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  • 1
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 48 (1995), S. 118-122 
    ISSN: 0006-3592
    Keywords: apoptosis ; bcl-2 ; hybridoma ; cell survival ; antibody productivity ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Human bcl-2 DNA was introduced into mouse hybridoma 2E3 cells and expressed at a high level by using BCMGSneo vector, which reportedly amplifies as multiple copies in the cells independently of their chromosomes. The high expression of bcl-2 in BCMGSneo-bcl-2 transfectants was confirmed by western blotting. In batch cultures, the overexpression of bcl-2 raised the maximum viable cell density by 45%, delayed the initiation of apoptosis by 2 days, and prolonged the viable culture period by 4 days. The delayed initiation of apoptosis was detected by emergence of the ladder pattern on DNA electrophoresis and increase of the dead cell number. The bcl-2 transfectants produced lgG1 fourfold per batch culture in comparison with 2E3 cells transfected with BCMGSneo but not with bcl-2: a little less than twofold due to the improved survival of the cells and more than twofold due to the enhanced lgG1 production rate per cell of the bcl-2 transfectants. The method to engineer hybridoma cells genetically with bcl-2 using BCMGSneo vector for increasing viability and productivity would be widely applied for improving antibody productivity of hybridoma cultures. © 1995 John Wiley & Sons, Inc.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 0006-3592
    Keywords: c-jun ; cell cycle ; apoptosis ; antisense ; growth deprivation ; F-MEL ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: F-MEL cells were transfected with the c-jun antisense gene located downstream of a glucocorticoid-inducible MMTV promoter, and the obtained cells were named c-jun AS cells. When the c-jun AS cells were treated with dexamethasone (DEX) in DMEM supplemented with 10% serum, the growth of the cells was completely suppressed for a duration of 16 days with a high cell viability exceeding 86%. The c-jun expression in the c-jun AS cells was suppressed moderately in the absence of DEX and strongly in the presence of DEX. The c-jun AS cells grew well and reached a density of 106 cells/mL without supplementation of any serum components. Viability was greater than 80% after the cells had been cultured for 8 days in the absence of DEX. The c-jun AS cells stayed at a constant cell density and high viability above 80% for 8 days when they were cultured in the presence of DEX under serum deprivation. In contrast, the wild type F-MEL cells were unable to grow and died by apoptosis in 3 days under serum deprivation. Internucleosomal cleavage of DNA, a landmark of apoptosis, was clearly detectable. Thus the c-jun AS cell line that is resistant to apoptosis induced by serum deprivation and can reversibly and viably be growth-arrested was established. A dual-signal model was proposed to explain the experimental result, the interlinked regulation of apoptosis, and growth by c-jun.© 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:65-72, 1998.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1573-0778
    Keywords: apoptosis ; bcl-2 ; COS cell ; myeloma
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract COS, myeloma and HeLa cells, which are commonly used for protein production by cell culture, were transfected with human bcl-2 gene encoded on the shuttle vector BCMGS. Expression of human bcl-2 improved survival of cells remarkably, mildly, or negligibly for COS, myeloma, and HeLa, respectively. Four clones were obtained from the human bcl-2 expressing cell population of COS cells. They expressed human bcl-2 almost at the same level. The viable cell numbers were 6, 2.5, 2.5, and 0.8 times as many for the clones #8, #5, #6, and #7, respectively, as for the control COS cells, when they were cultured at low (0.2%) serum concentration for 9 days. The bcl-2 overexpressing COS cells showed morphology different from that of the control COS cells in serum limited condition. When transfected with mouse lambda protein gene carried by an SV40-derived vector, clone #8 of the bcl-2 transfected COS cells continued the transient expression of lambda protein longer than the control COS cells.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 1573-0778
    Keywords: antisense ; apoptosis ; cell cycle ; c-jun ; protein production
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Expression of c-jun gene induces apoptosis ofcells cultured in serum-free medium. It also promotescell-cycling in serum-containing medium, leading cellsto die by overgrowth. Previously, we established anapoptosis-suppressible, cell-cycle arrestable cellline, c-jun AS, by transfecting Friend murineerythroleukemia (F-MEL) cells with adexamethasone-inducible antisense c-jun gene.Induction of the antisense c-jun transcriptionwith dexamethasone suppressed c-jun expression.As a result, c-jun AS cells survived inserum-free medium containing dexamethasone for a longtime, while F-MEL cells died quickly in the presenceor absence of dexamethasone. In serum-containingmedium, the growth of c-jun AS cells was viablyblocked by inducing antisense c-juntranscription, and the cells survived at thenon-growth state avoiding overgrowth. In the presentstudy, protein productivity of c-jun AS cellswas examined in comparison with that of wild typeF-MEL cells. C-jun AS and F-MEL cells werefurther transfected with a vector for expressingalkaline phosphatase as a protein to be produced, andnamed c-jun AS-SEAP and F-MEL-SEAP cells,respectively. In the serum-free medium withdexamethasone, c-jun AS-SEAP cells produced theprotein for up to 6 days, while F-MEL-SEAP cellsstopped production on day 3 due to cell death causedby serum deprivation. In the serum-containing mediumwith dexamethasone, c-jun AS-SEAP cells wereviably arrested in the cell cycle, and cell death dueto overgrowth was avoided. As the result, they couldproduce the protein for up to 18 days, whileF-MEL-SEAP cells stopped production within 7 days dueto cell death caused by overgrowth.
    Type of Medium: Electronic Resource
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  • 5
    ISSN: 1573-0778
    Keywords: antibody productivity ; apoptosis ; bcl-2 ; fed batchculture ; hybridoma
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Mouse hybridoma 2E3 transfected with human bcl-2 gene survived longer with increasing expression level of bcl-2 when cultured in DME medium supplemented with 9% serum. One of the transfectants, 2E3BCMGbcl-2, overexpressed bcl-2 and could maintain viable cell density higher than the initial density for more than four days at a low 0.5% serum concentration. In comparison a mock transfectant 2E3BCMG remained viable for only one day. However, both hybridomas died out within a day in serum-free medium. These results suggested that bcl-2 needed a small amount of some serum components to suppress apoptosis of the hybridoma. Overexpression of bcl-2 also suppressed apoptosis of the hybridoma induced by glutamine deprivation. When hybridoma 2E3BCMGbcl-2 was inoculated in DME medium supplemented with 9% serum and cultured for 10 d with additional 2% serum feed at day 4 of the culture, viable cell density increased 2-fold and antibody produced 3-fold, in comparison with mock transfected 2E3 cultured in the same manner. The mock transfectant with additional feed of serum at day 4 of the culture showed no difference in viable cell density and antibody production. These results suggested that the mock transfectant committed to apoptosis before day 4 of the culture and the additional serum at day 4 could not reverse the commitment.
    Type of Medium: Electronic Resource
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  • 6
    ISSN: 1573-0778
    Keywords: antibody productivity ; apoptosis ; BAG-1 ; Bcl-2 ; cell survival ; hybridoma
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Human bcl-2 and bag-1 DNA were introduced into mouse hybridoma 2E3- O cells and expressed. The expression of bcl-2 in BCMGneo-bcl2 transfectants was confirmed by ELISA and that of bag-1 in pZeo-bag1 was confirmed by western blotting. In batch cultures, the over-expression of bcl-2 prolonged the culture period by 2 days and co-expression of bcl-2 and bag-1 prolonged the culture period by 3 days. The delayed increase in the dead cell number in culture of the bcl-2 and bag-1 cotransfectant indicated the additional antiapoptosis effect of bcl-2 and bag-1 cotransfection in comparison with the bcl-2 only transfection. The bcl-2 transfectants (2E3O-Bcl2) produced antibody twofold per batch culture in comparison with 2E3-O cells transfected with BCMGSneo (2E3O-Mock). Enhancement of this MoAb production was due to the improved survival of the cells and was not due to stimulation of antibody production rate per cell by Bcl-2 expression. And the bcl-2 and bag-1 co-transfectant (2E3O-Bcl2-BAG1) produced antibody approximately fourfold of 2E3O-Mock per batch culture. Enhancement of this MoAb production was due to the improved survival of the cells and was partly due to stimulation of MoAb production rate per cell in the non-growing phase by the cotransfection. The method to engineer hybridoma cells genetically with bcl-2 and bag-1 for increasing viability and productivity would be widely applied for improving antibody productivity of hybridoma cultures.
    Type of Medium: Electronic Resource
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