JPHS : Vol. 103 (2007) , No. 2 pp.159-167

Caspase Cascade Proceeds Rapidly After Cytochrome c Release From Mitochondria in Tumor Necrosis Factor-α-Induced Cell Death

Hiroshi Kawai¹⁾³⁾, Takuo Suzuki¹⁾, Tetsu Kobayashi¹⁾, Akiko Ishii-Watabe¹⁾, Haruna Sakurai²⁾, Hisayuki Ohata²⁾, Kazuo Honda²⁾, Kazutaka Momose²⁾, Takao Hayakawa¹⁾ and Toru Kawanishi¹⁾

1) Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, Japan
2) Department of Pharmacology, School of Pharmaceutical Sciences, Showa University, Japan
3) Faculty of Pharmaceutical Sciences, Josai International University, Japan

ABSTRACT: The caspase activation cascade and mitochondrial changes are major biochemical reactions in the apoptotic cell death machinery. We attempted to clarify the temporal relationship between caspase activation, cytochrome c release, mitochondrial depolarization, and morphological changes that take place during tumor necrosis factor (TNF)-α-induced cell death in HeLa cells. These reactions were analyzed at the single-cell level with 0.5 – 1 min resolution by using green fluorescent protein (GFP)-variant-derived probes and chemical probes. Cytochrome c release, caspase activation, and cellular shrinkage were always observed in this order within 10 min in all dying cells. This sequence of events was thus considered a critical pathway of cell death. Mitochondrial depolarization was also observed in all dying cells observed, but frequently occurred after caspase activation and cellular shrinkage. Mitochondrial depolarization is therefore likely to be a reaction that does not induce caspase activation and subsequent cellular shrinkage. Mitochondrial changes are important for apoptotic cell death; moreover, cytochrome c release, and not depolarization, is a key reaction related to cell death. In addition, we also found that the apoptotic pathway proceeds only when cells are exposed to TNF-α. These findings suggest that the entire cell death process proceeds rapidly during TNF-α exposure.

Keywords:

tumor necrosis factor (TNF)-α, cytochrome c, mitochondrial depolarization, caspase, real-time imaging