Prevention of hybridoma cell death bybcl-2 during suboptimal culture conditions

Simpson, Nicholas H.; Milner, Andrew; Al‐Rubeai, Mohamed

doi:10.1002/(sici)1097-0290(19970405)54:1<1::aid-bit1>3.0.co;2-k

Cited by 134 publications

(90 citation statements)

References 36 publications

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“…These findings are very much in agreement with many previous studies that showed that Bcl-2 only exerts its influence in sub-optimal culture conditions and not during the exponential growth phase (Singh et al 1996;Simpson et al 1997;Tey 2000a). Studies where apoptosis was induced by nutrient deprivation revealed that Bcl-2 protected against some stimuli and not others.…”

Section: Discussionsupporting

confidence: 93%

“…Studies where apoptosis was induced by nutrient deprivation revealed that Bcl-2 protected against some stimuli and not others. In the absence of cystine and glucose, results strongly agree with previous findings (Singh et al 1996;Simpson et al 1997). In conditions of glutamine and serum deprivation, no protection is conferred suggesting that apoptosis under these conditions occurs via a pathway that is not associated with cytochrome c release and involvement of the Bcl-2 family.…”

Section: Discussionsupporting

confidence: 90%

“…Since its discovery as a regulator of apoptosis, bcl-2 has been seen as the prototype apoptosis suppressor gene and has been successfully overexpressed in many cell types including Burkitts lymphoma (Singh et al 1996), hybridoma (Simpson et al 1997(Simpson et al , 1998(Simpson et al , 1999Ishaque and AlRubeai 2002), myeloma (Suzuki et al 1997;Tey et al 2000a), CHO (Fussenegger et al 2000;Tey et al 2000b) and hepatic cells (Lacronique et al 1996). However, the assertion that over-expression of bcl-2 is the answer to the apoptosis problem in biotechnology has come under increasing scrutiny.…”

Section: Discussionmentioning

confidence: 99%

“…The Bcl-2 family has been implicated in other non-apoptotic roles including modifying cell cycle and cell growth profiles and increasing cell densities and specific productivity (O'Reilly et al 1996;Huang et al 1997;Vaux et al 1998;Simpson et al 1999). Bcl-2 has been extensively over-expressed in a number of cell lines including CHO, hybridoma, and myeloma cells and has been shown to suppress apoptosis, in response to many inducing agents (Singh et al 1996, Simpson et al 1997, 1999Suzuki et al 1997;Fussenegger et al 2000;Tey et al 2000a, b;Ishaque and Al-Rubeai 2002).…”

Section: Introductionmentioning

confidence: 99%

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Apoptosis and Its Suppression in Hepatocytes Culture

Mukwena

Al‐Rubeai

2004

Cytotechnology

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In order to achieve the goal of developing extracorporeal liver support devices, it is necessary to optimise bioprocess environment such that viability and function are maximised. Optimising culture medium composition and controlling the constitution of the cellular microenvironment within the bioreactor have for many years been considered vital to achieving these aims. Coupled to this is the need to understand apoptosis, the prime suspect in the demise of animal cultures, including those of hepatocytes. Results presented here show that absent nutrients including glucose and amino acids play a substantial part in the induction of apoptosis. The use of chemical apoptosis inhibitors was utilised to investigate key components of hepatic apoptosis where caspases, predominantly caspase 8, were implicated in staurosporine (STS)-induced HepZ apoptosis. Caspase 9 and 3 activation although recorded was of less significance. Interestingly, these results were not consistent with those of mitochondrial membrane depolarisation where inhibition of caspase activation appeared to drive depolarisation. Inhibition of mitochondrial permeability transition and use of anti-oxidants was unsuccessful in reducing apoptosis, caspase activation and mitochondrial membrane depolarisation. In further studies, the anti-apoptotic gene bcl-2 was over-expressed in HepZ, resulting in a cell line that was more robust and resistant to death induced by glucose and cystine deprivation and treatment with STS. Bcl-2 did not however show significant cytoprotectivity where apoptosis was stimulated by deprivation of glutamine and serum. Overall, results indicated that although apoptosis can be curbed by use of chemical inhibitors and genetic manipulation, their success is dependent on apoptotic stimuli.

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Section: Discussionsupporting

confidence: 93%

Section: Discussionsupporting

confidence: 90%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Apoptosis and Its Suppression in Hepatocytes Culture

Mukwena

Al‐Rubeai

2004

Cytotechnology

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“…The over-expression of the bcl-2 gene in plasmocytoma (myeloma) (Rabinder 1996), NS0 (Tey et al 2000b), CHO (Tey et al 2000a), and hybridoma (Simpson et al 1998) cells has imparted improved robustness to nutrient deprivation and toxin exposure, including longer survival in intensified culture systems (Tey and Al-Rubeai 2004). Some of the first studies showed that when hybridoma cells over-expressed the Bcl-2 protein it significantly increases cell viability and productivity (Simpson et al 1997). When myeloma cells, in perfusion culture, were engineered to express E1B-19K (the adenovirus Bcl-2 homologue) the resulted 2-fold increase in viable cell densities allowed for a 40% increase in monoclonal antibody yield (Mercille and Massie 1999).…”

Section: Apoptosis Engineeringmentioning

confidence: 99%

Using cell engineering and omic tools for the improvement of cell culture processes

et al. 2007

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Significant strides have been made in mammalian cell based biopharmaceutical process and cell line development over the past years. With several established mammalian host cell lines and expression systems, optimization of selection systems to reduce development times and improvement of glycosylation patterns are only some of the advances being made to improve cell culture processes. In this article, the advances pertaining to cell line development and cell engineering strategies are discussed. An overview of the cell engineering strategies to enhance cellular characteristics by genetic manipulation are illustrated, focusing on the use of genomics and proteomics tools and their application in such endeavors. Included in this review are some of the early studies using the 'omic' technique to understand cellular mechanisms of product synthesis and secretion, apoptosis, cell proliferation and the influence of the physicochemical environment. The article highlights the significance of integrating genomics and proteomics data with the vast amounts of bioprocess data for improved analysis of the biological pathways involved. Further improvements of the techniques and methodologies used are needed but ultimately, the new cell engineering strategies should provide great insight into the regulatory networks within the cell in a bioprocess environment and how to manipulate them to increase overall productivity.

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In hybridoma cultures, deprivation of any single amino acid leads to apoptotic death, which is suppressed by the expression of thebcl-2 gene

Simpson

Singh

Perani

et al. 1998

Biotechnol. Bioeng.

127

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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 bybcl-2 during suboptimal culture conditions

Cited by 134 publications

References 36 publications

Apoptosis and Its Suppression in Hepatocytes Culture

Apoptosis and Its Suppression in Hepatocytes Culture

Using cell engineering and omic tools for the improvement of cell culture processes

In hybridoma cultures, deprivation of any single amino acid leads to apoptotic death, which is suppressed by the expression of thebcl-2 gene

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