ALTERATIONS IN MITOCHONDRIAL STRUCTURE AND FUNCTION ARE EARLY EVENTS OF DEXAMETHASONE-INDUCED THYMOCYTE APOPTOSIS

In this paper we used a multiparametric approach to analyze extensivel y the events occurring during apoptotic cell death of thymocytes, and furthermore, we asked whether alterations in mitochondrial structure a nd function are occurring in early stages of apoptosis. A multiparamet ric quantitative analysis was performed on normal or apoptotic thymocy tes emerging from a few-hour culture performed in culture medium or in the presence of dexamethasone. Simultaneous detection of light scatte ring properties, integrity of plasma membrane (trypan blue exclusion), chromatin condensation (AO/EB staining of entire cells or PI staining of nuclei), and DNA fragmentation (in situ nick-translation in apopto tic cells) allowed a precise analysis of the preapoptotic and apoptoti c stages. Moreover a thorough study of mitochondrial transmembrane pot ential (Delta Psi(m)) assessed following in a time course study the up take by apoptotic cells of the cationic lipophilic dye DiOC(6)(3) or t he J-aggregate-forming cation JC-1, indicates that a drop in Delta Psi (m) occurs very early in thymocyte apoptosis, before DNA fragmentation . This is associated with alteration in mitochondrial structure assess ed by cytofluorimetric study of NAO uptake in apoptotic cells. Finally these dramatic alterations in mitochondrial structure and function oc curring in early stages of apoptosis were confirmed by confocal and el ectron microscopy analysis.