Co-purification of transforming growth factor like activity with pth-like and bone-resorbing activities from a tumor associated with humoral hypercalcemia of malignancy

Humoral hypercalcemia of malignancy (HHM) is caused by a circulating bone-resorbing factor or factors. Suggestions as to the nature of this factor include PTH-like proteins, transforming growth factors, and bone-resorbing factors distinct from either of the first two classes of polypeptides. We investigated the occurrence of these three activities in a highly purified extract of the H-500 Leydig cell tumor which causes HHM when implanted into Fisher rats. PTH-like adenylate cyclase-stimulating activity (ACSA) was extracted from tumor tissue by sequential treatment with urea/HCl and ethanol/NaCl. Tumor extract was further purified by hydrophobic-interaction, gel-filtration, and reverse-phase HPLC steps to a specific activity of 1038 ng eq bPTH(l–34)/mg protein. Only the fraction pool containing ACSA demonstrated significant bone-resorbing (1.78-fold over basal) and transforming growth factor activity (epidermal growth factor (EGF)-dependent colony formation in soft agar suspension by NRK-49F indicator cells). A subsequent reverse-phase HPLC step produced material which contained both ACSA and transforming growth factor beta (TGF β)-like activity in a single fraction. Whether the responsible mediator in this animal model has TGF β-like properties as well as PTH-like and bone-resorbing activity remains to be determined.