Bcl-2 over-expression reduces growth rate and prolongs G1 phase in continuous chemostat cultures of hybridoma cells

Simpson, Nicholas H.; Singh, R. P.; Emery, A. N.; Al‐Rubeai, Mohamed

doi:10.1002/(sici)1097-0290(19990720)64:2<174::aid-bit6>3.0.co;2-d

Cited by 76 publications

(56 citation statements)

References 31 publications

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“…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%

“…Cell suspensions were stained with equal volumes of dual staining solution containing acridine orange (AO, 10 lg ml À1 ; Sigma) and propidium iodide (PI, 10 lg ml À1 ; Sigma). Cells were classified by virtue of colour and chromatin morphology as previously described (Simpson et al 1999). …”

Section: Assessment Of Growth and Viabilitymentioning

confidence: 99%

“…Many theories exist on how Bcl-2 prevents caspase activation including dimerisation with pro-apoptotic members rendering them ineffective, preventing permeability transition (PT) pore formation on the outer mitochondrial membrane, thereby preventing cytochrome c release and unregulated influx and efflux of ions across these membranes (Antonsson et al 1997;Rao and White 1997;RobbGasper et al 1998). 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: Discussionmentioning

confidence: 99%

Section: Assessment Of Growth and Viabilitymentioning

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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“…Despite initial BrdU-and thymidinelabeling experiments suggesting BCL2 may be generally growth inhibitory, growth rate measurements in conventional and continuous chemostat cultures revealed that in cycling cells, BCL2 does not significantly affect growth rates under optimal conditions, but prolongs G1 in suboptimal conditions. 7,8,[10][11][12][13] It became increasingly clear that the cell cycle delay effect of BCL2 is selective for cell cycle re-entry from G0.…”

Section: Bcl2 Enhances G0 and Delays G0 To S Transitionmentioning

confidence: 99%

BCL2 family in DNA damage and cell cycle control

2006

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Individual BCL2 family members couple apoptosis regulation and cell cycle control in unique ways. Antiapoptotic BCL2 and BCL-x L are antiproliferative by facilitating G0. BAX is proapoptotic and accelerates S-phase progression. The dual functions in apoptosis and cell cycle are coordinately regulated by the multi-domain BCL2 family members (MCL-1) and suggest that survival is maintained at the expense of proliferation. The role of BH3-only molecules in cell cycle is more variable. BAD antagonizes both the cell cycle and antiapoptotic functions of BCL2 and BCL-x L through BH3 binding. BID has biochemically separable functions in apoptosis and S-phase checkpoint, determined by posttranslational modification. p53-induced PUMA is known only to have apoptotic function. Inhibition of apoptosis is oncogenic, whereas promotion of cell cycle arrest is tumor suppressive. Paradoxically, selected BCL2 family members can be both oncogenic and tumor suppressive. Which of the dual functions predominates is lineage specific and context dependent.

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“…Apart from CDKs and CKIs, expression of Bcl-2 can also cause cell cycle arrest in G1-phase of murine hybridoma cells (O'Reilly et al 1996;Simpson et al 1997Simpson et al , 1999. About 80% of hybridoma cells expressing Bcl-2 accumulated in G1 phase of the cell cycle (Simpson et al 1999).…”

Section: (Ii) Cell Engineering Based Approachesmentioning

confidence: 99%

Proliferation control strategies to improve productivity and survival during CHO based production culture

2007

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Chinese Hamster Ovary cells are the primary system for the production of recombinant proteins for therapeutic use. Protein productivity is directly proportional to viable biomass, viability and culture longevity of the producer cells and a number of approaches have been taken to optimise these parameters. Cell cycle arrest, particularly in G1 phase, typically using reduced temperature cultivation and nutritional control have been used to enhance productivity in production cultures by prolonging the production phase, but the mechanism by which these approaches work is still not fully understood. In this article, we analyse the public literature on proliferation control approaches as they apply to production cell lines with particular reference to what is known about the mechanisms behind each approach.

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Bcl-2 over-expression reduces growth rate and prolongs G1 phase in continuous chemostat cultures of hybridoma cells

Cited by 76 publications

References 31 publications

Apoptosis and Its Suppression in Hepatocytes Culture

Apoptosis and Its Suppression in Hepatocytes Culture

BCL2 family in DNA damage and cell cycle control

Proliferation control strategies to improve productivity and survival during CHO based production culture

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