Photodynamic therapy (PDT) of tumors involves light activation of a photosensitizer localized in host and malignant tumor cell membranes. The primary hypothesis is: The initial PDT-induced lesions activate membrane-localized enzymes, some of which are part of normal signal transduction pathways, and the action of these enzymes determines the fate of the cells. To test this hypothesis, Specific Aim 1 will determine the role of signal transduction in PDT-induced cell killing. Signalling reactions will be assessed in L5178Y cells, which undergo apoptosis in response to PDT, and RIF-1 fibrosarcoma cells, which do not undergo apoptosis upon PDT in vitro. Essential steps to be measured include protein tyrosine phosphorylation, breakdown of membrane phospholipids, release of Ca++, and activation and translocation of protein kinase C.
Aim 2 will elucidate the mechanism of the synergistic interaction between ionophores and PDT, examining the release of Ca++ and/or reduction of intracellular pH alone or with PDT.
Aim 3 will investigate the role of polyamine-like ligands in the efficient cell uptake and photocytotoxicity of certain of our lead phthalocyanines by modifying polyamine levels in the cells and by structure-function analysis with Project 1.
Aim 4 will evaluate the induction of apoptosis by conditions obtaining in PDT-treated tumors. Since PDT kills malignant tumor cells both by direct damage and by effects secondary to vascular collapse and inflammatory reactions, we will test the ability of hypoxia, low pH, and cytokines and eicosanoids identified in Project 3 to induce apoptosis in L5178Y cells and in RIF-1 cells in vitro.
Aim 5 will determine the relevance of apoptosis to the PDT response of RIF-1 tumor cells in vivo. The incidence of apoptosis vs. necrosis in PDT-treated RIF-1 tumors will be scored histologically and by flow cytometry. Apoptosis in RIF-1 cells whose DNA is tagged with the bacterial lac Z gene will be distinguished from apoptosis in infiltrating host cells. The mechanism for the in vivo apoptosis response will be explored by comparing tumors derived from RIF-1 cells expressing the human bcl-2 gene and from wild type RIF-1 cells with respect to (a) ability of PDT to induce apoptosis and (b) their relative sensitivity to PDT.
Aim 6 will evaluate the mutagenic potential of PDT in human cells and characterize PDT-induced mutations with respect to intergenic vs. intragenic events. The proposed investigations will contribute to an understanding of cellular responses and their role in determining the fate of PDT-treated tumors in vivo. The results should suggest new opportunities for modifying PDT responses in normal and tumor tissue.
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