This is a revised application for a 5-year study of cellular and molecular mechanisms of pthalocyanine-sensitized photodynamic therapy (PcPDT) of skin tumors and of the associated cutaneous photosensitivity. Pthalocyanins (Pcs) sensitize tumor cells to damage by subsequent red light exposure. The hypothesis to be examined is that PcPDT activates membrane-localized enzymes, some of which are components of normal signal transduction pathways, leading to cell death. It is postulated that pathways that cause direct vs. indirect cell death in tumors, and for tumor cell death vs. cutaneous photosensitivity may vary. Manipulations of such pathways to enhance cell death and minimize photosensitivity will be explored. The four specific aims remain the same, but have been reordered as suggested by the last review. Now, the first two aims will address the relevance of apoptosis in PcPDT. 1) Whether murine papillomas or squamous cell carcinomas induced by chemical carcinogens or by UVB radiation undergo apoptosis during PcPDT ablation will be examined by first optimizing conditions for production of apoptosis, then by attempting to block apoptosis by basic fibroblast growth factor or enhance it by dexamethasone, with assessment of apoptosis by DNA fragmentation, histology, direct immunoperoxidase staining of digoxigenin-labeled genomic DNA in sections, or flow cytometry. 2) The cellular target of apoptosis will be studied in RIF-1 tumors in which apoptosis occurs during PcPDT tumor shrinkage in vivo, but not in vitro. This disparity will be exploited to elucidate differences in in vitro vs. in vivo mechanisms of PcPDT cell killing. RIF-1 tumor cell DNA will be tagged with transfected bacterial lac-Z genes to permit identifying malignant vs. infiltrating host cells when tagged RIF-1 cells are implanted into host mice and grow into tumors. The occurrence of apoptosis and the cell lines involved will be explored in histologic sections, with X-gal incubation or in situ hybridization with lac-Z insert as probe to identify tagged cells, by assessing DNA fragmentation, and by flow cytometry. If RIF-1 cells show apoptosis in vivo, it will be concluded that culture conditions lack factors necessary for direct PDT induction of apoptosis. Next, the mechanism for in vivo apoptosis of RIF-1 cells will be studied by comparing the extent and time course of apoptosis in tumors derived from cells expressing transfected human bcl-2 gene vs wild type cells. In vitro expression of bcl-2 gene appears to prevent apoptosis is several cell types. Similar incidence of apoptosis in transfected and wild cells will indicate that bcl-2 protein does not block direct PDT causation of apoptosis; dissimilar incidence will be taken as evidence that mechanisms of cell killing differ in vivo and in vitro, and that vascular effects must figure importantly in PcPDT damage in vivo. 3) Signal transduction in PcPDT-induced cell killing will be investigated to define its mechanisms. Signalling events will be studied in human epidermoid squamous cell carcinoma A431 cells vs. normal human epidermal keratinocytes. Pathways to be studied include protein tyrosine phosphorylation, breakdown of membrane phospholipids, release of intracellular Ca++, and activation of protein kinase C (PKC). Essential signalling events will be identified by using inhibitors of specific metabolic events. 4) The mechanism of PcPDT-related cutaneous photosensitivity will be studied by evaluating the roles of various toxic oxygen intermediaries or vasoactive amines. Pc-sensitized normal or tumor-bearing C3H/HeN mice will be given quenchers of superoxide anion, hydroxyl radical, hydrogen peroxide, or singlet oxygen, with or without quencher-inhibitors, prior to irradiation, then ear swelling and footpad damage will be quantified to assess which oxygen species is/are produced. Histamine H1 and H2 blockers will be similarly tested for suppressive effects. Agents found to be effective will then be tested for abrogation of photosensitivity by administration to mice after Pc-PDT of skin tumors, and reexposure to light radiation vs. controls.
Showing the most recent 10 out of 42 publications
