HUMORAL FACTOR INTERACTIONS IN HYPERCALCEMIA OF CANCER

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The overall goal of this proposal is to investigate mechanisms which control parathyroid hormone-related protein (PTHrP) secretion and mRNA expression in normal and neoplastic keratinocytes. It is hypothesized that there are important differences in the pathophysiology of PTHrP regulation in normal compared to neoplastic keratinocytes. PTHrP mRNA will likely be expressed in a manner regulated by differentiation in normal keratinocytes and will demonstrate polarity of secretion (away from the basement membrane of the epidermis). In contrast, neoplastic keratinocytes likely have altered regulation of PTHrP mRNA secretion and expression and do not demonstrate polarity of secretion due to the lack of a basement membrane. The five-year proposal has three major objectives: (1) investigate the regulation of PTHrP mRNA expression, transcription rate, and stability by cytokines (TGFalpha, TGFbeta, and IL-1), 1,25-dihydroxyvitamin D, and calcium in normal human keratinocytes and squamous cell carcinoma cell lines grown in monolayer and 3-dimensional cell cultures, (2) investigate the regulation and polarity of PTHrP secretion by agents which affect protein kinase C and intracellular calcium, by inhibitors/stimulators of vesicular secretion, and by the presence of basement membrane components (3) determine the relationship of PTHrP mRNA expression to the regulation of tissue-specific gene expression during keratinocyte proliferation (c-myc and H3 histone genes) and differentiation (involucrin, filaggrin, and keratin 1 and 6 genes). The use of 3-dimensional cell cultures will be an important aspect of the investigations and will permit normal differentiation of epidermal keratinocytes in vitro and allow comparisons to neoplastic keratinocytes. These studies will define factors and conditions that regulate PTHrP synthesis and secretion. Information on the cellular biology of PTHrP is necessary to determine its role in the pathologic state of humoral hypercalcemia of malignancy, its role in the normal physiology of epithelial cells, and mechanisms by which its secretion and synthesis can be controlled.