Polyamine starvation causes accumulation of cadaverine and its derivatives in a polyamine-dependent strain of Chinese-hamster ovary cells.
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ABSTRACT:
SUBMITTER: Holtta E
PROVIDER: S-EPMC1154311 | biostudies-other | 1983 Mar
REPOSITORIES: biostudies-other
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Project description:We have used Chinese-hamster ovary (CHO) cells maintained in a chemically defined medium to study the regulation of ornithine decarboxylase (ODC) by polyamines. Cells maintained in the defined medium had no detectable putrescine, and approx. 1-3 units of ODC activity/10(6) cells, where 1 unit corresponds to 1 nmol of substrate decarboxylated in 30 min. The defined medium is ornithine-deficient, thus limiting the exogenous substrate for ODC, and subsequently decreasing intracellular polyamine accumulation. Restoration of intracellular putrescine and increased formation of spermidine by addition of exogenous ornithine or putrescine led to a marked decrease in ODC activity, which was paralleled by a decrease in a alpha-DL-difluoromethyl[3,4-3H]ornithine (DFMO)-binding protein of Mr approx. 53,000, which is precipitable with anti-ODC antibody. Calculation of DFMO binding per unit of activity showed no change in the specific activity of the enzyme. We identified [35S]methionine-labelled peptides corresponding to ODC by immunoprecipitation of radiolabeled whole cell proteins. Only one protein was precipitated, of Mr approx. 53 000, which co-migrated with the DFMO-binding protein. Immunoprecipitation of radiolabelled proteins from cells incubated in the presence of exogenous ornithine indicated that the observed activity decrease was not due to an inhibition of ODC protein synthesis. Analysis of immunoprecipitable ODC protein from cells that had been pulse-labelled with [35S]methionine, and then treated for 5 h with 100 microM-ornithine, -putrescine or -spermidine, revealed a distinct disappearance of labelled ODC protein after restoration of intracellular polyamine pools. No detectable turnover of ODC was observed in the absence of exogenous polyamine treatment. These data support the hypothesis that ODC protein, and subsequent activity, is regulated by intracellular polyamine content through mechanisms that influence turnover of the enzyme.
| S-EPMC1146847 | biostudies-other
Project description:A molecular-genetic approach towards isolating mammalian polyamine-transport genes and their encoded proteins was devised involving the production of Chinese-hamster ovary (CHO) cells expressing a human polyamine-transport protein. CHO cells and a polyamine-transport-deficient CHO mutant cell line (CHOMG) were equally sensitive to the antiproliferative effects of alpha-difluoromethylornithine (DFMO), which blocked endogenous polyamine synthesis. Exposure to exogenous polyamines increased intracellular polyamine levels and reversed this DFMO-induced cytostasis in the CHO cells, but not in the CHOMG cells. CHOMG cells were therefore transfected with human DNA (isolated from HT-29 colon carcinoma cells) and cells expressing the human polyamine-transport system were identified by the ability of these cells to grow in a medium containing DFMO and polyamines. A number of different positive clones were identified and shown to have the capacity for polyamine uptake and an increased sensitivity to the toxic effects of the polyamine analogue methylglyoxal bis(guanylhydrazone). Differences in these properties between the clones are consistent with a multiplicity of polyamine-transport systems. Some clones also showed a change in growth characteristics, which may indicate a relationship between genes involved in the polyamine-transport system and in cell proliferation.
| S-EPMC1133495 | biostudies-other
Project description:To complement the recent genomic sequencing of Chinese hamster ovary (CHO) cells, proteomic analysis was performed on CHO cells including the cellular proteome, secretome, and glycoproteome using tandem mass spectrometry (MS/MS) of multiple fractions obtained from gel electrophoresis, multidimensional liquid chromatography, and solid phase extraction of glycopeptides (SPEG). From the 120 different mass spectrometry analyses generating 682,097 MS/MS spectra, 93,548 unique peptide sequences were identified with at most 0.02 false discovery rate (FDR). A total of 6164 grouped proteins were identified from both glycoproteome and proteome analysis, representing an 8-fold increase in the number of proteins currently identified in the CHO proteome. Furthermore, this is the first proteomic study done using the CHO genome exclusively, which provides for more accurate identification of proteins. From this analysis, the CHO codon frequency was determined and found to be distinct from humans, which will facilitate expression of human proteins in CHO cells. Analysis of the combined proteomic and mRNA data sets indicated the enrichment of a number of pathways including protein processing and apoptosis but depletion of proteins involved in steroid hormone and glycosphingolipid metabolism. Five-hundred four of the detected proteins included N-acetylation modifications, and 1292 different proteins were observed to be N-glycosylated. This first large-scale proteomic analysis will enhance the knowledge base about CHO capabilities for recombinant expression and provide information useful in cell engineering efforts aimed at modifying CHO cellular functions.
| S-EPMC3772721 | biostudies-literature
Project description:Mitosis is a fundamental process in the development of all organisms. The mitotic spindle guides the cell through mitosis as it mediates the segregation of chromosomes, the orientation of the cleavage furrow, and the progression of cell division. Birth defects and tissue-specific cancers often result from abnormalities in mitotic events. Here, we report a proteomic study of the mitotic spindle from Chinese Hamster Ovary (CHO) cells. Four different isolations of metaphase spindles were subjected to Multi-dimensional Protein Identification Technology (MudPIT) analysis and tandem mass spectrometry. We identified 1155 proteins and used Gene Ontology (GO) analysis to categorize proteins into cellular component groups. We then compared our data to the previously published CHO midbody proteome and identified proteins that are unique to the CHO spindle. Our data represent the first mitotic spindle proteome in CHO cells, which augments the list of mitotic spindle components from mammalian cells.
| S-EPMC3103581 | biostudies-literature
Project description:The Chinese hamster ovary (CHO) cell line is a major expression system for the production of therapeutic proteins, the majority of which are glycoproteins, such as antibodies and erythropoietin (EPO). The characterization glycosylation profile of therapeutic proteins produced from engineered CHO cells and therapeutic functions, as well as side effects, are critical to understand the important roles of glycosylation. In this study, a large scale glycoproteomic workflow was established and applied to CHO-K1 cells expressing EPO. The workflow includes enrichment of intact glycopeptides from CHO-K1 cell lysate and medium using hydrophilic enrichment, fractionation of the obtained intact glycopeptides (IGPs) by basic reversed phase liquid chromatography (bRPLC), analyzing the glycopeptides using LC-MS/MS, and annotating the results by GPQuest 2.0. A total of 10 338 N-linked glycosite-containing IGPs were identified, representing 1162 unique glycosites in 530 glycoproteins, including 71 unique atypical N-linked IGPs on 18 atypical N-glycosylation sequons with an overrepresentation of the N-X-C motifs. Moreover, we compared the glycoproteins from CHO cell lysate with those from medium using the in-depth N-linked glycoproteome data. The obtained large scale glycoproteomic data from intact N-linked glycopeptides in this study is complementary to the genomic, proteomic, and N-linked glycomic data previously reported for CHO cells. Our method has the potential to monitor the production of recombinant therapeutic glycoproteins.
| S-EPMC6440468 | biostudies-literature
Project description:Cell-cycle-dependent protein levels and phosphorylation of DNA topoisomerase II in relation to its catalytic and cleavage activities were studied in Chinese-hamster ovary cells. Immunoreactive topoisomerase II protein levels were maximal in G2-phase cells, intermediate in S- and M-phase cells, and minimal in a predominantly G1-phase population. When the phosphorylation of topoisomerase II in vivo was corrected for differences in specific radioactivity of intracellular ATP, the apparent phosphorylation of S- and M-phase topoisomerase II was altered significantly. Relative phosphorylation in vivo was found to be greatest in M-phase cells and decreased in the other populations in the order: S > G2 > asynchronous. Phosphoserine was detected in every phase of the cell cycle, with a minor contribution of phosphothreonine demonstrated in M-phase cells. Topoisomerase II activity measured in vivo as 9-(4,6-O-ethylidene-beta-D-glucopyranosyl)-4'-demethylepipodophylloto xin (VP-16)-induced DNA double-strand breaks (determined by neutral filter elution) increased in the order: asynchronous < S < G2 < M. Topoisomerase II cleavage activity, assayed in vitro as the formation of covalent enzyme-DNA complexes, was lowest in S phase, intermediate in asynchronous and G2-phase cells, and maximal in M phase. Topoisomerase II decatenation activity was 1.6-1.8-fold greater in S-, G2- and M-phase populations relative to asynchronous cells. Therefore DNA topoisomerase II activity measured both in vivo and in vitro is maximal in M phase, that phase of the cell cycle with an intermediate level of immunoreactive topoisomerase II but the highest level of enzyme phosphorylation. The discordance between immunoreactive topoisomerase II protein levels, adjusted relative phosphorylation, catalytic activity, cleavage activity and amino acid residue(s) modified, suggests that the site of phosphorylation may be cell-cycle-dependent and critical in determining catalytic and cleavage activity.
| S-EPMC1134354 | biostudies-other
Project description:High concentrations of cytosolic Na(+) ions induce the time-dependent formation of an inactive state of the Na(+)/Ca(2+) exchanger (NCX), a process known as Na(+)-dependent inactivation. NCX activity was measured as Ca(2+) uptake in fura 2-loaded Chinese hamster ovary (CHO) cells expressing the wild-type (WT) NCX or mutants that are hypersensitive (F223E) or resistant (K229Q) to Na(+)-dependent inactivation. As expected, 1) Na(+)-dependent inactivation was promoted by high cytosolic Na(+) concentration, 2) the F223E mutant was more susceptible than the WT exchanger to inactivation, whereas the K229Q mutant was resistant, and 3) inactivation was enhanced by cytosolic acidification. However, in contrast to expectations from excised patch studies, 1) the WT exchanger was resistant to Na(+)-dependent inactivation unless cytosolic pH was reduced, 2) reducing cellular phosphatidylinositol-4,5-bisphosphate levels did not induce Na(+)-dependent inactivation in the WT exchanger, 3) Na(+)-dependent inactivation did not increase the half-maximal cytosolic Ca(2+) concentration for allosteric Ca(2+) activation, 4) Na(+)-dependent inactivation was not reversed by high cytosolic Ca(2+) concentrations, and 5) Na(+)-dependent inactivation was partially, but transiently, reversed by an increase in extracellular Ca(2+) concentration. Thus Na(+)-dependent inactivation of NCX expressed in CHO cells differs in several respects from the inactivation process measured in excised patches. The refractoriness of the WT exchanger to Na(+)-dependent inactivation suggests that this type of inactivation is unlikely to be a strong regulator of exchange activity under physiological conditions but would probably act to inhibit NCX-mediated Ca(2+) influx during ischemia.
| S-EPMC2575829 | biostudies-other
Project description:BackgroundChinese hamster ovary (CHO) cells are widely used for industrial production of biopharmaceuticals. Many genetic, chemical, and environmental approaches have been developed to modulate cellular pathways to improve titers. However, these methods are often irreversible or have off-target effects. Development of techniques which are precise, tunable, and reversible will facilitate temporal regulation of target pathways to maximize titers. In this study, we investigate the use of optogenetics in CHO cells. The light-activated CRISPR-dCas9 effector (LACE) system was first transiently transfected to express eGFP in a light-inducible manner. Then, a stable system was tested using lentiviral transduction.ResultsTransient transfections resulted in increasing eGFP expression as a function of LED intensity, and activation for 48 h yielded up to 4-fold increased eGFP expression compared to cells kept in the dark. Fluorescence decreased once the LACE system was deactivated, and a protein half-life of 14.9 h was calculated, which is in agreement with values reported in the literature. In cells stably expressing the LACE system, eGFP expression was confirmed, but there was no significant increase in expression following light activation.ConclusionsTaken together, these results suggest that optogenetics can regulate CHO cell cultures, but development of stable cell lines requires optimized expression levels of the LACE components to maintain high dynamic range.
| S-EPMC7863527 | biostudies-literature
Project description:Despite the abundance of available cell lines, nearly 70% of all recombinant therapeutic proteins today are produced in Chinese hamster ovary (CHO) cells. The impact of protein overproduction on the secretion of exosomes by CHO cells has been investigated here. Increased secretion of extracellular vesicles (EVs) by protein overexpressing CHO cells was demonstrated with protein content assay, nanoparticle tracking analysis, and capillary electrophoresis. Our results revealed that a protein overproduction might induce EVs secretion, which might be accompanied by the sequestration and loading of overexpressed proteins into the exosomes. These findings are of vital importance for the manufacturing of therapeutics in CHO expression systems due to the risk of product loss during downstream processing of culture medium as well as the application of exosomes as nanocarriers of therapeutic proteins. The study indicates also the importance of culturing process control.
| S-EPMC10287810 | biostudies-literature
Project description:Complete, accurate genome assemblies are necessary to design targets for genetic engineering strategies. Successful gene knockdowns and knockouts in Chinese hamster ovary (CHO) cells may prevent the expression of difficult-to-remove host cell proteins (HCPs). HCPs, if not removed, can cause problems in stability, safety, and efficacy of the biotherapeutic. A significantly improved Chinese hamster (CH) reference genome was used to identify new knockout targets with similar predicted functions and characteristics as the difficult-to-remove host cell lipases, LPL, PLBL2, and LPLA2. The CHO-K1 gene and protein sequences of several of these lipases were corrected using the updated CH genome. Sequence alignments were then used to identify conserved regions that may serve as possible targets for multiple simultaneous gene knockouts. Finally, comparison of the CHO-K1 lipase protein sequences to their human orthologs provided insight into which lipases, if persistent in the drug product, could possibly cause immunogenic responses in patients.
| S-EPMC6430115 | biostudies-literature