(86) STUDY OF APOPTOSIS IN SPLENOCYTES AND THYMOCYTES OBTAINED FROM GAMMA-GLUTAMYL TRANSPEPTIDASE DEFICIENCY KNOCKOUT MICE

Yvonne Will, Rhonda Kaetzel, M. Lieberman and D.J. Reed
Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR 97330

Our laboratory is interested in the relationship of cellular and mitochondrial glutathione status related to apoptotic events. In general, apoptosis is accompanied by a depletion of intracellular glutathione (GSH). Some studies concluded that intracellular GSH levels only affect the extent of apoptosis in the case of oxidative stress, other studies reported that not the treatment used to induce apoptosis, but apoptosis itself caused GSH depletion. In vitro treatment of cultured cells with GSH has been shown to prevent dexamethasone (DEX) induced apoptosis. In vitro depletion GSH depletion studies with BSO did not demonstrate any GSH effect on the extent or the kinetics of apoptosis. How GSH relates to apoptosis remains complex and not very well understood. Several studies suggested that maybe thiols other than GSH may play a key role in apoptosis. Kroemer et al, 1995 showed that thymocytes after DEX treatment undergo a step-wise dysregulation of mitochondrial function involving mitochondrial permeability transition (MPT). We suggest that mitochondrial GSH is involved either directly or indirectly in preventing MPT. To further elucidate the question of how cellular and mitochondrial GSH status relate to apoptosis we use gamma-glutamyl transpeptidase deficient knockout mice (GGT-KO). Due to the lack of GGT these animals show alteration of glutathione homeostasis in various organs. Glutathione is decreased between 25 and 40% in splenocytes when compared to control mice and 25% decreased in thymocytes. Mitochondrial GSH was measured in splenocytes and was shown to be decreased by 20%. In a pilot experiment GGT-KO mice were treated with DEX and splenocytes and thymocytes isolated. Flow cytometry was used to assess mitochondrial transmembrane potential (DiOC6, JC-1), ROS generation (HE), cytosolic calcium (calcium green-am), cardiolipin oxidation (NAO), and apoptosis (annexin/PI). We observed that untreated GGT-KO mice did not show any significant differences in any of the parameters when compared to wildtype mice. However, after treatment with DEX there was a higher number of cells in the GGT-KO knockout mice that showed alterations in these measured parameters. We are currently performing additional experiments to test our hypothesis that the decrease in cellular and mitochondrial GSH in GGT-KO mice will increase the susceptibility to MPT and apoptosis.