ANALYSIS OF THYMIC LYMPHOCYTE APOPTOSIS USING INVITRO TECHNIQUES

Thymocyte apoptosis was analyzed in 4-week-old chicks that were treate d with dexamethasone. Glucocorticoid treatment of birds resulted in th e internucleosomal cleavage of thymocyte DNA as detected by agarose ge l electrophoresis and the generation of a population of thymocyte nucl ei with a hypodiploid content of cellular DNA when analyzed by flow cy tometry. Visualization of apoptotic thymocytes at the ultrastructural level or via confocal microscopy revealed the typical morphological ch aracteristics of cells undergoing apoptosis. Similar techniques were e mployed to further analyze apoptosis using an in vitro approach where extracts of thymocyte nuclear proteins from glucocorticoid-treated bir ds were incubated with chicken red blood cell (cRBC) nuclei. The thymo cyte nuclear protein extract contained an endonuclease activity that d egraded chromatin at internucleosomal sites and generated an amplified population of hypodiploid cRBC nuclei, similar to that detected in vi vo in glucocorticoid-treated thymocytes. These data demonstrate a clea r correlation between the detection of apoptotic endonuclease activity generated in vivo, where endogenous thymocyte DNA was degraded, with an in vitro assay, where extracted thymocyte nuclear proteins demonstr ated a similar type of nuclease activity when cRBC nuclei were employe d as an exogenous chromatin substrate.