Linked Life Data

10699869

Source:http://linkedlifedata.com/resource/pubmed/id/10699869

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
2000-4-24
pubmed:abstractText
The influence of Bcl-2 expression on the robustness of a CHO cell line (22H11) developed for the industrial production of a chimeric antibody was evaluated. Western blot analysis following transfection with the expression vector unexpectedly revealed upregulation of endogenous Bcl-2 expression in the control (Neo) cell line in response to exposure to the selection drug G418. This indicated that geneticin may function by inducing apoptosis in cells not carrying the control plasmid or expressing very low levels of survival genes. Thus, exposure to the drug enriched the culture for a population of cells which expressed enhanced levels of endogenous Bcl-2. In batch cultures, ectopic bcl-2 expression resulted in a 75% increase in maximum viable cell density over control cultures. Moreover, the rate of decrease in viability in the Bcl-2 cultures was significantly lower than that in the control cultures. After 18 days, the Bcl-2 viability was around 90%, compared to 20% in the control cultures. Evaluation of the mechanism of cell death revealed very few cells with classical apoptotic morphology. Around 10% were clearly necrotic, but the majority of dead cells were seen as chromatin free but otherwise relatively intact structures. Because of the relatively low rate of cell death in both cell lines, few cells were observed in the transitional, easily identifiable early stages of apoptosis. However, DNA gel electrophoresis revealed a clear ladder-pattern, but only in the control cultures, thus confirming high levels of apoptotic death. Antibody concentrations during both sets of cultures were very similar, both during the growth and death phases, with a maximum titer of around 40 microgram/ml. Analysis of Bcl-2 expression by flow cytometry revealed that the cultures contained two populations of cells: a large population which expressed high levels of Bcl-2 and a relatively smaller low-expressing population. During the course of the batch, the smaller, low-expressing population declined in frequency, suggesting that these cells were more sensitive to cell death. In addition, the mean level of Bcl-2 expression in the overexpressing population also declined significantly, presumably reflecting the exhaustion of precursors for protein synthesis following nutrient depletion. Importantly, when cells were taken from day 40 of the significantly extended Bcl-2 batch cultures, they immediately proliferated, confirming that they had retained their replicative potential. Cultivation of the cells in basal medium lacking (individually) serum, all amino acids, glutamate/asparagine, and, finally, glucose, resulted in relatively lower viable cell numbers and viability in the control cell line compared to the Bcl-2 cell line. Exposure of cells to ammonia toxicity also revealed the relative robustness of the bcl-2 transfected cells. When growth was arrested by treatment with 4 mM thymidine, Bcl-2 overexpressing cells exhibit a viability of over 80% after 5 days in culture, compared to only 40% in the control cell line. However, under growth-arrested conditions, there was no major difference in antibody titer between the two cell lines.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Apr
pubmed:issn
0006-3592
pubmed:author
pubmed:copyrightInfo
Copyright 2000 John Wiley & Sons, Inc.
pubmed:issnType
Print
pubmed:day
5
pubmed:volume
68
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
31-43
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed-meshheading:10699869-Ammonia, pubmed-meshheading:10699869-Animals, pubmed-meshheading:10699869-Anti-Bacterial Agents, pubmed-meshheading:10699869-Antibodies, Monoclonal, pubmed-meshheading:10699869-Antibody Formation, pubmed-meshheading:10699869-Apoptosis, pubmed-meshheading:10699869-Biotechnology, pubmed-meshheading:10699869-Blotting, Western, pubmed-meshheading:10699869-CHO Cells, pubmed-meshheading:10699869-Cell Division, pubmed-meshheading:10699869-Cell Survival, pubmed-meshheading:10699869-Cricetinae, pubmed-meshheading:10699869-Culture Media, pubmed-meshheading:10699869-Flow Cytometry, pubmed-meshheading:10699869-Gene Expression, pubmed-meshheading:10699869-Gentamicins, pubmed-meshheading:10699869-Proto-Oncogene Proteins c-bcl-2, pubmed-meshheading:10699869-Recombinant Fusion Proteins, pubmed-meshheading:10699869-Transfection
pubmed:year
2000
pubmed:articleTitle
Influence of bcl-2 on cell death during the cultivation of a Chinese hamster ovary cell line expressing a chimeric antibody.
pubmed:affiliation
Animal Cell Technology Group, School of Chemical Engineering, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't