Members of time bcl-2 and caspase families regulate nuclear degeneration during chick lens fibre differentiation

The optical clarity of the lens is ensured by the programmed removal of nuc lei and other organelles from the lens fibre cells during development. The morphology of the degenerating nuclei is similar to that observed during ap optosis and is accompanied by DNA fragmentation. Proteins encoded by the bc l-2 proto-oncogene family are important in either promoting or inhibiting a poptosis, and caspases are, involved in downstream proteolytic events. Here , the expression of bcl-2 family members (bcl-2, bar, bad, and bcl-x(s/l)) and caspases-1, -2 -3, -4, and -6 was investigated through a range of stage s of chick lens development using immunocytochemistry, Western blotting, an d affinity labelling for caspases using biotinylated caspase inhibitors. Us ing differentiating lens epithelial cell cultures, it was demonstrated that the addition to cultures of synthetic, peptide inhibitors of caspases -1, -2, -4, -6, and -9 brought about a 50-70% reduction in the number of degene rating nuclei per unit area of culture, as assessed by image analysis. Thes e effects were comparable to those seen when general inhibitors of caspases were added to cultures. On the other hand, inhibitors of caspases-3 and -8 were not effective in significantly reducing the number of TUNEL-labelled nuclei. Expression of the caspase substrates poly(ADP-ribose) polymerase (P ARP) and the 45-kDa subunit of DNA. fragmentation factor (DFF 45) was also observed in the developing lens. Western blots of cultures to which caspase inhibitors were added revealed alterations in the PARP cleavage pattern, b ut not in that of DFF. These results demonstrate a role for members of the bcl-2 family and caspases in the degeneration of lens fibre cell nuclei dur ing chick secondary lens fibre development and support the proposal that th is process has many characteristics in common with apoptosis. (C) 1999 Acad emic Press.