This proposal is designed to study the biosynthesis, secretion, cell- specific processing, and gene expression of the parathyroid hormone-related protein (PTHrP) associated with the hypercalcemia of malignancy with immunochemical and nucleic acid hybridization procedures that can also be used in the clinical diagnosis, classification, and management of PTHrP- producing tumors. PTHrP is a 141 residue protein that is produced by tumors commonly associated with hypercalcemia. Of the first 13 amino acids of this protein, 8 are homologous to the amino terminus of human PTH, the biologically active fragment of PTH. The amino-terminal fragment of PTHrP, PTHrP1-34, reacts with the PTH receptor and produces most of the biological effects of native PTH, including hypercalcemia. We have developed the experimental procedures to study the cellular and molecular biology of PTHrP. We have developed several cell lines that produce multiple species of PTHrP mRNAS and PTHrP, itself. We have specific antibodies that identify PYHrP in tissues and biological fluids and we have nucleotide probes to study its gene expression. In immunohistology studies, we have been able to demonstrate PTHrP in a variety of tissues, including squamous cell lung cancer and also small-cell lung cancer. We propose to study the biosynthesis and secretion of PTHrP in these cell lines using radioimmunoassay and related immunochemical procedures. The radioimmunoassay procedures will additionally be performed in a two-site format in order to measure defined species of PTHrP. These assay studies will be conducted in conjunction with sizing procedures such as chromatography, Western Analysis, and pulse-chase experiments. The regulation of PTHrP mRNA will be evaluated by Northern hybridization. In addition to these human tissues, we have developed animal models for the study of PTHrP. These experimental tools will permit the basic regulatory studies that we propose. We also propose to study the clinical role of PTHrP as a tissue and serum marker for endocrine cancers with newly developed immunochemical procedures based on multiple epitope recognition. These studies should help elucidate the role of PTHrP in the molecular, cellular, and clinical biology of lung tumors with endocrine characteristics.
