The goals of this project are to determine how phthalocyanine (PC) based photodynamic therapy (PDT) can be used to purge tumor cells from bone marrow for use in high dose chemotherapy regimens with autologous bone marrow transplant and to further define the biochemical mechanisms of PC-based PDT killing of tumor cells under conditions useful for bone marrow purging. New PC derivatives, will be tested for selective cytotoxicity against tumor cells relative to normal bone marrow stem cells. Tumor cell lines to be studied have been chosen to reflect diseases which are current- ly being treated by high dose chemotherapy and commonly show marrow involvement. These include neuroblastoma, melanoma, breast cancer, small cell lung cancer, acute and chronic myelogenous leukemia and B and T cell acute lymphoblastic leukemia. Effective regimens will be evaluated to define biochemical consequences of PC based PDT in tumor cell lines and to evaluate the contributions of DNA damage and mitochondrial toxicity in the production of these metabolic alterations. We have shown that NAD and ATP depletion represent a final common pathway by which diverse acting agents lead to cell death. This pathway can be initiated by DNA strand breaks which activate poly(ADPR) polymerase with consequent depletion of cellular NAD and ATP pools. Mitochondrial damage can also cause ATP depletion without a primary effect on NAD. Agents employed in photodynamic therapy induce DNA strand breaks and also interfere with mitochondrial metabolism. Thus it is possible that they induce ATP depletion by either or both of these mechanisms. In these studies we will determine the effects of PC based PDT on NAD, ATP and other metabolites by microfluorometric enzymatic cycling assays. Inhibitors of poly(ADPR) polymerase, glycolysis and mitochondrial oxidative phosphorylation will be used to determine the contribution of each pathway to the metabolic consequences of PDT. Activation of poly(ADPR) synthesis will be determined by direct measurement of enzyme activity, substrate utilization and polymer accumulation. Enzymatic markers will be measured to ascertain damage to organelles where they are localized. These will include selective markers for mitochondria, cytoplasm and nucleus. This information will be used in the development and evaluation of new photosensitizers and in the optimization of PDT regimens for purging tumor cells from bone marrow.
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