APOPTOSIS OF L929 CELLS BY ETOPOSIDE - A QUANTITATIVE AND KINETIC APPROACH

Exponentially growing L929 cells were continuously exposed to 1 or 10 mu M etoposide (VP-16). The effects of such treatment on cell growth, cycle distribution, morphology, and selected biochemical events were e xamined, DNA synthesis rates were markedly decreased and the protein/D NA ratio increased (unbalanced growth). Growth was blocked, with most cells being cycle arrested by 24 h in (late S-)G2-M. An asynchronous p rocess of cell death then developed. Cells initially shrank into eosin ophilic, trypan blue-excluding bodies, which were then released into t he medium, and eventually became permeable to trypan blue. Transmissio n electron microscopy confirmed that dying cells acquired an apoptotic morphotype, with compaction and margination of chromatin, loss of mic rovilli, and shrinkage of cytoplasm and nucleus, Tissue transglutamina se activity and intensity of immunostaining rapidly increased in treat ed cultures. Internucleosomal DNA fragmentation could not be detected by agarose gel electrophoresis, yet flow cytometry revealed that the a poptotic bodies had a very low DNA fluorescence (less than or equal to 10% of the 2n value), In agreement with the microscopic findings, thi s suggested that extensive DNA degradation had occurred in dead cells. While rates of cell loss from the monolayer amounted to 21 and 57% da y(-1) (1 and 10 mu M VP-16, respectively), apoptotic indexes largely u nderestimated the extent of the process. These indexes only measured t he accumulation of apoptotic bodies, i.e., the balance between their g eneration and disposal. The latter occurred by mechanisms similar to t hose that operate in tissues: ''secondary necrosis'' or phagocytosis b y viable homotypic cells in the monolayer (''homophagy''). (C) 1996 Ac ademic Press, Inc.