Adenocarcinoma of the prostate exhibits a marked propensity to metastasize to the skeleton, where it commonly produces osteoblastic rather than osteolytic lesions. Although such bone-producing lesions may be due to inhibition of bone resorbing cells (directly or via stimulation of osteoclast-inhibiting agents such as calcitonin), most evidence suggests that osteoblast numbers and activity are directly enhanced. Preliminary evidence indicates this might be due to the production by prostate cancer of osteoblast-stimulating humoral factors. The proposed studies are directed at isolating and chemically purifying such humoral factors from freshly isolated human prostate tissue (normal, hyperplastic and neoplastic), from human prostate cancer cell lines, from transplantable rat prostate cancers grown in vivo and in vitro, and from conditioned tissue culture medium. As bioassays for osteoblast-stimulating activity, proliferation and function of isolated fetal or neonatal rat calvarial cells grown in monolayer culture, will be examined. As indices of cell proliferation, [H3] thymidine incorporation, and cell numbers will be assessed; as indices of cell function, [14C] proline incorporation into collagenase-digestible protein, synthesis of type I collagen, alkaline phosphatase activity, and production of Gamma-carboxyglutamic acid containing protein will be determined. Additionally, rat osteogenic sarcoma cell lines (osteoblast in origin) will be employed as an alternate bioassay system, and similar indices followed. Tissue and medium will be extracted in hydrophobic and hydrophilic solvents. Hydrophilic stimulatory extracts will be purified by gel filtration and reversed-phase liquid chromatography and/or iron exchange chromatography. Lipophilic stimulatory extracts will be purified by gel chromatography and normal phase high pressure liquid chromatography. Sufficient purification for development of a radioimmunoassay and for structural analysis (amino acid analysis and ultimately sequence analysis for a peptide factor) will be attempted. Additionally skeletal lesions produced by transplantable rat tumors in vivo will be examined histologically and for their capacity to alter osteoclast numbers and osteoclast binding by calcitonin. The studies therefore seek to examine the mechanisms whereby metastatic prostatic adenocarcinoma alter skeletal function, and aim to chemically characterize humoral factors which may mediate effects on the skeleton.
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