MULTIPLE PROTEASES ARE INVOLVED IN THYMOCYTE APOPTOSIS

To investigate the involvement of proteases in apoptosis, rat thymocyt es were treated with the glucocorticoid hormone methylprednisolone or the topoisomerase II inhibitor etoposide in the presence of selective substrate inhibitors of either interleukin-1 beta-converting enzyme (I CE), (Z-Val-Ala-Asp-chloromethylketone, VADcmk) or Ca2+-regulated seri ne protease (Suc-Ala-Ala-Pro-Phe-chloromethylketone, AAPFcmk), VADcmk protected from lamin proteolysis, chromatin fragmentation, cell shrink age, and formation of apoptotic nuclei in both methylprednisolone-and etoposide-treated thymocytes when present during the initiation of the apoptotic process, AAPFcmk prevented lamin breakdown, chromatin fragm entation, and apoptotic morphological changes in thymocytes treated wi th methylprednisolone, but not with etoposide. Both MPS- and etoposide -treated thymocytes exhibited enhanced ICE-like protease activity whic h was maximal 1 h after treatment, This increase in proteolytic activi ty was blocked by VADcmk, but not AAPFcmk, Our findings suggest that I CE-like protease activity is critically involved in the early phase of both methylprednisolone- and etoposide-induced apoptosis in thymocyte , whereas the Ca2+-regulated serine protease is an obligatory componen t of the proteolytic cascade in methylprednisolone-induced apoptosis. (C) 1995 Academic Press, Inc.