Photodynamic therapy (PDT) is a relatively new cancer treatment modality that exploits the ability of certain dyes to localize in vivo or ex vivo in tumor cells and to preferentially destroy these cells when excited by visible light of appropriate wavelengths. One such dye is merocyanine 540 (MC54O), an amphiphilic compound being evaluated for clinical use in the selective eradication of neoplastic cells in autologous remission bone marrow grafts. Pathways of dye-sensitized cell killing are still poorly understood, as are the means by which cells cope with potentially lethal photodamage. Previous studies in the parent project have shown that glutathione (GSH) and GSH-dependent selerioperoxidases (SePXs) play an important role in suppressing lipid hydroperoxide (LOOH) accumulation in MC540/light-treated leukemia cells (L121O, K562) and also in protecting the cells against photokilling. Thus, Se-deficient [Se(-)] cells exhibited higher steady state LOOH levels during irradiation than Se-satisfied (Se( +)] cells, and died off faster. Moreover, GSH-deficient Se( +) cells were more photosensitive than GSH-normal controls. Our working hypothesis in this proposal is that cells will respond to a photooxidative stress by mobilizing glutathione cycle activity in an effort to detoxify damaging LOOHs. In the process, oxidized glutathione (GSSG) would be recycled to GSH at the expense of NADPH generated via the pentose phosphate pathway. We will test this hypothesis by monitoring GSH and GSSG levels in glucose- depleted cells (L1210, K562) exposed to increasing light doses in the presence of MC54O. [GSH] and [GSSG] will be determined by high performance liquid chromatography with electrochemical detection. SePX involvement in cytoprotection will be checked by comparing GSH/GSSG changes in Se( +) cells with those in Se(-) cells. Any non-metabolic alterations in [GSH] due to direct photooxidation will be assessed, using cell-free model systems or SePX-depleted cells. The relationship between GSH/GSSG ratio, LOOH levels, and cell survival will be examined for the different cell types. This study will contribute to our basic understanding of how cells cope with photooxidative stress and in particular how tumor cells might resist PDT treatment.
