, Volume 4, Issue 5, pp 335-355

Role of Ca2+, Mg2+ and K+ ions in determining apoptosis and extent of suppression afforded by bcl-2 during hybridoma cell culture

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Abstract

We have addressed the possibility that Ca2+, Mg2+ and K+ ions play a central role in governing the morphological and biochemical changes attributed to apoptotic cell death. By removing Ca2+, Mg2+ or K+ ions from the cell culture medium we were able to assess the contribution of each ion to hybridoma cell growth and viability. The differences were explained in terms of a possible reduction in their respective intracellular levels. From several lines of evidence, the deprivation of K+ ions was the most detrimental to cellular growth and viability and induced significant levels of early apoptotic cells. Another effect of this deprivation was to weaken the plasma membranes without causing membrane breakdown; exposure to high agitation rates confirmed fragility of the cell membranes. Removal of Mg2+ caused a reduction in the levels of early apoptotic cells and predisposed cells to high levels of primary necrotic death. The lower levels of apoptotic cells failed to demonstrate the classic nuclear morphology associated with apoptosis, while retaining other apoptotic features. These results highlighted the importance of utilizing several assays for the determination of apoptosis. The absence of Ca2+ appeared to be the mildest insult, but its deprivation did accelerate a significant decline in culture by increasing apoptotic death. Hybridoma cells overexpressing the apoptotic suppresser gene bcl-2 were protected from the predominantly necrosis inducing effects of Mg2+ ion deprivation and apoptosis inducing effects of Ca2+ ion deprivation. However, apoptosis was not as effectively suppressed in bcl-2 cells responding to incubation in K+ free medium. The inclusion of bcl-2 activity in the mechanisms of Ca2+ Mg2+ or K+ deprivation induced cell death emphasizes a close relationship between ionic dissipation and the apoptotic process.