Application of a fluorometric assay to detect caspase activity in thymus tissue undergoing apoptosis in vivo

To date, in vivo apoptosis within the thymus has been assessed using morpho logical criteria and/or detection of a DNA ladder indicative of oligonucleo somal fragmentation of the DNA. Here, we have used a fluorometric method to investigate activation of the caspase protease family in the thymus follow ing in vivo induction of apoptosis by injection of the synthetic glucocorti coid hydrocortisone. Cleavage of DEVD-MCA by caspase-3 and other group II c aspases releases free MCA which can be detected fluorimetrically. We demons trate a time-dependent increase in DEVD-MCA cleavage activity within this t issue indicating the activation of caspase-3 like enzymes. This activity wa s inhibited by the specific group II caspase inhibitor DEVD-CHO. The interp retation of increased caspase activity was confirmed by immunoblot analysis to reveal cleavage of the caspase-3 substrate, fodrin. In addition, agaros e gel electrophoresis of the DNA yielded a ladder pattern, confirming the o ccurrence of apoptosis. This study demonstrates that DEVD-MCA cleavage acti vity may be a useful quantitative method for the analysis of apoptosis in t hymus tissue. It is a relatively rapid procedure not requiring thymocyte is olation or gel electrophoresis and detects fairly early biochemical changes occurring during apoptosis. In the present study we have used this method to demonstrate the involvement of caspases in thymocyte apoptotic death ind uced in vivo by glucocorticoids. Thus, measurement of caspase activity in t hymus tissue may have applications for studying the in vivo effects of immu notoxicants. (C) 1999 Elsevier Science B.V. All rights reserved.