Parathyroid hormone-related protein (PTHrP) Is a recently identified hormone which mimics many of the renal and skeletal actions of parathyroid hormone. This protein has been isolated from a number of human and animal tumors associated with hypercalcemia, and has been identified as a causative factor in the pathogenesis of humoral hypercalcemia of malignancy (HHM). PTHrP has also been Isolated from a number of normal tissues including normal human keratinocytes. The function of PTHrP in these tissues is not known and little is known about the regulation of synthesis and secretion of this new hormone. A thorough knowledge of the regulation of PTHrP production and secretion is necessary in order to understand it's role in normal physiology as well as in HHM. It is currently believed that the regulation of PTHrP production and secretion is related to differentiation. The observations that PTHrP is preferentially expressed in undifferentiated tissues, and factors known to regulate PTHrP are also known to influence the differentiation of certain PTHrP-producing cells, support this hypothesis. This study will examine the regulation of PTHrP production and secretion as it relates to the differentiated state of epithelial cells. Differences in the regulation of PTHrP between normal and neoplastic tissue should provide insight into the pathogenesis of HHM as well as the role of PTHrP in normal physiology of cells in the skin. Experiments in phase I will focus on the intracellular production and secretion of PTHrP in cultures of normal human keratinocytes and squamous cell carcinomas. Initial studies will utilize normal cell culture conditions in order to ascertain baseline measurements of PTHrP production. Thereafter, factors known to influence the growth and differentiation of cells will be examined for their effects on PTHrP production and secretion, and will be compared to their effects on growth and differentiation. Phase 11 will focus on the control of PTHrP RNA expression in epithelial cultures. Northern blot hybridization, pulse-chase labelling, and nuclear run-off assays will be employed as measures of total RNA, RNA stability, and transcription rate, respectively. In addition, intracellular PTHrP and PTHrP RNA production will be examined within individual cells using combined immunohistochemistry and in situ hybridization.
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