This project is designed to provide information on mechanism(s) of cell death induced by photodynamic therapy (PDT). The long-range goal is an improved understanding of the selective tumor eradication mediated by PDT, including neoplastic cells resistant to chemotherapy or ionizing radiation. While it was initially believed that PDT caused a necrotic response, PDT-induced apoptosis has now been described in several animal tumor systems, both in vivo and in cell culture. Although PDT does not always appear to initiate an apoptotic response, this may be related to the assay procedure, or differences in sites of photodamage. They therefore plan to use a variety of methods to assess both effects of PDT and mode(s) of cell death. Their current hypothesis is that (1) expression of apoptosis after PDT depends on the subcellular site(s) of photodamage; (2) mitochondrial photodamage leads to very rapid apoptotic response; (3) the latter is triggered by release of cytochrome c and other factors from mitochondrial membranes which activate cytosolic caspases; (4) concurrent membrane photodamage will delay or abolish the apoptotic response as release of membrane-derived products, e.g. ceramide, is impaired. They propose that the latter factors can alter the apoptotic threshold or accelerate the program. High PDT doses may inhibit apoptosis, as specificity is lost. Their hypothesis suggests that the role of protein phosphorylation in the PDT- induced apoptotic program may be peripheral, with certain kinase and phosphatase inhibitors responsible for non-specific cytotoxicity or alterations in the apoptotic threshold.
Three aims are proposed.
Aim 1 is designed to provide a better understanding of site(s) of photodamage as a determinant of the apoptotic response. They will amplify studies already done with the murine leukemias, and extend the scope of work to determine if a different set of localization criteria apply to cell types of non-lymphoid origin.
Aim 2 examines the mechanism whereby mitochondrial photodamage elicits an apoptotic response in the murine leukemias, and concurrent membrane photodamage can delay this response.
In Aim 3, they turn to the study of the PDT- induced apoptotic response in cell lines non-responsive to conventional chemotherapy and sublines varying in p53 and bcl-2 expression. Ca PDT initiate apoptosis at such a late stage in the program as to circumvent drug resistance associated with a high apoptotic threshold? Since this proposal involves a change in focus from their previous research direction, three consultants (two locals) have been solicited to aid in the design and interpretation of experimental results.
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