Role of the multidrug resistance-associated proteins (MRP/ABCC) in glutathione transport during apoptosis

Thesis (Ph. D.)--University of Rochester School of Medicine & Dentistry. Dept. of Environmental Medicine, 2007.Glutathione (GSH) is important for normal cellular functions and recent observations suggest that GSH also plays a key role in apoptotic cell death. During apoptosis, intracellular GSH is rapidly depleted due to its enhanced export from cells. The precise mechanism behind the GSH efflux and its significance to the apoptotic process are not understood. Although loss of intracellular GSH may simply be a result of apoptosis, recent studies suggest that GSH depletion is required for the activation of specific signaling pathways, including those mediated by reactive oxygen species. The overall goal of the presented work is to determine whether members of the multidrug resistance-associated protein family (MRP/ABCC) mediate GSH release during apoptosis, and whether this release is important for the progression of apoptosis. Fas antibody and staurosporine were used to induce apoptosis in three cell types: Jurkat cells, Raji cells, and HEK293 cells overexpressing MRP1. Apoptotic GSH release in Jurkat cells was associated with an increase in apoptosis because inhibiting GSH release decreased apoptosis. Conversely, Raji cells did not release GSH nor externalize phosphatidylserine when treated with Fas antibody or staurosporine. Additionally, apoptosis was slower in Raji cells when compared to the Jurkat cells, suggesting that GSH release is important for the progression of apoptosis and may be associated with phosphatidylserine externalization. MRP expression and function were measured in Jurkat and Raji cells to further characterize the role of MRPs in GSH transport. Although both cell lines expressed similar levels of MRPs, they were not functional in the Raji cells. MRP1 vii was further analyzed because it is implicated in GSH transport and is ubiquitously expressed in mammalian cells. Decreasing MRP1 expression in Jurkat cells inhibited both basal and apoptotic GSH release, providing direct evidence for MRP1 in mediating GSH transport. Furthermore, HEK293 cells overexpressing MRP1 had elevated basal and apoptotic GSH release; however, apoptosis was not increased in these cells, an effect that is most likely explained by an enhanced rate of GSH synthesis in these cells. Overall, these studies support a role for the MRPs, more specifically MRP1, in GSH release and indicate that GSH release is important for the progression of apoptosis