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  • 1
    Electronic Resource
    Electronic Resource
    Cell suicide in starving hybridoma culture: survival-signal effect of some amino acids (1997)
    Springer
    Cytotechnology 23 (1997), S. 231-239 
    Details
    ISSN: 1573-0778
    Keywords: apoptosis ; hybridoma ; amino acids ; starvation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Two mouse hybridoma cell lines cultured in different basal media withthe iron-rich protein-free supplement were subjected to deliberatestarvation by inoculation into media diluted with saline to 50% or less.In the diluted media the growth was markedly suppressed and a largefraction of cells died by apoptosis. The cells could be rescued fromapoptotic death by individual additions of amino acids, such as glycine,L-alanine, L-serine, L-threonine, L-proline, L-asparagine, L-glutamine,L-histidine, D-serine, β-alanine or taurine. Amino acids withhydrophobic or charged side chains were without effect. The apoptosispreventing activity manifested itself even in extremely diluted media,down to 10% of the standard medium. The activity of L-alanine in theprotection of cells starving in 20% medium was shown also in semicontinuousculture. In the presence of 2 mM L-alanine the steady-state viable cell density more than doubled, with respect to control, andthe apoptotic index dropped from 37% in the control to 16%. It wasconcluded that the apoptosis-preventing amino acids acted as signalmolecules, rather than nutrients, and that the signal had a character ofa survival factor. The specificity of present results, obtained with twodifferent hybridomas, supports our view (Franěk and Chládková-Šrámková, 1995) that the membranetransport macromolecules themselves may play the role of therecognition elements in a signal transduction pathway controlling thesurvival of hybridoma cells.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/B:CYTO.0000010400.89582.b8
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  • 2
    Electronic Resource
    Electronic Resource
    New insights into the kinetic resistance to anticancer agents (1998)
    Springer
    Cytotechnology 27 (1998), S. 225-235 
    Details
    ISSN: 1573-0778
    Keywords: anticancer drugs ; apoptosis ; cell cycle ; drug resistance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Kinetic resistance plays a major role in the failure of chemotherapy towards many solid tumors. Kinetic resistance to cytotoxic drugs can be reproduced in vitro by growing the cells as multicellular spheroids (Multicellular Resistance) or as hyperconfluent cultures (Confluence-Dependent Resistance). Recent findings on the cell cycle regulation have permitted a better understanding why cancer cells which arrest in long quiescent phases are poorly sensitive to cell-cycle specific anticancer drugs. Two cyclin-dependent kinase inhibitors (CDKI) seem particularly involved in the cell cycle arrest at the G1 to S transition checkpoint: the p53-dependent p21cip1 protein which is activated by DNA damage and the p27kip1 which is a mediator of the contact inhibition signal. Cell quiescence could alter drug-induced apoptosis which is partly dependent on an active progression in the cell cycle and which is facilitated by overexpression of oncogenes such as c-Myc or cyclins. Investigations are yet necessary to determine the influence of the cell cycle on the balance between antagonizing (bcl-2, bcl-XL...) or stimulating (Bax, Bcl-XS, Fas...) factors in chemotherapy-induced apoptosis. Quiescent cells could also be protected from toxic agents by an enhanced expression of stress proteins, such as HSP27 which is induced by confluence. New strategies are required to circumvent kinetic resistance of solid tumors: adequate choice of anticancer agents whose activity is not altered by quiescence (radiation, cisplatin), recruitment from G1 to S/G2 phases by cell pretreatment with alkylating drugs or attenuation of CDKI activity by specific inhibitors.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008025124242
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  • 3
    Electronic Resource
    Electronic Resource
    Reinforcing apoptosis-resistance of COS and myeloma cells by transfecting with bcl-2 gene (1997)
    Springer
    Cytotechnology 25 (1997), S. 25-33 
    Details
    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
    URL: http://dx.doi.org/10.1023/A:1007935026770
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  • 4
    Electronic Resource
    Electronic Resource
    Concanamycin A, a vacuolar type H+-ATPase inhibitor, induces cell death in activated CD8+ CTL (1997)
    Springer
    Cytotechnology 25 (1997), S. 127-135 
    Details
    ISSN: 1573-0778
    Keywords: apoptosis ; CD8+T cell ; cell death ; concanamycin A
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Concanamycin A (CMA) and concanamycin B (CMB) are specific inhibitors of vacuolar type H+-ATPase (V-ATPase). In our previous studies, intraperitoneal injection of CMB was shown to suppress the increase in CD8+ CTL population, but not to affect CD4+ and B220+ populations, in mice immunized with allogeneic tumors. To clarify the molecular basis of the selective decrease in the CD8+ CTL population by CMB, we have performed a series of in vitro experiments with use of CMA. Cell viability of the CD8+ population prepared from the immunized mice was preferentially decreased by CMA treatment. Moreover, in the CD8+ CTL clone, CMA induced a marked DNA fragmentation and nuclear condensation characteristic of apoptosis. Anti-CD3 or phorbol ester accelerated the CMA-induced reduction in cell viability of the CD8+ CTL clone, but not CD4+ T cell clones. However, this rapid cell death was not accompanied by DNA fragmentation and nuclear condensation. Perforin and granzyme B were unlikely to be involved in such cell death. Thus, our data suggest that V-ATPase activity is essential for survival of CD8+ CTL especially when activated.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1007995212658
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  • 5
    Electronic Resource
    Electronic Resource
    Regulation of caspase activation in apoptosis: implications for transformation and drug resistance (1998)
    Springer
    Cytotechnology 27 (1998), S. 309-320 
    Details
    ISSN: 1573-0778
    Keywords: apoptosis ; caspases ; cell death ; proteases ; proteolysis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Recent developments in the apoptosis field have uncovered a family of cysteine proteases, the Caspases, that act as signalling components as well as effectors of the cell death machinery. Caspases are constitutively present as inactive precursors within most cells and undergo proteolytic processing in response to diverse death-inducing stimuli to initiate the death programme. Active caspases can process other caspases of the same type as well as process caspases further downstream in the pathway that ultimately leads to collapse of the cell. This cellular collapse is thought to occur as a consequence of caspase-mediated cleavage of a diverse array of cellular substrates. Regulation of entry into the death programme is controlled at a number of levels by members of the Bcl-2 family, as well as by other cell death regulatory proteins. Recent data has shed light upon the mechanism of action of these regulatory molecules and suggests that the point of caspase activation is a major checkpoint in the cell death programme. Because many transformed cell populations possess derangements in cell death-regulatory genes, such as bcl-2, such cells frequently exhibit elevated resistance to cytotoxic chemotherapy. Thus, a deeper understanding of how apoptosis is normally regulated has therapeutic implications for disease states where the normal controls on the cell death machinery have been subverted.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008014215581
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  • 6
    Electronic Resource
    Electronic Resource
    Variable functions of bcl-2 in mediating bioreactor stress- induced apoptosis in hybridoma cells (1998)
    Springer
    Cytotechnology 28 (1998), S. 177-188 
    Details
    ISSN: 1573-0778
    Keywords: apoptosis ; bcl-2 ; cell death ; hybridoma ; osmolarity ; pH ; shear ; stress
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract It has been demonstrated that the cell lines used for production of biopharmaceuticals are highly susceptible to apoptosis, and that over-expression of the bcl-2 oncogene can protect cells from death. Stress associated with the deprivation of nutrients has been shown to be the main cause of apoptosis in culture. We have extended these studies by investigating the mechanism of cell death under conditions of sub-optimal pH, shear stress and hyperosmolarity, and the protective action of bcl-2 over-expression. At pH 6, there was no clear evidence of protection from cell death. However, at pH 8, the viability of the bcl-2 transfected cells was about 20% higher relative to the control cells. Cultivation of control cells in a flat bottomed bioreactor with a magnetic stirrer bar without a pivot ring resulted in exposure of the cells to a high attrition effect. As a result, cell growth was retarded and a high level of cell death by apoptosis was observed. Under the same conditions, the bcl-2 transfected cell line exhibited a nearly five fold increase in viable cell number. This finding indicates that under apoptosis-suppressed conditions, shear stress can stimulate cell growth. Batch cultivation of both control and bcl-2 transfected cells in 350 and 400 mOsm media resulted in suppression of cell growth, athough the effect was most marked in the control cell line. Adaptation of control cells to 400 mOsm proved to be impossible to achieve. However, the bcl-2 transfected cells exhibited resistance to the osmotic stress resulting in long term adaptation to a high salt environment. Specific productivity of bcl-2 transfected cells grown in high osmolarity medium was 100% higher than that produced by non- adapted bcl-2 transfected cells grown in normal osmolarity medium. These results demonstrate that bcl-2 has a beneficial effect on hybridoma cultivation under a wide range of culture stresses.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008002319400
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  • 7
    Electronic Resource
    Electronic Resource
    Apoptosis and nutrition: Involvement of amino acid transport system in repression of hybridoma cell death (1995)
    Springer
    Cytotechnology 18 (1995), S. 113-117 
    Details
    ISSN: 1573-0778
    Keywords: apoptosis ; hybridoma cells ; amino acids
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Mouse hybridoma cells cultured on the verge of starvation-induced apoptosis, i.e. in a medium diluted with saline, proved to serve as a sensitive screening system for apoptosis-suppressing activity of nutrient medium components. Conventional amino acid mixtures were found to suppress the starvation-induced apoptosis, whereas a vitamin mixture was ineffective. (Franěk F (1995) Biotechnol. Bioeng. 45: 86–90). Recent experiments showed that suppression of apoptosis, and concurrent resumption of growth, could be achieved by addition of single substances at millimolar concentrations. The set of active substances included certain coded L-amino acids (glycine, alanine, serine, threonine, proline, asparagine, glutamine, histidine), non-coded amino acids (β-alanine, taurine, 4-aminobutyric acid), and a non-metabolizable analogue (2-aminoisobutyric acid). This finding shows that some amino acids do not act solely as nutrients, but also as specific signal molecules. The specificity of the effect points to the involvement of adaptively regulated amino acid transport systems A and N in maintaining the balance between triggering and suppression of starvation-induced apoptosis.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00744326
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  • 8
    Electronic Resource
    Electronic Resource
    Effects of temperature shift on cell cycle, apoptosis and nucleotide pools in CHO cell batch cultues (1997)
    Springer
    Cytotechnology 23 (1997), S. 47-54 
    Details
    ISSN: 1573-0778
    Keywords: apoptosis ; programmed cell death ; nucleotides ; energy charge ; CHO cells ; batch culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Temperature reduction in CHO cell batch culture may be beneficial in the production of recombinant protein and in maintenance of viability. The effects on cell cycle, apoptosis and nucleotide pools were studied in cultures initiated at 37°C and temperature shifted to 30 °C after 48 hours. In control cultures maintained at 37 °C, viable cells continued to proliferate until the termination of the culture, however, temperature reduction caused a rapid decrease in the percent of cells in S phase and accumulation of cells in G-1. This was accompanied by a concurrent reduction in U ratio (UTO/UDP-GNAc), previously shown to be a sensitive indicator of growth rate. Culture viability was extended following temperature shift, as a result of delayed onset of apoptosis, however, once initiated, the rate and manner of cell death was similar to that observed at 37 °C. All nucleotide pools were similarly degraded at the time of apoptotic cell death. Temperature reduction to 30 °C did not decrease the energy charge of the cells, however, the overall rate of metabolism was reduced. The latter may be sufficient to extend culture viability via a reduction in toxic metabolites and/or limitation of nutrient deprivation. However, the possibility remains that the benefits of temperature reduction in terms of both viability and productivity are more directly associated with cultures spending extended time in G-1.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1007919921991
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  • 9
    Electronic Resource
    Electronic Resource
    Establishment of an apoptosis-resistant and growth-controllable cell line by transfecting with inducible antisense c-Jun gene (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 58 (1998), S. 65-72 
    Details
    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.
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  • 10
    Electronic Resource
    Electronic Resource
    Use of ribozymes and antisense oligodeoxynucleotides to investigate mechanisms of drug resistance (1998)
    Springer
    Cytotechnology 27 (1998), S. 113-136 
    Details
    ISSN: 1573-0778
    Keywords: antisense ; apoptosis ; multidrug resistance (MDR) ; multidrugresistance-related protein (MRP) ; P-glycoprotein (Pgp) ; ribozyme
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Chemotherapy can cure a number of human cancers but resistance (either intrinsic or acquired) remains a significant problem in many patients and in many types of solid tumour. Combination chemotherapy (using drugs with different cellular targets/mechanisms) was introduced in order to kill cells which had developed resistance to a specific drug, and to allow delivery of a greater total dose of anti-cancer chemicals by combining drugs with different side-effects (Pratt et al., 1994). Nearly all anti-cancer drugs kill tumour cells by activating an endogenous bio-chemical pathway for cell suicide, known as programmed cell death or apoptosis.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008052401952
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  • 11
    Electronic Resource
    Electronic Resource
    Apoptosis-resistant NS/0 E1B-19K myelomas exhibit increased viability and chimeric antibody productivity under cell cycle modulating conditions (1998)
    Springer
    Cytotechnology 28 (1998), S. 189-203 
    Details
    ISSN: 1573-0778
    Keywords: apoptosis ; cell cycle ; E1B-19K ; hydroxyurea ; hyperosmosis ; hypertonic ; monoclonal antibodies ; OptiMAbTM ; thymidine
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Lymphoid cells expressing sufficient levels of Bcl-2 or E1B-19K are known to resist to induction of apoptosis in glutamine-free or nutrient-limited batch cultures. However, despite the increased viability and prolonged stationary phase achieved in batch culture, product yields are not necessarily improved. Here we have found that expression of E1B-19K in NS/0 myeloma cells cultivated in the presence of certain cell cycle modulators could result in a significant increase in MAb productivity as compared to untransfected control cells. The use of E1B-19K significantly enhanced cell survival in the presence of osmolytes (sorbitol, NaCl), DNA synthesis inhibitors (hydroxyurea, excess thymidine), and the cell culture additive OptiMAb™. E1B-19K myelomas cultivated in the presence of NaCl or OptiMAb™ accumulated in the G1 phase, while those arrested with excess thymidine were blocked in all phases. Interestingly, control NS/0 cells treated with these agents were found to die in a cell-cycle specific manner. Thus, while all G1 and most S phase cells quickly underwent apoptosis, G2/M cells remained alive and maintained MAb secretion for more than 10 days if supplied with adequate nutrients. For both control and E1B-19K cells, incubation with sorbitol or hydroxyurea was detrimental for MAb secretion, while addition of NaCl, excess thymidine and OptiMAb™ resulted in an increased specific MAb productivity as compared to the batch culture. However, this increase resulted in an improvement of final MAb yields only in the case of OptiMAb™. The extension of viability conferred by E1B-19K allowed to further improve the final MAb yield obtained using OptiMAb™ with a 3.3-fold increase for E1B-19K cells as compared to 1.8-fold for control NS/0 cells.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008054403470
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  • 12
    Electronic Resource
    Electronic Resource
    Characterization and fed-batch culture of hybridoma overexpressing apoptosis suppressing gene bcl-2 (1997)
    Springer
    Cytotechnology 24 (1997), S. 135-141 
    Details
    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
    URL: http://dx.doi.org/10.1023/A:1007922104344
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  • 13
    Electronic Resource
    Electronic Resource
    Effect of Bcl-2 Expression on Hybridoma Cell Growth in Serum-Supplemented, Protein-Free and Diluted Media (1998)
    Springer
    Cytotechnology 26 (1998), S. 219-225 
    Details
    ISSN: 1573-0778
    Keywords: apoptosis ; Bcl-2 ; diluted medium ; hybridoma ; protein-free medium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Two transfected hybridoma cell lines TB/C3-bcl2 (overexpressing the Bcl-2 protein) and TB/C3-pEF (control cell line), were compared in batch suspension cultures using a medium supplemented either with horse serum or with a protein-free, iron-rich supplement. The membrane intact index (percentage of cells with intact membranes determined by trypan blue staining) of the TB/C3-bcl2 cell line decreased much slower than that of the control cell line during the dying phase of the cultures. No significant difference in antibody, lactate and ammonia production as well as glucose and glutamine consumption was noted in the exponential phase of the experiments. Both cell lines were also compared in batch experiments using media diluted with saline to further investigate the effect of Bcl-2 under sub-optimal conditions. The Bcl-2 overexpressing cell line again exhibited a higher membrane intact index at increasing dilution steps.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1007914619219
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  • 14
    Electronic Resource
    Electronic Resource
    The role of oncogenes in drug resistance (1998)
    Springer
    Cytotechnology 27 (1998), S. 283-292 
    Details
    ISSN: 1573-0778
    Keywords: apoptosis ; cell-cycle control ; Growth factor receptors ; signal transduction ; transcription factors
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Increasing evidences indicate that oncogenes can directly or indirectly impact on cancer-cell drug resistance. This chapter provides a conceptual review regarding the role of oncogenes in drug resistance. The review is focused on drug resistance mediated by oncogenes encoding growth factor receptors, signaling molecules, transcription factors, cell-cycle regulators, and apoptosis regulators. It is my hope that better undertsnading on the role of oncogenes in drug resistance will invoke ideas on new approaches to enhance the cytotoxicity of the standard chemotherapeutic agents by functional perturbation of resistance-inducing oncogenes.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008053913764
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  • 15
    Electronic Resource
    Electronic Resource
    Starvation-induced programmed death of hybridoma cells: Prevention by amino acid mixtures (1995)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 45 (1995), S. 86-90 
    Details
    ISSN: 0006-3592
    Keywords: hybridoma ; nutrition ; cell death ; apoptosis ; monoclonal antibody ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Association of the availability of nutrients with the phenomenon of programmed cell death - apoptosis - was investigated using hybridoma cells cultured in protein-free medium under conditions of starvation, i.e., in RPMl-1640 medium diluted to 50% with saline. Amino acid mixtures, such as MEM essential amino acids or MEM nonessential amino acids were found to prevent starvation death significantly when added to the diluted medium in 1 to 2 mM concentrations, the MEM vitamin mixture was ineffective, and glutamine displayed a moderate growth-supporting effect. The specific monoclonal antibody production rate in cultures supplemented with amino acid mixtures was strikingly low, whereas supplementation with glutamine alone or simultaneously with other amino acids resulted in a specific antibody production rate comparable with the rate observed in undiluted medium. © 1995 John Wiley & Sons, Inc.
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    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260450112
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  • 16
    Electronic Resource
    Electronic Resource
    Death mechanisms of animal cells in conditions of intensive agitation (1995)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 45 (1995), S. 463-472 
    Details
    ISSN: 0006-3592
    Keywords: apoptosis ; animal cell death ; hybridoma cells ; agitation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The question is addressed as to whether cells which are subject to high-energy dissipation rates in agitated bioreactors show an apoptotic response. Murine hybridoma cells in batch culture were agitated in bench-scale (1-L) bioreactors without gas sparging. At an energy dissipation rate of 1.5 W m-3 there was no apparent damage. At 320 W m-3 cell viability declined, and increasing proportions of the dead cells displayed the morphological features of apoptosis, but necrosis also remained as a significant mechanism of death. When cells were subjected to the intensive energy dissipation rate of 1870 W m-3 in a bioreactor without gas headspace, the cell number dropped by 50% within 2 h and a subpopulation of smaller-sized cells emerged. This excluded trypan blue but showed some apoptotic characteristics such as reduced and condensed DNA content and low F-actin content. The incidence of apoptotic activity was further demonstrated by the appearance of numerous apoptotic bodies. Analysis of the cell cycles of both small and normal size populations indicated that greater proportions of S and G2 cells had become apoptotic and there was evidence of preferential survival of G1 cells. It is suggested that two mechanisms of cell death are apparent in hydrodynamically stressful situations, but their relative expression depends on the energy dissipation rate. © 1995 John Wiley & Sons, Inc.
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    URL: http://dx.doi.org/10.1002/bit.260450602
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  • 17
    Electronic Resource
    Electronic Resource
    Overexpression of bcl-2, apoptosis suppressing gene: Prolonged viable culture period of hybridoma and enhanced antibody production (1995)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 48 (1995), S. 118-122 
    Details
    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.
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    URL: http://dx.doi.org/10.1002/bit.260480205
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    A combined cell-cycle and metabolic model for the growth of hybridoma cells in steady-state continuous culture (1995)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 48 (1995), S. 49-65 
    Details
    ISSN: 0006-3592
    Keywords: cell cycle ; apoptosis ; hybridoma ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The Model presented in this work demonstrates the combination of cell-cycle model with a model describing the growth and conversion kinetics of hybridoma cells in a steady-state continuous culture. The cell-cycle model is based upon a population balance model as described by Cazzador et al. and assumes the existence of a cycling-and apoptotic-cell population, which together form the viable-cell population. In this part the fraction of apoptotic cells, the age distribution of the cycling and apoptotic-cell population, the mean volume and biomass content per cell of the cycling, apoptotic, and viable cells, and the specific growth and death rates of the cells are calculated. The metabolic part consists of a Monod-type growth equation, four elemental balances, an equation assuming a constant yield of ammonia on glutamine, an equation for product formation, and the relation of Glacken for energy production. Furthermore, a maintenance-energy model for the consumption of glucose and glutamine is introduced, which combines the approaches of Herbert and Pirt into one model in a way similar to Beeftink et al. For energy consumption a Pirt model is assumed. The model is capable of predicting trends in steady-state vaues of a large number of variables of interest like specific growth rate, specific death rate, viability, cell numbers, mean viable-cell volume, and concentrations and conversion rates of product, glucose, glutamine, lactate, and ammonia. Also the concentrations and conversion rates of oxygen and carbon dioxide are qualitatively predicted. The values of the model predictions are generally close to experimental data obtained from literature. © 1995 John Wiley & Sons, Inc.
    Additional Material: 8 Ill.
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    URL: http://dx.doi.org/10.1002/bit.260480109
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    Electronic Resource
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    Cell cycle and cell size dependence of susceptibility to hydrodynamic forces (1995)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 46 (1995), S. 88-92 
    Details
    ISSN: 0006-3592
    Keywords: cell cycle ; hydrodynamic forces ; apoptosis ; cell culture ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Exposure of animal cells to intense hydrodynamic forces exerted in turbulent capillary flow, and by controiled agitation and aeration, resulted in preferential destruction of S and G2 cells and the extent of destruction of these cells was dependent upon the intensity of the action. The loss of these cells was possibly due to their larger size. However, the appearance of large numbers of membrane-bound vesicular structures similar to apoptotic bodies as well as cells with low DNA stainability (in a sub-G1 peak) suggested that the action of adverse hydrodynamic forces on these large cells may at least in part be to induce an apoptotic response. © 1995 John Wiley & Sons, Inc.
    Additional Material: 6 Ill.
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    URL: http://dx.doi.org/10.1002/bit.260460112
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    Electronic Resource
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    Extension of Sp2/0 hybridoma cell viability through interleukin-6 supplementation (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 55 (1997), S. 439-446 
    Details
    ISSN: 0006-3592
    Keywords: cell culture viability ; apoptosis ; IL-6 ; hybridomas ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Sp2/0 hybridoma cells die principally by apoptosis in batch culture. We have found that cultures of the Sp2/0 hybridoma exhibit increased viability in response to interleukin 6 (IL-6) supplementation relative to control cultures during serum shiftdown experiments. When shifted from a medium containing 10% fetal bovine serum (FBS) to a medium with 1% FBS, IL-6 supplemented cultures displayed viabilities and viable cell densities similar to control cultures containing 10% FBS. The degree of the survival response induced varied in accordance with the severity of the shiftdown, as cells resuspended in a high serum medium showed little observable enhancement in viability. The extension in culture viability was not accompanied by an observable decrease in growth relative to control cultures, indicating that the effect was not a consequence of growth inhibition. These results suggest the existence of serum components with behavior functionally similar to IL-6, with respect to enhancing cell survival, and that under certain experimental conditions IL-6 serves as a survival factor. In contrast to the extended viability displayed by cultures supplemented with IL-6, Sp2/0 cultures transfected with IL-6 cDNA expression vectors displayed a growth inhibitory response relative to control cultures. This inhibitory response was characterized by an extended lag phase following inoculation, and a decrease in batch culture cell yield. The depression in cell yield varied with serum concentration, with the largest depression occurring at high serum concentrations. We conclude that interactions between components in serum, presumably growth factors, and cytokines play an important role in altering the behavior of industrially relevant cell lines in culture. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 439-446, 1997.
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    In hybridoma cultures, deprivation of any single amino acid leads to apoptotic death, which is suppressed by the expression of the bcl-2 gene (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 59 (1998), S. 90-98 
    Details
    ISSN: 0006-3592
    Keywords: apoptosis ; necrosis ; bcl-2 ; amino acids ; cell culture ; cell death ; hybridoma ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The transfection of murine hybridomas with the apoptosis suppressor gene bcl-2 has been reported to result in the extension of batch culture duration, leading to significant improvements in culture productivity. In the present study, the effect of deprivation, individually, of each amino acid found in culture medium was examined to characterize the chemical environment of the culture in terms of its propensity to induce apoptosis. When cells were deprived of each amino acid, individually for 48 h, the majority of cell deaths in each case occurred by apoptosis, with essential amino acids being clearly most effective. For nearly all the amino acids, the viability of the bcl-2 cell line cultures was greater than 70% after 48 h, representing a substantial improvement in viability over control cell line cultures. Time course studies revealed that the induction of death could be divided into two phases. Initially, following the deprivation of a single essential amino acid, there was a period of time during which all the control cell line cultures retained high viability. The duration of this phase varied from 15 h in the case of lysine deprivation, through to 40 h in the case methionine deprivation. In the second phase of deprivation, the cultures exhibited an abrupt and rapid collapse in viability. The time taken for the viability to fall to 50% was similar for each amino acid. In every case, the duration of both phases of the bcl-2 cultures was considerably extended. Specific utilization rates were increased during the control cultures relative to the bcl-2 cultures for both the growth phase (ranging between 2% and 57% higher than the bcl-2 cultures) and the death phase (ranging between 172% to 1900% higher than the bcl-2 culture). © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:90-98, 1998.
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    Prevention of hybridoma cell death by bcl-2 during suboptimal culture conditions (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 54 (1997), S. 1-16 
    Details
    ISSN: 0006-3592
    Keywords: apoptosis ; hypoxia ; hyperoxia ; growth arrest ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: TB/C3 hybridoma cells were transected with either pEF-MClneopA or pEF bcl2-MClneopA vectors to produce a control cell line (TB/C3 pEF) and a cell line that overexpresses the “antiapoptotic” human bcl-2 protein (TB/C3 bcl2). Flow cytometry analysis of intracellular bcl-2 protein levels enabled near on-line monitoring of the stability of bcl-2 expression in the absence of drug selection. It was possible to maintain spontaneous selection of cells with the overexpression of bcl-2 protein during semicontinuous cultures at very low dilution rates, where cells were subjected to the selective conditions of nutrient limitation and high toxic metabolite concentrations. Interestingly, cells that overexpressed bcl-2 were adapted to suspension culture conditions significantly faster than control cells. Dual fluorescence staining with acridine orange and propidium iodide allowed for discrimination between viable, apoptotic, secondary necrotic, and necrotic cells, respectively. Compared with the usual trypan blue method of establishing culture viability, dual staining demonstrated that under stressful conditions a significant proportion of cells that excluded trypan blue were also undergoing cell death through apoptosis. In batch cultures the overexpression of bcl-2 more than doubled the membrane intact (MI) cell productive period (the integral of Ml cell density with respect to culture time) and increased the monoclonal antibody (mAb) production by approximately 40% when compared with the control cell line. The overexpression of bcl-2 protein also significantly extended the cell integrity and viability by the suppression of apoptosis in conditions of hypoxia, hyperoxia, glutamine deprivation, glucose deprivation, and serum limitation. The suppression of apoptosis in anaerobic conditions suggests that bcl-2 exerts its antiapoptotic activity by a mechanism that does not involve an oxidative reactive pathway. In conditions of excess thymidine, which suppressed cell proliferation, Ml cell density and specific mAb productivity were further enhanced by the overexpression of bcl-2, which suggests the possibility of accomplishing a controlled proliferation in immortalized cell lines without invoking cell death. Cell size and intracellular mAb were increased for TB/C3 bcl2 cells compared with TB/C3 pEF control cells when analyzed by flow cytometry. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 1-16, 1997.
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    Growth factor and Bcl-2 mediated survival during abortive proliferation of hybridoma cell line (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 57 (1998), S. 164-171 
    Details
    ISSN: 0006-3592
    Keywords: cell death ; apoptosis ; bcl -2 ; cell culture ; cell viability ; growth factors ; survival factors ; abortive proliferation ; hybridomas ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Cultures of the CRL-1606 hybridoma (ATCC) have been reported to undergo continuous proliferation with simultaneous death during nutrient limited fed-batch fermentations. The bcl-2 proto-oncogene has been shown to prevent cell death under a variety of otherwise death inducing conditions. We were interested in elucidating the nature of the massive death observed in cultures of CRL-1606, specifically with respect to the possible environmental causes, and the ability of overexpressed human bcl-2 (hbcl-2) to mitigate cell death. Abortive proliferation, or continuous proliferation in the presence of continuous death, could be induced in serum free cultures of CRL-1606 through the withdrawal of insulin provided the culture was competent for cell proliferation. Culture competency for proliferation was found to be solely determined by the presence of cell culture nutrients. Abortive proliferation was defective in cultures transfected with hbcl-2 and the enhanced viability observed resulted from an increased viable cell population and at the expense of the nonviable cell population normally found in untransfected cultures. Abortive proliferation was also observed in serum containing cultures upon serum shiftdowns. Like the insulin-supplemented serum free culture system, hbcl-2 transfected cultures exhibited defects in the abortive proliferation process. These results suggest that the massive death observed during nutrient-limited fed-batch fermentation originate, in part, from growth or survival factor limitations. Hence, approaches to design cell culture media that account for the cell's proliferation requirements without accounting for the cell's survival requirements may represent a cell death sentence. Given the transformed nature of the hybridomas, we conclude that the abortive proliferation of CRL-1606 is a consequence of inappropriate cell cycle entry in a survival factor limited environment. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 164-171, 1998.
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