TGF-β1 is an apoptogenic agent for mammary epithelial cells (MEC). The molecular mechanism of the TGF-β1-induced apoptosis remains, however, obscure. In the present study we used laser scanning cytometry, confocal microscopy and immunogold electron microscopy to analyze the expression, aggregation and co-localization of caspase-8, Bid, Bax and VDAC-1. These proteins are regarded as the most important factors involved in the regulatory phase of TGF-β1-induced apoptosis. Apoptosis in HC11 mouse MEC manifested with a simultaneous increase in expression and subcellular aggregation of caspase-8, Bid, Bax and VDAC-1. Confocal microscopy revealed a strong pattern of co-localization of examined proteins during both early and late apoptosis. Experiments with double- and triple-staining immunoelectron microscopy showed a co-localization of Bax/Bid, caspase-8/Bax/Bid, and Bax/VDAC-1, on the membranes of mitochondria, Golgi apparatus, rough endoplasmic reticulum, nuclear envelope, nuclear pore, and within the nucleus. In conclusion, the observed pattern of changes in aggregation and subcellular localization of caspase-8, Bid, Bax and VDAC-1 during TGF-β1-induced apoptosis in HC11 mouse MEC suggests an interaction between these proteins and formation of multimeric complexes on organellar membranes, thus controlling their permeability for intracellular mediators of apoptosis.
- Mammary epithelial cells