Apoptosis of L929 cells by etoposide: A quantitative and kinetic approach

Gabriella Bonelli, Maria Cristina Sacchi, Giuseppe Barbiero, Federica Duranti, Giovanna Goglio, Ludovica Verdun Di Cantogno, Joseph Salvatore Amenta, Mauro Piacentini, Carlo Tacchetti, Francesco Maria Baccino

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Abstract

Exponentially growing L929 cells were continuously exposed to 1 or 10 μM etoposide (VP-16). The effects of such treatment on cell growth, cycle distribution, morphology, and selected biochemical events were examined. DNA synthesis rates were markedly decreased and the protein/DNA ratio increased (unbalanced growth). Growth was blocked, with most cells being cycle arrested by 24 h in (late S-)G2-M. An asynchronous process of cell death then developed. Cells initially shrank into eosinophilic, trypan blue-excluding bodies, which were then released into the medium, and eventually became permeable to trypan blue. Transmission electron microscopy confirmed that dying cells acquired an apoptotic morphotype, with compaction and margination of chromatin, loss of microvilli, and shrinkage of cytoplasm and nucleus. Tissue transglutaminase activity and intensity of immunostaining rapidly increased in treated cultures. Internucleosomal DNA fragmentation could not be detected by agarose gel electrophoresis, yet flow cytometry revealed that the apoptotic bodies had a very low DNA fluorescence (≤ 10% of the 2n value). In agreement with the microscopic findings, this suggested that extensive DNA degradation had occurred in dead cells. While rates of cell loss from the monolayer amounted to 21 and 57% day-1 (1 and 10 μm VP-16, respectively), apoptotic indexes largely underestimated the extent of the process. These indexes only measured the accumulation of apoptotic bodies, i.e., the balance between their generation and disposal. The latter occurred by mechanisms similar to those that operate in tissues: 'secondary necrosis' or phagocytosis by viable homotypic cells in the monolayer ('homophagy').

Original languageEnglish
Pages (from-to)292-305
Number of pages14
JournalExperimental Cell Research
Volume228
Issue number2
DOIs
Publication statusPublished - Nov 1 1996

ASJC Scopus subject areas

  • Cell Biology

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    Bonelli, G., Sacchi, M. C., Barbiero, G., Duranti, F., Goglio, G., Di Cantogno, L. V., Amenta, J. S., Piacentini, M., Tacchetti, C., & Baccino, F. M. (1996). Apoptosis of L929 cells by etoposide: A quantitative and kinetic approach. Experimental Cell Research, 228(2), 292-305. https://doi.org/10.1006/excr.1996.0329