scholarly journals Effects of the S-adenosylmethionine decarboxylase inhibitor, 5′-{[(Z)-4-amino-2-butenyl]methylamino)-5′-deoxyadenosine, on cell growth and polyamine metabolism and transport in Chinese hamster ovary cell cultures

1994 ◽  
Vol 303 (1) ◽  
pp. 89-96 ◽  
Author(s):  
T L Byers ◽  
R S Wechter ◽  
R H Hu ◽  
A E Pegg
Keyword(s):  
Cell Growth ◽  
Transport System ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Polyamine Metabolism ◽  
Decarboxylase Inhibitor ◽  
Transport Activity ◽  
Polyamine Transport

The regulation of polyamine transport and the roles of polyamine transport and synthesis in cell growth were investigated using cultured Chinese hamster ovary (CHO) cells and CHOMG cells which are mutants lacking polyamine-transport activity. Metabolically stable methylated polyamine analogues were used to measure polyamine accumulation, and the irreversible S-adenosyl-L-methionine decarboxylase inhibitor, 5′-([(Z)-4-amino-2-butenyl]methylamino)-5′-deoxyadenosine (AbeAdo), was used to inhibit synthesis. Exposure to AbeAdo lead to a dose-dependent decrease in growth for both cell lines, although CHOMG cells were more sensitive. Intracellular putrescine levels were greatly increased in AbeAdo-treated CHO cells and to a lesser extent in CHOMG cells, whereas intracellular spermidine and spermine levels were substantially reduced in both. Treatment with AbeAdo increased putrescine content in the culture medium to a much greater extent in CHOMG cultures indicating that a portion of the excess putrescine synthesized in response to AbeAdo treatment is excreted, but that CHO cells salvage this putrescine whereas it is lost to CHOMG cells which cannot take up polyamines. AbeAdo treatment increased polyamine transport into CHO cells despite high intracellular putrescine, suggesting that spermidine and/or spermine, and not putrescine, are the major factors regulating transport activity. The accumulation of either 1-methylspermidine or 1,12-dimethylspermine was significantly increased by AbeAdo treatment. Accumulation was increased even further when protein synthesis was blocked by cycloheximide, indicating that a short-lived protein is involved in the regulation of polyamine uptake. In the presence of cycloheximide and AbeAdo or alpha-difluoromethylornithine, methylated polyamine derivatives accumulated to very high levels leading to cell death. These results show that the polyamine-transport system plays an important role in retaining intracellular polyamines and that down-regulation of the transport system in response to increased intracellular polyamine content is necessary to prevent accumulation of toxic levels of polyamines.

scholarly journals Expression of a human gene for polyamine transport in Chinese-hamster ovary cells

1989 ◽  
Vol 263 (3) ◽  
pp. 745-752 ◽  
Author(s):  
T L Byers ◽  
R Wechter ◽  
M E Nuttall ◽  
A E Pegg
Keyword(s):  
Transport System ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster Ovary Cells ◽  
Mutant Cell ◽  
Molecular Genetic ◽  
Chinese Hamster ◽  
Transport Systems ◽  
Polyamine Transport System ◽  
Polyamine Transport

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.

Chinese hamster ovary cell growth and interferon production kinetics in stirred batch culture

1991 ◽  
Vol 34 (5) ◽  
pp. 559-564 ◽  
Author(s):  
P. M. Hayter ◽  
E. M. A. Curling ◽  
A. J. Baines ◽  
N. Jenkins ◽  
I. Salmon ◽  
...  
Keyword(s):  
Cell Growth ◽  
Batch Culture ◽  
Chinese Hamster Ovary Cell ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Interferon Production ◽  
Production Kinetics

scholarly journals Isolation and characterization of Chinese hamster ovary cell variants defective in adhesion to fibronectin-coated collagen.

1980 ◽  
Vol 87 (3) ◽  
pp. 755-763 ◽  
Author(s):  
P A Harper ◽  
R L Juliano
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Divalent Cations ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Human Diploid Fibroblasts ◽  
Isolation And Characterization ◽  
A Cell ◽  
Cold Insoluble Globulin

Variant clones of Chinese hamster ovary (CHO) cells were selected for reduced adhesion to serum-coated tissue culture plates. These clones also displayed reduced adhesion to substrata composed of collagen layers coated with bovine serum or with fibronectin (cold-insoluble globulin). Wild-type (WT) and adhesion variant (ADv) cells grew at comparable rates in suspension culture, but the adhesion variants could not be grown in monolayer culture because of their inability to attach to the substratum. The adhesion deficit in these cells was not corrected by raising the concentration of divalent cations or of serum to levels 10-fold greater than those normally utilized in cell culture. However, both WT and ADv clones could adhere, spread, and attain a normal CHO morphology on substrata coated with concanavalin A or poly-L-lysine. In addition, the adhesion variants could attach to substrata coated with "footpad" material (substratum-attached material) derived from monolayers of human diploid fibroblasts or WT CHO cells. These observations suggest that the variant clones may have a cell surface defect that prevents them from utilizing exogeneous fibronectin as an adhesion-promoting ligand; however the variants seem to have normal cytoskeletal and metabolic capacities that allow them to attach and spread on substrata coated with alternative ligands. These variants should be extremely useful in studying the molecular basis of cell adhesion.

scholarly journals Amino Acid Requirements of the Chinese Hamster Ovary Cell Metabolism during Recombinant Protein Production

2019 ◽  
Author(s):  
Bergthor Traustason
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Recombinant Protein ◽  
Recombinant Proteins ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Scale Model ◽  
Amino Acid Requirements

SummaryMajority of biopharmaceutical drugs today are produced by Chinese hamster ovary (CHO) cells, which have been the standard industry host for the past decades. To produce and secrete a substantial amount of the target recombinant proteins the CHO cells must be provided with suitable growth conditions and provided with the necessary nutrients. Amino acids play a key role in this as the building blocks of proteins, playing important roles in a large number of metabolic pathways and being important sources of nitrogen as well as carbon under certain conditions. In this study exploratory analysis of the amino acid requirements of CHO cells was carried out using metabolic modelling approaches. Flux balance analysis was employed to evaluate the optimal distribution of fluxes in a genome-scale model of CHO cells to gain information on the cells’ metabolic response in silico.The results showed that providing non-essential amino acids (NEAAs) has a positive effect on CHO cell biomass production and that cysteine as well as tyrosine play a fundamental role in this. This implies that extracellular provision of NEAAs limits the extent of energy loss in amino acid biosynthetic pathways and renders additional reducing power available for other biological processes. Detailed analysis of the possible secretion and uptake of D-serine in the CHO model was also performed and its influence on the rest of the metabolism mapped out, which revealed results matching various existing literature. This is interesting since no mention of D-serine in regard to CHO cells was found in current literature, as well as the fact that this opens up the possibility of using the model for better understanding of certain disorders in higher up organisms that have been implicated with D-serine, such as motor neuron and cognitive degeneration. Finally, outcome from the model optimisation of different recombinant proteins demonstrated clearly how the difference in protein structure and size can influence the production outcome. These results show that systematic and model-based approaches have great potential for broad de novo exploration as well as being able to handle the cellular burden associated with the production of different types of recombinant protein.

Chinese hamster ovary mRNA-dependent, Na(+)-independent L-leucine transport in Xenopus laevis oocytes

1992 ◽  
Vol 12 (12) ◽  
pp. 5281-5287
Author(s):  
T Z Su ◽  
C D Logsdon ◽  
D L Oxender
Keyword(s):  
Amino Acids ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Xenopus Laevis Oocytes ◽  
Transport Activity ◽  
Leucine Uptake ◽  
System L ◽  
Cationic Amino Acids ◽  
Leucine Transport

In freshly prepared uninjected folliculated oocytes, Na(+)-independent leucine uptake is mediated predominantly by a system L-like transport system. Removal of follicular cells, however, results in an irreversible loss of this transport activity. When total poly(A)+ mRNA derived from Chinese hamster ovary (CHO) cells was injected into prophase-arrested stage V or VI Xenopus laevis oocytes, enhanced expression of Na(+)-independent leucine transport was observed. The injected mRNAs associated with increased levels of leucine uptake were between 2 and 3 kb in length. The newly expressed leucine transport activity exhibited important differences from the known characteristics of system L, which is the dominant Na(+)-independent leucine transporter in CHO cells as well as in freshly isolated folliculated oocytes. The CHO mRNA-dependent leucine uptake in oocytes was highly sensitive to the cationic amino acids lysine, arginine, and and ornithine (> 95% inhibition). As with the leucine uptake, an enhanced lysine uptake was also observed in size-fractionated CHO mRNA-injected oocytes. The uptakes of leucine and lysine were mutually inhibitable, suggesting that the newly expressed transporter was responsible for uptakes of both leucine and lysine. The inhibition of uptake of lysine by leucine was Na+ independent, thus clearly distinguishing it from the previously reported endogenous system y+ activity. Furthermore, the high sensitivity to tryptophan of the CHO mRNA-dependent leucine transport was in sharp contrast to the properties of the recently cloned leucine transport-associated gene from rat kidney tissue, although leucine transport from both sources was sensitive to cationic amino acids. Our results suggest that there may be a family of leucine transporters operative in different tissues and possibly under different conditions.

scholarly journals Expanding the Chinese hamster ovary cell long non-coding RNA transcriptome using RNASeq

2019 ◽  
Author(s):  
Krishna Motheramgari ◽  
Ricardo Valdés-Bango Curell ◽  
Ioanna Tzani ◽  
Clair Gallagher ◽  
Marina Castro Rivadeneyra ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Cell Biology ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Cho Cell ◽  
Transcriptomic Response ◽  
Protein Coding ◽  
Non Coding Rnas

AbstractOur ability to study Chinese hamster ovary (CHO) cell biology has been revolutionised over the last decade with the development of next generation sequencing and the publication of reference DNA sequences for CHO cells and the Chinese hamster. RNA sequencing has not only enabled the association of transcript expression with bioreactor conditions and desirable bioprocess phenotypes but played a key role in the characterisation of protein coding and small non-coding RNAs. The annotation of long non-coding RNAs, and therefore our understanding of their role in CHO cell biology, has been limited to date. In this manuscript, we use high resolution RNASeq data to more than double the number of annotated lncRNA transcripts for the CHOK1 genome. In addition, the utilisation of strand specific sequencing enabled the identification of more than 1,000 new lncRNAs located antisense to protein coding genes. The utility of monitoring lncRNA expression is demonstrated through an analysis of the transcriptomic response to a reduction of cell culture temperature and identification of simultaneous sense/antisense differential expression for the first time in CHO cells. To enable further studies of lncRNAs, the transcripts annotated in this study have been made available for the CHO cell biology community.

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