This is a proposal to use cellular differentiation as a way to modify ALA-mediated photodynamic therapy (PDT) in the treatment of cancer and other hyperproliferative diseases. Differentiation therapy is the principle of converting cells to a more differentiated state in order to enhance the response to specific therapies. Here, we take advantage of the fact that differentiated cells are more susceptible to ALA-dependent PDT than are undifferentiated cells. ALA is the initial precursor for heme biosynthesis within cells. When administered exogenously t o cells, ALA is taken up and metabolized by endogenous enzymes of the heme synthetic pathway to produce a photosensitizing intermediate, protoporphyrin IX (PPIX) which can be exploited for cell killing. Preliminary data in several cell types demonstrate that accumulation of PPIX during ALA exposure is greater in terminally-differentiated cells than in undifferentiated cells. Further, PPIX accumulates in the differentiated cells largely as a result of increased transcription of coproporphyrinogen oxidase (CPO), one of the heme-synthetic enzymes. Proposed studies would seek to optimize the expression of CPO, through the understanding and manipulation of differentiation-specific mechanisms regulating CPO gene expression. One physiologically normal cell type (skin keratinocytes) and one neoplastic cell type (LNCaP prostate carcinoma) will be used as models for study. Epidermal keratinocytes were chosen as the normal model because the keratinocyte differentiation program is well-defined both in vitro and in vivo, and because C/EBP transcription factors have been shown to regulate differentiation- associated genes in the skin. Similarly, prostate carcinoma (LNCaP) cells are useful because their state of differentiation can be hormonally altered by androgens, retinoids, or vitamin D. Using these two cell systems, the role of C/EBPs in regulating CPO gene expression during differentiation will be studied. Ways to increase photosensitized cell death by upregulating CPO, perhaps via C/EBPs, will be sought. Ultimately, we will test the feasibility of exploiting differentiation as a means for enhancing ALA-PDT efficacy for neoplastic conditions in which cells are well-differentiated, or in which differentiation can be transiently induced. Overall, these studies may fill a gap in current cancer therapy, providing new way to effectively target cells that are resistant to standard radiation or chemotherapy regimens.
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