The long term goal of our application is to contribute to the advancement of Photodynamic Therapy (PDT) by increasing the molecular understanding of PDT mediated cytotoxicity and by developing strategies to enhance the local tumoricidal response following PDT. The investigators will build upon recently completed studies to accomplish our objectives. The investigators have isolated mouse RIF fibrosarcoma cells exhibiting a stable PDT resistant phenotype. The investigators have also demonstrated that PDT mediated oxidative stress is a strong transcriptional inducer of stress proteins belonging to the glucose regulated protein (GRP) family and the heat shock protein (HSP) family. The investigators hypothesize that analyzing PDT resistance will provide insights into basic mechanisms of PDT induced cytotoxicity and thereby lead to strategies to improve PDT. The investigators also hypothesize that targeted gene therapy using recombinant constructs with PDT inducible promoters to drive high level local expression of cytotoxins and/or immuno-modulators will enhance PDT tumoricidal action.
Two specific aims will address the hypotheses: 1.) to examine the molecular, cellular and in-vivo parameters associated with our PDT resistant cells. The investigators will identify and characterize differentially expressed genes in parental and PDT resistant RIF tumor cells as well as in cells exposed to PDT mediated cytotoxicity. The roles of identified genes in modulating PDT sensitivity and photosensitizer localization will be evaluated using vector mediated gene transfer and subcellular confocal fluorescence microscopy. 2.) to use PDT inducible promoters to selectively drive expression of target genes. The investigators will use grp-78 and hsp-70 promoter/reporter gene constructs to examine PDT parameters required for optimal in-vitro and in-vivo gene expression. The investigators will then evaluate grp-78 and hsp-70 promoter/TNFalpha fusion constructs for PDT induced expression of TNFalpha in RIF tumor cells grown in culture or transplanted into C3H mice. The investigators will also examine the effectiveness of PDT inducible promoter/TNFalpha expression constructs to enhance the tumoricidal effectiveness of PDT.
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