Fragmented DNA and apoptotic bodies document the programmed way of cell death in hybridoma cultures

Abstract

Markers of apoptosis were followed in batch hybridoma cultures carried out in protein-free medium. Samples were collected on day 0, representing early exponential phase (viability 91%), and on day 8, corresponding to late stationary phase (viability 8%). The apoptotic index reflecting the relative number of bodies insoluble in 6 M guanidinium hydrochloride in the culture of day 8 (30%) exceeded markedly the index in the culture of day 0 (2.5%). A gel chromatography on Sepharose 2B was developed for quantitative evaluation of fragmented cellular DNA. This analysis, including a correction for nonspecific fragmentation, showed that on day 8 more than 30% of cellular DNA was fragmented, whereas on day 0 it was less than 5%. Control necrotic cells prepared by rapid killing in 1% sodium azide displayed a low apoptotic index (2.4%) and low DNA fragmentation. Electrophoretic patterns in agarose gel showed a typical "ladder" of fragments in the DNA sample of day 8. The demonstration of fragmented cellular DNA and of the high incidence of apoptotic bodies at late stationary phase adds substantial weight to the view that in hybridoma cultures apoptosis represents the prevalent mode of cell death.

Cell death in bioreactors: A role for apoptosis

“…Further studies provided additional evidence for the induction of this process (again during the cultivation of hybridoma cells) based on the appearance of apoptotic bodies and the endonuclease-mediated fragmentation of DNA. 13 The purpose of the study presented here was to confirm the importance of apoptotic death during hydridoma culture. Additionally, we hoped to establish how widespread a mechanism it is by considering other commercially important cell lines.…”

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

“…Transglutaminase activation during apoptosis causes crosslinking of cytoplasmic proteins resulting in the formation of rigid apoptotic bodies that retain plasma membrane integrity (Fesus et al, 1991). Another feature of apoptosis, although not universal, is the activation of Mg/Ca-dependent endonucleases that cleave double stranded DNA at internucleosomal regions into approximately 180 base pair oligomers (Arends et al, 1990;Cohen et al, 1992;Franek and Dolnikova, 1991;Franek et al, 1992;Ucker et al, 1992). In vivo, apoptotic bodies are removed by phagocytosis.…”

Pro-haloacetate Nanoparticles for Efficient Cancer Therapy via Pyruvate Dehydrogenase Kinase Modulation

“…Thus, apoptotic analyses revealed the better efficiency of MCA compared to DCA and DBA while Pro-MCA-NP was found to be the best among all the used formulations. Pyruvate dehydrogenase (PDH) is responsible for the rate-limiting conversion of pyruvate to acetyl-CoA, while PDK phosphorylates PDH and inhibits its enzymatic activity20. DCA inactivates PDK, leading to reactivation of PDH.…”