Photodynamic therapy (PDT) of tumors begins with photodynamic activation of a photosensitizer localized in malignant cells or in cells within the tumor vasculature and subsequent biochemical reactions. The primary hypothesis is: The initial PDT-induced membrane lesions activate membrane- localized enzymes, some of which are part of normal signal transduction pathways, and these reactions determine the fate of the cells. These pathways may vary for direct vs. indirect malignant cell death within tumors, and manipulation of the pathways may allow modulation of PDT responses.
Specific Aim 1 : The role of signal transduction in PDT-induced killing will be determined in L5178Y Cells, which undergo apoptosis in response to PDT, and in RIF-1 fibrosarcoma cells, which do not undergo apoptosis upon PDT in vitro. Essential steps, to be identified with inhibitors and measurement of pertinent reactions, include protein tyrosine phosphorylation, breakdown of membrane phospholipids, elevation of intracellular Ca++, and activation and translocation of protein kinase C.
Specific Aim 2 : The mechanism of the synergistic interaction between ionophores and PDT will be elucidated by examining (a) the release of Ca++ and breakdown of membrane phospholipids or reduction of intracellular pH and (b) whether observed signals are unique to cells induced to undergo apoptosis.
Specific Aim 3 : Apoptosis will be measured in malignant and host cells within PDT-treated RIF-1 tumors in vivo, scoring the incidence of apoptosis vs. necrosis in histological sections and by DNA fragmentation. For this purpose, tumor cell DNA will be tagged with the bacterial lac Z gene to differentiate malignant from host cells. Since RIF-1 cells do not undergo apoptosis after PDT in vitro, but appear to do so in vivo, the mechanism for the in vivo response will be explored. Tumors derived from RIF-1 cells expressing the human bcl-2 gene will be compared to tumors derived from wild type RIF-1 cells with respect to (a) the ability of PDT to induce apoptosis and (b) the relative sensitivity of these tumors to PDT.
Specific Aim 4 : The mutagenic potential of PDT in human cells will be evaluated and PDT-induced mutations characterized. PDT has been shown to be mutagenic in L5178Y cell lines. Because of the specific properties of the tk locus in these cells (murine origin; highly mutable by clastogens), it is essential that the mutagenicity of PDT in human cells be assessed. The mutational spectra will be determined 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 will hopefully suggest new opportunities for modifying PDT responses in normal and tumor tissue.
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