Process of apoptosis induced by TNF-α in murine fibroblast Ltk-cells: Continuous observation with video enhanced contrast microscopy

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Abstract
Apoptosis is originally defined by unique morphological changes of dying cells, and the biochemical hallmark associated with apoptosis is internucleosomal DNA fragmentation. However, few report has shown the precise time course of the apoptotic events. The present study was designed to try to clarify apoptotic processes using a video-enhanced contrast-differential interference contrast (VEC-DIC) microscopy. The morphological changes of murine fibroblast Ltk-cells treated with TNF-α were divided into four stages: (i) pre-apoptotic, (ii) cytoplasmic shrinkage, (iii) membrane blebbing, and (iv) ballooning. Almost of the cells underwent cytoplasmic shrinkage and membrane blebbing within 6 hours after TNF-α exposure, and at about 9 hours, they were in the ballooning stage. Based on these data, we investigated the relationship between morphological changes and other biochemical features. The earliest event was exposure of phosphatidyl-serine at the cytoplasmic membrane, which was already observed in the pre-apoptotic stage. Loss of mitochondrial membrane potential was observed in the cytoplasmic shrinkage stage. Caspase-8/-3 activities already started increasing in the pre-apoptotic stage, and reached their peak at 6 hours after TNF-α exposure. DNA fragmentation occurred in the late phase of the membrane blebbing.

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