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

Research output: Contribution to journalArticle

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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

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Etoposide
Apoptosis
Trypan Blue
DNA
Cell Cycle
Growth
Agar Gel Electrophoresis
DNA Fragmentation
Microvilli
Transmission Electron Microscopy
Phagocytosis
Chromatin
Flow Cytometry
Cytoplasm
Cell Death
Necrosis
Fluorescence
Proteins

ASJC Scopus subject areas

  • Cell Biology

Cite this

Bonelli, G., Sacchi, M. C., Barbiero, G., Duranti, F., Goglio, G., Di Cantogno, L. V., ... 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

Apoptosis of L929 cells by etoposide : A quantitative and kinetic approach. / Bonelli, Gabriella; Sacchi, Maria Cristina; Barbiero, Giuseppe; Duranti, Federica; Goglio, Giovanna; Di Cantogno, Ludovica Verdun; Amenta, Joseph Salvatore; Piacentini, Mauro; Tacchetti, Carlo; Baccino, Francesco Maria.

In: Experimental Cell Research, Vol. 228, No. 2, 01.11.1996, p. 292-305.

Research output: Contribution to journalArticle

Bonelli, G, Sacchi, MC, Barbiero, G, Duranti, F, Goglio, G, Di Cantogno, LV, Amenta, JS, Piacentini, M, Tacchetti, C & Baccino, FM 1996, 'Apoptosis of L929 cells by etoposide: A quantitative and kinetic approach', Experimental Cell Research, vol. 228, no. 2, pp. 292-305. https://doi.org/10.1006/excr.1996.0329
Bonelli G, Sacchi MC, Barbiero G, Duranti F, Goglio G, Di Cantogno LV et al. Apoptosis of L929 cells by etoposide: A quantitative and kinetic approach. Experimental Cell Research. 1996 Nov 1;228(2):292-305. https://doi.org/10.1006/excr.1996.0329
Bonelli, Gabriella ; Sacchi, Maria Cristina ; Barbiero, Giuseppe ; Duranti, Federica ; Goglio, Giovanna ; Di Cantogno, Ludovica Verdun ; Amenta, Joseph Salvatore ; Piacentini, Mauro ; Tacchetti, Carlo ; Baccino, Francesco Maria. / Apoptosis of L929 cells by etoposide : A quantitative and kinetic approach. In: Experimental Cell Research. 1996 ; Vol. 228, No. 2. pp. 292-305.
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