Effects of the Inhibitors of Dynamics of Cytoskeletal Structures on the Development of Apoptosis Induced by the Tumor Necrosis Factor

L. V. Domnina^*, O. Yu. Ivanova, B. V. Cherniak, V. P. Skulachev, and J. M. Vasiliev

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119899 Russia; fax: (095) 939-3181; E-mail: Domnina@mbio.genebee.msu.su

^* To whom correspondence should be addressed.

Received December 19, 2001; Revision received March 12, 2002

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Changes in cytoskeletal structures have been investigated during apoptosis of epithelial HeLa cells induced by tumor necrosis factor-alpha (TNF-alpha). Shape and surface cell activity were investigated by time-lapse video microscopy, and changes of the cytoskeletal structure were studied by immune fluorescent microscopy. Addition of TNF-alpha to HeLa cell culture caused early disruption of the actin cytoskeleton and vinculin-containing focal contacts, keratin filaments, and microtubules. Rounding of cells, general blebbing, and nuclear fragmentation were observed at the terminal apoptotic stages. Actomyosin complex inhibitors, H7 and HA1077, suppressed blebbing (but not cell rounding) and activated the development of apoptosis. The latter suggests that in contrast to blebbing the general rounding does not depend on increased contractility of actomyosin cortex. These cytoskeletal inhibitors accelerated the development of apoptosis of HeLa cells and increased sensitivity of HeLa-Bcl-2 cells (transfected with DNA encoding antiapoptotic protein Bcl-2) to TNF-induced apoptosis. Damage of cytoskeletal structures significantly attenuated antiapoptotic activity of Bcl-2 in the HeLa-Bcl-2 cells. It is suggested that the stimulation of apoptosis by cytoskeletal inhibitors may be attributed to the altered distribution of cell organelles, especially, mitochondria.

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KEY WORDS: TNF, apoptosis, actin, cytoskeleton, microtubules, vinculin, blebbing, mitochondria

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