu, Qiufang; Qi, Ming; Lu, Shuai; Xi, Qi; Cai, Yuan

    2011-05-10

    Bystander effect (BE) can be induced by ionizing radiation and chemicals, including alkylating agents. Ionizing radiation mostly induces the bystander effect by causing double-strand DNA breakage in the exposed cells. However, the chemical-induced bystander effect is poorly studied. Here we chose actinomycin D (ACTD), a genotoxic chemotherapeutic drug, to investigate whether it could cause bystander effect in Chinese hamster V79 cells. Results are that (1) ACTD induced apoptosis in V79 cells and an optimal apoptosis model in V79 cells was established with ACTD (4 mg/L, 1h); (2) using apoptosis rate, chromosome aberration, and ultrastructure changes as endpoints of bystander effect, ACTD could induce bystander effect in V79 cells; (3) as in the exposed cells, ACTD mainly induced apoptosis in bystander V79 cells cultured in different period conditioned medium; (4) the strongest bystander effect was induced by 4 h conditioned medium collected from cells treated with ACTD. It suggests that ACTD could cause BE through the medium soluble factors excreted from exposed cells during apoptosis and ACTD-induced BE was a novel quantitative and kinetic response.

  9. DNA conformation of Chinese hamster V79 cells and sensitivity to ionizing radiation

    SciTech Connect

    Olive, P.L.; Hilton, J.; Durand, R.E.

    1986-07-01

    Chinese hamster V79 cells grown for 20 h in suspension culture form small clusters of cells (spheroids) which are more resistant to killing by ionizing radiation than V79 cells grown as monolayers. This resistance appears to be due to the greater capacity of cells grown in contact to repair radiation damage. Attempts to relate this ''contact effect'' to differences in DNA susceptibility or DNA repair capacity have provided conflicting results. Two techniques, alkaline sucrose gradient sedimentation and alkaline elution, show no difference in the amounts of radiation-induced DNA single-strand breakage or its repair between suspension or monolayer cells. However, using the alkali-unwinding assay, the rate of DNA unwinding is much slower for suspension cells than for monolayer cells. Interestingly, a decrease in salt concentration or in pH of the unwinding solution eliminates these differences in DNA unwinding kinetics. A fourth assay, sedimentation of nucleoids on neutral sucrose gradients, also shows a significant decrease in radiation damage produced in suspension compared to monolayer cultures. It is believed that this assay measures differences in DNA conformation (supercoiling) as well as differences in DNA strand breakage. We conclude from these four assays that the same number of DNA strand breaks/Gy is produced in monolayer and spheroid cells. However, changes in DNA conformation or packaging occur when cells are grown as spheroids, and these changes are responsible for reducing DNA damage by ionizing radiation.

  10. Piwi is required in multiple cell types to control germline stem cell lineage development in the Drosophila ovary.

    PubMed

    Ma, Xing; Wang, Su; Do, Trieu; Song, Xiaoqing; Inaba, Mayu; Nishimoto, Yoshiya; Liu, Lu-ping; Gao, Yuan; Mao, Ying; Li, Hui; McDowell, William; Park, Jungeun; Malanowski, Kate; Peak, Allison; Perera, Anoja; Li, Hua; Gaudenz, Karin; Haug, Jeff; Yamashita, Yukiko; Lin, Haifan; Ni, Jian-quan; Xie, Ting

    2014-01-01

    The piRNA pathway plays an important role in maintaining genome stability in the germ line by silencing transposable elements (TEs) from fly to mammals. As a highly conserved piRNA pathway component, Piwi is widely expressed in both germ cells and somatic cells in the Drosophila ovary and is required for piRNA production in both cell types. In addition to its known role in somatic cap cells to maintain germline stem cells (GSCs), this study has demonstrated that Piwi has novel functions in somatic cells and germ cells of the Drosophila ovary to promote germ cell differentiation. Piwi knockdown in escort cells causes a reduction in escort cell (EC) number and accumulation of undifferentiated germ cells, some of which show active BMP signaling, indicating that Piwi is required to maintain ECs and promote germ cell differentiation. Simultaneous knockdown of dpp, encoding a BMP, in ECs can partially rescue the germ cell differentiation defect, indicating that Piwi is required in ECs to repress dpp. Consistent with its key role in piRNA production, TE transcripts increase significantly and DNA damage is also elevated in the piwi knockdown somatic cells. Germ cell-specific knockdown of piwi surprisingly causes depletion of germ cells before adulthood, suggesting that Piwi might control primordial germ cell maintenance or GSC establishment. Finally, Piwi inactivation in the germ line of the adult ovary leads to gradual GSC loss and germ cell differentiation defects, indicating the intrinsic role of Piwi in adult GSC maintenance and differentiation. This study has revealed new germline requirement of Piwi in controlling GSC maintenance and lineage differentiation as well as its new somatic function in promoting germ cell differentiation. Therefore, Piwi is required in multiple cell types to control GSC lineage development in the Drosophila ovary.

  11. DNA (deoxyribonucleic acid) synthesis following microinjection of heterologous sperm and somatic cell nuclei into hamster oocytes

    SciTech Connect

    Naish, S.J.; Perreault, S.D.; Zirkin, B.R.

    1987-01-01

    The authors investigated the ability of the hamster oocyte to initiate DNA synthesis in nuclei differing in basic protein content. DNA synthesis was studied by autoradiography in oocytes that had been incubated in /sup 3/H-thymidine after being parthenogenetically activated by sham microinjection, or microinjected with hamster, mouse, rabbit, or fish sperm nuclei, or hamster hepatocyte nuclei. Within 6 hr of sham or nucleus microinjection, nuclei of each type underwent transformation into pronuclei and synthesized DNA. These results demonstrated that the hamster egg can access and utilize its own and each type of template provided, whether homologous or heterologous. However, pronuclei derived from hamster sperm nuclei were more likely to be synthesizing DNA at 6 hr than pronuclei derived from sperm nuclei of other species. The authors conclude that the mechanisms employed by the hamster oocyte to transform hamster sperm nuclei into pronuclei and to effect DNA synthesis in these nuclei are not specific for the hamster sperm nucleus. Nevertheless, these mechanisms apparently operate more efficiently when the hamster sperm nucleus, rather than a heterologous sperm nucleus, is present.

  12. Quercetin protects hamster spermatogenic cells from oxidative damage induced by diethylstilboestrol.

    PubMed

    Li, G; Ma, Aituan; Shi, W; Zhong, Xiuhui

    2010-10-01

    Quercetin has been reported to be an efficient antioxidant which protects chicken spermatogonial cells from oxidative damage through increasing intracellular antioxidants and decreasing lipid peroxidation. Exposure to diethylstilboestrol (DES) could cause reproductive damage in males, which is associated with oxidative stress. This study was conducted to investigate the protective effects of quercetin on DES-induced oxidative damage in cultured hamster spermatogenic cells. The cells were treated with different concentrations of DES, and their growth status was observed under inverted microscope. The viability of spermatogenic cells was detected by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT). The contents of superoxide dismutase (SOD) in supernatants and glutathione peroxidase (GSH-Px) in cells were detected with spectrophotography. The results showed that quercetin significantly inhibited the DES-induced damage on spermatogenic cells, with the exception of the low-dose group in which no significant difference was observed. The cell survival rate increased significantly in the middle- and high-dose groups. The contents of SOD and GSH-Px were significantly elevated after medication with quercetin (P < 0.01). It can be concluded that quercetin protects spermatogenic cells against DES-induced oxidative damage through increasing intracellular antioxidants and decreasing lipid peroxidation. Quercetin plays a very important role in ameliorating reproductive toxicity induced by environmental oestrogens.

  13. Rat bladder cell-mediated mutagenesis of Chinese hamster V79 cells and metabolism of benzo(a)pyrene

    SciTech Connect

    Langenbach, R.; Malick, L.; Nesnow, S.

    1981-05-01

    Primary rat bladder epithelial cells were cocultivated with Chinese hamster V79 cells in the presence of carcinogens, and the induction of 6-thioguanine resistance in the V79 cells was used as a market of cell-mediated mutagenesis. The carcinogens dimethylnitrosamine, 7,12-dimethylbenz(a)anthracene, and benzo(a)pyrene (BP) were mutagenic to V79 cells in the presence of bladder cells but not in their absence. Analysis of BP metabolites (formed by bladder cells indicated that 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene, 9,10-dihydro-9,10-dihydroxybenzo(a)pyrene, benzo(a)pyrene-3,6-quinone, and 9-hydroxybenzo(a)pyrene were the major organic-soluble metabolites formed. The finding that rat bladder peithelium can metabolize some carcinogens offers new possibilities for the mechanism of initiation of bladder cancer.

  14. Phagocytosis genes nonautonomously promote developmental cell death in the Drosophila ovary

    PubMed Central

    Timmons, Allison K.; Mondragon, Albert A.; Schenkel, Claire E.; Yalonetskaya, Alla; Taylor, Jeffrey D.; Moynihan, Katherine E.; Etchegaray, Jon Iker; Meehan, Tracy L.; McCall, Kimberly

    2016-01-01

    Programmed cell death (PCD) is usually considered a cell-autonomous suicide program, synonymous with apoptosis. Recent research has revealed that PCD is complex, with at least a dozen cell death modalities. Here, we demonstrate that the large-scale nonapoptotic developmental PCD in the Drosophila ovary occurs by an alternative cell death program where the surrounding follicle cells nonautonomously promote death of the germ line. The phagocytic machinery of the follicle cells, including Draper, cell death abnormality (Ced)-12, and c-Jun N-terminal kinase (JNK), is essential for the death and removal of germ-line–derived nurse cells during late oogenesis. Cell death events including acidification, nuclear envelope permeabilization, and DNA fragmentation of the nurse cells are impaired when phagocytosis is inhibited. Moreover, elimination of a small subset of follicle cells prevents nurse cell death and cytoplasmic dumping. Developmental PCD in the Drosophila ovary is an intriguing example of nonapoptotic, nonautonomous PCD, providing insight on the diversity of cell death mechanisms. PMID:26884181

  15. Synchronization of mitochondrial DNA synthesis in Chinese hamster cells (line CHO) deprived of isoleucine.

    PubMed

    Ley, K D; Murphy, M M

    1973-08-01

    Mitochondrial DNA (mit-DNA) synthesis was compared in suspension cultures of Chinese hamster cells (line CHO) whose cell cycle events had been synchronized by isoleucine deprivation or mitotic selection. At hourly intervals during cell cycle progression, synchronized cells were exposed to tritiated thymidine ([(3)H]TdR), homogenized, and nuclei and mitochondria isolated by differential centrifugation. Mit-DNA and nuclear DNA were isolated and incorporation of radioisotope measured as counts per minute ([(3)H]TdR) per microgram DNA. Mit-DNA synthesis in cells synchronized by mitotic selection began after 4 h and continued for approximately 9 h. This time-course pattern resembled that of nuclear DNA synthesis. In contrast, mit-DNA synthesis in cells synchronized by isoleucine deprivation did not begin until 9-12 h after addition of isoleucine and virtually all [(3)H]TdR was incorporated during a 3-h interval. We have concluded from these results that mit-DNA synthesis is inhibited in CHO cells which are arrested in G(1) because of isoleucine deprivation and that addition of isoleucine stimulates synchronous synthesis of mit-DNA. We believe this method of synchronizing mit-DNA synthesis may be of value in studies of factors which regulate synthesis of mit-DNA.

  16. Transitional cell carcinoma of the ovary: A rare case and review of literature

    PubMed Central

    2010-01-01

    Introduction Transitional cell carcinoma (TCC) of the ovary is a rare, recently recognized, subtype of ovarian surface epithelial cancer. Case presentation A 69-year-old postmenopausal woman presented with a 2-year history of progressive enlargement of an abdominal mass. Abdominal computed tomography showed a pelvic mass. CA-125 was normal. A staging operation with total abdominal hysterectomy, bilateral salpingo-oophorectomy, infracolic omentectomy and pelvic lymph node dissection was performed. After surgery, the pathologic report of the right ovarian tumour was TCC, grade 3, stage IC. The patient underwent 3 cycles of chemotherapy: carboplatin and paclitaxel. She is regularly followed up and has been disease free for 10 months Conclusion Transitional cell carcinoma (TCC) of the ovary is a rare subtype of epithelial ovarian cancer. Surgical resection is the primary therapeutic approach, and patient outcomes after chemotherapy are better than for other types of ovarian cancers. PMID:21073751

  17. Identifying Early Target Cells of Nipah Virus Infection in Syrian Hamsters

    PubMed Central

    Baseler, Laura; Scott, Dana P.; Saturday, Greg; Horne, Eva; Rosenke, Rebecca; Thomas, Tina; Meade-White, Kimberly; Haddock, Elaine; Feldmann, Heinz

    2016-01-01

    Background Nipah virus causes respiratory and neurologic disease with case fatality rates up to 100% in individual outbreaks. End stage lesions have been described in the respiratory and nervous systems, vasculature and often lymphoid organs in fatal human cases; however, the initial target organs of Nipah virus infection have not been identified. Here, we detected the initial target tissues and cells of Nipah virus and tracked virus dissemination during the early phase of infection in Syrian hamsters inoculated with a Nipah virus isolate from Malaysia (NiV-M) or Bangladesh (NiV-B). Methodology/Principal Findings Syrian hamsters were euthanized between 4 and 48 hours post intranasal inoculation and tissues were collected and analyzed for the presence of viral RNA, viral antigen and infectious virus. Virus replication was first detected at 8 hours post inoculation (hpi). Nipah virus initially targeted type I pneumocytes, bronchiolar respiratory epithelium and alveolar macrophages in the lung and respiratory and olfactory epithelium lining the nasal turbinates. By 16 hpi, virus disseminated to epithelial cells lining the larynx and trachea. Although the pattern of viral dissemination was similar for both virus isolates, the rate of spread was slower for NiV-B. Infectious virus was not detected in the nervous system or blood and widespread vascular infection and lesions within lymphoid organs were not observed, even at 48 hpi. Conclusions/Significance Nipah virus initially targets the respiratory system. Virus replication in the brain and infection of blood vessels in non-respiratory tissues does not occur during the early phase of infection. However, virus replicates early in olfactory epithelium and may serve as the first step towards nervous system dissemination, suggesting that development of vaccines that block virus dissemination or treatments that can access the brain and spinal cord and directly inhibit virus replication may be necessary for preventing central

  18. Adhesion of phospholipid vesicles to Chinese hamster fibroblasts: Role of cell surface proteins

    PubMed Central

    Pagano, RE; Takeichi, M

    1977-01-01

    The adhesion of artificially generated lipid membrane vesicles to Chinese hamster V79 fibroblasts in suspension was used as a model system for studying membrane interactions. Below their gel-liquid crystalline phase transition temperature, vesicles comprised of dipalmitoyl lecithin (DPL) or dimyristoyl lecithin (DML) absorbed to the surfaces of EDTA- dissociated cells. These adherent vesicles could not be removed by repeated washings of the treated cells but could be released into the medium by treatment with trypsin. EM autoradiographic studies of cells treated with[(3)H]DML or [(3)H]DPL vesicles showed that most of the radioactive lipids were confined to the cell periphery. Scanning electron microscopy and fluorescence microscopy further confirmed the presence of adherent vesicles at the cell surface. Adhesion of DML or DPL vesicles to EDTA-dissociated cells modified the lactoperoxidase-catalyzed iodination pattern of the cell surface proteins; the inhibition of labeling of two proteins with an approximately 60,000- dalton mol wt was particularly evident. Incubation of cells wit h (3)H-lipid vesicles followed by sodium dodecyl sulfate (SDS)- polyacrylamide gel electrophoresis showed that some of the (3)H-lipid migrated preferentially with these approximately 60,000-mol wt proteins. Studies of the temperature dependence of vesicle uptake and subsequent release by trypsin showed that DML or DPL vesicle adhesion to EDTA- dissociated cells increased with decreasing temperatures. In contrast, cells trypsinized before incubation with vesicles showed practically no temperature dependence of vesicle uptake. These results suggest two pathways for adhesion of lipid vesicles to the cell surface-a temperature-sensitive one involving cell surface proteins, and a temperature-independent one. These findings are discussed in terms of current models for cell-cell interactions. PMID:407233

  19. Recommended protocol for the Syrian hamster embryo (SHE) cell transformation assay.

    PubMed

    Maire, Marie-Aline; Pant, Kamala; Phrakonkham, Pascal; Poth, Albrecht; Schwind, Karl-Rainer; Rast, Claudine; Bruce, Shannon Wilson; Sly, Jamie E; Bohnenberger, Susanne; Kunkelmann, Thorsten; Schulz, Markus; Vasseur, Paule

    2012-04-11

    The Syrian hamster embryo (SHE) cell transformation assay (CTA) is a short-term in vitro assay recommended as an alternative method for testing the carcinogenic potential of chemicals. SHE cells are "normal" cells since they are diploid, genetically stable, non-tumourigenic, and have metabolic capabilities for the activation of some classes of carcinogens. The CTA, first developed in the 1960s by Berwald and Sachs (1963,1964) [3,4], is based on the change of the phenotypic feature of cell colonies expressing the first steps of the conversion of normal to neoplastic-like cells with oncogenic properties. Pienta et al. (1977) [22] developed a protocol using cryopreserved cells to enhance practicality of the assay and limit sources of variability. Several variants of the assay are currently in use, which mainly differ by the pH at which the assay is performed. We present here the common version of the SHE pH 6.7 CTA and SHE pH 7.0 CTA protocols used in the ECVAM (European Centre for the Validation of Alternative Methods) prevalidation study on CTA reported in this issue. It is recommended that this protocol, in combination with the photo catalogues presented in this issue, should be used in the future and serve as a basis for the development of the OECD test guideline.

  20. Novel population of small tumour-initiating stem cells in the ovaries of women with borderline ovarian cancer

    PubMed Central

    Virant-Klun, Irma; Stimpfel, Martin

    2016-01-01

    Small stem cells with diameters of up to 5 μm previously isolated from adult human ovaries indicated pluripotency and germinal lineage, especially primordial germ cells, and developed into primitive oocyte-like cells in vitro. Here, we show that a comparable population of small stem cells can be found in the ovarian tissue of women with borderline ovarian cancer, which, in contrast to small stem cells in “healthy” ovaries, formed spontaneous tumour-like structures and expressed some markers related to pluripotency and germinal lineage. The gene expression profile of these small putative cancer stem cells differed from similar cells sorted from “healthy” ovaries by 132 upregulated and 97 downregulated genes, including some important forkhead box and homeobox genes related to transcription regulation, developmental processes, embryogenesis, and ovarian cancer. These putative cancer stem cells are suggested to be a novel population of ovarian tumour-initiating cells in humans. PMID:27703207

  1. The influence of ciprofloxacin on hamster ovarian cancer cell line CHO AA8.

    PubMed

    Kloskowski, Tomasz; Olkowska, Joanna; Nazlica, Aybars; Drewa, Tomasz

    2010-01-01

    Abstract: Ciprofloxacin is a chinolone antibiotic, which is used mainly in the treatment of urinary tract infections but also in pulmonary tract, prostate gland, bone and bone marrow infection. Ciprofloxacin is also known for its anticancer in vitro properties. In this study hamster ovarian cancer line CHO AA8 was used for evaluation of cytotoxic properties of ciprofloxacin against neoplastic cells. For this purpose we used different concentrations of ciprofloxacin range from 10 to 1000 microg/mL. Cell viability was counted using trypan blue assay. Ciprofloxacin induced morphological changes and decreased viability in a concentration and time dependent manner within CHO AA8 cells. In low concentrations cytotoxic effect of ciprofloxacin is weak only after 24 h incubation. In the highest concentration of ciprofloxacin, after 24, 48 and 72 h incubation only a very small number of living cells (not exceeding 1%) was observed. No living cells were observed after 96 h of incubation times and ciprofloxacin concentrations of 800 and 1000 micrpg/mL. These promising results deserved future studies on chinolones and ovarian cancer.

  2. Most of the G1 period in hamster cells is eliminated by lengthening the S period.

    PubMed Central

    Stancel, G M; Prescott, D M; Liskay, R M

    1981-01-01

    Two Chinese hamster cell lines, G1+-1 and CHO, have been grown in the presence of low concentrations of hydroxyurea to determine how a slowing DNA synthesis (i.e., a lengthening of the S period) affects the length of the G1 period. Hydroxyurea concentrations of approximately 10 microM do not alter the generation times of these cell lines but do cause increases in S with corresponding decreases in G1. In both cell lines, 10 microM hydroxyurea reduces G1 to an absolute value of 1 hr, which represents decreases of 70% (G1+-1) and 60% (CHO) from control values. Higher concentrations of hydroxyurea increase the generation times and lengths of S for both cell lines but do not reduce G1 below the minimum value of 1 hr. These observations indicate that the majority of G1 is expendable and most of G1 therefore cannot contain specific events required for the initiation of DNA synthesis. This result supports the hypothesis that G1 is a portion of the cell growth cycle but not of the chromosome cycle. PMID:6947230

  3. Specific estrogen-induced cell proliferation of cultured Syrian hamster renal proximal tubular cells in serum-free chemically defined media

    SciTech Connect

    Oberley, T.D.; Lauchner, L.J.; Pugh, T.D.; Gonzalez, A.; Goldfarb, S. ); Li, S.A.; Li, J.J. )

    1989-03-01

    It has long been recognized that the renal proximal tubular epithelium of the hamster is a bona fide estrogen target tissue. The effect of estrogens on the growth of proximal tubule cell explants and dissociated single cells derived from these explant outgrowths has been studied in culture. Renal tubular cells were grown on a PF-HR-9 basement membrane under serum-free chemically defined culture conditions. At 7-14 days in culture, cell number was enhanced 3-fold in the presence of either 17{beta}-estradiol or diethylstilbestrol. A similar 3-fold increase in cell number was also seen at 1 nM 17{beta}-estradiol in subcultured dissociated single tubular cells derived from hamster renal tubular explant outgrowths at 21 days in culture. Concomitant exposure of tamoxifen at 3-fold molar excess in culture completely abolished the increase in cell number seen with 17{beta}-estradiol. The proliferation effect of estrogens on proximal tubular cell growth appears to be species specific since 17{beta}-estradiol did not alter the growth of either rat or guinea pig proximal tubules in culture. In addition, at 7-10 days in culture in the presence of 17{beta}-estradiol, ({sup 3}H)thymidine labeling of hamster tubular cells was enhanced 3-fold. These results clearly indicate that estrogens can directly induce primary epithelial cell proliferation at physiologic concentrations and provide strong additional evidence for an important hormonal role in the neoplastic transformation of the hamster kidney.

  4. Perfluorooctanoic acid (PFOA) acts as a tumor promoter on Syrian hamster embryo (SHE) cells.

    PubMed

    Jacquet, N; Maire, M A; Rast, C; Bonnard, M; Vasseur, P

    2011-08-01

    Perfluorooctane sulfonate (PFOS) (C(8)F(17)SO(3)) and perfluorooctanoic acid (PFOA) (C(8)HF(15)O(2)) are synthetic chemicals widely used in industrial applications for their hydrophobic and oleophobic properties. They are persistent, bioaccumulative, and toxic to mammalian species. Their widespread distribution on earth and contamination of human serum raised concerns about long-term side effects. They are suspected to be carcinogenic through a nongenotoxic mode of action, a mechanism supported by recent findings that PFOS induced cell transformation but no genotoxicity in Syrian hamster embryo (SHE) cells. In the present study, we evaluated carcinogenic potential of PFOA using the cell transformation assay on SHE cells. The chemical was applied alone or in combination with a nontransformant concentration of benzo[a]pyrene (BaP, 0.4 μM) in order to detect PFOA ability to act as tumor initiator or tumor promoter. The results showed that PFOA tested alone in the range 3.7 × 10(-5) to 300 μM did not induce SHE cell transformation frequency in a 7-day treatment. On the other side, the combination BaP/PFOA induced cell transformation at all PFOA concentrations tested, which revealed synergistic effects. No genotoxicity of PFOA on SHE cells was detected using the comet assay after 5 and 24 h of exposure. No significant increase in DNA breakage was found in BaP-initiated cells exposed to PFOA in a 7-day treatment. The whole results showed that PFOA acts as a tumor promoter and a nongenotoxic carcinogen. Cell transformation in initiated cells was observed at concentrations equivalent to the ones found in human serum of nonoccupationally and occupationally exposed populations. An involvement of PFOA in increased incidence of cancer recorded in occupationally exposed population cannot be ruled out.

  5. Differentiation and diversification of follicular cells in polytrophic ovaries of crane flies (Diptera: Nematocera: Tipulomorpha and Trichoceridae).

    PubMed

    Mazurkiewicz, Marta; Kubrakiewicz, Janusz

    2005-10-01

    To gain insight into the evolution of differentiation pathways that are involved in the follicular cells' morphogenesis in dipteran ovaries we have undertaken the comparative morphological analysis of the follicular cell behavior in crane flies, representatives of more ancestral nematocerous flies. This analysis revealed that initially the organization of the follicular epithelium in the species under study shows significant similarities to that reported in the ovaries of true flies (Brachycera), indicating that the ancestors of dipterans must have evolved a common and specific system of the early patterning of their follicular epithelium. On the other hand, in contrast to Drosophila and other advanced brachycerans, the follicular cells in the studied nematoceran ovaries do not exhibit any migratory activity. Instead, they were found to change their relative position but only within the epithelial layer. These "translocations" appeared to depend merely on cell shape changes. Although the "immobility" of the follicular cells in the ovaries of crane flies results in the lower number of their specialized subgroups when compared with the true flies, the functional homology between particular subsets of follicular cells can be postulated. We suggest that the anterior polar cells and the micropyle forming anterior terminal follicular cells in crane fly ovaries have their counterparts in the brachyceran anterior polar cells and border cells, respectively.

  6. A flow cytometric study of chromosomes from rat kangaroo and Chinese hamster cells.

    PubMed

    Stöhr, M; Hutter, K J; Frank, M; Futterman, G; Goerttler, K

    1980-01-01

    Chromosomes from rat kangaroo (PTK) and chinese hamster (CHV 79) cells have been prepared for quantitative flow-cytometric analysis. The preparation time was otimized down to 30 (PTK) and 40 min (CHV 79). DAPI was used as a AT-sensitive fluorescent dye to stain for monoparameter DNA measurements. Simultaneous two-parameter DNA-protein analysis was carried out with DAPI and SR 101 (as a general protein fluorochrome) in combination. The karyotype of the PTK cells with 13 (14) chromosomes was separated into 10DNA peaks. The X-chromosome bearing the nucleolus organizer region generates a distinct peak. The karyotype of the CHV 79 cells with 22 chromosomes was separated inot 15 peaks. The DNA profile obtained indicates a geometric grading of the chromosomal amount of AT components in teh karyotype of this particular cell line. The simultaneous DNA-protein analysis performed show enough sensitivity of the instrument utilizing hihg power UV excitation illumination to discriminate the two color emission consisting of blue (DAPI) and red (SR 101) fluorescence. Color overlapping could be completely avoided. Additionally, the quality (number, location, and resolution of peaks) of the DNA distribution was not influences by the simultaneous application of a second fluorescent stain. Fluorescence activated electronic sorting applied on chromosomal fluorescence distributions providing purified fractions of chromosomes for subsequent biochemical and biological determinations is discussed.

  7. CD10/neutral endopeptidase inhibition augments pulmonary neuroendocrine cell hyperplasia in hamsters treated with diethylnitrosamine and hyperoxia.

    PubMed

    Willett, C G; Shahsafei, A; Graham, S A; Sunday, M E

    1999-07-01

    In previous studies, we demonstrated that pulmonary neuroendocrine cell (PNEC) hyperplasia in hamsters treated with diethylnitrosamine (DEN) plus 65% hyperoxia (DEN/O2) reflects predominantly neuroendocrine cell differentiation. Several peptides implicated in non-neoplastic PNEC hyperplasia are hydrolyzed by CD10/neutral endopeptidase 24.11 (CD10/NEP), an enzyme known to downregulate neurogenic inflammation of the lung by modulating locally effective concentrations of multiple bioactive peptides. In fetal mice, we observed that CD10/NEP inhibition by SCH32615 potentiates cell proliferation and type II cell differentiation in the lung in utero. Further, CD10/NEP messenger RNA levels parallelled relative PNEC numbers in DEN/O2-treated hamster lung, suggesting that the enzyme might mediate spontaneous regression of PNEC hyperplasia. The goals of the present study were: (1) to determine whether CD10/NEP inhibition would alter the extent of PNEC hyperplasia occurring in these hamsters, and (2) to analyze cellular mechanisms potentially involved in altering numbers of PNECs in this model. We administered SCH32615 chronically to a subset of DEN/O2-treated hamsters. Immunostaining of lungs from the CD10/ NEP-inhibited subset demonstrated significant acceleration of the development of PNEC hyperplasia, increased PNEC proliferation, and diminished PNEC apoptosis as compared with animals receiving no SCH32615. These observations indicate that PNEC hyperplasia can occur as a result of multiple cellular processes, including increased neuroendocrine cell differentiation, proliferation, and survival. CD10/NEP modulates PNEC numbers primarily by promoting cell differentiation and proliferation during lung injury, probably via increasing the half-life of bioactive peptides in the lung.

  8. Study origin of germ cells and formation of new primary follicles in adult human and rat ovaries.

    PubMed

    Bukovsky, Antonin; Gupta, Satish K; Virant-Klun, Irma; Upadhyaya, Nirmala B; Copas, Pleas; Van Meter, Stuart E; Svetlikova, Marta; Ayala, Maria E; Dominguez, Roberto

    2008-01-01

    The central thesis regarding the human ovaries is that, although primordial germ cells in embryonal ovaries are of extraovarian origin, those generated during the fetal period and in postnatal life are derived from the ovarian surface epithelium (OSE) bipotent cells. With the assistance of immune system-related cells, secondary germ cells and primitive granulosa cells originate from OSE stem cells in the fetal and adult human gonads. Fetal primary follicles are formed during the second trimester of intrauterine life, prior to the end of immune adaptation, possibly to be recognized as self-structures and renewed later. With the onset of menarche, a periodical oocyte and follicular renewal emerges to replace aging primary follicles and ensure that fresh eggs for healthy babies are always available during the prime reproductive period. The periodical follicular renewal ceases between 35 and 40 yr of age, and the remaining primary follicles are utilized during the premenopausal period until exhausted. However, the persisting oocytes accumulate genetic alterations and may become unsuitable for ovulation and fertilization. The human OSE stem cells preserve the character of embryonic stem cells, and they may produce distinct cell types, including new eggs in vitro, particularly when derived from patients with premature ovarian failure or aging and postmenopausal ovaries. Our observations also indicate that there are substantial differences in follicular renewal between adult human and rat ovaries. As part of this chapter, we present in detail protocols utilized to analyze oogenesis in humans and to study interspecies differences when compared to the ovaries of rat females.

  9. FACS-sorted putative oogonial stem cells from the ovary are neither DDX4-positive nor germ cells.

    PubMed

    Zarate-Garcia, Larissa; Lane, Simon I R; Merriman, Julie A; Jones, Keith T

    2016-06-15

    Whether the adult mammalian ovary contains oogonial stem cells (OSCs) is controversial. They have been isolated by a live-cell sorting method using the germ cell marker DDX4, which has previously been assumed to be cytoplasmic, not surface-bound. Furthermore their stem cell and germ cell characteristics remain disputed. Here we show that although OSC-like cells can be isolated from the ovary using an antibody to DDX4, there is no good in silico modelling to support the existence of a surface-bound DDX4. Furthermore these cells when isolated were not expressing DDX4, and did not initially possess germline identity. Despite these unremarkable beginnings, they acquired some pre-meiotic markers in culture, including DDX4, but critically never expressed oocyte-specific markers, and furthermore were not immortal but died after a few months. Our results suggest that freshly isolated OSCs are not germ stem cells, and are not being isolated by their DDX4 expression. However it may be that culture induces some pre-meiotic markers. In summary the present study offers weight to the dogma that the adult ovary is populated by a fixed number of oocytes and that adult de novo production is a rare or insignificant event.

  10. Detection of G1 proteins in Chinese hamster cells synchronized by isoleucine deprivation or mitotic selection.

    PubMed

    Ley, K D

    1975-07-01

    Examination of labeling patterns of proteins in Chinese hamster cells(line CHO) revealed the presence of a class of protein(s) that is synthesized during G1 phase of the cell cycle. Cells arrested in G1 by isoleucine (Ile) deprivation were prelabeded with [14-C]Ile, induced to traverse G1 by addition of unlabeled Ile, and labeled with [3-H]Ile at hourly intervals. Cells were fractionated into neclear and cytoplasmic portions, and proteins were separated by sodium dodecyl sulfate-polyacrylamide get electrophoresis. Gel profiles of proteins in the 45,000-160,000 mol wt range from the cytoplasm of cells in G1 were similar to those from cells arrested in G1 except for the presence of a mojor peak of [1-H]Ile incorporated into a protein(s) of approximately 80,000 mol wt. Peaks of net [3-H]Ile incorporation were not detected in neclear preparations. Cellular fractionation by differential centrifugation showed the peak I protein was located in the soluble supernatant fraction of the cytoplasm. Time-course studies showed that synthesis of this protein began 1-2 h after initiation of G1 traverse; the protein reached maximum levels in 4-6 h and was reduced to undetectable levels by 9 h. A cytoplasmic protein with similar electrophoretic mobility was found in G1 phase of cells synchronized by mitotic selection. This class of proteins is synthesized by cells before entry into S phase and may be involved in initiation of DNA synthesis.

  11. Effect of photoperiod on the rate of 3H-thymidine incorporation of epididymal principal cells in adult Syrian hamsters

    SciTech Connect

    Johnson, L.; Bartke, A. )

    1991-04-01

    Photoperiod-induced cycles of gonadal regression and recrudescence in the Syrian hamster were used to determine if epididymal growth in adults involves mitotic activity of principal cells. In Experiment 1, the following groups of adult hamsters were examined: induced recrudescing (5L:19D (5 hr light and 19 hr dark) for 13 wk followed by 14L:10D for at least 3 wk), spontaneous recrudescing (5L:19D for 25 wk), and active gonadal state (14:10D). In Experiment 2, adult hamsters were divided into the following groups: induced recrudescing, active, and regressed (5L:19D for 16 wk). Hamsters received subcutaneous injections of 0.5 microCi 3H-thymidine/g body weight three times/wk for 3 wk. The epididymis was fixed in a glutaraldehyde followed by osmium, embedded in Epon 812, and sectioned at 1 micron. Slides were dipped in Kodak NTB-3 emulsion, exposed for 2 or 3 months, developed, and evaluated for isotopic labeling of principal and basal cell nuclei by scoring 500 to 1,000 nuclei. In Experiment 1, the percentages of labeled principal cell nuclei for the induced recrudescing, spontaneous recrudescing, and active groups were 26 {plus minus} 2%, 23 {plus minus} 5%, and 9 {plus minus} 1%, respectively. Considering the intermittent availability of 3H-thymidine during 21 days, this represents daily recruitment of 6.3%, 5.6%, and 2.2%, respectively. In Experiment 2, the percentages of labeled principal cell nuclei for induced recrudescing, active, and regressed groups were 12 {plus minus} 4%, 3 {plus minus} 1%, and 4 {plus minus} 1%, respectively. There was no effect of photoperiod on labeling pattern of basal cells (1.5 {plus minus} 0.6%, 1.2 {plus minus} 0.1%, 0.4 {plus minus} 0.1% for the three photoperiod groups, respectively).

  12. Rotational diffusion of TEMPONE in the cytoplasm of Chinese hamster lung cells.

    PubMed Central

    Lepock, J R; Cheng, K H; Campbell, S D; Kruuv, J

    1983-01-01

    The correlation time for rotational diffusion (tau R) of 2,2,6,6-tetramethyl-4-piperidone-N-oxide (TEMPONE) in Chinese hamster lung (V79) cells has been measured. For these cells in an isosmotic solution at 20 degrees C, tau R = 4.18 X 10(-11) s, approximately 3.6 times greater than tau R = 1.17 X 10(-11) s in water. The relationship between tau R and viscosity was investigated in a number of glycerol-water (0-50%) and sucrose-water (20-40%) solutions and a constant Stokes-Einstein volume of 44 A3 was found for TEMPONE in solutions of less than 20% glycerol and sucrose. This gives an average shear viscosity (for rotation of a small molecule) of 0.038 poise for the cytoplasm. When nonsecular terms were used in the calculation of tau R, the activation energies for rotation of TEMPONE in the above solutions correlated well with the activation energies for shear viscosity. The viscosity increases as the cell is shrunk in hypertonic solutions. It also increases with decreasing temperature with an activation energy of 3.7 kcal/mol, about the same as the activation energy for the viscosity of pure water. The rotational correlation times were carefully calculated considering inhomogeneous line broadening, non-Lorentzian line shapes, the need for accurate tensor values and nonsecular terms. PMID:6318842

  13. Immunostaining of germline stem cells and the niche in Drosophila ovaries.

    PubMed

    Luo, Lichao; Chai, Phing Chian; Cai, Yu

    2013-01-01

    Stem cells have the ability to switch between proliferative (self-renewal) and differentiation modes. The Drosophila germarium is a well-established in vivo model for the study of communication between stem cells and their niche. One commonly used technique for such study is immunostaining that allows examination of protein localization at a fixed time point. This chapter provides a detailed protocol for immunofluorescence staining of Drosophila ovaries. This protocol has been optimized to enable explicit visualization of the niche structure, as well as to maximize the degree of multiplexing for protein labeling and detection.

  14. Assessing in vivo microRNA function in the germline stem cells of the Drosophila ovary.

    PubMed

    Chan, Kin; Ruohola-Baker, Hannele

    2010-01-01

    A more complete understanding of the biology of adult stem cells could yield important insights toward devising effective cell-based regenerative therapies to treat disease. The germline stem cells (GSCs) in the fruit fly Drosophila melanogaster are an excellent in vivo model for the study of adult stem cell biology. There is increasing evidence from a growing field that microRNAs (miRNAs) play important roles in controlling many aspects of stem-cell biology. Using straightforward genetic manipulations combined with well-established cell biological analysis techniques, we and others have found that the miRNA pathway regulates the cell division rate of Drosophila GSCs as well as the maintenance of the GSCs in their niche. In this chapter, we offer a detailed, self-contained description of a general method to assess the in vivo functions of miRNAs in the GSCs of the Drosophila ovary.

  15. VEGF is a Promising Therapeutic Target for the Treatment of Clear Cell Carcinoma of the Ovary

    PubMed Central

    Mabuchi, Seiji; Kawase, Chiaki; Altomare, Deborah A.; Morishige, Kenichirou; Hayashi, Masami; Sawada, Kenjiro; Ito, Kimihiko; Terai, Yoshito; Nishio, Yukihiro; Klein-Szanto, Andres J.; Burger, Robert A.; Ohmichi, Masahide; Testa, Joseph R.; Kimura, Tadashi

    2010-01-01

    This study examined the role of VEGF as a therapeutic target in clear cell carcinoma (CCC) of the ovary, which has been regarded as a chemoresistant histological subtype. Immunohistochemical analysis using tissue microarrays of 98 primary ovarian cancers revealed that VEGF was strongly expressed both in early stage and advanced stage CCC of the ovary. In early stage CCCs, patients who had tumors with high levels of VEGF had significantly shorter survival than those with low levels of VEGF. In vitro experiments revealed that VEGF expression was significantly higher in cisplatin-refractory human clear cell carcinoma cells (RMG1-CR and KOC7C-CR), compared to the respective parental cells (RMG1 and KOC7C) in the presence of cisplatin. In vivo treatment with bevacizumab markedly inhibited the growth of both parental CCC cells-derived (RMG1 and KOC7C) and cisplatin-refractory CCC cells-derived (RMG1-CR and KOC7C-CR) tumors as a result of inhibition of tumor angiogenesis. The results of the current study indicate that VEGF is frequently expressed and can be a promising therapeutic target in the management of CCC. Bevacizumab may be efficacious not only as a first-line treatment but also as a second-line treatment of recurrent disease in patients previously treated with cisplatin. PMID:20663925

  16. Immunohistochemical demonstration of Clara cell antigen in lung tumors of bronchiolar origin induced by N-nitrosodiethylamine in Syrian golden hamsters.

    PubMed Central

    Rehm, S.; Takahashi, M.; Ward, J. M.; Singh, G.; Katyal, S. L.; Henneman, J. R.

    1989-01-01

    Both alveolar type II cells and Clara cells have been suggested as cells of origin of human bronchioloalveolar lung carcinomas and other pulmonary neoplasms, based on the presence of cell specific markers identified by immunocytochemical methods. Alveolar type II cell origin of solid and papillary lung tumors of the mouse has been demonstrated, and Clara cells have been suggested as cell of origin for hamster pulmonary neoplasms. Therefore, chemically induced bronchiolar hyperplasias and pulmonary neoplasms of Syrian golden hamsters were analyzed by avidin-biotin immunohistochemistry to localize a hamster-specific Clara cell antigen (CCA) and keratin. The hamsters had been treated subcutaneously with multiple doses of N-nitrosodiethylamine (NDEA). Proliferative lesions of low cuboidal, tall columnar, or pleomorphic cells were present within bronchioles or adjacent to airways in the alveolar parenchyma. Frequently areas of squamous cell differentiation were present focally or diffusely that were immunoreactive for cytokeratin. Immunoreactivity for cytokeratin was also noted for hyperplastic bronchiolar neuroepithelial bodies. Cellular hyperplasias extending out into the alveolar parenchyma contained ciliated cells and frequently consisted of cells immunoreactive for CCA, showing them to be of bronchiolar Clara cell origin. Tumors developed from bronchiolar cell hyperplasias localized within bronchioles and from bronchiolar cells lining former alveolar walls. Neoplastic growth patterns were tubulo-papillary, forming loose networks or densely cellular areas. Immunoreactivity for cytoplasmic CCA was found in 50% of the tumors and was seen most frequently in small cuboidal cells and larger, vacuolated cells scattered throughout the neoplasms. In summary, evidence is presented that NDEA-induced pulmonary tumors of the Syrian golden hamster originated from cells lining bronchioles and from extrabronchiolar Clara cell hyperplasias of the terminal bronchioles. As the

  17. Histological study of cell migration in the dermis of hamsters after immunisation with two different vaccines against visceral leishmaniasis.

    PubMed

    Moreira, Nádia das Dores; Giunchetti, Rodolfo Cordeiro; Carneiro, Cláudia Martins; Vitoriano-Souza, Juliana; Roatt, Bruno Mendes; Malaquias, Luiz Cosme Cotta; Corrêa-Oliveira, Rodrigo; Reis, Alexandre Barbosa

    2009-04-15

    Vaccine candidates, including live and/or killed parasites, Leishmania-purified fractions, defined recombinant antigens and antigen-encoding DNA-plasmids have been proposed to use as vaccine anti-Leishmania. More recently, the hamsters have been used to pre-selection of antigens candidate to apply in further experiments using canine model. In this report we evaluated the kinetics of cell migration in dermal inflammatory infiltrate, circulating leukocytes and the presence of nitric oxide (NO)/induced nitric oxide synthase during the early (1-24h) and late (48-168h) periods following inoculation of hamsters with antigenic components of anti-canine visceral leishmaniasis vaccines Leishmune and Leishmania braziliensis antigen (LB) with and without saponin (Sap) adjuvant. Our results show that LB caused an early reduction of lymphocytes in the dermis while Sap and LBSap triggered a late recruitment, suggesting the role of the adjuvant in the traffic of antigen-presenting cells and the induction of lymphocyte migration. In that manner our results suggest that the kinetics of cell migration on hamster model may be of value in the selection of vaccine antigens prior the tests in dogs particularly in respect of the toxicity of the preparations.

  18. The Stage- and Cell Type-Specific Localization of Fragile X Mental Retardation Protein in Rat Ovaries.

    PubMed

    Takahashi, Noriyuki; Tarumi, Wataru; Itoh, Masanori T; Ishizuka, Bunpei

    2015-12-01

    Premutations of the fragile X mental retardation 1 (FMR1) gene are associated with increased risk of primary ovarian insufficiency. Here we examined the localization of the Fmr1 gene protein product, fragile X mental retardation protein (FMRP), in rat ovaries at different stages, including fetus, neonate, and old age. In ovaries dissected from 19 days postcoitum embryos, the germ cells were divided into 2 types: one with decondensed chromatin in the nucleus was FMRP positive in the cytoplasm, but the other with strongly condensed chromatin in the nucleus was FMRP negative in the cytoplasm. The FMRP was predominantly localized to the cytoplasm of oocytes in growing ovarian follicles. Levels of FMRP in oocytes from elderly (9 or 14 months of age) ovaries were lower than in those from younger ovaries. These results suggest that FMRP is associated with the activation of oogenesis and oocyte function. Especially, FMRP is likely to be implicated in germline development during oogenesis.

  19. Ascorbate enhances u. v. -mutagenesis in E. coli but inhibits it in Chinese hamster cells

    SciTech Connect

    Rossman, T.G.; Klein, C.B.; Naslund, M.

    1986-05-01

    Ascorbic acid (vitamin C) causes an increase in the mutation frequency of u.v.-irradiated Escherichia coli WP2. The enhancement occurs at all u.v. fluences, and is dependent upon the ascorbate concentration in the medium. A maximum effect (approximately 8- to 13-fold) is seen at 100-150 micrograms/ml, although some enhancement can be seen even at 10 micrograms/ml. The comutagenic effect of ascorbate with u.v. in E. coli is dependent upon peptone, a constituent of nutrient broth. The enhancement of u.v.-mutagenesis by ascorbate is absent in strains WP2s (uvrA) and WP6 (polA), suggesting that ascorbate affects the repair of pyrimidine dimers. The opposite results are observed for u.v.-mutagenesis in Chinese hamster V79 cells. The presence of ascorbate (50 micrograms/ml) during u.v. irradiation does not enhance the u.v. effect, but rather decreases it approximately 30%. These results are discussed with regard to differences in the mechanism of u.v.-mutagenesis and DNA repair in bacterial and mammalian cells.

  20. Laser microirradiation of Chinese hamster cells at wavelength 365 nm: effects of psoralen and caffeine

    SciTech Connect

    Cremer, T.; Peterson, S.P.; Cremer, C.; Berns, M.W.

    1981-03-01

    Cells of a V79 subline of the Chinese hamster were microirradiated at wavelength 365 nm in the presence of the psoralen derivative, trioxsalen. Microirradiation was accomplished by a pulsed argon laser microbeam either in anaphase or in interphase 3 h after mitosis. Inhibition of clonal growth and formation of micronuclei at the first postirradiation mitosis were observed after microirradiation of anaphase chromosomes and of small parts of the interphase nucleus. Microirradiation of the cytoplasm beside the interphase nucleus or between the sets of chromosomes moving apart from each other in anaphase did not produce these effects. Anaphase experiments showed that only the daughter cell which received microirradiated chromatin exhibited an abnormal growth pattern. Most interestingly, shattering of the whole chromosome complement could be induced by microirradiation of small parts of the interphase nucleus and post-treatment with caffeine. Since microirradiation of chromatin in the absence of psoralen was not effective, we consider formation of psoralen photoadducts to nucleic acids in microirradiated chromatin to be the specific cause of the effects. We suggest that DNA photolesions in chromosome segments present in the microirradiated part of the nucleus can induce shattering of all the chromosomes in the microirradiated nucleus. Several possibilities are discussed to explain this unexpected finding.

  1. Concomitant loss of SMARCA2 and SMARCA4 expression in small cell carcinoma of the ovary, hypercalcemic type.

    PubMed

    Jelinic, Petar; Schlappe, Brooke A; Conlon, Niamh; Tseng, Jill; Olvera, Narciso; Dao, Fanny; Mueller, Jennifer J; Hussein, Yaser; Soslow, Robert A; Levine, Douglas A

    2016-01-01

    Small cell carcinoma of the ovary, hypercalcemic type is an aggressive tumor generally affecting young women with limited treatment options. Mutations in SMARCA4, a catalytic subunit of the SWI/SNF chromatin remodeling complex, have recently been identified in nearly all small cell carcinoma of the ovary, hypercalcemic type cases and represent a signature molecular feature for this disease. Additional biological dependencies associated with small cell carcinoma of the ovary, hypercalcemic type have not been identified. SMARCA2, another catalytic subunit of the SWI/SNF complex mutually exclusive with SMARCA4, is thought to be post-translationally silenced in various cancer types. We analyzed 10 archival small cell carcinoma of the ovary, hypercalcemic type cases for SMARCA2 protein expression by immunohistochemistry and found that SMARCA2 expression was lost in all but one case. None of the 50 other tumors that primarily or secondarily involved the ovary demonstrated concomitant loss of SMARCA2 and SMARCA4. Deep sequencing revealed that this loss of SMARCA2 expression is not the result of mutational inactivation. In addition, we established a small cell carcinoma of the ovary, hypercalcemic type patient-derived xenograft and confirmed the loss of SMARCA2 in this in vitro model. This patient-derived xenograft model, established from a recurrent tumor, also had unexpected mutational features for this disease, including functional mutations in TP53 and POLE. Taken together, our data suggest that concomitant loss of SMARCA2 and SMARCA4 is another hallmark of small cell carcinoma of the ovary, hypercalcemic type-a finding that offers new opportunities for therapeutic interventions.

  2. Persistent competition among stem cells and their daughters in the Drosophila ovary germline niche.

    PubMed

    Rhiner, Christa; Díaz, Begoña; Portela, Marta; Poyatos, Juan F; Fernández-Ruiz, Irene; López-Gay, Jesús M; Gerlitz, Offer; Moreno, Eduardo

    2009-03-01

    Cell competition is a short-range cell-cell interaction leading to the proliferation of winner cells at the expense of losers, although either cell type shows normal growth in homotypic environments. Drosophila Myc (dMyc; Dm-FlyBase) is a potent inducer of cell competition in wing epithelia, but its role in the ovary germline stem cell niche is unknown. Here, we show that germline stem cells (GSCs) with relative lower levels of dMyc are replaced by GSCs with higher levels of dMyc. By contrast, dMyc-overexpressing GSCs outcompete wild-type stem cells without affecting total stem cell numbers. We also provide evidence for a naturally occurring cell competition border formed by high dMyc-expressing stem cells and low dMyc-expressing progeny, which may facilitate the concentration of the niche-provided self-renewal factor BMP/Dpp in metabolically active high dMyc stem cells. Genetic manipulations that impose uniform dMyc levels across the germline produce an extended Dpp signaling domain and cause uncoordinated differentiation events. We propose that dMyc-induced competition plays a dual role in regulating optimal stem cell pools and sharp differentiation boundaries, but is potentially harmful in the case of emerging dmyc duplications that facilitate niche occupancy by pre-cancerous stem cells. Moreover, competitive interactions among stem cells may be relevant for the successful application of stem cell therapies in humans.

  3. Follicular cell differentiation in polytrophic ovaries of a moth midge, Tinearia alternata.

    PubMed

    Mazurkiewicz, Marta; Kubrakiewicz, Janusz

    2008-01-01

    Dipteran ovaries consist of structural-functional units termed egg chambers. Each egg chamber is composed of a cluster of germ cells enveloped by a simple somatic follicular epithelium. With the progress of oogenesis, initially an almost uniform population of follicular cells (FCs) becomes diversified into a few subgroups, which significantly differ in their function and behaviour. From the extensive genetic and molecular studies on Drosophila it became evident that the mode of diversification of FCs and the interactions between distinct FC subpopulations and the germ-line cells are essential for a proper course of oogenesis and the generation of oocyte/embryo polarity. Recent comparative studies showed that major dipteran lineages may significantly differ in the mode of FC differentiation. The most essential difference occurs in the ability of the FCs to undertake migrations within the egg chamber. In contrast to long distance, invasive migrations characteristic of distinct FC subgroups in the egg chambers of the most derived flies (Brachycera), including Drosophila, the FCs in the ovaries of more ancestral Nematocera lack migratory activity and change their location only within the epithelial layer. Comparative analyses indicate that the FCs in the representatives of particular evolutionary lineages within Nematocera may differ in their behaviour during oogenesis. In this report we describe the FC differentiation pathway in the egg chambers of a moth midge, T. alternata (Psychodomorpha). Comparison with representatives of craneflies (Nematocera: Polyneura) showed that differences in the behaviour of FCs and in the number of FC subpopulations between Polyneura and Psychodomorpha, may depend on different oogenesis dynamics. In spite of the observed differences, some functional homologies between distinct subsets of the FCs in dipteran ovaries are postulated.

  4. Follicular growth and atresia in mammalian ovaries: regulation by survival and death of granulosa cells.

    PubMed

    Matsuda, Fuko; Inoue, Naoko; Manabe, Noboru; Ohkura, Satoshi

    2012-01-01

    The mammalian ovary is an extremely dynamic organ in which a large majority of follicles are effectively eliminated throughout their reproductive life. Due to the numerous efforts of researchers, mechanisms regulating follicular growth and atresia in mammalian ovaries have been clarified, not only their systemic regulation by hormones (gonadotropins) but also their intraovarian regulation by gonadal steroids, growth factors, cytokines and intracellular proteins. Granulosa cells in particular have been demonstrated to play a major role in deciding the fate of follicles, serving molecules that are essential for follicular growth and maintenance as well as killing themselves by an apoptotic process that results in follicular atresia. In this review, we discuss the factors that govern follicular growth and atresia, with a special focus on their regulation by granulosa cells. First, ovarian folliculogenesis in adult life is outlined. Then, we explain about the regulation of follicular growth and atresia by granulosa cells, in which hormones, growth factors and cytokines, death ligand-receptor system and B cell lymphoma/leukemia 2 (BCL2) family members (mitochondria-mediated apoptosis) are further discussed.

  5. Expression of c-myc gene in human ovary carcinoma cells treated with vanadate

    SciTech Connect

    Itkes, A.V.; Imamova, L.R.; Alexandrova, N.M.; Favorova, O.O.; Kisselev, L.L. )

    1990-05-01

    The widely accepted hypothesis of vanadate action on cells postulates that this ion inhibits protein phosphatase(s) that dephosphorylates protein phosphotyrosine residues. This inhibition causes tyrosine hyperphosphorylation of cell proteins followed by changes in physiological action of phosphoproteins resulting in stimulation of cell proliferation, expression of protooncogenes, and transient cell transformation. The authors have found that treatment of human ovary carcinoma (CaOv) cells with vanadate causes the increase in total protein phosphorylation from 1.5- to 2.0-fold whereas the ratio between phosphoserine, phosphothreonine, and phosphotyrosine content remains unchanged. At the same time, enhancement of c-myc gene expression (not c-fos) was observed. Hence, the increase in the ratio of phosphotyrosine to phosphoserine and phosphothreonine is not an obligatory intermediate stage before vanadate-dependent activation of c-myc expression.

  6. Changes in follicular cells architecture during vitellogenin transport in the ovary of social Hymenoptera.

    PubMed

    Ronnau, Milton; Azevedo, Dihego Oliveira; Fialho, Maria do Carmo Queiroz; Gonçlaves, Wagner Gonzaga; Zanuncio, José Cola; Serrão, José Eduardo

    2016-05-01

    Vitellogenins are the major yolk proteins, synthesized in the fat body, released into the hemolymph and captured by the developing oocytes, but the mechanisms by which these proteins cross the follicular cell layer are still poorly understood. This study describes the actin distribution in follicular cells during vitellogenin transport to the oocyte in social Hymenoptera represented by bees Apis mellifera and Melipona quadrifasciata, the wasp Mischocyttarus cassununga and the ant Pachycondyla curvinodis. In oocytic chambers of vitellogenic follicles, vitellogenin was found within the follicular cells, perivitelline space and oocyte, indicating a transcellular route from the hemolymph to the perivitelline space. The cortical actin cytoskeleton in follicular cells underwent reorganization during transport of vitellogenin across this epithelium suggesting that in the ovary of social hymenopterans, vitellogenin delivery to oocytes requires a dynamic cytoskeletal rearrangement of actin filaments in the follicular cells.

  7. Regulation of c-jun by lung carcinogens in Clara cells of hamsters.

    PubMed

    Dolan, L R; Rutberg, S E; Amin, S; Emura, M; Mohr, U; Kraft, A; Yokoyama, K; Ronai, Z

    1994-12-01

    In vitro differentiated hamster Clara cells were used to study the effects of lung carcinogens on the regulation of the c-jun oncogene. Northern blot analysis revealed a decrease in the expression of jun transcripts 24 h following the exposure of Clara cells to the direct acting forms of benzo[a]pyrene (BPDE*) or 5-methylchrysene (5MeCDE). To determine whether this decrease was mediated at the transcriptional level, we have used CAT reporter constructs driven by nested deletions of the 5' non-coding regulatory region of the c-jun oncogene. While BPDE was capable of activating certain regulatory domains of the c-jun promoter, this activation was not observed with either 5MeCDE or the less active lung carcinogens BADE or 6MeCDE. Analysis of enhancer elements identified the SP1 target site as a strong silencer after BPDE treatment. While positive regulatory element(s) mediating activation of c-jun by BPDE were localized within the promoter region up to -1639, further upstream sequences reduced this transcriptional activation. Thus, when the complete promoter region, up to -4500, was tested, no transcriptional activation was noted following BPDE treatment. These observations suggest that the regulation of c-jun in Clara cells exposed to potent lung carcinogens is mediated at the post-transcriptional level, possibly by reducing the stability and, in turn, the half life of c-jun mRNA. Overall, in contrast to the response of c-jun to numerous carcinogens and stress inducing agents noted in various other cell systems, our findings suggest the existence of a tissue-specific regulatory response for c-jun.

  8. Spermatogonial multiplication in the Chinese hamster. I. Cell cycle properties and synchronization of differentiating spermatogonia.

    PubMed

    Lok, D; de Rooij, D G

    1983-01-01

    The cell cycle properties of the six successive generations of differentiating spermatogonia in the Chinese hamster were analysed by the fraction of labelled mitoses technique (FLM). Except for the A1 spermatogonia most of which have a longer cell cycle time (Tc), Tc was found to be c. 60 hr for all types of differentiating spermatogonia. As in the mouse and the rat this represents c. 14% of the duration of the cycle of the seminiferous epithelium. With ongoing differentiation, ts of the differentiating spermatogonia increases from 14 to 25 hr, while tG2 shortens from 22 to 10 hr, ts + tG2 remaining at around 35 hr throughout. Autoradiography of whole mounted seminiferous tubules at 1 hr after injection of [3H]thymidine, and experiments with Ara-C revealed that the differentiating spermatogonia traverse S in sharply defined tubular segments. Thus adjacent clones of differentiating spermatogonia start and finish their S phase at virtually the same moment. This synchronization is not yet fully established among the first generation, as clones of A1 spermatogonia in the S phase were found intermingled with A1 cells in other phases of the cell cycle. Since there is little variation in tS and tG2 in the A1 spermatogonia, it was concluded that adjacent clones of A2 spermatogonia do not always arise at the same moment. Yet A2 spermatogonia do start S synchronously, and the FLM study confirms the expected variability in their tG1. A hypothesis is proposed that each generation of differentiating spermatogonia receives a stimulus to divide from outside the spermatogonial compartment. This would ensure the synchronous behaviour of adjacent clones and the strict relationship of the pattern of proliferation to the stages of the cycle of the seminiferous epithelium.

  9. Interlaboratory comparison of transformation in Syrian hamster embryo cells with model and coded chemicals

    SciTech Connect

    Tu, A.; Hallowell, W.; Pallotta, S.; Sivak, A.; Lubet, R.A.; Curren, R.D.; Avery, M.D.; Jones, C.; Sedita, B.A.; Huberman, E.

    1986-01-01

    Three independent laboratories tested eight model and five coded chemicals in the Syrian hamster embryo clonal transformation assay system to establish the intra- and interlaboratory reproducibility of the system and to identify sources of variability. When a common cell pool and the same lot of fetal calf serum were used, the three laboratories obtained consensus on the activity of eight model chemicals; five chemicals (benzo(a)pyrene, 7,12-dimethylbenz(a)anthracene, N-methyl-N'-nitro-N-nitrosoguanidine, nitroquinoline-N-oxide, and lead chromate) induced morphological transformation without exogenous metabolic activation and three (N-2-fluorenylacetamide, pyrene, and anthracene) produced no transformation response. Five coded chemicals (2,6-dichloro p-phenylenediamine, 4,4'-oxydianiline, cinnamyl anthranilate, dichlorvos, and reserpine), representative of environmental chemical classes, but not necessarily strong carcinogens, produced more equivocal responses in this interlaboratory study. Efforts to increase the transformation frequency or to amplify the expression of the transformed phenotype constitute some of the approaches which should be explored in order to overcome these limitations.

  10. HPRT mutations in V79 Chinese hamster cells induced by accelerated Ni, Au and Pb ions.

    PubMed

    Stoll, U; Barth, B; Scheerer, N; Schneider, E; Kiefer, J

    1996-07-01

    Mutation induction by accelerated heavy ions to 6-TG resistance (HPRT system) in V79 Chinese hamster cells was investigated with Ni (6-630 Me V/u), Au (2.2, 8.7 Me V/u) and Pb ions (11.6-980 Me V/u) corresponding to a LET range between 180 and 12895 ke V/microns. Most experiments could only be performed once due to technical limitations using accelerator beam times. Survival curves were exponential, mutation induction curves linear with fluence. From their slopes inactivation- and mutation-induction cross-sections were derived. If they are plotted versus LET, single, ion-specific curves are obtained. It is shown that other parameters like ion energy and effective charge play an important role. In the case of Au and Pb ions the cross-sections follow a common line, since these ions have nearly the same atomic weight, so that they should have similar spatial ionization patterns in matter at the same energies. Calculated RBEs were higher for mutation induction than for killing for all LETs.

  11. Permeabilization of ultraviolet-irradiated chinese hamster cells with polyethylene glycol and introduction of ultraviolet endonuclease from Micrococcus luteus

    SciTech Connect

    Yarosh, D.B.; Setlow, R.B.

    1981-03-01

    Chinese hamster V-79 cells were made permeable by treatment with polyethylene glycol and then incubated with a Micrococcus luteus extract containing ultraviolet-specific endonuclease activity. This treatment introduced nicks in irradiated, but not in unirradiated, deoxyribonucleic acid. The nicks remained open for at least 3 h; there was no loss of endonuclease-sensitive sites, and no excision of dimers as measured by chromatography was detected. In addition, there was no increase in ultraviolet resistance in treated cells. This suggests that the absence of a significant amount of excision repair in rodent cells is due to the lack of both incision and excision capacity.

  12. Augmenting Chinese hamster genome assembly by identifying regions of high confidence.

    PubMed

    Vishwanathan, Nandita; Bandyopadhyay, Arpan A; Fu, Hsu-Yuan; Sharma, Mohit; Johnson, Kathryn C; Mudge, Joann; Ramaraj, Thiruvarangan; Onsongo, Getiria; Silverstein, Kevin A T; Jacob, Nitya M; Le, Huong; Karypis, George; Hu, Wei-Shou

    2016-09-01

    Chinese hamster Ovary (CHO) cell lines are the dominant industrial workhorses for therapeutic recombinant protein production. The availability of genome sequence of Chinese hamster and CHO cells will spur further genome and RNA sequencing of producing cell lines. However, the mammalian genomes assembled using shot-gun sequencing data still contain regions of uncertain quality due to assembly errors. Identifying high confidence regions in the assembled genome will facilitate its use for cell engineering and genome engineering. We assembled two independent drafts of Chinese hamster genome by de novo assembly from shotgun sequencing reads and by re-scaffolding and gap-filling the draft genome from NCBI for improved scaffold lengths and gap fractions. We then used the two independent assemblies to identify high confidence regions using two different approaches. First, the two independent assemblies were compared at the sequence level to identify their consensus regions as "high confidence regions" which accounts for at least 78 % of the assembled genome. Further, a genome wide comparison of the Chinese hamster scaffolds with mouse chromosomes revealed scaffolds with large blocks of collinearity, which were also compiled as high-quality scaffolds. Genome scale collinearity was complemented with EST based synteny which also revealed conserved gene order compared to mouse. As cell line sequencing becomes more commonly practiced, the approaches reported here are useful for assessing the quality of assembly and potentially facilitate the engineering of cell lines.

  13. Development of pulmonary neuroendocrine cells of fetal hamster in explant culture.

    PubMed

    Ito, T; Nogawa, H; Udaka, N; Kitamura, H; Kanisawa, M

    1997-11-01

    Fetal hamster lung explant was cultured in serum-free medium on gestational Day 11-2 days before the appearance of pulmonary neuroendocrine cells (PNEC)--and the development and differentiation of PNEC from immature fetal lung epithelium was examined through immunostaining for neural cell adhesion molecule (NCAM) to establish an in vitro system to study the mechanisms involved. PNEC were present in the main bronchus after 2 days of culture. Thereafter, NCAM-positive clusters of PNEC increased and were distributed from the large bronchus to the terminal bronchiole with a proximal-to-distal wave. To elucidate the role of NCAM in the fetal development of PNEC, whole fetal lung was cultured on gestational Day 11 with an anti-NCAM antibody. This antibody slightly inhibited the growth and branching morphogenesis of the lung and disturbed the formation of PNEC clusters. NCAM may function to form clusters of PNEC known as neuroepithelial bodies. We cultured fetal lung epithelial explant at gestational Day 11 after removing mesenchyme, including nerve tissue, with dispase digestion. Immunohistochemical staining for NCAM revealed that PNEC were induced in cultured fetal epithelium without mesenchymal tissue, but basement membrane Matrigel was necessary to maintain cultured epithelium. In conclusion, PNEC derive from immature airway epithelial cells. This organ culture system, therefore, is a useful experimental model and should facilitate further investigations of the development and differentiation of PNEC. Mesenchymal and neural tissues are not always necessary for the development of PNEC, but matrix substance and/or growth factors may be required to induce or maintain PNEC.

  14. Nicotine, acetylcholine and bombesin are trophic growth factors in neuroendocrine cell lines derived from experimental hamster lung tumors

    SciTech Connect

    Schueller, H.M.; Nylen, E.; Park, P.; Becker, K.L. George Washington Univ., Washington, DC )

    1990-01-01

    Neuroendocrine hamster lung tumors, induced by exposure to 60% hyperoxia and subcutaneous administration of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) for 12 weeks, were placed in cell culture. By subsequent selective transfer of epithelial cells and maintenance in an atmosphere of 8% CO{sub 2}, cell lines with characteristics of neuroendocrine cells were established. The neuroendocrine markers expressed by these cell lines included electron dense neuroendocrine secretion granules as well as secretion of calcitonin and mammalian bombesin. In keeping with data previously reported for a human neuroendocrine lung tumor cell line, nicotine, acetylcholien, and mammalian bombesin (MB) acted as strongrowth factors in these neuroendocrine hamster tumor lines. The mitogenic effect of nicotine an acetylcholine was abolished by nicotinic receptor inhibition while the effects of mammalian bombesin were inhibited by an antagonist of MB receptors. Our data suggest that a receptor-mediated mitogenic effect of nicotine on neuroendocrine lung cells may be instrumental in the induction of smoking-associated small cell lung cancer.

  15. Large cell neuroendocrine carcinoma of the ovary: A pathologic entity in search of clinical identity.

    PubMed

    Voutsadakis, Ioannis A

    2014-05-10

    Large cell neuroendocrine carcinoma (LCNEC) of the ovary is a rare diagnosis and only a few dozen cases have been reported in the literature. It is characterized by large pleiomorphic cells with large round or oval nuclei, presence of mitoses and staining for neuroendocrine (NE) markers such as chromogranin A, synaptophysin, neuron specific enolase. This editorial gives a brief overview of this histologic type of ovarian carcinomas. LCNEC of the ovary is a pathologic entity that may not be diagnosed purely on clinical grounds due to the similarity of its clinical features with those of the more common epithelial ovarian cancers. Nevertheless the diagnosis is worth-making from a practical point of view in order to consider treatments tailored towards the NE component if it is dominant or it becomes dominant during the natural evolution of the disease. Establishment of an international tumor registry with an accompanying tumor tissue bank of ovarian LCNEC could be a means of obtaining further knowledge on clinical characteristics and advance research on this rare entity. This will further inform on treatment strategies and could identify future molecular treatment targets.

  16. Photo catalogue for the classification of cell colonies in the Syrian hamster embryo (SHE) cell transformation assay at pH 7.0.

    PubMed

    Maire, Marie-Aline; Rast, Claudine; Vasseur, Paule

    2012-04-11

    This catalogue is a display of Syrian hamster embryo (SHE) cell colony photos representative of the cell transformation assay (CTA) carried out at pH 7.0. It is intended as a visual aid for the identification and the scoring of cell colonies in the conduct of the assay. A proper training from experienced personnel together with the protocol reported in this issue and the present photo catalogue will support method transfer and consistency in the assay results.

  17. A distinct G1 step required to specify the Chinese hamster DHFR replication origin.

    PubMed

    Wu, J R; Gilbert, D M

    1996-03-01

    Nuclei isolated from Chinese hamster ovary (CHO) cells at various times during the G1 phase of the cell cycle were stimulated to enter S phase by incubation in Xenopus egg cytosol. Replication of DNA initiated within the dihydrofolate reductase (DHFR) origin locus in nuclei isolated late in G1, but at random sites in nuclei isolated early in G1. A discrete transition point occurred 3 to 4 hours after metaphase. Neither replication licensing nor nuclear assembly was sufficient for origin recognition. Thus, a distinct cell cycle-regulated event in the nucleus restricts the initiation of replication to specific sites downstream of the DHFR gene.

  18. The Wnt pathway limits BMP signaling outside of the germline stem cell niche in Drosophila ovaries.

    PubMed

    Mottier-Pavie, Violaine I; Palacios, Victor; Eliazer, Susan; Scoggin, Shane; Buszczak, Michael

    2016-09-01

    The mechanisms that modulate and limit the signaling output of adult stem cell niches remain poorly understood. To gain further insights into how these microenvironments are regulated in vivo, we performed a candidate gene screen designed to identify factors that restrict BMP signal production to the cap cells that comprise the germline stem cell (GSC) niche of Drosophila ovaries. Through these efforts, we found that disruption of Wnt4 and components of the canonical Wnt pathway results in a complex germ cell phenotype marked by an expansion of GSC-like cells, pre-cystoblasts and cystoblasts in young females. This phenotype correlates with an increase of decapentaplegic (dpp) mRNA levels within escort cells and varying levels of BMP responsiveness in the germline. Further genetic experiments show that Wnt4, which exhibits graded expression in somatic cells of germaria, activates the Wnt pathway in posteriorly positioned escort cells. The activation of the Wnt pathway appears to be limited by the BMP pathway itself, as loss of Mad in escort cells results in the expansion of Wnt pathway activation. Wnt pathway activity changes within germaria during the course of aging, coincident with changes in dpp production. These data suggest that mutual antagonism between the BMP and Wnt pathways in somatic cells helps to regulate germ cell differentiation.

  19. Induction by inorganic metal salts of sister chromatid exchanges and chromosome aberrations in human and Syrian hamster cell strains

    SciTech Connect

    Larramendy, M.L.; Popescu, N.C.; DiPaolo, J.A.

    1981-01-01

    Sister chromatid exchange (SCE) and chromosome aberration induction were determined for several inorganic metal salts. Arsenic, nickel, and beryllium salts at concentrations effective in causing transformation of Syrian hamster cells (HEC) induced SCE and chromosome aberrations of HEC and human lymphocytes, whereas sodium tungstate, a non-transforming chemical, neither induced SCE nor chromosome aberrations. Normal human and hamster cells exhibited equal sensitivity to SCE induction; nontoxic concentrations of sodium arsenite, beryllium sulfate, and nickel sulfate caused an increase of 8-10 SCE/cell over control values. Sodium arsenite, a trivalent arsenic, and sodium arsenate, a pentavalent arsenic, produced increases in SCE but the former was effective at lower concentrations. Both arsenic salts were less efficient in inducing SCE in human whole blood than in purified lymphocyte cultures. Sodium arsenite, sodium arsenate, nickel sulfate, and beryllium sulfate also caused damage consisting primarily of chromatid type of aberrations. In HEC, with doses most effective in SCE induction , all four metals produced aberrations in 16-21% of cells. In human lymphocytes, 34 and 30% of the cells had chromosome damage after sodium arsenite and sodium arsenate, respectively, whereas beryllium sulfate or nickel sulfate caused damage in about 10% of the cells. The induction of SCE and chromosomal aberrations by metals reemphasizes the sensitivity of cytological assays and their importance for detecting genetic damage caused by carcinogens.

  20. Histochemical study of brown-fat cells in the golden hamster (Mesocricetus auratus) in cultures

    SciTech Connect

    Sokolov, V.E.; Boyadzhieva-Mikhailova, A.; Koncheva, L.; Angelova, P.; Evgen'eva, T.P.

    1985-11-01

    The authors undertake the task of studying the synthesis of certain hormones by brown-fat cells. The authors used brown-fat cells from the golden hamster. The metabolism of brown-fat cells was studied on precultured cells, which made it possible to detect the synthesis of the studied substances rather than their accumulation in the organ. The authors conducted three experiments. First, fragments of brown fat were cultivated in diffusion chambers in vivo. Pieces of brown fat were cultivated in parallel in vitro on agar (organotypic cultures) and on plasma (histotypic cultures). During cultivation in diffusion chambers, the chambers were implanted in the abdominal cavity of young white rats. For in vitro cultivation, TCM 199 plus 15-20% calf serum was used. A total of 36 cultures with 12 cultures in each series of experiments were performed. The auto-radiographic studies of brown-fat cells were conducted on 24-hour cultures and on brown-fat fragments taken from the intact animal. The cultures were incubated with isotopes for 1 h. Either (/sup 3/H)lysine (87.3 Ci/mM specific activity), (/sup 3/H)arginine (16.7 Ci/mM), (/sup 3/H)glycerol (43 Ci/mM), or (/sup 3/H)cholesterol (43 Ci/mM) were added to the medium. After incubation, the cultures were washed three times in pure medium, fixed in Sierra fluid, and embedded in paraffin. The paraffin sections were covered with Ilford K/sub 2/ emulsion, and the preparations were exposed for 20 days at 4/sup 0/C temperature. Radio-immunological methods were used to study the accumulation of estradiol-17-beta in the culture medium by the Dobson method and that of testerone. The culture medium was taken on cultivation days 2,4,6,8, and 10. The medium was changed during cultivation every third day, which made it possible to judge the rates of accumulation of material with increase in the cultivation times.

  1. Endothelial Progenitor Cell Dysfunction in Polycystic Ovary Syndrome: Implications for The Genesis of Cardiovascular Diseases

    PubMed Central

    Kao, Yu-Hsun; Chiu, Wan-Chun; Hsu, Ming-I; Chen, Yi-Jen

    2013-01-01

    Polycystic ovary syndrome (PCOS), the most common endocrine disorder affecting women of reproductive age, is characterized by hyperandrogenism and insulin resistance. Women with PCOS have a higher risk for cardiovascular diseases (CVDs) and endothelial dysfunction. The mechanisms underlying these risks are unclear. Human peripheral blood contains circulating endothelial progenitor cells (EPCs) derived from bone marrow that have the ability to proliferate and differentiate into mature endothelial cells, which may contribute to vessel homeostasis and repair. PCOS is associated with insulin resistance, hyperinsulinemia, and dyslipidemia, which may result in EPC dysfunction. In this review, we summarize the potential mechanisms of EPC dysfunction in PCOS, which possibly result in a higher genesis of CVDs in PCOS-affected subjects. PMID:24520442

  2. Low doses of alpha particles do not induce sister chromatid exchanges in bystander Chinese hamster cells defective in homologous recombination

    SciTech Connect

    Nagasawa, H; Wilson, P F; Chen, D J; Thompson, L H; Bedford, J S; Little, J B

    2007-10-26

    We reported previously that the homologous recombinational repair (HRR)-deficient Chinese hamster mutant cell line irs3 (deficient in the Rad51 paralog Rad51C) showed only a 50% spontaneous frequency of sister chromatid exchange (SCE) as compared to parental wild-type V79 cells. Furthermore, when irradiated with very low doses of alpha particles, SCEs were not induced in irs3 cells, as compared to a prominent bystander effect observed in V79 cells (Nagasawa et al., Radiat. Res. 164, 141-147, 2005). In the present study, we examined additional Chinese hamster cell lines deficient in the Rad51 paralogs Rad51C, Rad51D, Xrcc2, and Xrcc3 as well as another essential HRR protein, Brca2. Spontaneous SCE frequencies in non-irradiated wild-type cell lines CHO, AA8 and V79 were 0.33 SCE/chromosome, whereas two Rad51C-deficient cell lines showed only 0.16 SCE/chromosome. Spontaneous SCE frequencies in cell lines defective in Rad51D, Xrcc2, Xrcc3, and Brca2 ranged from 0.23-0.33 SCE/chromosome, 0-30% lower than wild-type cells. SCEs were induced significantly 20-50% above spontaneous levels in wild-type cells exposed to a mean dose of 1.3 mGy of alpha particles (<1% of nuclei traversed by an alpha particle). However, induction of SCEs above spontaneous levels was minimal or absent after {alpha}-particle irradiation in all of the HRR-deficient cell lines. These data suggest that Brca2 and the Rad51 paralogs contribute to DNA damage repair processes induced in bystander cells (presumably oxidative damage repair in S-phase cells) following irradiation with very low doses of alpha particles.

  3. Morphological transformation of Syrian hamster embryo cells by low doses of fission neutrons delivered at different dose rates

    SciTech Connect

    Jones, C.A.; Sedita, B.A. ); Hill, C.K. . Cancer Research Lab.); Elkind, M.M. . Dept. of Radiology and Radiation Biology)

    1991-01-01

    Both induction of cell transformation and killing were examined with Syrian hamster embryo (SHE) fibroblasts exposed to low doses of JANUS fission-spectrum neutrons delivered at high (10.3 cGy/min) and low (0.43 and 0.086 cGy/min) dose rates. Second-passage cells were irradiated in mass cultures, then cloned over feeder cells. Morphologically transformed colonies were identified 8-10 days later. Cell killing was independent of dose rate, but the yield of transformation was greater after low-dose-rate irradiations. Decreasing the neutron dose-rate from 10.3 to 0.086 cGy/min resulted in a two- to threefold increase in the yield of transformation for neutron exposures below 50 cGy, and enhancement which was consistently observed in repetitive experiments in different radiosensitive SHE cell preparations. 43 refs., 5 figs., 1 tab.

  4. Fractalkine restores the decreased expression of StAR and progesterone in granulosa cells from patients with polycystic ovary syndrome

    PubMed Central

    Huang, Shuo; Pang, Yanli; Yan, Jie; Lin, Shengli; Zhao, Yue; Lei, Li; Yan, Liying; Li, Rong; Ma, Caihong; Qiao, Jie

    2016-01-01

    Low progesterone levels are associated with luteal phase deficiency in women with polycystic ovary syndrome (PCOS). The mechanisms regulating progesterone biosynthesis in the granulosa cells from women with PCOS is largely unknown. Fractalkine is expressed in human ovaries, and is reported to regulate progesterone production in granulosa cells of healthy women. In the current study, we aimed to examine the role of fractalkine in women with PCOS. Reduced fractalkine levels were found in follicular fluid and granulosa cells, accompanied by decreased progesterone production and reduced steroidogenic acute regulatory protein (StAR) expression in the granulosa cells of patients with PCOS. Administration of fractalkine reversed the inhibition of progesterone and StAR expression. The mechanism mediating these effects may be associated with the inhibition of ERK activity in the granulosa cells from women with PCOS. Our findings revealed that fractalkine regulated steroidogenesis in follicular granulosa cells of women with PCOS. PMID:27386819

  5. Clear cell carcinoma of the ovary: molecular insights and future therapeutic perspectives

    PubMed Central

    2016-01-01

    Clear cell carcinoma (CCC) of the ovary is known to show poorer sensitivity to chemotherapeutic agents and to be associated with a worse prognosis than the more common serous adenocarcinoma or endometrioid adenocarcinoma. To improve the survival of patients with ovarian CCC, the deeper understanding of the mechanism of CCC carcinogenesis as well as the efforts to develop novel treatment strategies in the setting of both front-line treatment and salvage treatment for recurrent disease are needed. In this presentation, we first summarize the mechanism responsible for carcinogenesis. Then, we highlight the promising therapeutic targets in ovarian CCC and provide information on the novel agents which inhibit these molecular targets. Moreover, we discuss on the cytotoxic anti-cancer agents that can be best combined with targeted agents in the treatment of ovarian CCC. PMID:27029752

  6. Presenilin 1 overexpressions in Chinese hamster ovary (CHO) cells decreases the phosphorylation of retinoblastoma protein: relevance for neurodegeneration.

    PubMed

    Prat, María I; Adamo, Ana M; González, Silvia A; Affranchino, José L; Ikeda, Masaki; Matsubara, Etsuro; Shoji, Mikio; Smith, Mark A; Castaño, Eduardo M; Morelli, Laura

    2002-06-21

    Mutations in the presenilin 1 (PS1) gene have been associated to familial Alzheimer disease although the exact pathogenic mechanism is unclear. We report that stable overexpression of wild type PS1 led to a decrease in cyclin-dependent kinase 4 (CDK 4) activity and retinoblastoma tumor suppressor protein (pRb) phosphorylation that correlated with decreased levels of beta-catenin and cyclin D1. PS1 mutant D385A also precipitated a similar effect suggesting that gamma-secretase cleavage is not essential for PS1-mediated CDK 4 inhibition. We postulate that PS1 overexpression may balance the hyperphosphorylation of pRb associated with death of post mitotic neurons after injury.

  7. The Notch pathway regulates both the proliferation and differentiation of follicular cells in the panoistic ovary of Blattella germanica.

    PubMed

    Irles, Paula; Elshaer, Nashwa; Piulachs, Maria-Dolors

    2016-01-01

    The Notch pathway is an essential regulator of cell proliferation and differentiation during development. Its involvement in insect oogenesis has been examined in insect species with meroistic ovaries, and it is known to play a fundamental role in cell fate decisions and the induction of the mitosis-to-endocycle switch in follicular cells (FCs). This work reports the functions of the main components of the Notch pathway (Notch and its ligands Delta and Serrate) during oogenesis in Blattella germanica, a phylogenetically basal species with panoistic ovary. As is revealed by RNAi-based analyses, Notch and Delta were found to contribute towards maintaining the FCs in an immature, non-apoptotic state. This ancestral function of Notch appears in opposition to the induction of transition from mitosis to endocycle that Notch exerts in Drosophila melanogaster, a change in the Notch function that might be in agreement with the evolution of the insect ovary types. Notch was also shown to play an active role in inducing ovarian follicle elongation via the regulation of the cytoskeleton. In addition, Delta and Notch interactions were seen to determine the differentiation of the posterior population of FCs. Serrate levels were found to be Notch-dependent and are involved in the control of the FC programme, although they would appear to play no crucial role in panoistic ovary oogenesis.

  8. DNA double-strand breaks measured in individual cells subjected to gel electrophoresis

    SciTech Connect

    Olive, P.L.; Wlodek, D.; Banath, J.P. )

    1991-09-01

    Microscopic examination of individual mammalian cells embedded in agarose, subjected to electrophoresis, and stained with a fluorescent DNA-binding dye provides a novel way of measuring DNA damage and more importantly, of assessing heterogeneity in DNA damage within a mixed population of cells. With this method, DNA double-strand breaks can be detected in populations of cells exposed to X-ray doses as low as 5 Gy. The radiation dose-response relationship for initial formation of double-strand breaks was identical for cell lines irradiated in G1, regardless of their sensitivity to killing by ionizing radiation. However, for cells irradiated in S phase, DNA migration was significantly reduced. For Chinese hamster V79 cells, Chinese hamster ovary cells, WiDr human colon carcinoma cells, and L5178Y-R mouse lymphoblastoid cells, S-phase DNA appeared to be about 3 times less sensitive to X-ray damage than DNA from other phases of the cell cycle. However, for the very radiosensitive L5178Y-S cells, the migration of replicating DNA was reduced only slightly. For Chinese hamster V79 and Chinese hamster ovary cells, damage was repaired at a similar rate in all cells of the population, and 85% of the breaks were rejoined within 2 h after irradiation. The radiosensitive L5178Y-S cells repaired damage more slowly than V79 or Chinese hamster ovary cells; 2 h after exposure to 50 Gy, approximately 50% of the damage was still present.

  9. Cytotoxicity of liposomal α-tocopheryl succinate towards hamster cheek pouch carcinoma (HCPC-1) cells in culture

    PubMed Central

    Gu, Xinbin; Schwartz, Joel L.; Pang, Xiaowu; Zhou, Yanfei; Sirois, David A.; Sridhar, Rajagopalan

    2007-01-01

    There is compelling evidence for the cancer chemopreventive effects of vitamin E and related compounds. Of all the vitamin E derivatives that have been investigated to date, vitamin E acid succinate is the most effective anti-cancer agent. This report describes the preparation and testing of liposomal formulation of mono α-tocopheryl ester of succinic acid (α-TOS) for cytotoxicity against hamster cheek pouch carcinoma cell line (HCPC-1). Small unilamellar vesicles (SUV) of phosphatidylcholine incorporating 70 μM α-TOS were superior to α-TOS alone or SUV without incorporated α-TOS, as inducers of apoptosis in HCPC-1 cells. Liposomal α-TOS perturbed the lipid structure in cells, promoted apoptosis, and decreased cell viability. The mechanism of action of α-TOS appears to involve membrane damage and induction of ceramide mediated apoptosis. PMID:16271438

  10. Immune response to acute virus infection in the Syrian hamster. II. Studies on the identity of virus-induced cytotoxic effector cells

    SciTech Connect

    Nelles, M.J.; Duncan, W.R.; Streilein, J.W.

    1981-01-01

    The identity of the effector cell(s) mediating vaccinia virus-induced cytotoxic activity in Syrian hamsters undergoing acute virus infection has been investigated. Two different approaches have been utilized in this regard. Although T cells do not mediate vaccinia virus-induced cytotoxic activity directly, functional T cells were required for the in vivo development of a significant portion of vaccinia virus-induced cytotoxic activity. In addition, incorporation of aggregated gamma-globulins as well as anti-immunoglobulin reagents into the in vitro 51 Cr release assay inhibited a significant proportion of the cytotoxic activity mediated by spleen cells obtained from acutely infected hamsters possessing an intact thymus. Both approaches have yielded information consistent with the idea that a sizable portion of vaccinia virus-induced cytotoxic activity in the Syrian hamster is effected by K cells mediating antibody-dependent cell-mediated cytotoxicity (ADCC). The significance of this observation is discussed with regard to hamster viral immunity in general.

  11. Androgen and FSH synergistically stimulate lipoprotein degradation and utilization by ovary granulosa cells

    SciTech Connect

    Schreiber, J.R.; Nakamura, K.; Schmit, V.; Weinstein, D.B.

    1984-01-01

    Androgen can directly modulate the induction of steroidogenic enzymes by FSH (follicle stimulating hormone) in ovary granulosa cells. In studies of its mechanism of action, the authors examined the androgen effect on granulosa cell interaction with lipoproteins, the physiologic source of cholesterol. After granulosa cells were cultured for 48 hours with and without androgen and/or FSH, the cells were incubated for 24 hours with /sup 125/I-lipoproteins (human high density lipoprotein (HDL), rat HDL, or human low density lipoprotein (LDL)). The media were then analyzed for lipoprotein protein coat degradation products (mainly /sup 125/I-monoiodotyrosine) and progestin (mainly 20 alpha-dihydroprogesterone (20 alpha-DHP)). In the absence of FSH and androgen, 2 X 10(5) granulosa cells degraded basal levels of all three lipoproteins, but produced no measurable 20 alpha-DHP. The addition of 10(-7) M androstenedione (A), testosterone (T), or 5 alpha-dihydrotestosterone (DHT) had no effect on lipoprotein protein degradation or 20 alpha-DHP production. FSH alone stimulated lipoprotein protein degradation by 50 to 300% while the addition of androgen synergistically augmented the FSH-stimulated 20 alpha-DHP production as well as protein coat degradation of all three lipoproteins. DHT and T were both effective, indicating that androgens themselves, and not estrogen products, were responsible for the effect on lipoprotein protein degradation and 20 alpha-DHP production.

  12. In vitro comet and micronucleus assays do not predict morphological transforming effects of silica particles in Syrian Hamster Embryo cells.

    PubMed

    Darne, Christian; Coulais, Catherine; Terzetti, Francine; Fontana, Caroline; Binet, Stéphane; Gaté, Laurent; Guichard, Yves

    2016-01-15

    Crystalline silica particles and asbestos have both been classified as carcinogenic by the International Agency for Research on Cancer (IARC). However, because of the limited data available, amorphous silica was not classifiable. In vitro, the carcinogenic potential of natural crystalline and amorphous silica particles has been revealed by the Syrian Hamster Embryo (SHE) cell transformation assay. On the other hand, the genotoxic potential of those substances has not been investigated in SHE cells. And yet, genotoxicity assays are commonly used for hazard evaluation and they are often used as in vitro assays of reference to predict a possible carcinogenic potential. The main objective of this study was to compare the genotoxic potential and the carcinogenic potential of different crystalline and amorphous silica particles in SHE cells. Three silica samples of different crystallinity were used: natural amorphous silica, partially crystallized silica and quartz silica particles. Their genotoxicity were tested through the in vitro micronucleus assay and the comet assay in SHE, and their carcinogenic potential through the SHE transformation assay. In addition, silica samples were also tested with the same genotoxicity assays in V79 hamster-lung cells, a common in vitro model for particle exposure. Results obtained in the micronucleus and the comet assays show that none of the silica was capable of inducing genotoxic effects in SHE cells and only the amorphous silica induced genotoxic effects in V79 cells. However in the SHE cell transformation assays, the partially crystallized and quartz silica were able to induce morphological cell transformation. Together, these data suggest that, in vitro, the short-term genotoxic assays alone are not sufficient to predict the hazard and the carcinogenic potential of this type of particles; SHE transformation assay appears a more reliable tool for this purpose and should be included in the "in vitro battery assays" for hazard

  13. Pro-nerve growth factor in the ovary and human granulosa cells

    PubMed Central

    Meinel, Sabine; Blohberger, Jan; Berg, Dieter; Berg, Ulrike; Dissen, Gregory A.; Ojeda, Sergio R.; Mayerhofer, Artur

    2016-01-01

    Background Pro-nerve growth factor must be cleaved to generate mature NGF, which was suggested to be a factor involved in ovarian physiology and pathology. Extracellular proNGF can induce cell death in many tissues. Whether extracellular proNGF exists in the ovary and may play a role in the death of follicular cells or atresia was unknown. Material and Methods Immunohistochemistry of human and Rhesus monkey ovarian sections was performed. IVF-derived follicular fluid and human granulosa cells were studied by RT-PCR, qPCR, Western blotting, ATP- and caspase-assays. Results and Conclusions Immunohistochemistry of ovarian sections identified proNGF in granulosa cells and Western blotting of human isolated granulosa cells confirmed the presence of proNGF. Ovarian granulosa cells thus produce proNGF. Recombinant human proNGF even at high concentrations did not affect the levels of ATP or the activity of caspase 3/7, indicating that in granulosa cells proNGF does not induce death. In contrast, mature NGF, which was detected previously in follicular fluid, may be a trophic molecule for granulosa cells with unexpected functions. We found that in contrast to proNGF, NGF increased the levels of the transcription factor early growth response 1 and of the enzyme choline acetyl-transferase. A mechanism for the generation of mature NGF from proNGF in the follicular fluid may be extracellular enzymatic cleavage. The enzyme MMP7 is known to cleave proNGF and was identified in follicular fluid and as a product of granulosa cells. Thus the generation of NGF in the ovarian follicle may depend on MMP7. PMID:26457789

  14. Retinal ganglion cell projections to the hamster suprachiasmatic nucleus, intergeniculate leaflet, and visual midbrain: bifurcation and melanopsin immunoreactivity

    NASA Technical Reports Server (NTRS)

    Morin, Lawrence P.; Blanchard, Jane H.; Provencio, Ignacio

    2003-01-01

    The circadian clock in the suprachiasmatic nucleus (SCN) receives direct retinal input via the retinohypothalamic tract (RHT), and the retinal ganglion cells contributing to this projection may be specialized with respect to direct regulation of the circadian clock. However, some ganglion cells forming the RHT bifurcate, sending axon collaterals to the intergeniculate leaflet (IGL) through which light has secondary access to the circadian clock. The present studies provide a more extensive examination of ganglion cell bifurcation and evaluate whether ganglion cells projecting to several subcortical visual nuclei contain melanopsin, a putative ganglion cell photopigment. The results showed that retinal ganglion cells projecting to the SCN send collaterals to the IGL, olivary pretectal nucleus, and superior colliculus, among other places. Melanopsin-immunoreactive (IR) ganglion cells are present in the hamster retina, and some of these cells project to the SCN, IGL, olivary pretectal nucleus, or superior colliculus. Triple-label analysis showed that melanopsin-IR cells bifurcate and project bilaterally to each SCN, but not to the other visual nuclei evaluated. The melanopsin-IR cells have photoreceptive characteristics optimal for circadian rhythm regulation. However, the presence of moderately widespread bifurcation among ganglion cells projecting to the SCN, and projection by melanopsin-IR cells to locations distinct from the SCN and without known rhythm function, suggest that this ganglion cell type is generalized, rather than specialized, with respect to the conveyance of photic information to the brain. Copyright 2003 Wiley-Liss, Inc.

  15. Influence of DMSO on Carbon K ultrasoft X-rays induced chromosome aberrations in V79 Chinese hamster cells.

    PubMed

    Natarajan, Adayapalam T; Palitti, Fabrizio; Hill, Mark A; Stevens, David L; Ahnström, Gunnar

    2010-09-10

    Ultrasoft X-rays have been shown to be very efficient in inducing chromosomal aberrations in mammalian cells. The present study was aimed to evaluate the modifying effects of DMSO (a potent scavenger of free radicals) on the frequencies of chromosome aberrations induced by soft X-rays. Confluent held G1 Chinese hamster cells (V79) were irradiated with Carbon K ultrasoft X-rays in the presence and absence of 1M DMSO and frequencies of chromosome aberrations in the first division cells were determined. DMSO reduced the frequencies of exchange types of aberrations (dicentrics and centric rings) by a factor of 2.1-3.5. The results indicate that free radicals induced by ultrasoft X-rays contribute to a great extent to the induction of chromosome aberrations. The possible implications of these results in interpreting the mechanisms involved in the high efficiency of ultrasoft X-rays in the induction of chromosome aberrations are discussed.

  16. Relative biological effectiveness of accelerated heavy ions for induction of morphological transformation in Syrian hamster embryo cells.

    PubMed

    Han, Z B; Suzuki, H; Suzuki, F; Suzuki, M; Furusawa, Y; Kato, T; Ikenaga, M

    1998-09-01

    Syrian hamster embryo cells were used to study the morphological transformation induced by accelerated heavy ions with different linear energy transfer (LET) ranging from 13 to 400 keV/micron. Exponentially growing cells were irradiated with 12C or 28Si ion beams generated by the Heavy Ion Medical Accelerator in Chiba (HIMAC), then inoculated to culture dishes. Morphologically altered colonies were scored as transformants. Over the LET range examined, the frequency of transformation induced by the heavy ions increased sharply at very low doses no greater than 5 cGy. The relative biological effectiveness (RBE) of the heavy ions relative to X-rays first increased with LET, reached a maximum value of about 7 at 100 keV/micron, then decreased with the further increase of LET. Our findings confirmed that high LET heavy ions are much more effective than X-rays for the induction of in vitro cell transformation.

  17. Pulmonary toxicity of methylcyclopentadienyl manganese tricarbonyl: nonciliated bronchiolar epithelial (Clara) cell necrosis and alveolar damage in the mouse, rat, and hamster

    SciTech Connect

    Hakkinen, P.J.; Haschek, W.M.

    1982-01-01

    Methylcyclopentadienyl manganese tricarbonyl manganese tricarbonyl (MMT) was administered ip to young female BALB/c mice (120 mg MMT/kg), S/A albino rats (5 mg MMT/kg), or LV/sub 6//LAK Syrian hamsters (180 mg MMT/kg). This administration resulted in lung cell damage followed by cellular proliferation, which was quantified by measuring increases in thymidine incorporation into DNA (mouse, rat, and hamster) and by labeling indices (LI) determined from cell kinetic studies (mouse and rat). Thymidine incorporation into pulmonary DNA was significantly elevated within 1 to 2 days following MMT treatment in all three species, with peak incorporation occurring on Day 2 in the rat and hamster, and Day 4 in the mouse. Both bronchiolar and parenchymal LI were elevated at this time. Alveolar damage and nonciliated bronchiolar epithelial (Clara) cell necrosis were evident within 1 day of injection. This finding was followed by type II epithelial and Clara cell proliferation. Ultrastructurally, in the mouse, mitochondrial swelling and degeneration preceded Clara cell necrosis. Bronchiolar damage was most severe in the mouse, whereas parenchymal damage was most severe in the rat. These results suggest that the mouse, rat, and hamster have different susceptibilities to MMT-induced injury.

  18. Identification of Polycystic Ovary Syndrome (PCOS) Specific Genes in Cumulus and Mural Granulosa Cells.

    PubMed

    Aydos, Alp; Gurel, Aykut; Oztemur Islakoglu, Yasemin; Noyan, Senem; Gokce, Bagdagul; Ecemis, Tolga; Kaya, Cemil; Aksu, Arif Tarik; Gur Dedeoglu, Bala

    2016-01-01

    Polycystic ovary syndrome (PCOS) is a metabolic and endocrine disorder which affects women of reproductive age with prevalence of 8-18%. The oocyte within the follicle is surrounded by cumulus cells (CCs), which connect with mural granulosa cells (MGCs) that are responsible for secreting steroid hormones. The main aim of this study is comparing gene expression profiles of MGCs and CCs in PCOS and control samples to identify PCOS-specific differentially expressed genes (DEGs). In this study, two microarray databases were searched for mRNA expression microarray studies performed with CCs and MGCs obtained from PCOS patients and control samples. Three independent studies were selected to be integrated with naive meta-analysis since raw meta-data from these studies were found to be highly correlated. DEGs in these somatic cells were identified for PCOS and control groups. This study enabled us to reveal dysregulation in MAPK (mitogen activated protein kinase), insulin and Wnt signaling pathways between CCs and MGCs in PCOS. The meta-analysis results together with qRT-PCR validations provide evidence that molecular signaling is dysregulated through MGCs and CCs in PCOS, which is important for follicle and oocyte maturation and may contribute to the pathogenesis of the syndrome.

  19. Identification of Polycystic Ovary Syndrome (PCOS) Specific Genes in Cumulus and Mural Granulosa Cells

    PubMed Central

    Aydos, Alp; Gurel, Aykut; Oztemur Islakoglu, Yasemin; Noyan, Senem; Gokce, Bagdagul; Ecemis, Tolga; Kaya, Cemil; Aksu, Arif Tarik

    2016-01-01

    Polycystic ovary syndrome (PCOS) is a metabolic and endocrine disorder which affects women of reproductive age with prevalence of 8–18%. The oocyte within the follicle is surrounded by cumulus cells (CCs), which connect with mural granulosa cells (MGCs) that are responsible for secreting steroid hormones. The main aim of this study is comparing gene expression profiles of MGCs and CCs in PCOS and control samples to identify PCOS-specific differentially expressed genes (DEGs). In this study, two microarray databases were searched for mRNA expression microarray studies performed with CCs and MGCs obtained from PCOS patients and control samples. Three independent studies were selected to be integrated with naive meta-analysis since raw meta-data from these studies were found to be highly correlated. DEGs in these somatic cells were identified for PCOS and control groups. This study enabled us to reveal dysregulation in MAPK (mitogen activated protein kinase), insulin and Wnt signaling pathways between CCs and MGCs in PCOS. The meta-analysis results together with qRT-PCR validations provide evidence that molecular signaling is dysregulated through MGCs and CCs in PCOS, which is important for follicle and oocyte maturation and may contribute to the pathogenesis of the syndrome. PMID:27997581

  20. Functional Geonic Analysis of Breast Cancer Cell Tumorigenicity Using a Noval Gene Silencing Resource

    DTIC Science & Technology

    2007-04-01

    is downregulated. In the studies in Chinese hamster ovary cells , intracellular palmitate accumulation induces apoptosis through the generation of...15. SUBJECT TERMS FUNCTIONAL GENOMICS , CELL SIGNALING, GENE THERAPY, RNAI 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18...American Cancer Society “Wellness Day” May 2007 Functional genomics in mammalian cells (invited presentation), University of Pittsburgh Cancer

  1. WNT/β-catenin and p27/FOXL2 differentially regulate supporting cell proliferation in the developing ovary.

    PubMed

    Gustin, Sonja E; Hogg, Kirsten; Stringer, Jessica M; Rastetter, Raphael H; Pelosi, Emanuele; Miles, Denise C; Sinclair, Andrew H; Wilhelm, Dagmar; Western, Patrick S

    2016-04-15

    Sexual development is initiated through differentiation of testicular Sertoli cells or ovarian granulosa cells. Although these supporting cells are considered to develop from common bipotential precursors, recent evidence suggests that distinct supporting cell populations are present in the ovary, with one providing granulosa cells of the medullary follicles and the other providing granulosa cells of the cortical follicles, the latter of which support lifelong fertility. Here, we demonstrate that XX fetal gonads contain GATA4 expressing supporting cells that either enter mitotic arrest, or remain proliferative. Blocking WNT signalling reduces XX supporting cell proliferation, while stabilising β-catenin signalling promotes proliferation, indicating that the renewal of pre-granulosa cells is dependent on WNT/β-catenin signalling in the proliferative supporting cell population. In contrast, XX supporting cells express p27 and FOXL2 and are maintained in mitotic arrest. Although FOXL2 is required for maintaining high levels of p27 expression, it is dispensable for entry and maintenance of mitotic arrest in XX supporting cells. Combined our data suggest that both medullary and cortical precursors arise from a common GATA4 expressing cell type. In addition, this work indicates that a balance between supporting cell self-renewal and differentiation is maintained in the developing ovary by relative WNT/β-catenin and p27/FOXL2 activities. This study provides significant new insights into the origin and formation of ovarian follicles and evidence supporting a common fetal origin of medullary and cortical granulosa cells.

  2. A comparison of cell killing by heat and/or X rays in Chinese hamster V79 cells, Friend erythroleukemia mouse cells, and human thymocyte MOLT-4 cells.

    PubMed

    Raaphorst, G P; Szekely, J; Lobreau, A; Azzam, E I

    1983-05-01

    The radiation and/or heat sensitivity of Chinese hamster V79 cells, Friend erythroleukemia (FELC) mouse cells, and MOLT-4 human transformed thymocytes were compared. MOLT-4 cells were more radiosensitive (D0 = 0.50 Gy) than FELC (D0 = 0.65 Gy) and V79 cells (D0 = 1.43 Gy). Arrhenius analysis showed that MOLT-4 cells were more heat sensitive than FELC or V79 cells below 42.0 degrees C, but more heat resistant at higher temperatures. In addition, the MOLT-4 cells showed a single-heat inactivation energy between 41.0 and 45.0 degrees C, while FELC and V79 cells both showed a transition in the inactivation energy at about 43.0 and 43.5 degrees C, respectively. These differences may be related to the fact that the upper temperature limit for the development of thermal tolerance during continuous heating was lower for MOLT-4 cells than for FELC or V79 cells. Killing of FELC and V79 cells was dependent on the sequence in which heat and X rays were applied, but the greatest effect was obtained when both treatments were given simultaneously. Recovery occurred when treatments were separated by incubation at 37.0 degrees C. The MOLT-4 cells did not show a sequence dependence for heating and irradiation. Survival of MOLT-4 cells after heating and/or irradiation was compared using trypan blue dye exclusion or colony formation. Both assays showed similar qualitative responses, but survival levels measured by the trypan blue assay were much higher than those determined from the colony-forming assay.

  3. Juvenile granulosa cell tumour of the ovary presenting with hyperprolactinaemic amenorrhoea and galactorrhoea

    PubMed Central

    Iqbal, Ahmed; Lubina-Solomon, Alexandra; Kew, Fiona M; Webster, Jonathan

    2016-01-01

    Summary Secondary amenorrhoea and galactorrhoea represent a common endocrine presentation. We report a case of an oestrogen-producing juvenile granulosa cell tumour (JGCT) of the ovary in a 16-year-old post-pubertal woman with hyperprolactinaemia amenorrhoea and galactorrhoea which resolved following surgical resection of the tumour. This patient presented with a 9-month history of secondary amenorrhoea and a 2-month history of galactorrhoea. Elevated serum prolactin at 7081 mIU/l and suppressed gonadotropins (LH <0.1 U/l; FSH <0.1 U/l) were detected. Serum oestradiol was significantly elevated at 7442 pmol/l with undetectable β-human chorionic gonadotropin. MRI showed a bulky pituitary with no visible adenoma. MRI of the abdomen showed a 4.8 cm mass arising from the right ovary with no evidence of metastatic disease. Serum inhibin B was elevated at 2735 ng/l. A right salpingo-oophorectomy was performed, and histology confirmed the diagnosis of a JGCT, stage International Federation of Gynaecology and Obstetrics 1A. Immunohistochemical staining for prolactin was negative. Post-operatively, oestrogen and prolactin levels were normalised, and she subsequently had a successful pregnancy. In summary, we present a case of an oestrogen-secreting JGCT with hyperprolactinaemia manifesting clinically with galactorrhoea and secondary amenorrhoea. We postulate that observed hyperprolactinaemia was caused by oestrogenic stimulation of pituitary lactotroph cells, a biochemical state analogous to pregnancy. To the best of our knowledge, this is the first report of hyperprolactinaemia as a result of excessive oestrogen production in the context of a JGCT. Learning points Hyperprolactinaemia with bilateral galactorrhoea and secondary amenorrhoea has a wide differential diagnosis and is not always caused by a prolactin secreting pituitary adenoma.Significantly elevated serum oestradiol levels in the range seen in this case, in the absence of pregnancy, are indicative

  4. Chromatid breaks induced in Chinese hamster cells by low doses (12--100 rad) of pi--mesons.

    PubMed

    Mindek, G; Blattmann, H; Cordt, I; Fritz-Niggli, H

    1979-08-10

    Monolayer cultures of the fibroblast-like Chinese hamster cell-line 19/1 were irradiated in the G2-phase of the cell cycle by pi--mesons (6 rad/min peak-pion dose rate). Frequencies of induced single- and isochromatid breaks, acentric fragments and interchanges were compared with data obtained from 140 kV X-rays. The RBE-values were for the pion dose peak between 0.8--1.2 and for the pion dose plateau 0.5--0.9. Whereas for single chromatid breaks there was no significant difference between X-rays and peak pions for identical physical doses, the isochromatid breaks alone showed a significantly higher frequency for 100 rad peak pions.

  5. Influence of dose rate on the transformation of Syrian hamster embryo cells by fission-spectrum neutrons

    SciTech Connect

    Jones, C.A.; Sedita, B.A.; Hill, C.K.; Elkind, M.M.

    1988-01-01

    Several explanations for this neutron dose-rate effect have been proposed, but further investigation is necessary to determine the mechanisms involved. In all cell transformation studies to date the immortalized, aneuploid 10T1/2 cell-line has been used. These cells may be premalignant; thus their response characteristics and, in particular, the nature of the transformation event, might differ from that in a normal, fibroblast cell. One reason for the present study was to determine whether the low-dose-rate effect of fission neutrons could be demonstrated in normal cells. If so, a normal cell system, which would more closely resemble a normal in vivo system, could be used for mechanistic studies. We chose Syrian hamster embryo (SHE) fibroblasts which are normal, diploid cells with a limited life span in culture. Upon exposure to low doses of ionizing radiation, the fraction of the cells that are transformed can be identified in a standard 8--10 day colony assay by examining their clonal morphology. Transformed cells form colonies with a dense, criss-crossed or piled-up structure. A high percentage of the transformed colonies can be further propagated and will acquire additional neoplastic characteristics; i.e., anchorage independence, immortality, altered proteolytic activity, karyotype alterations, and finally, tumorigenicity.

  6. Do OH radicals react with organic substances in gamma-irradiated frozen cells of golden hamster embryo?

    NASA Astrophysics Data System (ADS)

    Yoshimura, Toru; Miyazaki, Tetsuo; Mochizuki, Shigehiro; Suzuki, Keiji; Watanabe, Masami

    Reactivity of OH radicals, produced by γ-irradiation, in golden hamster embryo (GHE) cells and in phosphate-buffered saline (PBS) solutions containing nuclei of the GHE cells was studied at 77 and 111 K by ESR spectroscopy. Since OH radicals do not diffuse in frozen cells at 77 K, they do not react with organic substances, such as protein and DNA, in the cells at 77 K. The efficiency of production and trapping of OH radicals at high concentration of organic substances in cells are the same as that at low concentration. Thus OH radicals produced near the organic substances do not react with them, being trapped at 77 K. When GHE cells or PBS-nuclei solutions are irradiated by γ-rays at 77 K and then warmed to 111 K, OH radicals decay fast, while the amounts of organic radicals remain constant at this temperature. These results indicate that OH radicals do not react with organic substances in γ-irradiated cells during warming of the irradiated cells. Therefore it was concluded that OH radicals are not the main reactive species responsible for biological effects in γ-irradiated frozen cells.

  7. Functional analysis of the RNAi response in ovary-derived silkmoth Bm5 cells.

    PubMed

    Kolliopoulou, Anna; Swevers, Luc

    2013-08-01

    Experiments of dsRNA-mediated gene silencing in lepidopteran insects in vivo are characterized by high variability although lepidopteran cell cultures have shown an efficient response to RNAi in transfection experiments. In order to identify the core RNAi factors that regulate the RNAi response of Lepidoptera, we employed the silkmoth ovary-derived Bm5 cells as a test system since this cell line is known to respond potently in silencing after dsRNA transfection. Two parallel approaches were used; involving knock-down of the core RNAi genes or over-expression of the main siRNA pathway factors, in order to study possible inhibition or stimulation of the RNAi silencing response, respectively. Components from all three main small RNA pathways (BmAgo-1 for miRNA, BmAgo-2/BmDcr-2 for siRNA, and BmAgo-3 for piRNA) were found to be involved in the RNAi response that is triggered by dsRNA. Since BmAgo-3, a factor in the piRNA pathway that functions independent of Dicer in Drosophila, was identified as a limiting factor in the RNAi response, sense and antisense ssRNA was also tested to induce gene silencing but proved to be ineffective, suggesting a dsRNA-dependent role for BmAgo-3 in Bombyx mori. After efficient over-expression of the main siRNA factors, immunofluorescence staining revealed a predominant cytoplasmic localization in Bm5 cells. This is the first study in Lepidoptera to provide evidence for possible overlapping of all three known small RNA pathways in the regulation of the dsRNA-mediated silencing response using transfected B. mori-derived Bm5 cells as experimental system.

  8. Amino acid sequence and posttranslational modifications of human factor VII sub a from plasma and transfected baby hamster kidney cells

    SciTech Connect

    Thim, L.; Bjoern, S.; Christensen, M.; Nicolaisen, E.M.; Lund-Hansen, T.; Pedersen, A.H.; Hedner, U. )

    1988-10-04

    Blood coagulation factor VII is a vitamin K dependent glycoprotein which in its activated form, factor VII{sub a}, participates in the coagulation process by activating factor X and/or factor IX in the presence of Ca{sup 2+} and tissue factor. Three types of potential posttranslational modifications exist in the human factor VII{sub a} molecule, namely, 10 {gamma}-carboxylated, N-terminally located glutamic acid residues, 1 {beta}-hydroxylated aspartic acid residue, and 2 N-glycosylated asparagine residues. In the present study, the amino acid sequence and posttranslational modifications of recombinant factor VII{sub a} as purified from the culture medium of a transfected baby hamster kidney cell line have been compared to human plasma factor VII{sub a}. By use of HPLC, amino acid analysis, peptide mapping, and automated Edman degradation, the protein backbone of recombinant factor VII{sub a} was found to be identical with human factor VII{sub a}. Asparagine residues 145 and 322 were found to be fully N-glycosylated in human plasma factor VII{sub a}. In the recombinant factor VII{sub a}, asparagine residue 322 was fully glycosylated whereas asparagine residue 145 was only partially (approximately 66%) glycosylated. Besides minor differences in the sialic acid and fucose contents, the overall carbohydrate compositions were nearly identical in recombinant factor VII{sub a} and human plasma factor VII{sub a}. These results show that factor VII{sub a} as produced in the transfected baby hamster kidney cells is very similar to human plasma factor VII{sub a} and that this cell line thus might represent an alternative source for human factor VII{sub a}.

  9. The insecticide buprofezin induces morphological transformation and kinetochore-positive micronuclei in cultured Syrian hamster embryo cells in the absence of detectable DNA damage.

    PubMed

    Herrera, L A; Ostrosky-Wegman, P; Schiffmann, D; Chen, Q Y; Ziegler-Skylakakis, K; Andrae, U

    1993-11-01

    The insecticide buprofezin was examined for its genotoxicity in cultured Syrian hamster embryo cells in order to better understand the mechanisms underlying the genotoxicity of the compound in mammalian cells. Exposure to buprofezin concentrations of 12.5-100 microM did not significantly affect the colony-forming ability of the cells, but did result in increased frequencies of morphologically transformed colonies. Treatment with buprofezin did not cause a detectable induction of DNA repair synthesis, an indicator of DNA damage, but significantly increased the frequency of micronuclei. Immunostaining of the cells with antikinetochore antibody (CREST antibody) showed that essentially all of the buprofezin-induced micronuclei were kinetochore-positive. The results suggest that morphological transformation of Syrian hamster embryo cells by buprofezin results from an interaction of the compound or a metabolite of it with the mitotic apparatus rather than from DNA damage.

  10. In vitro cytotoxicity and transforming potential of industrial carbon dust (fibers and particles) in syrian hamster embryo (SHE) cells.

    PubMed

    Darne, C; Terzetti, F; Coulais, C; Fournier, J; Guichard, Y; Gaté, L; Binet, S

    2010-07-01

    Carbon fibers have many applications, mainly in high-tech industries such as the aviation industry. Eleven carbon samples (fibers and particles) coming from an aeronautic group were tested for their cytotoxicity and carcinogenic potential using in vitro short-term assays in Syrian hamster embryo cells. These samples were taken during each important step of the process, i.e. from the initial heating of polyacrylonitrile fibers to pure carbon fibers. They were compared to an asbestos fiber, an amorphous silica, and two commercial graphite powders. Their physical-chemical characteristics and their capacity to release reactive oxygen species (ROS) were determined. This study showed that none of the carbon samples was able to generate ROS as measured by Electron Paramagnetic Resonance analysis, and in our biological assays, they demonstrated no morphological transformation potential and low cytotoxicity compared to positive control (chrysotile asbestos).

  11. Hammerhead ribozyme cleavage of hamster prion pre-mRNA in complex cell-free model systems.

    PubMed

    Denman, R B; Purow, B; Rubenstein, R; Miller, D L

    1992-07-31

    The cleavage properties of a trans-acting hammerhead ribozyme targeted 51 bases upstream of the putative splicing branch point in the hamster prion pre-mRNA intron were investigated in cell-free model systems in vitro. The specificity of cleavage was demonstrated by the inability of this ribozyme to cleave a non-homologous synthetic message encoding part of the beta amyloid peptide precursor, beta APP, and by the inability of the prion pre-mRNA to be cleaved by a ribozyme targeted to beta amyloid peptide precursor mRNA. Also, the addition of total RNA isolated from rat brain had only a minimal effect on the cleavage of the prion substrate pre-mRNA by the ribozyme. Finally neither the presence of 100 ng of nuclear or cytoplasmic proteins were found to affect the rate of cleavage in vitro.

  12. Suppressive action of near-ultraviolet light on ouabain resistance induced by far-ultraviolet light in Chinese hamster cells.

    PubMed

    Suzuki, F; Han, A; Hill, C K; Elkind, M M

    1983-03-01

    The interaction between ultraviolet light (UV-C) from germicidal lamps (254 nm) and near-ultraviolet light (UV-B) from Westinghouse Sun Lamps (290-345 nm) was studied in Chinese hamster V79 cells by measuring the effectiveness of combined exposures to induce the resistance to 6-thioguanine or to ouabain. Exposure of cells to a conditioning dose of UV-B (approximately 70% survival) results in significant inhibition of the induction by UV-C of cells resistant to ouabain. The inhibition is lost, however, if cells are incubated for 12 h at 37 degrees C between exposures. Inhibition is also observed when cells are preirradiated with a dose of UV-B filtered with polystyrene (300-345 nm) which, in itself, has no effect on cell killing. Conditioning exposures of unfiltered or filtered UV-B light do not inhibit the induction of 6-thioguanine-resistant cells by UV-C light, and the effects of UV-B and UV-C light are largely independent.

  13. A comparison of enzyme activity mutation frequencies in germ cells of mice (Mus musculus) and golden hamsters (Mesocricetus auratus) after exposure to 2 + 2 Gy gamma-irradiation.

    PubMed

    Pretsch, W; Neuhäuser-Klaus, A; Favor, J

    2000-01-01

    The radiation-induced germ cell mutation rate has been investigated in two species of mammals. Mice and golden hamsters of both sexes were exposed to 2 + 2 Gy gamma-irradiation with a 24 h fractionation interval and mated to untreated partners. In mice, specific locus mutations were examined as positive controls and the obtained mutation rates (per locus and gamete x10(-5)) were 51.4, 10.1, 13.6 and 17.4 for irradiated post-spermatogonia, spermatogonia and 1-7 and >7 days post-treatment oocytes, respectively. Offspring of mice and golden hamsters were screened for activity alterations of 10 erythrocyte enzymes coded by at least 14 loci. The observed mutation rates per locus per gamete x10(-5) for treated post-spermatogonial stages, spermatogonia and oocytes 1-7 and >7 days post-treatment were 6.5, 1.5, 8.8 and 7.0, respectively, for mice and 16.7, 0, 7.6 and 0, respectively, for golden hamsters. There is a significant difference for mutation rates in mouse oocytes 1-7 days post-treatment compared with the control. No differences in the frequencies of mutations in the various germ cell stages could be observed between mice and golden hamsters. A critical assumption for the extrapolation of experimental mutagenesis studies to humans is that no species effects exist in sensitivity to mutation induction by irradiation. Our results do not contradict this assumption.

  14. Role of Adjuvant Radiotherapy in Granulosa Cell Tumors of the Ovary

    SciTech Connect

    Hauspy, Jan; Beiner, Mario E.; Harley, Ian; Rosen, Barry; Murphy, Joan; Chapman, William; Le, Lisa W.; Fyles, Anthony; Levin, Wilfred

    2011-03-01

    Purpose: To review the role of adjuvant radiotherapy (RT) in the outcome and recurrence patterns of granulosa cell tumors (GCTs) of the ovary. Methods and Materials: The records of all patients with GCTs referred to the Princess Margaret Hospital University Health Network between 1961 and 2006 were retrospectively reviewed. The patient, tumor, and treatment factors were assessed by univariate and multivariate analyses using disease-free survival (DFS) as the endpoint. Results: A total of 103 patients with histologically confirmed GCTs were included in the present study. The mean duration of follow-up was 100 months (range, 1-399). Of the 103 patients, 31 received adjuvant RT. A total of 39 patients developed tumor recurrence. The tumor size, incidence of intraoperative rupture, and presence of concurrent endometrial cancer were not significant risk factors for DFS. The median DFS was 251 months for patients who underwent adjuvant RT compared with 112 months for patients who did not (p = .02). On multivariate analysis, adjuvant RT remained a significant prognostic factor for DFS (p = .004). Of the 103 patients, 12 had died and 44 were lost to follow-up. Conclusion: Ovarian GCTs can be indolent, with patients achieving long-term survival. In our series, adjuvant RT resulted in a significantly longer DFS. Ideally, randomized trials with long-term follow-up are needed to define the role of adjuvant RT for ovarian GCTs.

  15. Ci antagonizes Hippo signaling in the somatic cells of the ovary to drive germline stem cell differentiation.

    PubMed

    Li, Chaoyi; Kan, Lijuan; Chen, Yan; Zheng, Xiudeng; Li, Weini; Zhang, Wenxin; Cao, Lei; Lin, Xiaohui; Ji, Shanming; Huang, Shoujun; Zhang, Guoqiang; Liu, Xiaohui; Tao, Yi; Wu, Shian; Chen, Dahua

    2015-10-01

    Many stem cell populations are tightly regulated by their local microenvironment (niche), which comprises distinct types of stromal cells. However, little is known about mechanisms by which niche subgroups coordinately determine the stem cell fate. Here we identify that Yki, the key Hippo pathway component, is essential for escort cell (EC) function in promoting germline differentiation in Drosophila ovary. We found that Hedgehog (Hh) signals emanating primarily from cap cells support the function of ECs, where Cubitus interruptus (Ci), the Hh signaling effector, acts to inhibit Hippo kinase cascade activity. Mechanistically, we found that Ci competitively interacts with Hpo and impairs the Hpo-Wts signaling complex formation, thereby promoting Yki nuclear localization. The actions of Ci ensure effective Yki signaling to antagonize Sd/Tgi/Vg-mediated default repression in ECs. This study uncovers a mechanism explaining how subgroups of niche cells coordinate to determine the stem cell fate via Hh-Hippo signaling crosstalk, and enhances our understanding of mechanistic regulations of the oncogenic Yki/YAP signaling.

  16. Persistence of sister chromatid exchanges and in vitro morphological transformation of Syrian hamster fetal cells by chemical and physical carcinogens

    SciTech Connect

    Popescu, N.C.; Amsbaugh, S.C.; DiPaolo, J.A.

    1985-11-01

    The induction of neoplastic cell transformation is closely associated with DNA alterations which occur shortly after carcinogen exposure. Sister chromatid exchange (SCE) formation is a sensitive indicator of carcinogen-DNA interaction and correlates with the induction of morphological cell transformation. The persistence of lesions generating SCE produced by chemical and physical carcinogens and its relevance to the induction of morphologic transformation was evaluated in coordinated experiments with cultured Syrian hamster fetal cells (HFC). Exponentially growing HFC were exposed for 1 h to benzo(a)pyrene (BP), methyl-methanesulfonate (MMS), cis-platinum (II) diaminedichloride (cis Pt II), N-methyl-N'-nitrosourea (MNU), mitomycin C (MMC), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-acetoxy-2-fluorenyl-acetamide (AcAAF) or u.v. light irradiated. SCE analysis demonstrates that for a period of 48 h after carcinogen exposure, during which time the cells undergo at least four replicative cycles, DNA damage generating SCE induced by all chemical carcinogens either persisted or was partially removed, whereas u.v.-induced lesions were completely removed. An elevated SCE frequency persisted after two additional cell cycles after treatment with BP, AcAAF or MMC without increased cell lethality as compared to other carcinogens whose lesions were completely eliminated during the same period.

  17. Effects of low level laser treatment on the survival of axotomized retinal ganglion cells in adult Hamsters

    PubMed Central

    So, Kwok-Fai; Leung, Mason Chin Pang; Cui, Qi

    2014-01-01

    Injury to axons close to the neuronal bodies in the mammalian central nervous system causes a large proportion of parenting neurons to degenerate. It is known that optic nerve transection close to the eye in rodents leads to a loss of about half of retinal ganglion cells in 1 week and about 90% in 2 weeks. Using low level laser treatment in the present study, we demonstrated that treatment with helium-neon (660 nm) laser with 15 mW power could delay retinal ganglion cell death after optic nerve axotomy in adult hamsters. The effect was most apparent in the first week with a short period of treatment time (5 minutes) in which 65–66% of retinal ganglion cells survived the optic nerve axotomy whereas 45–47% of retinal ganglion cells did so in optic nerve axotomy controls. We also found that single dose and early commencement of laser irradiation were important in protecting retinal ganglion cells following optic nerve axotomy. These findings thus convincingly show that appropriate laser treatment may be neuroprotective to retinal ganglion cells. PMID:25558230

  18. Cell proliferation as a requirement for development of the contact effect in Chinese hamster V79 spheroids

    SciTech Connect

    Olive, P.L.

    1989-01-01

    Chinese hamster V79 cells grown for several hours in suspension culture form spheroids which are more resistant to killing by ionizing radiation than cells grown on petri dishes, a phenomenon known as the contact effect. Previous results using the alkali-unwinding assay as a measure of DNA damage have implicated differences in DNA conformation as contributing to this effect; spheroid DNA denatures more slowly in dilute alkali than monolayer DNA, perhaps due to the presence of constraints to DNA unwinding. In this paper, the rate of development of radiation resistance is shown to be similar when either cell survival or DNA unwinding is used as an end point. At the midpoint for development of resistance, approximately 10 h, the unwinding kinetics indicate that either half of the cells contain constraints to DNA unwinding, or half of the DNA in all of the cells contains constraints. The latter explanation appears more likely since all cells seem to develop these constraints at the same rate, regardless of position in the cell cycle or the degree of contact with other cells. Results using the microelectrophoresis assay to measure damage to individual nuclei confirm the fact that 10-h cultures show a homogeneous radiation response intermediate between that of monolayers and spheroids. Incubation of cells at room temperature or in the presence of drugs which inhibit cell cycle progression prevents full development of the contact effect. Conversely, incubation of cells in medium containing inhibitors of polyamine synthesis, adenylcyclase, glutathione synthesis, poly(ADP-ribose)polymerase, topoisomerase II, or cell-cell communication does not inhibit development of the contact effect as measured by DNA-unwinding kinetics.

  19. Cell-Type Specific Increases in Female Hamster Nucleus Accumbens Spine Density following Female Sexual Experience

    PubMed Central

    Staffend, Nancy A.; Hedges, Valerie L.; Chemel, Benjamin R.; Watts, Val J.; Meisel, Robert L.

    2013-01-01

    Female sexual behavior is an established model of a naturally motivated behavior which is regulated by activity within the mesolimbic dopamine system. Repeated activation of the mesolimbic circuit by female sexual behavior elevates dopamine release and produces persistent postsynaptic alterations to dopamine D1 receptor signaling within the nucleus accumbens. Here we demonstrate that sexual experience in female Syrian hamsters significantly increases spine density and alters morphology selectively in D1 receptor expressing medium spiny neurons within the nucleus accumbens core, with no corresponding change in dopamine receptor binding or protein expression. Our findings demonstrate that previous life experience with a naturally motivated behavior has the capacity to induce persistent structural alterations to the mesolimbic circuit that can increase reproductive success and are analogous to the persistent structural changes following repeated exposure to many drugs of abuse. PMID:23934655

  20. Effect of mitochondrial inhibitors on metaphase-telophase progression and nuclear membrane formation in Chinese hamster cells.

    PubMed

    Chai, L S; Schumer, J M; Sandberg, A A

    1985-01-01

    Chinese hamster Don cells in log-phase were exposed to Colcemid during the G2 period with and without a combination of divalent cation chelators and mitochondrial inhibitors. Isolated metaphase cells were incubated as follows: (i) without Colcemid but with other agents and the progression was monitored from metaphase (M) to telophase (Tel) and to cell division; (ii) with Colcemid and other agents and the rate of micronuclei formation in the absence of anaphase was studied. Both EDTA and EGTA accelerated the progression from M to Tel, but did not affect the overall rate of cell division. Chloramphenicol (CAP), an inhibitor of mitochondrial protein synthesis, blocked the effect of the chelators and also retarded the progression. An inhibitor of mitochondrial respiration, Antimycin A (AA), also retarded the progression in the absence of the chelators and prevented the promoting effect of the chelators. A stimulator of ATPase for ATP breakdown. 2,4-dinitrophenol (DNP), accelerated the M to Tel progression. Chloramphenicol (CAP) and AA, as well as DNP, appeared to have little effect on the formation of micronuclei in the presence of Colcemid. EGTA, which affects cell surface Ca2+, stimulated the formation of micronuclei. This study indicates that Ca2+ ions and mitochondrial function are involved in the regulation of a certain segment of mitosis beyond metaphase, with Ca2+ sequestration in the mitochondria and chelation of Ca2+ by EGTA as dominant factors.

  1. Endogenous ADP-ribosylation of elongation factor 2 in polyoma virus-transformed baby hamster kidney cells

    SciTech Connect

    Fendrick, J.L.; Iglewski, W.J. )

    1989-01-01

    Polyoma virus-transformed baby hamster kidney (pyBHK) cells were cultured in medium containing ({sup 32}P)orthophosphate and 105 (vol/vol) fetal bovine serum. A {sup 32}P-labeled protein with an apparent molecular mass of 97 kDa was immunoprecipitated from cell lysates with antiserum to ADP-ribosylated elongation factor 2 (EF-2). The {sup 32}P labeling of the protein was enhanced by culturing cells in medium containing 2% serum instead of 10% serum. The {sup 32}P label was completely removed from the protein by treatment with snake venom phosphodiesterase and the digestion product was identified as ({sup 32}P)AMP, indicating the protein was mono-ADP-ribosylated. HPLC analysis of tryptic peptides of the {sup 32}P-labeled 97-kDa protein and purified EF-2, which was ADP-ribosylated in vitro with diphtheria toxin fragment A and ({sup 32}P)NAD, demonstrated an identical labeled peptide in the two proteins. The data strongly suggest that EF-2 was endogenously ADP-ribosylated in pyBHK cells. Maximum incorporation of radioactivity in EF-2 occurred by 12 hr and remained constant over the subsequent 12 hr. It was estimated that 30-35% of the EF-2 was ADP-ribosylated in cells cultured in medium containing 2% serum. When {sup 32}P-labeled cultures were incubated in medium containing unlabeled phosphate, the {sup 32}P label was lost from the EF-2 within 30 min.

  2. Effect of intercellular contact on DNA conformation, radiation-induced DNA damage, and mutation in Chinese hamster V79 cells

    SciTech Connect

    Olive, P.L.; Durand, R.E.

    1985-01-01

    Chinese hamster V79 cells, when grown as small spheroids in suspension culture, are more resistant to killing by ionizing radiation than when grown as monolayers. The authors have attempted to determine whether this enhanced survival following irradiation is reflected in DNA damage and repair at the structural level (by measuring alkali-induced DNA unwinding rates from strand breaks) and at the functional level (by measuring resistance to forward mutation at the HGPRT locus). For a given dose of radiation, the unwinding of DNA in high salt/weak alkali was less complete for spheroid DNA than for monolayer DNA, and the rate of repair of radiation damage was faster in spheroid DNA. These differential responses were lost 8 hr after separation of spheroids into single cells, coinciding with loss of radioresistance measured by clonogenicity. In addition, spheroid cells showed fewer numbers of induced mutants per Gray, although, for a given level of survival, the mutation frequency for monolayers and spheroids was identical. These results suggest that conformational changes in DNA resulting from cell growth as spheroids might enhance repair of radiation-induced lesions.

  3. Ability of 13 chemical agents used in dental practice to induce sister-chromatid exchanges in Syrian hamster embryo cells.

    PubMed

    Miyachi, Takashi; Tsutsui, Takeki

    2005-09-01

    To evaluate the genotoxic potential of 13 chemical agents used in dental practice, the abilities of these agents to induce sister-chromatid exchanges (SCEs) were examined using Syrian hamster embryo (SHE) cells. Statistically significant increases in the frequencies of SCEs were observed in SHE cells treated with all seven of the chemical agents used as endodontic medicaments: p-chlorophenol, m-cresol, formaldehyde, guaiacol, hydrogen peroxide, p-phenolsulfonic acid, and sodium hypochlorite (P < 0.01; Student t test). Assessment of two chemical agents that are applied to the oral mucosa as antiseptics showed that SCEs were induced by iodine (P < 0.01), but not by chlorhexidine. Of three chemical agents that are used as dyes for disclosing dental plaque, erythrosine B had no effect on SCE induction, while acid fuchsin and basic fuchsin increased the SCE frequencies in SHE cells (P < 0.01). Glutaraldehyde, which is used as a disinfectant for dental instruments and impressions, also induced SCEs (P < 0.01). Because SCE assays are used as a sensitive indicator for evaluating genetic toxicity of chemicals, the chemical agents that had a positive response in the present study are potentially genotoxic to mammalian cells.

  4. Differential expression of p53, p63 and p73 protein and mRNA for DMBA-induced hamster buccal-pouch squamous-cell carcinomas

    PubMed Central

    Chen, Yuk-Kwan; Huse, Shue-Sang; Lin, Li-Min

    2004-01-01

    Abnormalities in the p53 gene are regarded as the most consistent of the genetic abnormalities associated with oral squamous-cell carcinoma. Two related members of the p53 gene family, p73 and p63, have shown remarkable structural similarity to p53, suggesting possible functional and biological interactions. The purpose of this study was to investigate the differential expression of p73, p63 and p53 genes for DMBA-induced hamster buccal-pouch squamous-cell carcinoma. Immunohistochemical analysis for protein expression and reverse transcriptase-polymerase chain reaction (RT-PCR) for mRNA expression were performed for 40 samples of hamster buccal pouches, the total being separated into one experimental group (15-week DMBA-treated; 20 animals) and two control groups (untreated and mineral oil-treated; 10 animals each). Using immunohistochemical techniques, nuclear staining of p53 and p73 proteins was detected in a subset of hamster buccal-pouch tissue specimens treated with DMBA for a period of 15 weeks, whereas p63 proteins were noted for all of the 20 hamster buccal-pouch tissue specimens treated with DMBA for 15 weeks as well as for all of the untreated and mineral oil-treated hamster buccal-pouch tissue specimens. Differential expression of p63, p73 and p53 protein for the experimental group was as follows: p63+/p73+/p53+ (n = 14; 70%); p63+/p73+/p53− (n = 2; 10%); p63+/p73−/p53− (n = 4; 20%) and p63+/p73−/p53− (untreated [n = 10] and mineral oil-treated mucosa [n = 10]; 100% each). Upon RT-PCR, ΔNp63mRNA was detected within all of the 20 hamster buccal-pouch tissue specimens treated with DMBA for 15 weeks, whereas expression of TAp63 was not detected. Furthermore, p73 mRNA was identified for 16 of the hamster buccal-pouch tissue specimens treated with DMBA for 15 weeks, whereas p53 mRNA was noted for 14 15-week DMBA-treated pouches. The proportional (percentage) expression of ΔNp63, p73 and p53 mRNA for the hamster buccal-pouch tissue specimens

  5. Cryopreservation of ovaries from neonatal marmoset monkeys

    PubMed Central

    Motohashi, Hideyuki H.; Ishibashi, Hidetoshi

    2016-01-01

    The ovary of neonatal nonhuman primates contains the highest number of immature oocytes, but its cryopreservation has not yet been sufficiently investigated in all life stages. In the current study, we investigated cryodamage after vitrification/warming of neonatal ovaries from a nonhuman primate, the common marmoset (Callithrix jacchus). A Cryotop was used for cryopreservation of whole ovaries. The morphology of the vitrified/warmed ovaries was found to be equivalent to that of fresh ovaries. No significant difference in the number of oocytes retaining normal morphology per unit area in histological sections was found between the two groups. In an analysis of dispersed cells from the ovaries, however, the cell viability of the vitrified/warmed group tended to be decreased. The results of a comet assay showed no significant differences in DNA damage. These results show that cryopreservation of neonatal marmoset ovaries using vitrification may be useful as a storage system for whole ovaries. PMID:26876597

  6. Measurement of Bluetongue Virus Binding to a Mammalian Cell Surface Receptor by an In Situ Immune Fluorescent Staining Technique

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A quantifiable in situ immune fluorescent assay (IFA) was developed to measure bluetongue virus (BTV) binding to mammalian cells. The utility of the assay was demonstrated with both Chinese hamster ovary (CHO) and bovine pulmonary artery endothelial (CPAE) cells. Since heparin sulfate (HS) has been ...

  7. Effects of magnetized water on ovary, pre-implantation stage endometrial and fallopian tube epithelial cells in mice

    PubMed Central

    Hafizi, Leili; Gholizadeh, Mostafa; Karimi, Mohammad; Hosseini, Golkoo; Mostafavi-Toroghi, Hesam; Haddadi, Mehdi; Rezaiean, Amin; Ebrahimi, Mahmoud; Emami Meibodi, Neda

    2014-01-01

    Background: Magnetized water has made many improvements in industry, agriculture and medicine. However its utilization in medicine still remains controversial. Objective: In this study we aimed to investigate the effects of magnetized water on height of epithelial cells in pre-implantation stage endometrium and fallopian tube and number of corpus lutea in female mice. Materials and Methods: Eighty female NRMI mice were recruited to this experimental study and randomly divided into two groups: the control group which drank normal water and the experimental (case) group which drank magnetized water for 2 weeks. Super-ovulation was induced in these mice and then they were mated with male mice as well. Samples of ovary, uterus and fallopian tube were obtained at the pre-implantation stage. Then, after preparation, the number of corpus lutea in each ovary was counted and the height of fallopian and endometrial epithelial cells was measured by light microscopy. Results: Data analysis showed a significant increase in the mean number of corpus lutea and the height of epithelial cells in fallopian tube comparing the case with the control group (p=0.01, p=0.002 respectively) whereas uterus epithelial cells of the case group showed insignificant increase in height, in compare with the control group (p=0.052). Conclusion: Our results suggest that magnetized water intake increases the number of corpus lutea and the height of fallopian tube epithelial cells. Further research is needed to determine whether this will increase in the success rate of fertility. PMID:24976818

  8. gone early, a novel germline factor, ensures the proper size of the stem cell precursor pool in the Drosophila ovary.

    PubMed

    Matsuoka, Shinya; Gupta, Swati; Suzuki, Emiko; Hiromi, Yasushi; Asaoka, Miho

    2014-01-01

    In order to sustain lifelong production of gametes, many animals have evolved a stem cell-based gametogenic program. In the Drosophila ovary, germline stem cells (GSCs) arise from a pool of primordial germ cells (PGCs) that remain undifferentiated even after gametogenesis has initiated. The decision of PGCs to differentiate or remain undifferentiated is regulated by somatic stromal cells: specifically, epidermal growth factor receptor (EGFR) signaling activated in the stromal cells determines the fraction of germ cells that remain undifferentiated by shaping a Decapentaplegic (Dpp) gradient that represses PGC differentiation. However, little is known about the contribution of germ cells to this process. Here we show that a novel germline factor, Gone early (Goe), limits the fraction of PGCs that initiate gametogenesis. goe encodes a non-peptidase homologue of the Neprilysin family metalloendopeptidases. At the onset of gametogenesis, Goe was localized on the germ cell membrane in the ovary, suggesting that it functions in a peptidase-independent manner in cell-cell communication at the cell surface. Overexpression of Goe in the germline decreased the number of PGCs that enter the gametogenic pathway, thereby increasing the proportion of undifferentiated PGCs. Inversely, depletion of Goe increased the number of PGCs initiating differentiation. Excess PGC differentiation in the goe mutant was augmented by halving the dose of argos, a somatically expressed inhibitor of EGFR signaling. This increase in PGC differentiation resulted in a massive decrease in the number of undifferentiated PGCs, and ultimately led to insufficient formation of GSCs. Thus, acting cooperatively with a somatic regulator of EGFR signaling, the germline factor goe plays a critical role in securing the proper size of the GSC precursor pool. Because goe can suppress EGFR signaling activity and is expressed in EGF-producing cells in various tissues, goe may function by attenuating EGFR signaling

  9. The death of sertoli cells and the capacity to phagocytize elongated spermatids during testicular regression due to short photoperiod in Syrian hamster (Mesocricetus auratus).

    PubMed

    Seco-Rovira, Vicente; Beltrán-Frutos, Esther; Ferrer, Concepción; Sáez, Francisco José; Madrid, Juan Francisco; Pastor, Luis Miguel

    2014-05-01

    In the Syrian hamster (Mesocricetus auratus), an animal that displays testicular regression due to short photoperiod, germ cells are removed by apoptosis during this process and the apoptotic remains are phagocytized by Sertoli cells. The aim of this work was to investigate morphologically whether the testicular regression process due to short photoperiod leads to the apoptosis of Sertoli cells, and whether, during testicular regression, the elongated spermatids are eliminated through phagocytosis by Sertoli cells. To this end, we studied testis sections during testicular regression in Syrian hamster subjected to short photoperiod by means of several morphological techniques using conventional light microscopy (hematoxylin and eosin [H&E], semi-thin section vimentin, immunohistochemistry, SBA lectin, and TUNEL staining), fluorescence microscopy, and transmission electron microscopy (TEM). H&E and semi-thin sections identified Sertoli cells with a degenerated morphology. Greater portion of Sertoli cells that were positive for TUNEL staining were observed especially during the mild regression (MR) and strong regression (SR) phases. In addition, TEM identified the characteristic apoptotic changes in the nucleus and cytoplasm of Sertoli cells. Moreover, during testicular regression and using light microscopy, some elongated spermatids were seen in basal position next to the Sertoli cell nucleus. This Sertoli phagocytic activity was higher in MR and SR phases. TEM confirmed this to be the result of the phagocytic activity of Sertoli cells. In conclusion, during testicular regression in Syrian hamster due to short photoperiod, when germ cells are known to be lost through apoptosis, there is morphological evidences that Sertoli cells are also lost through apoptosis, while some elongated spermatids are phagocytized and eliminated by the Sertoli cells.

  10. Indium chloride-induced micronuclei via reactive oxygen species in Chinese hamster lung fibroblast V79 cells.

    PubMed

    Lin, Ruey-Hseng; Yang, Ming-Ling; Li, Yi-Ching; Chang, Hui-Min; Kuan, Yu-Hsiang

    2013-10-01

    We study the cytotoxicity of indium chloride (InCl₃) in Chinese hamster lung fibroblasts, the V79 cells, using MTT assay. The results showed that InCl₃ did not induce significant cytotoxicity at various concentrations tested. In addition, the frequency of micronuclei (MN) was assayed to evaluate the genotoxic effects of InCl₃ in V79 cells. InCl₃ at concentrations ranged 0.1-1 μM significantly increased MN frequency in a concentration-dependent manner. Both catalase and superoxide dismutase at concentrations of 75 and 150 μg/mL significantly inhibited InCl₃-induced MN. Similarly, Germanium oxide (GeO₂) and dimercaprol expressed antigenotoxic effects. From these findings, it is concluded that InCl₃ is a potent genotoxic chemical, which may be mediated partly by inducing oxidative stress. The significance of this study shows that the workers in the semiconductor factories should be cautious in exposing to the hazardous genotoxic InCl₃.

  11. Interlaboratory studies with the Chinese hamster V79 cell metabolic cooperation assay to detect tumor-promoting agents

    SciTech Connect

    Bohrman, J.S.; Burg, J.R.; Elmore, E.; Gulati, D.K.; Barfknecht, T.R.; Niemeier, R.W.; Dames, B.L.; Toraason, M.; Langenbach, R.

    1988-01-01

    Three laboratories participated in an interlaboratory study to evaluate the usefulness of the Chinese hamster V79 cell metabolic cooperation assay to predict the tumor-promoting activity of selected chemical. Twenty-three chemicals of different chemical structures (phorbol esters, barbiturates, phenols, artificial sweeteners, alkanes, and peroxides) were chosen for testing based on in vivo promotion activities, as reported in the literature. Assay protocols and materials were standardized, and the chemicals were coded to facilitate unbiased evaluation. A chemical was tested only once in each laboratory, with one of the three laboratories testing only 15 out of 23 chemicals. Dunnett's test was used for statistical analysis. Chemicals were scored as positive (at least two concentration levels statistically different than control), equivocal (only one concentration statistically different), or negative. For 15 chemicals tested in all three laboratories, there was complete agreement among the laboratories for nine chemicals. For the 23 chemicals tested in only two laboratories, there was agreement on 16 chemicals. With the exception of the peroxides and alkanes, the metabolic cooperation data were in general agreement with in vivo data. However, an overall evaluation of the V79 cell system for predicting in vivo promotion activity was difficult because of the organ specificity of certain chemicals and/or the limited number of adequately tested nonpromoting chemicals.

  12. The effect of pinealectomy, melatonin and leptin hormones on ovarian follicular development in female Syrian hamsters (Mesocricetus auratus).

    PubMed

    Karakaş, A; Kaya, Aliye; Gündüz, B

    2010-12-01

    We studied the effects of melatonin and leptin hormones on ovarian follicular development in intact and pinealectomized female Syrian hamsters. We first monitored the oestrous cycle of the hamsters by the vaginal smear samples throughout a ten day period to start the injections simultaneously in all groups and performed saline, melatonin and leptin hormone injection groups for both control and pinealectomized hamsters. Then the injections were applied for four days starting the oestrus phase of the cycle and the ovaries were removed for preparation of histological analysis. We measured the diameters and the numbers of the follicles and we classified the follicles according to the number of the granulosa cell layer. Leptin hormone injection increased melatonin hormone injection decreased the number and the diameter of the follicles. The stimulating effect of the leptin hormone was more pronounced in the pinealectomized group. The results of the present study indicate that the removal of the pineal gland and leptin hormone administration are playing a stimulatory while melatonin hormone administration is playing an inhibitory role on the follicular development in female Syrian hamsters.

  13. Rapid turn-over of plasma membrane sphingomyelin and cholesterol in baby hamster kidney cells after exposure to sphingomyelinase.

    PubMed

    Slotte, J P; Härmälä, A S; Jansson, C; Pörn, M I

    1990-12-14

    Plasma membrane sphingomyelin in baby hamster kidney (BHK-21) cells was hydrolyzed with sphingomyelinase (Staphylococcus aureus) and the effects on membrane cholesterol translocation and the properties of membrane bound adenylate cyclase and Na+/K(+)-ATPase were determined. Exposure of confluent BHK-21 cells to 0.1 U/ml of sphingomyelinase led to the degradation (at 37 degrees C) of about 60% of cell sphingomyelin. No simultaneous hydrolysis of phosphatidylcholine occurred. The hydrolysis of sphingomyelin subsequently led to the translocation (within 40 min) of about 50-60% of cell [3H]cholesterol from a cholesterol oxidase susceptible pool to an oxidase resistant compartment. The translocation of [3H]cholesterol from the cell surface to intracellular membranes was accompanied by a paralleled increase in [3H]cholesterol ester formation. When cells were first exposed to sphingomyelinase (to degrade sphingomyelin) and then incubated without the enzyme in serum-free media, the mass of cell sphingomyelin decreased initially (by 60%), but then began to increase and reached control levels within 3-4 h. The rapid re-synthesis of sphingomyelin was accompanied by an equally rapid normalization of cell [3H]cholesterol distribution. The re-formation of cell sphingomyelin also led to a decreased content of cellular [3H]cholesterol esters, indicating that unesterified [3H]cholesterol was pulled out of the cholesterol ester cycle and transported to the cell surface. Exposure of BHK-21 cells to sphingomyelinase further led to a dramatically decreased activity of ouabain-sensitive Na+/K(+)-ATPase, whereas forskolin-stimulated adenylate cyclase activity was not affected. The activity of Na+/K(+)-ATPase returned to normal in parallel with the normalization of cell sphingomyelin mass and cholesterol distribution. We conclude that sphingomyelin has profound effects on the steady-state distribution of cell cholesterol, and that manipulations of cell sphingomyelin levels directly and

  14. Gene mutations, chromosome aberrations and survivability after X-ray irradiation of Chinese hamster cell culture under conditions of cysteamine protection

    SciTech Connect

    Yesilova, T.V.; Feoktistova, T.P.

    1984-06-01

    Experimental results were reported to the determination of protective action of cysteamine on the yield of genetic mutations, chromosome aberrations and cell kill during reproduction, evidently due to damage of genetic structures. The experiments were performed on transplanted fibroblast cells of Chinese hamsters, clone 431 in which 80% of the cells had pseudodiloidy. A dose-modifying factor of 2 was established for chromosome aberrations and cell inactivation and a factor of 2.8 for the gene mutations. The data obtained led to a conclusion that there are general protective mechanisms which include the reaction of cysteamine on the radiation-chemical level and possible effect on the reparative processes.

  15. Classification of agents using Syrian hamster embryo (SHE) cell transformation assay (CTA) with ATR-FTIR spectroscopy and multivariate analysis.

    PubMed

    Ahmadzai, Abdullah A; Trevisan, Júlio; Pang, Weiyi; Riding, Matthew J; Strong, Rebecca J; Llabjani, Valon; Pant, Kamala; Carmichael, Paul L; Scott, Andrew D; Martin, Francis L

    2015-09-01

    The Syrian hamster embryo (SHE) cell transformation assay (pH 6.7) has a reported sensitivity of 87% and specificity of 83%, and an overall concordance of 85% with in vivo rodent bioassay data. To date, the SHE assay is the only in vitro assay that exhibits multistage carcinogenicity. The assay uses morphological transformation, the first stage towards neoplasm, as an endpoint to predict the carcinogenic potential of a test agent. However, scoring of morphologically transformed SHE cells is subjective. We treated SHE cells grown on low-E reflective slides with 2,6-diaminotoluene, N-nitroso-N-ethylnitroguanidine, N-nitroso-N-methylurea, N-nitroso-N-ethylurea, EDTA, dimethyl sulphoxide (DMSO; vehicle control), methyl methanesulfonate, benzo[e]pyrene, mitomycin C, ethyl methanesulfonate, ampicillin or five different concentrations of benzo[a]pyrene. Macroscopically visible SHE colonies were located on the slides and interrogated using attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy acquiring five spectra per colony. The acquired IR data were analysed using Fisher's linear discriminant analysis (LDA) followed by principal component analysis (PCA)-LDA cluster vectors to extract major and minor discriminating wavenumbers for each treatment class. Each test agent vs. DMSO and treatment-induced transformed cells vs. corresponding non-transformed were classified by a unique combination of major and minor discriminating wavenumbers. Alterations associated with Amide I, Amide II, lipids and nucleic acids appear to be important in segregation of classes. Our findings suggest that a biophysical approach of ATR-FTIR spectroscopy with multivariate analysis could facilitate a more objective interrogation of SHE cells towards scoring for transformation and ultimately employing the assay for risk assessment of test agents.

  16. Activation of mitochondrial promoter P{sub H}-binding protein in a radio-resistant Chinese hamster cell strain associated with Bcl-2

    SciTech Connect

    Roychoudhury, Paromita; Ghosh, Utpal . E-mail: keyachaudhuri@yahoo.com

    2006-11-17

    The cellular response to ionizing radiation is mediated by a complex interaction of number of proteins involving different pathways. Previously, we have shown that up regulation of mitochondrial genes ND1, ND4, and COX1 transcribed from the heavy strand promoter (P{sub H}) has been increased in a radio-resistant cell strain designated as M5 in comparison with the parental Chinese hamster V79 cells. These genes are also up regulated in Chinese hamster V79 cells VB13 that express exogenous human Bcl2. In the present study, the expression of the gene ND6 that is expressed from the light strand promoter (P{sub L}) was found to be similar in both the cell lines, as determined by RT-PCR. To test the possibility that this differential expression of mitochondrial genes under these two promoters was mediated by differences in proteins' affinity to interact with these promoters, we have carried out electrophoretic mobility shift assay (EMSA) using mitochondrial cell extracts from these two cell lines. Our result of these experiments revealed that two different proteins formed complex with the synthetic promoters and higher amount of protein from M5 cell extracts interacted with the P{sub H} promoter in comparison to that observed with cell extracts from Chinese hamster V79 cells. The promoter-specific differential binding of proteins was also observed in VB13. These results showed that differential mitochondrial gene expression observed earlier in the radio-resistant M5 cells was due to enhanced interaction proteins with the promoters P{sub H} and mediated by the expression of Bcl2.

  17. Simultaneous Gene Deletion of Gata4 and Gata6 Leads to Early Disruption of Follicular Development and Germ Cell Loss in the Murine Ovary1

    PubMed Central

    Padua, Maria B.; Fox, Shawna C.; Jiang, Tianyu; Morse, Deborah A.; Tevosian, Sergei G.

    2014-01-01

    ABSTRACT Granulosa cell formation and subsequent follicular assembly are important for ovarian development and function. Two members of the GATA family of transcription factors, GATA4 and GATA6, are expressed in ovarian somatic cells early in development, and their importance in adult ovarian function has been recently highlighted. In this study, we demonstrated that the embryonic loss of Gata4 and Gata6 expression within the ovary results in a strong down-regulation of genes involved in the ovarian developmental pathway (Fst and Irx3) as well as diminished expression of the pregranulosa and granulosa cell markers SPRR2 and FOXL2, respectively. Postnatal ovaries deficient in both Gata genes show impaired somatic cell proliferation and arrested follicular development at the primordial stage, where oocytes are either enclosed by one layer of squamous granulosa cells or remain in germ cell nests/clusters. Furthermore, germ cell nests and primordial follicles are predominantly localized to the central region of the Sf1Cre; Gata4flox/flox Gata6flox/flox ovaries, where the boundary between the medulla and cortex is almost nonexistent. Lastly, most of the oocytes are lost early in development in conditional double mutant ovaries, which confirms the importance of normally differentiated granulosa cells as supporting cells for oocyte survival. Thus, both GATA4 and GATA6 proteins are fundamental regulators of granulosa cell differentiation and proliferation, and consequently of proper follicular assembly during normal ovarian development and function. PMID:24899573

  18. SMARCA4-deficient undifferentiated carcinoma of the ovary (small cell carcinoma, hypercalcemic type): clinicopathologic and immunohistochemical study of 3 cases.

    PubMed

    Agaimy, Abbas; Thiel, Falk; Hartmann, Arndt; Fukunaga, Masaharu

    2015-10-01

    Small cell carcinoma of the ovary, hypercalcemic type is a very rare aggressive neoplasm of unknown histogenesis, affecting mainly girls and young women. Recently, inactivating mutations in SMARCA4 (BRG1), a member of the switch/sucrose nonfermenting chromatin remodeling complex, has been identified as driver events in most cases. We herein describe 3 cases in 34, 34, and 37-year-old women. Symptoms were mainly abdominal pain and mass. One patient was normocalcemic, and the other 2 had no preoperative serum calcium values available. All patients received radical hysterectomy with salpingo-oophorectomy, lymphadenectomy, and variable multimodality therapy. Two developed abdominal recurrences/metastases and died of disease at 4 and 12 months. One patient was alive without disease 17 months after surgery and radiochemotherapy. Histologic examination showed undifferentiated neoplasms composed of diffuse sheets, nests and cords of noncohesive monomorphic small blue/basaloid cells (classic variant, 1 case), and large undifferentiated/rhabdoid cells with abundant cytoplasm (large cell/rhabdoid variant, 2 case) admixed with minor small cell areas. One case contained rare isolated goblet cells, but true glandular component was absent. All tumors expressed vimentin and variably pancytokeratin and WT1. Nuclear SMARCB1 was intact in all cases (1 case showed small foci with mosaic loss). All tumors showed complete loss of SMARCA4. In conclusion, SMARCA4 immunohistochemistry represents a highly valuable emerging tool in identifying small cell carcinoma of the ovary, hypercalcemic type in routine practice. Distinguishing this aggressive neoplasm from juvenile granulosa cell tumor and other undifferentiated ovarian cancers is mandatory in selecting appropriate chemotherapeutic regimens and would allow better characterization of this entity, for which targeted molecular therapy still remains to be established.

  19. Adult granulosa cell tumor of the ovary: fine-needle-aspiration cytology of 10 cases and review of literature.

    PubMed

    Ali, Sarfraz; Gattuso, Paolo; Howard, Allison; Mosunjac, Marina B; Siddiqui, Momin T

    2008-05-01

    Adult granulosa cell tumor (GCT) of the ovary is mostly diagnosed in postmenopausal women. They typically secrete estrogen, which stimulates the endometrium to proliferate and cause abnormal bleeding. This study reviews the cytologic features of adult GCT of the ovary diagnosed by fine-needle aspiration (FNA). We reviewed slides from ten cases diagnosed by CT guided FNA from 1995 to 2007 at our institutions. Smears were stained with Diff-Quik and Papanicolaou stains. Patient's history and histologic diagnosis were also available and reviewed for all cases. The patients ranged in age from 39 to 83 yr. All 10 cases were hypercellular with both large and small overlapping cell clusters and individual cells. The cytologic features identified included: naked nuclei (10/10 cases), Call-Exner bodies (7/10 cases), blood vessels with prominent perivascular tumor cell growth (4/10 cases), spindle-shaped hyperchromatic stromal cells within cellular clusters (6/10 cases), mixed inflammation (3/10 cases), tumor cell necrosis (1/10 cases), and prominent metachromatic stroma seen in association with blood vessels (1/10 cases). Moderate to scant delicate cytoplasm was also seen (10/10 cases). Small, punctuate cytoplasmic vacuoles were also noted (7/10 cases) and were occasionally prominent (3/10 cases). In general nuclear to cytoplasmic ratios were high although lower than those typically seen in a lymphoma or small-cell carcinoma. Nuclei were generally centrally located although eccentrically located nuclei were consistently seen in a minority of cells. Nuclei were monotonous in size showing slightly convoluted (occasional rentiform and fetiform nuclei) to polygonal outlines. Prominent, central nucleoli were also seen (4/10 cases). Nuclear grooves were also seen (9/10 cases). No atypical mitotic activity was identified in any of the 10 cases (0/10 cases). In summary, the above cytologic features can also help in the cytologic diagnosis of adult GCTs.

  20. Use of the Syrian hamster embryo cell transformation assay for carcinogenicity prediction of chemical currently being tested by the National Toxicology Program in rodent bioassays

    SciTech Connect

    Kerckaert, G.A.; LeBoeuf, R.A.; Isfort, R.J.; Brauninger, R.

    1996-10-01

    The Syrian hamster embryo (SHE) cell transformation assay was used to predict the carcinogenicity of 26 chemicals currently being tested in the rodent bioassay by the National Toxicology Program as part of its program titled {open_quotes}Strategies for Predicting Chemical Carcinogenesis in Rodents.{close_quotes} Of these 26 chemicals, 17 were found to be positive in the SHE cell transformation assay while 9 were negative. Carcinogenicity predictions were made for these chemicals, based upon the SHE cell transformation assay results. Our predictions will be compared with the rodent bioassay results as they become available. 11 refs., 2 tabs.

  1. Different catalytic properties and inhibitor responses of the goldfish brain and ovary aromatase isozymes.

    PubMed

    Zhao, J; Mak, P; Tchoudakova, A; Callard, G; Chen, S

    2001-08-01

    The brain and ovarian aromatase isozymes of goldfish (Carassius auratus) are encoded by different CYP19 genes. This study measured aromatase activity in the goldfish brain tissues. For a direct comparison of the properties of the two aromatase isozymes, Chinese hamster ovary cells were stably transfected with brain- and ovary-derived cDNAs (respectively, p450 arom B and -A) and the properties of the expressed isozymes were compared. The kinetic parameters of the two isozymes were determined using androstenedione and testosterone as substrates and compared to those of human aromatase. Inhibition profile analyses on the two isozymes were performed using seven inhibitors [4-hydroxyandrostenedione, 7 alpha-(4'-amino)phenylthio-1,4-androstadiene-3,17-dione, bridge (2,19-methyleneoxy)androstene-3,17-dione, aminoglutethimide (AG), CGS 20267, ICI D1033, and vorozole]. Except for AG, the compounds tested were found to be much stronger inhibitors against the ovary enzyme than the brain enzyme. In addition, the ovary isoform was more sensitive to two phytoestrogens, chrysin and 7,8-dihydroxyflavone, than the brain form. These studies reveal that catalytic properties of the goldfish aromatase isoforms are significantly different from those of human aromatase. In addition, differences in the K(i) values of aromatase inhibitors for the two goldfish isoforms suggest structural variance in the active sites of these isozymes.

  2. Use of DEAD-box polypeptide-4 (Ddx4) gene promoter-driven fluorescent reporter mice to identify mitotically active germ cells in post-natal mouse ovaries

    PubMed Central

    Park, Eun-Sil; Tilly, Jonathan L.

    2015-01-01

    Several laboratories have independently isolated mitotically active germ cells, termed female germline stem cells or oogonial stem cells (OSCs), from adult mouse ovaries. However, a recent study using Ddx4-Cre;Rosa26 reporter mice concluded that such germ cells do not exist. Given the disparity in conclusions drawn in this study compared with others, we felt it was important to re-assess the utility of Ddx4-Cre;Rosa26 reporter mice for identification of OSCs in adult mouse ovaries. Transgenic Ddx4-Cre mice were crossed with Rosa26tdTm/tdTm mice to drive restricted tomato red (tdTm) gene expression in cells in which the Ddx4 gene promoter has been activated. Crude dispersion of ovaries from recombined offspring generated cell fractions containing tdTm-positive immature oocytes, which are incapable of proliferation and thus probably represent the uncharacterized reporter-positive ovarian cells identified in the paper Zhang et al. (2012) as being mitotically inactive. Dispersed ovaries further subjected to fluorescence-activated cell sorting yielded a large population of non-germline tdTm-positive cells, indicative of promoter ‘leakiness’ in the Ddx4-Cre mouse line. Nonetheless, a small percentage of these tdTm-positive cells exhibited externalized (extracellular, ec) expression of Ddx4 protein (ecDdx4-positive), expressed markers of primitive germ cells but not of oocytes, and actively proliferated in culture, all of which are characteristic features of OSCs. Thus, crude dispersion of ovaries collected from Ddx4 gene promoter-driven reporter mice is not, by itself, a reliable approach to identify OSCs, whereas the same ovarian dispersates further subjected to cell sorting strategies yield purified OSCs that can be expanded in culture. PMID:25147160

  3. Use of DEAD-box polypeptide-4 (Ddx4) gene promoter-driven fluorescent reporter mice to identify mitotically active germ cells in post-natal mouse ovaries.

    PubMed

    Park, Eun-Sil; Tilly, Jonathan L

    2015-01-01

    Several laboratories have independently isolated mitotically active germ cells, termed female germline stem cells or oogonial stem cells (OSCs), from adult mouse ovaries. However, a recent study using Ddx4-Cre;Rosa26 reporter mice concluded that such germ cells do not exist. Given the disparity in conclusions drawn in this study compared with others, we felt it was important to re-assess the utility of Ddx4-Cre;Rosa26 reporter mice for identification of OSCs in adult mouse ovaries. Transgenic Ddx4-Cre mice were crossed with Rosa26(tdTm/tdTm) mice to drive restricted tomato red (tdTm) gene expression in cells in which the Ddx4 gene promoter has been activated. Crude dispersion of ovaries from recombined offspring generated cell fractions containing tdTm-positive immature oocytes, which are incapable of proliferation and thus probably represent the uncharacterized reporter-positive ovarian cells identified in the paper Zhang et al. (2012) as being mitotically inactive. Dispersed ovaries further subjected to fluorescence-activated cell sorting yielded a large population of non-germline tdTm-positive cells, indicative of promoter 'leakiness' in the Ddx4-Cre mouse line. Nonetheless, a small percentage of these tdTm-positive cells exhibited externalized (extracellular, ec) expression of Ddx4 protein (ecDdx4-positive), expressed markers of primitive germ cells but not of oocytes, and actively proliferated in culture, all of which are characteristic features of OSCs. Thus, crude dispersion of ovaries collected from Ddx4 gene promoter-driven reporter mice is not, by itself, a reliable approach to identify OSCs, whereas the same ovarian dispersates further subjected to cell sorting strategies yield purified OSCs that can be expanded in culture.

  4. Dimorphic ovary differentiation in honeybee (Apis mellifera) larvae involves caste-specific expression of homologs of ark and buffy cell death genes.

    PubMed

    Dallacqua, Rodrigo Pires; Bitondi, Márcia Maria Gentile

    2014-01-01

    The establishment of the number of repeated structural units, the ovarioles, in the ovaries is one of the critical events that shape caste polyphenism in social insects. In early postembryonic development, honeybee (Apis mellifera) larvae have a pair of ovaries, each one consisting of almost two hundred ovariole primordia. While practically all these ovarioles continue developing in queen-destined larvae, they undergo massive programmed cell death (PCD) in worker-destined larvae. So as to gain insight into the molecular basis of this fundamental process in caste differentiation we used quantitative PCR (qPCR) and fluorescent in situ hybridization (FISH) to investigate the expression of the Amark and Ambuffy genes in the ovaries of the two honeybee castes throughout the fifth larval instar. These are the homologs of ark and buffy Drosophila melanogaster genes, respectively, involved in activating and inhibiting PCD. Caste-specific expression patterns were found during this time-window defining ovariole number. Amark transcript levels were increased when ovariole resorption was intensified in workers, but remained at low levels in queen ovaries. The transcripts were mainly localized at the apical end of all the worker ovarioles, but appeared in only a few queen ovarioles, thus strongly suggesting a function in mediating massive ovariolar cell death in worker larvae. Ambuffy was mainly expressed in the peritoneal sheath cells covering each ovariole. The levels of Ambuffy transcripts increased earlier in the developing ovaries of queens than in workers. Consistent with a protective role against cell death, Ambuffy transcripts were localized in practically all queen ovarioles, but only in few worker ovarioles. The results are indicative of a functional relationship between the expression of evolutionary conserved cell death genes and the morphological events leading to caste-specific ovary differentiation in a social insect.

  5. Non-thermal effects of 2.45 GHz microwaves on spindle assembly, mitotic cells and viability of Chinese hamster V-79 cells.

    PubMed

    Ballardin, Michela; Tusa, Ignazia; Fontana, Nunzia; Monorchio, Agostino; Pelletti, Chiara; Rogovich, Alessandro; Barale, Roberto; Scarpato, Roberto

    2011-11-01

    The production of mitotic spindle disturbances and activation of the apoptosis pathway in V79 Chinese hamster cells by continuous 2.45 GHz microwaves exposure were studied, in order to investigate possible non-thermal cell damage. We demonstrated that microwave (MW) exposure at the water resonance frequency was able to induce alteration of the mitotic apparatus and apoptosis as a function of the applied power densities (5 and 10mW/cm(2)), together with a moderate reduction in the rate of cell division. After an exposure time of 15 min the proportion of aberrant spindles and of apoptotic cells was significantly increased, while the mitotic index decreased as well, as compared to the untreated V79 cells. Additionally, in order to understand if the observed effects were due to RF exposure per se or to a thermal effect, V79 cells were also treated in thermostatic bath mimicking the same temperature increase recorded during microwave emission. The effect of temperature on the correct assembly of mitotic spindles was negligible up to 41°C, while apoptosis was induced only when the medium temperature achieved 40°C, thus exceeding the maximum value registered during MW exposure. We hypothesise that short-time MW exposures at the water resonance frequency cause, in V79 cells, reversible alterations of the mitotic spindle, this representing, in turn, a pro-apoptotic signal for the cell line.

  6. Development of mammalian ovary.

    PubMed

    Smith, Peter; Wilhelm, Dagmar; Rodgers, Raymond J

    2014-06-01

    Pre-natal and early post-natal ovarian development has become a field of increasing importance over recent years. The full effects of perturbations of ovarian development on adult fertility, through environmental changes or genetic anomalies, are only now being truly appreciated. Mitigation of these perturbations requires an understanding of the processes involved in the development of the ovary. Herein, we review some recent findings from mice, sheep, and cattle on the key events involved in ovarian development. We discuss the key process of germ cell migration, ovigerous cord formation, meiosis, and follicle formation and activation. We also review the key contributions of mesonephric cells to ovarian development and propose roles for these cells. Finally, we discuss polycystic ovary syndrome, premature ovarian failure, and pre-natal undernutrition; three key areas in which perturbations to ovarian development appear to have major effects on post-natal fertility.

  7. Establishment and characterization of a new marine fish cell line from ovary of barfin flounder ( Verasper moseri)

    NASA Astrophysics Data System (ADS)

    Xu, Xiaohui; Fan, Tingjun; Jiang, Guojian; Yang, Xiuxia

    2015-12-01

    A novel continuous ovary cell line from barfin flounder ( Verasper moseri) (BFO cell line) was established with its primitive application in transgenic expression demonstrated in this study. Primarily cultured cells grew well at 22°C in Dulbecco's modified Eagle medium/F12 medium (DMEM/F12, 1:1; pH 7.2) supplemented with 20% fetal bovine serum (FBS), carboxymethyl chitooligosaccharide, basic fibroblast growth factor (bFGF) and insulin-like growth factor-I (IGF-I). The primary BFO cells in fibroblastic morphology proliferated into a confluent monolayer about 2 weeks later, and were able to be subcultured. Impacts of medium and temperature on the growth of the cells were examined. The optimum growth was found in DMEM/F12 with 20% FBS and at 22°C. The BFO cells can be continuously subcultured to Passage 120 steadily with a population doubling time of 32.7 h at Passage 60. Chromosome analysis revealed that 72% of BFO cells at Passage 60 maintained the normal diploid chromosome number (46) with a normal karyotype of 2st+44t. The results of gene transformation indicated that green fluorescence protein (GFP) positively expressed in these cells after being transformed with pcDNA3.1-GFP. Therefore, a continuous and transformable BFO cell line was successfully established, which may serve as a useful tool for cytotechnological manipulation and transgenic modification of this fish.

  8. Simvastatin, an HMG-CoA reductase inhibitor, induces the synthesis and secretion of apolipoprotein AI in HepG2 cells and primary hamster hepatocytes.

    PubMed

    Bonn, Victoria; Cheung, Raphael C; Chen, Biao; Taghibiglou, Changiz; Van Iderstine, Stephen C; Adeli, Khosrow

    2002-07-01

    Clinical studies have recently suggested that statin treatment may beneficially elevate plasma concentrations of high density lipoprotein (HDL)-cholesterol in patients with hyperlipidemia. Here, we have investigated the effect of a potent inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase on the synthesis and secretion of apolipoprotein AI (apoAI) in two model systems, HepG2 cells and primary hamster hepatocytes. Cultured cells were incubated with different doses of simvastatin (0.1-10 microM) for a period of 18 h. A dose-dependent increase in synthesis and secretion of apoAI was observed in both cell types. There was a significant increase in the synthesis of apoAI in HepG2 cells (44.3+/-12.1%), and hamster hepatocytes (212+/-2%) after treatment with 10 microM of the statin. The increase in apoAI synthesis appeared to result in a higher level of apoAI secreted into the culture media in both cell types (49.2+/-7.8% in HepG2, 197+/-0.2% in hamster hepatocytes). ApoAI mRNA levels were also significantly increased in both cell types in response to statin treatment. Control experiments with transferrin confirmed specificity of the effect on apoAI secretion. Analysis of a density fraction containing HDL particles in culture media revealed an increase in HDL-associated apoAI of 94.3+/-2.1% in HepG2 cells and 27.0+/-0.03% in hamster hepatocytes following 10 microM simvastatin-treatment. Comparative studies of simvastatin and lovastatin indicated a differential ability to induce apoAI synthesis and secretion, with simvastatin having a more significant effect. Thus, acute statin treatment of cultured hepatocytes (transformed as well as primary) resulted in a significant upregulation of apoAI mRNA and apoAI synthesis, causing oversecretion of apoAI and HDL extracellularly. The stimulatory effect on apoAI synthesis and secretion may thus explain the clinical observation of an elevated plasma HDL-cholesterol level in hyperlipidemic patients treated with

  9. Expression of human basic fibroblast growth factor cDNA in baby hamster kidney-derived cells results in autonomous cell growth

    PubMed Central

    1988-01-01

    Growth factor over-production by responsive cells might contribute to their autonomous proliferation as well as their acquisition of a transformed phenotype in culture. Basic fibroblast growth factor (bFGF) has been shown to induce transient changes in cell behavior that resemble those encountered in transformed cells. In addition, several types of human tumor cells have been shown to produce bFGF. To determine directly the role that bFGF might play in the induction of the transformed phenotype, we have introduced a human bFGF cDNA expression vector into baby hamster kidney-derived (BHK-21) fibroblasts. One of the BHK transfectants, termed clone 19, expresses the bFGF mRNA and produces biologically active bFGF that accumulates to a high concentration inside the cells. These properties correlate with the ability of the cells to grow in serum-free medium without the addition of exogenous bFGF. Clone 19 cells also proliferated in soft agar, indicating that constitutive expression of the bFGF gene results in a loss of anchorage-dependent growth. PMID:3360856

  10. Stable expression of rat cytochrome P-450IIB1 cDNA in Chinese hamster cells (V79) and metabolic activation of aflatoxin B sub 1

    SciTech Connect

    Doehmer, J.; Dogra, S.; Friedberg, T.; Monier, S.; Adesnik, M.; Glatt, H.; Oesch, F. )

    1988-08-01

    V79 Chinese hamster fibroblasts are widely used for mutagenicity testing but have the serious limitation that they do not express cytochromes P-450, which are needed for the activation of many promutagens to mutagenic metabolites. A full-length cDNA clone encoding the monooxygenase cytochrome P-450IIB1 under control of the simian virus 40 early promoter was constructed and cointroduced with the selection marker neomycin phosphotransferase (conferring resistance to G418) into V79 Chinese hamster cells. G418-resistant cells were selected, established as cell lines, and tested for cytochrome P-450IIB1 expression and enzymatic activity. Two cell lines (SD1 and SD3) were found that stably produce cytochrome P-450IIB1. Although purified cytochromes P-450 possess monooxygenase activity only after reconstitution with cytochrome P-450 reductase and phospholipid, the gene product of the construct exhibited this activity. This implies that the gene product is intracellularly localized in a way that allows access to the required components. If compared with V79 cells, the mutation rate for the hypoxanthine phosphoribosyltranferase (HPRT) locus in SD1 cells is markedly increased when exposed to aflatoxin B{sub 1}, which is activated by this enzyme.

  11. Effect of VULM 1457, an ACAT inhibitor, on serum lipid levels and on real time red blood cell flow in diabetic and non-diabetic hamsters fed high cholesterol-lipid diet.

    PubMed

    Vojtassáková, E; Syneková, M; Tazká, D; Mátyás, S; Hózová, R; Sadlonová, I; Svec, P

    2007-12-01

    Acyl-coenzyme A: cholesterol O-acyltransferase (ACAT) catalyzes the formation of cholesterol/fatty acyl-coenzyme A esters. Accumulation of cholesterol esters leads to pathological changes connected with atherosclerosis. We have evaluated effects of a newly synthesized ACAT inhibitor, 1-(2,6-diisopropyl-phenyl)-3-[4-(4'-nitrophenylthio)phenyl] urea (VULM 1457), on serum lipid (cholesterol and triglycerides) levels and velocity of red blood cells (RBC) in non-diabetic and diabetic hamsters fed on high cholesterol-lipid (HCHL) diet during 3 months. The VULM 1457 effects on the paw microcirculation were assessed using capillary microscopy by measuring (RBC) velocity in vivo. Hamsters fed on HCHL diet became hypercholesterolemic with a dramatic increase in serum lipids accompanied with significantly decreased RBC velocity. Diabetic hamsters fed on HCHL diet had further increased serum lipids with reduction of RBC velocity. The VULM 1457 inhibitor lowered cholesterol levels in both non-diabetic and diabetic hamsters fed on HCHL diet. The greater VULM 1457 effect was shown in diabetic hamsters fed on HCHL diet where VULM 1457 expressed hypotriglycerides effects, too. An improved RBC velocity-pronounced effect was observed in diabetic hamsters fed on HCHL diet treated with VULM 1457. These results suggest that the ACAT inhibitor, VULM 1457, is a prospective hypolipidemic and anti-atherogenic drug which treats diabetes.

  12. Protective Effect of Boric Acid on Oxidative DNA Damage In Chinese Hamster Lung Fibroblast V79 Cell Lines

    PubMed Central

    Yılmaz, Sezen; Ustundag, Aylin; Cemiloglu Ulker, Ozge; Duydu, Yalcın

    2016-01-01

    Objective Many studies have been published on the antioxidative effects of boric acid (BA) and sodium borates in in vitro studies. However, the boron (B) concentrations tested in these in vitro studies have not been selected by taking into account the realistic blood B concentrations in humans due to the lack of comprehensive epidemiological studies. The recently published epidemiological studies on B exposure conducted in China and Turkey provided blood B concentrations for both humans in daily life and workers under extreme exposure conditions in occupational setting. The results of these studies have made it possible to test antioxidative effects of BA in in vitro studies within the concentra- tion range relevant to humans. The aim of this study was to investigate the protective ef- fects of BA against oxidative DNA damage in V79 (Chinese hamster lung fibroblast) cells. The concentrations of BA tested for its protective effect was selected by taking the blood B concentrations into account reported in previously published epidemiological studies. Therefore, the concentrations of BA tested in this study represent the exposure levels for humans in both daily life and occupational settings. Materials and Methods In this experimental study, comet assay and neutral red uptake (NRU) assay methods were used to determinacy to toxicity and genotoxicity of BA and hydrogen peroxide (H2O2). Results The results of the NRU assay showed that BA was not cytotoxic within the tested concentrations (3, 10, 30, 100 and 200 µM). These non-cytotoxic concentrations were used for comet assay. BA pre-treatment significantly reduced (P<0.05, one-way ANOVA) the DNA damaging capacity of H2O2 at each tested BA concentrations in V79 cells. Conclusion Consequently, pre-incubation of V79 cells with BA has significantly reduced the H2O2-induced oxidative DNA damage in V79 cells. The protective effect of BA against oxidative DNA damage in V79 cells at 5, 10, 50, 100 and 200 μM (54, 108, 540

  13. Regulation of initiation of DNA synthesis in Chinese hamster cells. I. Production of stable, reversible G1-arrested populations in suspension culture.

    PubMed

    Tobey, R A; Ley, K D

    1970-07-01

    Suspension cultures of Chinese hamster cells (line CHO) were grown to stationary phase (approximately 8-9 x 10(5) cells/ml) in F-10 medium. Cells remained viable (95%) for at least 80 hr in stationary phase, and essentially all of the cells were in G(1) Upon resuspension or dilution with fresh medium, the cells were induced to resume traverse of the life cycle in in synchrony, and the patterns of DNA synthesis and division were similar to those observed in cultures prepared by mitotic selection. Immediately after dilution, the rates of synthesis of RNA and protein increased threefold. This system provides a simple technique for production of large quantities of highly synchronized cells and may ultimately provide information on the biochemical mechanisms regulating cell-cycle traverse.

  14. [The influence of sodium nitrite on the osmoresistance of Chinese hamster cells].

    PubMed

    Bol'shakova, O I; Sverdlov, A G; Timoshenko, S I; Nikanorova, N G; Grachev, S A

    2007-01-01

    We have previously demonstrated that incubation of V-79 cells in the medium containing the nitric oxide donor, NaNO2, increases cell resistance to damaging effect of gamma-rays, UV radiation and hyperthermia. In the present study, we investigated the effects of the nitric oxide donor on the sensitivity of V-79 cells to changes in osmomolarity of the medium by adding different amounts of sodium chloride or water. We found that pretreatment of the cells with NaNO2 resulted in a significant increase in the number of growing cells in 48 h after the treatment. The osmomolarity-dependent morphological changes in cultured cells were also substantially diminished following NaNO2 treatment. This effect could be observed under both hyper- and hypoosmosis, and was dependent on concentration of sodium chloride in hypertonic medium (being maximal under 0.17 M NaCl) and on the amount of water in hypotonic medium (being maximal under 1.1 times the dilution with water). In the experiments with increased osmomolarity, we found that the observed increase in the number of growing cells following NaNO2 treatment was accompanied with a significant increase of the mitotic index. These findings indicate that nitric oxide increases cell resistance to the damaging effects of osmotic shock in the way which is similar to the protective effect of these molecules against radiation and hyperthermia. Similarities in the effects of NaNO2 under different conditions leading to cell damage suggest that nitric oxide might serve as the universal factor participating in recovery of damaged cells and mediating increased cellular resistance to the damaging conditions.

  15. Detection, Characterization, and Spontaneous Differentiation In Vitro of Very Small Embryonic-Like Putative Stem Cells in Adult Mammalian Ovary

    PubMed Central

    Parte, Seema; Telang, Jyoti; Daithankar, Vinita; Salvi, Vinita; Zaveri, Kusum; Hinduja, Indira

    2011-01-01

    The present study was undertaken to detect, characterize, and study differentiation potential of stem cells in adult rabbit, sheep, monkey, and menopausal human ovarian surface epithelium (OSE). Two distinct populations of putative stem cells (PSCs) of variable size were detected in scraped OSE, one being smaller and other similar in size to the surrounding red blood cells in the scraped OSE. The smaller 1–3 μm very small embryonic-like PSCs were pluripotent in nature with nuclear Oct-4 and cell surface SSEA-4, whereas the bigger 4–7 μm cells with cytoplasmic localization of Oct-4 and minimal expression of SSEA-4 were possibly the tissue committed progenitor stem cells. Pluripotent gene transcripts of Oct-4, Oct-4A, Nanog, Sox-2, TERT, and Stat-3 in human and sheep OSE were detected by reverse transcriptase–polymerase chain reaction. The PSCs underwent spontaneous differentiation into oocyte-like structures, parthenote-like structures, embryoid body-like structures, cells with neuronal-like phenotype, and embryonic stem cell-like colonies, whereas the epithelial cells transformed into mesenchymal phenotype by epithelial–mesenchymal transition in 3 weeks of OSE culture. Germ cell markers like c-Kit, DAZL, GDF-9, VASA, and ZP4 were immuno-localized in oocyte-like structures. In conclusion, as opposed to the existing view of OSE being a bipotent source of oocytes and granulosa cells, mammalian ovaries harbor distinct very small embryonic-like PSCs and tissue committed progenitor stem cells population that have the potential to develop into oocyte-like structures in vitro, whereas mesenchymal fibroblasts appear to form supporting granulosa-like somatic cells. Research at the single-cell level, including complete gene expression profiling, is required to further confirm whether postnatal oogenesis is a conserved phenomenon in adult mammals. PMID:21291304

  16. Detection, characterization, and spontaneous differentiation in vitro of very small embryonic-like putative stem cells in adult mammalian ovary.

    PubMed

    Parte, Seema; Bhartiya, Deepa; Telang, Jyoti; Daithankar, Vinita; Salvi, Vinita; Zaveri, Kusum; Hinduja, Indira

    2011-08-01

    The present study was undertaken to detect, characterize, and study differentiation potential of stem cells in adult rabbit, sheep, monkey, and menopausal human ovarian surface epithelium (OSE). Two distinct populations of putative stem cells (PSCs) of variable size were detected in scraped OSE, one being smaller and other similar in size to the surrounding red blood cells in the scraped OSE. The smaller 1-3 μm very small embryonic-like PSCs were pluripotent in nature with nuclear Oct-4 and cell surface SSEA-4, whereas the bigger 4-7 μm cells with cytoplasmic localization of Oct-4 and minimal expression of SSEA-4 were possibly the tissue committed progenitor stem cells. Pluripotent gene transcripts of Oct-4, Oct-4A, Nanog, Sox-2, TERT, and Stat-3 in human and sheep OSE were detected by reverse transcriptase-polymerase chain reaction. The PSCs underwent spontaneous differentiation into oocyte-like structures, parthenote-like structures, embryoid body-like structures, cells with neuronal-like phenotype, and embryonic stem cell-like colonies, whereas the epithelial cells transformed into mesenchymal phenotype by epithelial-mesenchymal transition in 3 weeks of OSE culture. Germ cell markers like c-Kit, DAZL, GDF-9, VASA, and ZP4 were immuno-localized in oocyte-like structures. In conclusion, as opposed to the existing view of OSE being a bipotent source of oocytes and granulosa cells, mammalian ovaries harbor distinct very small embryonic-like PSCs and tissue committed progenitor stem cells population that have the potential to develop into oocyte-like structures in vitro, whereas mesenchymal fibroblasts appear to form supporting granulosa-like somatic cells. Research at the single-cell level, including complete gene expression profiling, is required to further confirm whether postnatal oogenesis is a conserved phenomenon in adult mammals.

  17. Effect of Saw Palmetto Supplements on Androgen-Sensitive LNCaP Human Prostate Cancer Cell Number and Syrian Hamster Flank Organ Growth

    PubMed Central

    Opoku-Acheampong, Alexander B.; Penugonda, Kavitha; Lindshield, Brian L.

    2016-01-01

    Saw palmetto supplements (SPS) are commonly consumed by men with prostate cancer. We investigated whether SPS fatty acids and phytosterols concentrations determine their growth-inhibitory action in androgen-sensitive LNCaP cells and hamster flank organs. High long-chain fatty acids-low phytosterols (HLLP) SPS ≥ 750 nM with testosterone significantly increased and ≥500 nM with dihydrotestosterone significantly decreased LNCaP cell number. High long-chain fatty acids-high phytosterols (HLHP) SPS ≥ 500 nM with dihydrotestosterone and high medium-chain fatty acids-low phytosterols (HMLP) SPS ≥ 750 nM or with androgens significantly decreased LNCaP cell number (n = 3; p < 0.05). Five- to six-week-old, castrated male Syrian hamsters were randomized to control (n = 4), HLLP, HLHP, and HMLP SPS (n = 6) groups. Testosterone or dihydrotestosterone was applied topically daily for 21 days to the right flank organ; the left flank organ was treated with ethanol and served as the control. Thirty minutes later, SPS or ethanol was applied to each flank organ in treatment and control groups, respectively. SPS treatments caused a notable but nonsignificant reduction in the difference between left and right flank organ growth in testosterone-treated SPS groups compared to the control. The same level of inhibition was not seen in dihydrotestosterone-treated SPS groups (p < 0.05). Results may suggest that SPS inhibit 5α-reductase thereby preventing hamster flank organ growth. PMID:27272436

  18. Effect of Saw Palmetto Supplements on Androgen-Sensitive LNCaP Human Prostate Cancer Cell Number and Syrian Hamster Flank Organ Growth.

    PubMed

    Opoku-Acheampong, Alexander B; Penugonda, Kavitha; Lindshield, Brian L

    2016-01-01

    Saw palmetto supplements (SPS) are commonly consumed by men with prostate cancer. We investigated whether SPS fatty acids and phytosterols concentrations determine their growth-inhibitory action in androgen-sensitive LNCaP cells and hamster flank organs. High long-chain fatty acids-low phytosterols (HLLP) SPS ≥ 750 nM with testosterone significantly increased and ≥500 nM with dihydrotestosterone significantly decreased LNCaP cell number. High long-chain fatty acids-high phytosterols (HLHP) SPS ≥ 500 nM with dihydrotestosterone and high medium-chain fatty acids-low phytosterols (HMLP) SPS ≥ 750 nM or with androgens significantly decreased LNCaP cell number (n = 3; p < 0.05). Five- to six-week-old, castrated male Syrian hamsters were randomized to control (n = 4), HLLP, HLHP, and HMLP SPS (n = 6) groups. Testosterone or dihydrotestosterone was applied topically daily for 21 days to the right flank organ; the left flank organ was treated with ethanol and served as the control. Thirty minutes later, SPS or ethanol was applied to each flank organ in treatment and control groups, respectively. SPS treatments caused a notable but nonsignificant reduction in the difference between left and right flank organ growth in testosterone-treated SPS groups compared to the control. The same level of inhibition was not seen in dihydrotestosterone-treated SPS groups (p < 0.05). Results may suggest that SPS inhibit 5α-reductase thereby preventing hamster flank organ growth.

  19. Cytochrome c release and caspase-3 activation in retinal ganglion cells following different distance of axotomy of the optic nerve in adult hamsters.

    PubMed

    He, M H; Cheung, Z H; Yu, E H; Tay, D K C; So, K F

    2004-11-01

    This study examined the relationship between the distance of axotomy and the death of injured retinal ganglion cells (RGCs) in adult hamsters and the relationship of cytochrome c and caspase-3 on the death pathway of RGCs. The left optic nerve (ON) of adult hamsters was transected either at 1 or 3 mm away from the optic disc, and retrogradely labeled with Flurogold on the ON stump. After a predetermined period of postoperative time, the surviving RGCs were counted by retina flat-mount, and the activation of cytochrome c and caspase-3 were investigated by immunohistochemistry. Cell loss was found to be much faster (P < 0.01), more cells with cytochrome c were observed (P < 0.05) and the activation of caspase-3 was earlier when ON was transected 1 mm away from the optic disc than when was transected 3 mm away from the optic disc. Distance of axotomy affects the axotomized cell death rate where more RGCs died when the ON transection was applied closer to the eye. The timing of activation of caspase-3 in the RGCs may be linked to the distance of axotomy.

  20. Ultraviolet light action spectra for neoplastic transformation and lethality of Syrian hamster embryo cells correlate with spectrum for pyrimidine dimer formation in cellular DNA.

    PubMed Central

    Doniger, J; Jacobson, E D; Krell, K; DiPaolo, J A

    1981-01-01

    Action spectra were determined for neoplastic transformation, production of pyrimidine dimers, and lethality in Syrian hamster embryo cells. Of wavelengths between 240 and 313 nm, the most effective were 265 and 270. The relative sensitivities per quantum for transformation, pyrimidine dimer production, and lethality were essentially the same at each of the wavelengths tested. This action spectrum for transformation, which is relevant to carcinogenesis, is similar to spectra obtained previously by measuring other cellular responses in either microbial or mammalian systems. Because the action spectra for cytotoxicity and transformation are the same as the spectrum for dimer production, DNA is suggested as the target for all these processes. PMID:6941297

  1. Equine herpesvirus type 1 tegument protein VP22 is not essential for pathogenicity in a hamster model, but is required for efficient viral growth in cultured cells.

    PubMed

    Okada, Ayaka; Izume, Satoko; Ohya, Kenji; Fukushi, Hideto

    2015-10-01

    VP22 is a major tegument protein of Equine herpesvirus type 1 (EHV-1) that is a conserved protein among alphaherpesviruses. However, the roles of VP22 differ among each virus, and the roles of EHV-1 VP22 are still unclear. Here, we constructed an EHV-1 VP22 deletion mutant and a revertant virus to clarify the role of VP22. We found that EHV-1 VP22 was required for efficient viral growth in cultured cells, but not for virulence in a hamster model.

  2. Restoration of Chinese hamster cell radiation resistance by the human repair gene ERCC-5 and progress in molecular cloning of this gene

    SciTech Connect

    Strniste, G.F.; Chen, D.J.; deBruin, D.; McCoy, L.S.; Luke, J.A.; Mudgett, J.S.; Nickols, J.W.; Okinaka, R.T.; Tesmer, J.G.; MacInnes, M.A.

    1988-01-01

    The uv-sensitive Chinese hamster cell uv-135 is being used to identify and isolate the human gene, ERCC-5, which corrects nucleotide excision repair in this incision-defective mutant. A cosmid library, constructed from a 3/sup 0/ transformant of uv-135, has been screened for transfected gpt and human Alu family sequences. An ordered physical map of overlapping positives cosmids has been determined. Molecular evidence suggests a region of this map of <40 Kbp contains the ERCC-5 gene. 10 refs., 2 figs.

  3. Abundant primary piRNAs, endo-siRNAs, and microRNAs in a Drosophila ovary cell line

    PubMed Central

    Lau, Nelson C.; Robine, Nicolas; Martin, Raquel; Chung, Wei-Jen; Niki, Yuzo; Berezikov, Eugene; Lai, Eric C.

    2009-01-01

    Piwi proteins, a subclass of Argonaute-family proteins, carry ∼24–30-nt Piwi-interacting RNAs (piRNAs) that mediate gonadal defense against transposable elements (TEs). We analyzed the Drosophila ovary somatic sheet (OSS) cell line and found that it expresses miRNAs, endogenous small interfering RNAs (endo-siRNAs), and piRNAs in abundance. In contrast to intact gonads, which contain mixtures of germline and somatic cell types that express different Piwi-class proteins, OSS cells are a homogenous somatic cell population that expresses only PIWI and primary piRNAs. Detailed examination of its TE-derived piRNAs and endo-siRNAs revealed aspects of TE defense that do not rely upon ping-pong amplification. In particular, we provide evidence that a subset of piRNA master clusters, including flamenco, are specifically expressed in OSS and ovarian follicle cells. These data indicate that the restriction of certain TEs in somatic gonadal cells is largely mediated by a primary piRNA pathway. PMID:19541914

  4. Expression of potentially lethal damage in Chinese hamster cells exposed to hematoporphyrin derivative photodynamic therapy.

    PubMed

    Gomer, C J; Rucker, N; Ferrario, A; Murphree, A L

    1986-07-01

    Experiments were performed to determine whether the expression and/or repair of potentially lethal damage could be observed in mammalian cells exposed to hemataporphyrin derivative (HPD) photodynamic therapy (PDT). Photodynamic therapy was combined with posttreatment protocols known to inhibit the repair of potentially lethal damage in cells treated with X-rays, ultraviolet radiation, or alkylating agents. Potentiation of lethal damage from photodynamic therapy was induced by hypothermia (4 degrees C) following short (1 h) or extended (16 h) HPD incubation conditions. Caffeine potentiated the lethal effects of PDT only when cells were incubated with HPD for extended time periods. However, 3-aminobenzamide had no effect on the cytotoxic actions of PDT following either short or extended HPD incubations. Recovery from potentially lethal damage expressed by posttreatment hypothermia was complete within 1 h, while recovery from potentially lethal damage expressed by posttreatment caffeine required time periods of up to 24 h. The lack of effect of 3-aminobenzamide on expression of potentially lethal damage following photodynamic therapy may be related to direct inhibition of adenosine diphosphoribose transferase by photodynamic therapy. These results indicate that the expression and repair of potentially lethal damage can be observed in cells treated with PDT and will vary as a function of porphyrin incubation conditions.

  5. Irinotecan metabolite SN38 results in germ cell loss in the testis but not in the ovary of prepubertal mice

    PubMed Central

    Lopes, Federica; Smith, Rowena; Nash, Sophie; Mitchell, Rod T.; Spears, Norah

    2016-01-01

    STUDY QUESTION Does the Irinotecan metabolite 7-ethyl-10-hydroxycamptothecan (SN38) damage the gonads of male and female prepubertal mice? SUMMARY ANSWER The Irinotecan metabolite SN38 reduces germ cell numbers within the seminiferous tubules of mouse testes at concentrations that are relevant to cancer patients, while in contrast it has little if any effect on the female germ cell population. WHAT IS KNOWN ALREADY Little is known about the role of the chemotherapeutic agent Irinotecan on female fertility, with only one article to date reporting menopausal symptoms in perimenopausal women treated with Irinotecan, while no data are available either on adult male fertility or on the impact of Irinotecan on the subsequent fertility of prepubertal cancer patients, female or male. STUDY DESIGN SIZE, DURATION Male and female gonads were obtained from postnatal day 5 C57BL/6 mice and exposed in vitro to a range of concentrations of the Irinotecan metabolite SN38: 0.002, 0.01, 0.05, 0.1 or 1 µg ml–1 for the testis and 0.1, 1, 2.5 or 5 µg ml–1 for the ovary, with treated gonads compared to control gonads not exposed to SN38. SN38 was dissolved in 0.5% dimethyl sulfoxide, with controls exposed to the same concentration of diluent. The number of testis fragments used for each analysis ranged between 3 and 9 per treatment group, while the number of ovaries used for each analysis ranged between 4 and 12 per treatment group. PARTICIPANTS/MATERIALS, SETTING, METHODS Neonatal mouse gonads were developed in vitro, with tissue analysed at the end of the 4–6 day culture period, following immunofluorescence or hematoxylin and eosin staining. Statistical analyses were performed using one-way ANOVA followed by Bonferroni post-hoc test for normally distributed data and Kruskal-Wallis test followed by Dunns post-test for non-parametric data. MAIN RESULTS AND THE ROLE OF CHANCE Abnormal testis morphology was observed when tissues were exposed to SN38, with a smaller seminiferous

  6. Metabolism of Benzo(A)Pyrene and Diethylnitrosamine by Lung Cells of Rats, Mice, and Hamsters.

    DTIC Science & Technology

    1985-07-01

    carcinogens in one species, but produce little effect in others. These species differences appear to involve humans as well. The working hypothesis...morphological studies of human lung :- tumors have strongly suggested that these cell types are among the targets for carcinogenic chemicals. The results...from experimental systems could then be compared with metabolic pathways in human tissues obtained at autopsy or as specimens at surgery, in order to

  7. Isolation and preliminary characterization of 10-nm filaments from baby hamster kidney (BHK-21) cells.

    PubMed Central

    Starger, J M; Goldman, R D

    1977-01-01

    A procedure for isolating 10-nm filaments as juxtanuclear caps from normal and colchicine-treated BHK-21 cells is described. These aggregates of 10-nm filaments retain their birefringence and their structural integrity. The major proteins comprising the filament preparations can be resolved as two bands on sodium dodecyl sulfate gels, corresponding to approximate molecular weights of 54,000 and 55,000. Images PMID:329284

  8. Inhibition of calcium channels by neurokinin receptor and signal transduction in hamster submandibular ganglion cells.

    PubMed

    Yamada, T; Endoh, T; Suzuki, T

    1999-04-16

    Both substance P (SP) and neurokinin A (NKA) are known as neurotransmitters of the submandibular ganglion (SMG) neurons. SP released from collaterals of the sensory nerves also regulates the excitability of SMG neurons. It has recently been shown that neurokinins (NK) inhibit calcium channels in various neurons. In this study, the effects of NK on voltage-dependent calcium channel current (I(Ca)) in SMG cells were investigated using the whole-cell patch-clamp recording method. NK-1 receptor agonist and SP caused inhibition of I(Ca) in SMG cells in a dose-dependent manner. NK-1 receptor agonist inhibited L-, N- and P/Q-type I(Ca) components. GDP-beta-S included in the pipette solution reduced the NK-1 receptor agonist-induced inhibition of I(Ca). In addition, NK-1 receptor agonist-induced inhibition of I(Ca) was reduced by stimulation of protein kinase C (PKC) but not cyclic AMP-dependent protein kinase (PKA). The results provided evidence for a signal transduction pathway in which calcium channel inhibition by NK receptors required activation of G-protein and PKC-affected step phosphorylation in SMG neurons.

  9. Tomato ovary-to-fruit transition is characterized by a spatial shift of mRNAs for cell wall invertase and its inhibitor with the encoded proteins localized to sieve elements.

    PubMed

    Palmer, William M; Ru, Lei; Jin, Ye; Patrick, John W; Ruan, Yong-Ling

    2015-02-01

    Central to understanding fruit development is to elucidate the processes mediating a successful transition from pre-pollination ovaries to newly set fruit, a key step in establishing fruit yield potential. In tomato, cell wall invertase (CWIN) LIN5 and its inhibitor INH1 are essential for fruit growth. However, the molecular and cellular basis by which they exert their roles in ovary-to-fruit transition remains unknown. To address this issue, we conducted a study focusing on ovaries and fruitlets at 2 days before and 2 days after anthesis, respectively. In situ hybridization analyses revealed that LIN5 and INH1 exhibited a dispersed expression in ovaries compared with their phloem-specific expression in fruitlets. Remarkably, LIN5 and INH1 proteins were immunologically co-localized to cell walls of sieve elements (SEs) in ovaries immediately prior to anthesis and in young fruitlets, but were undetectable in provascular bundles of younger ovaries. A burst in CWIN activity occurred during ovary-to-fruit transition. Interestingly, the ovaries, but not the fruitlets, exhibited high expression of a defective invertase, SldeCWIN1, an ortholog of which is known to enhance inhibition of INH on CWIN activity in tobacco. Imaging of a fluorescent symplasmic tracer indicated an apoplasmic phloem unloading pathway operated in ovaries, contrary to the previously observed symplasmic unloading pathway in fruit pericarp. These new data indicate that (1) a phloem-specific patterning of the CWIN and INH mRNAs is induced during ovary-to-fruit transition, and (2) LIN5 protein functions specifically in walls of SEs and increases its activity during ovary-to-fruit transition, probably to facilitate phloem unloading and to generate a glucose signal positively regulating cell division, hence fruit set.

  10. Two Case Reports of a Malignant Germ Cell Tumor of Ovary and a Granulosa Cell Tumor: Interest of Tumoral Immunochemistry in the Identification and Management

    PubMed Central

    Bouquet de Jolinière, J.; Ben Ali, N.; Fadhlaoui, A.; Dubuisson, J. B.; Guillou, L.; Sutter, A.; Betticher, D.; Hoogewoud, H. M.; Feki, A.

    2014-01-01

    Objective: In this article, we present two case reports. The first case was a malignant germ cell tumor of the right ovary in a 23-year old woman and the second case was a bilateral undifferentiated granulosa cell tumor in a 71-year old woman. The aim of these reports is to illustrate the interest of the immunohistochemical analysis to define the correct diagnosis, to better classify these ovarian tumors and improve their management. Methods: In this study, we report two cases. The first case concerns a 23-year old woman (A) with a mixed germ cell tumor of the right ovary [dysgerminoma (75%), yolk sac tumor (20%), and a mature teratoma (5%)], and the second case concerns a 71-year old woman (B) with a bilateral non-differentiated and necrotic granulosa cell tumor of both ovaries. The staging system was used according to both the classifications: International Federation of Gynaecology and Obstetrics 1987 for ovarian cancer and TNM code 2009. Results: The immunostaining establishes the malignancy and the immunochemistry contributes to confirm effectively the right diagnosis (Tables 2 and 3). Conclusion: An immunohistochemical analysis is mandatory for the choice of chemotherapy to obtain a better response of the disease and improve the survival prognosis. The efficiency of the chemotherapy authorizes a conservative surgery including a unilateral salpingo-oophorectomy preserving fertility (A). Concerning the non-dysgerminoma tumor (B), and after a surgical staging and debulking, chemotherapy was recommended. The type of tumor and its histological feature conditioned the choice of treatment. The benefit of the immunohistological analysis in this case allowed the right adjuvant treatment. PMID:24982844

  11. Evidence for Integrin - Venus Kinase Receptor 1 Alliance in the Ovary of Schistosoma mansoni Females Controlling Cell Survival.

    PubMed

    Gelmedin, Verena; Morel, Marion; Hahnel, Steffen; Cailliau, Katia; Dissous, Colette; Grevelding, Christoph G

    2017-01-01

    In metazoan integrin signaling is an important process of mediating extracellular and intracellular communication processes. This can be achieved by cooperation of integrins with growth factor receptors (GFRs). Schistosoma mansoni is a helminth parasite inducing schistosomiasis, an infectious disease of worldwide significance for humans and animals. First studies on schistosome integrins revealed their role in reproductive processes, being involved in spermatogenesis and oogenesis. With respect to the roles of eggs for maintaining the parasite´s life cycle and for inducing the pathology of schistosomiasis, elucidating reproductive processes is of high importance. Here we studied the interaction of the integrin receptor Smβ-Int1 with the venus kinase receptor SmVKR1 in S. mansoni. To this end we cloned and characterized SmILK, SmPINCH, and SmNck2, three putative bridging molecules for their role in mediating Smβ-Int1/SmVKR1 cooperation. Phylogenetic analyses showed that these molecules form clusters that are specific for parasitic platyhelminths as it was shown for integrins before. Transcripts of all genes colocalized in the ovary. In Xenopus oocytes germinal vesicle breakdown (GVBD) was only induced if all members were simultaneously expressed. Coimmunoprecipitation results suggest that a Smβ-Int1-SmILK-SmPINCH-SmNck2-SmVKR1 complex can be formed leading to the phosphorylation and activation of SmVKR1. These results indicate that SmVKR1 can be activated in a ligand-independent manner by receptor-complex interaction. RNAi and inhibitor studies to knock-down SmILK as a representative complex member concurrently revealed effects on the extracellular matrix surrounding the ovary and oocyte localization within the ovary, oocyte survival, and egg production. By TUNEL assays, confocal laser scanning microscopy (CLSM), Caspase-3 assay, and transcript profiling of the pro-apoptotic BCL-2 family members BAK/BAX we obtained first evidence for roles of this signaling

  12. Evidence for Integrin – Venus Kinase Receptor 1 Alliance in the Ovary of Schistosoma mansoni Females Controlling Cell Survival

    PubMed Central

    Gelmedin, Verena; Morel, Marion; Hahnel, Steffen; Cailliau, Katia; Dissous, Colette; Grevelding, Christoph G.

    2017-01-01

    In metazoan integrin signaling is an important process of mediating extracellular and intracellular communication processes. This can be achieved by cooperation of integrins with growth factor receptors (GFRs). Schistosoma mansoni is a helminth parasite inducing schistosomiasis, an infectious disease of worldwide significance for humans and animals. First studies on schistosome integrins revealed their role in reproductive processes, being involved in spermatogenesis and oogenesis. With respect to the roles of eggs for maintaining the parasite´s life cycle and for inducing the pathology of schistosomiasis, elucidating reproductive processes is of high importance. Here we studied the interaction of the integrin receptor Smβ-Int1 with the venus kinase receptor SmVKR1 in S. mansoni. To this end we cloned and characterized SmILK, SmPINCH, and SmNck2, three putative bridging molecules for their role in mediating Smβ-Int1/SmVKR1 cooperation. Phylogenetic analyses showed that these molecules form clusters that are specific for parasitic platyhelminths as it was shown for integrins before. Transcripts of all genes colocalized in the ovary. In Xenopus oocytes germinal vesicle breakdown (GVBD) was only induced if all members were simultaneously expressed. Coimmunoprecipitation results suggest that a Smβ-Int1-SmILK-SmPINCH-SmNck2-SmVKR1 complex can be formed leading to the phosphorylation and activation of SmVKR1. These results indicate that SmVKR1 can be activated in a ligand-independent manner by receptor-complex interaction. RNAi and inhibitor studies to knock-down SmILK as a representative complex member concurrently revealed effects on the extracellular matrix surrounding the ovary and oocyte localization within the ovary, oocyte survival, and egg production. By TUNEL assays, confocal laser scanning microscopy (CLSM), Caspase-3 assay, and transcript profiling of the pro-apoptotic BCL-2 family members BAK/BAX we obtained first evidence for roles of this signaling

  13. Ovary of Fundulus heteroclitus

    SciTech Connect

    Brummett, A.R.; Dumont, J.N.; Larkin, J.R.

    1982-01-01

    Scanning and transmission electron microscopy, together with dissection and light microscopy, have produced heretofore unavailable structural detail of the ovary of Fundulus heteroclitus. Structural and functional interrelationships among developing follicles and other histological elements, particularly as they might relate to vascularization of follicles, oocyte development, and ovulation, are described and discussed. Mature eggs, ovulated into the ovarian lumen, accumulate in the posterior ovisac region of the ovary prior to oviposition. This ovisac region is thin-walled and apparently nongerminal. The temporary retention of ovulated eggs permits cyclical oviposition even though oogenesis and ovulation are asynchronous. The histological differences between the ovisac and the anterior ovigerous region of the ovary are described. The lumenal epithelium of the ovisac displays a localized population of unusual cells with long cytoplasmic extensions. The ultrastructure of these cells suggests that they might function in the transport of ovulated eggs into the oviduct and/or in secreting the substance (jelly) which forms the surface coat of extruded eggs.