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

Abstract

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.

Apoptosis and Its Suppression in Hepatocytes Culture

“…Amino acids considered essential for cellular proliferation were not always revealed as essential for survival, in particular glutamine. This discovery was in stark contrast to reports describing the induction of apoptosis in the absence of glutamine from the culture of many cell lines (Mecille and Massie 1994;Singh et al 1994;Simpson et al 1998;Sanfelui and Stephanopoulos 1999). Only cells that endogenously express GS synthetase or those genetically engineered to do so would be expected to survive such conditions as they are able to convert glutamic acid and ammonia to glutamine.…”

“…The question of the mechanisms at play in the regulation of apoptosis by amino acids has been an area of study that has ignited much research interest. Pathways proposed have implicated failure of nucleotide and protein synthesis, expression of 'new' pro-apoptotic genes, reduction of intracellular ATP pools due redox imbalance and collapse of mitochondrial membrane (Rabinovitz 1992;Mecille and Massie 1994;Galle et al 1995;Xie and Wang 1996;Simpson et al 1998). Clearly, amino acid deprivation can elicit an apoptotic response using several mechanisms.…”

“…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.…”

“…Studies carried out in hybridoma culture were mimicked in HepZ culture where the effect of deprivation of a single amino acid on induction of HepZ apoptosis was investigated (Simpson et al 1998). Stock cultures from mid-exponential pellets were resuspended in amino acid deprived medium and seeded onto 6-well plates to a final density of 2 · 10 4 cells ml À1 .…”

“…Although early interest in apoptosis stemmed from cancer research, the discovery of apoptosis in hybridoma cultures highlighted the significance of apoptosis to the biotechnology industry (Al-Rubeai et al 1990). Within the bioreactor environment apoptosis can be triggered by a number of stimuli, including exhaustion of essential nutrients (amino acids, serum components, and glucose), accumulation of metabolic by-products, and alterations in pH, dissolved oxygen content and medium osmolarity (Singh et al 1994;Simpson et al 1998;Al-Rubeai 1999, 2002). Additional environmental factors that lead to apoptotic cell death in large-scale mammalian cell cultures include those caused by mechanical stresses (Al-Rubeai et al 1995).…”