A B S T R A C T The culture media of three cell lines, a human prostate carcinoma (PC3), a rat Leydig cell tumor (Rice-500), and a rat carcinosarcoma , that were derived from tumors associated with humoral hypercalcemia of malignancy (HHM), were examined for stimulation of adenylate cyclase in ROS 17/2.8 osteoblastic cells and for bone resorptive activity in culture. Cells from a nonhypercalcemic variant of the WRC 256 tumor served as control. Extracts from three solid human tumors, a lung adenocarcinoma from a patient with HHM and two adendcarcinomata from normnocalcemic patients (lung and colon), were also examined for adenylate cyclase stimulation. We found excellent correlation between stimulation of cyclic AMP accumulation in ROS 17/2.8 cells and bone resorbing activity in culture, or production of HHM in vivo. Stimulation of adenylate cyclase by HHM factors was inhibited by the parathyroid hormone competitive inhibitor, [8norleucyl, '8norleucyl, 34tyrosinyl] bovine parathyroid hormone (3-34) amide.

Human recombinant transforming growth factor a (TGFa), which binds to the epidermal growth factor (EGF) receptor and causes several biological effects similar to those caused by EGF, was compared with murine EGF for its effects on a number of parameters of bone cell metabolism. TGFa stimulated bone resorption in two organ culture systems, the fetal rat long bone and neonatal mouse calvarial systems. TGFa stimulated bone resorption at concentrations as low as 0.1 ng/ml. TGFa effects on bone resorption in mouse calvariae were inhibited by indomethacin, suggesting that, like EGF, its effects were mediated by prostaglandin synthesis. TGFa had a different time course of action on bone resorption from that of EGF, causing more rapid release of previously incorporated 45Ca from bone cultures, suggesting that TGFa does not function on bone as a simple EGF analogue. TGFa also caused effects on osteoblast function resembling those of EGF. It inhibited alkaline phosphatase activity in cultured rat osteosarcoma cells with the osteoblast phenotype and inhibited collagen synthesis in fetal rat calvaria at concentrations of 1.0 ng/ml. The lowest concentration of TGFa (expressed as nanogram equivalents of EGF per ml) required to produce a response in all of the systems tested was about 1/10th of that needed for EGF to produce a similar effect. These results indicate that TGFa is a potent stimulator of bone resorption and inhibitor of bone formation as assessed by inhibition of collagen synthesis and alkaline phosphatase activity and are consistent with the hypothesis that TGFa may be responsible, at least in part, for the bone resorption associated with some tumors.Transforming growth factor alpha (TGFa) is secreted by a variety of human and rodent tumor cells and binds to the epidermal growth factor (EGF) receptor. TGFa causes many of the known biological effects of EGF (1-3). Multiple peptides of different molecular weights with TGFa activity (as assessed by inhibition of EGF binding and stimulation of anchorage-independent growth) have been detected in culture media and extracts of tumor cells. A small species of 50 amino acids has been purified (4, 5). It is about 40% homologous with EGF (4, 6). Cloning of the cDNA reveals that it is initially synthesized as a larger precursor, which undergoes subsequent posttranslational cleavage. Proper engineering of the sequence coding for the 50 amino acid TGFa has allowed the expression of biologically active human TGFa in Escherichia coli (6).EGF causes osteoclastic bone resorption (7,8) and inhibits bone formation as assessed by collagen synthesis in organ cultures of fetal rat calvariae (7-9) and alkaline phosphatase activity in osteoblast-like cells (10). Since EGF stimulates osteoclastic bone resorption and TGFa competes with EGF for the same receptor, it has been suggested that tumor production of TGFa could be responsible for the bone destruction that is associated with some neoplasms (11,12). Partially purified bone-resorbing activity associated with an animal tumor co...

Transforming growth factor-beta (TGF beta), a polypeptide that controls growth and differentiation in many cell types and has recently been found in abundant amounts in bone, was examined for its effects on cells with the osteoblast phenotype using the clonal osteoblastic osteosarcoma cell line ROS 17/2.8. TGF beta increased alkaline phosphatase (AP) activity and the rate of collagen synthesis per cell. Cell proliferation was inhibited, and the morphological appearance of the cells was markedly changed. All effects were observed at concentrations as low as 0.1 ng/ml TGF beta. Increases in AP activity were detectable after 24 h and increased progressively with time. TGF beta increased AP activity under serum-free conditions and during thymidine-induced inhibition of DNA synthesis. The increase in AP activity mediated by TGF beta could be completely inhibited with actinomycin D and cycloheximide. 1,25-Dihydroxyvitamin D3 at 10(-7) M slightly increased AP activity in ROS 17/2.8 cells, but strongly inhibited AP activity when the cells were pretreated with TGF beta. The data suggest that TGF beta stimulates expression of the osteoblastic phenotype in ROS 17/2.8 cells and that TGF beta may be an important regulator of local bone remodeling.

The calcitonin gene-related peptide (CGRP) is a peptide which normally circulates. It is encoded by the calcitonin gene, whose precise function is unknown. Since it has recently been shown that human CGRP (hCGRP) lowers plasma calcium levels in both the rat and the rabbit, we examined the in vitro effects of human synthetic CGRP on bone resorption (as measured by 45Ca release) stimulated by PTH, prostaglandin E2, and 1,25-dihydroxyvitamin D3. CGRP caused a dose-dependent inhibition of PTH-stimulated resorption, with 50% inhibition at approximately 5 X 10(-8) M CGRP. The inhibitory effects of CGRP on PTH-mediated bone resorption were not due to toxic effects, since bones preincubated with CGRP for 48 h were subsequently able to respond to PTH. The inhibitory activity of CGRP in the rat was approximately 3 orders of magnitude less potent than that of human calcitonin. In contrast to the effects of calcitonin, a marked inhibition of PTH-stimulated bone resorption was still observed after 96 h in the continued presence of CGRP. CGRP (10(-6)-10(-8) M) also inhibited resorption stimulated by prostaglandin and 1,25-dihydroxyvitamin D3 in a dose-dependent manner, but had no significant effect on basal bone resorption. In conclusion, these data show that hCGRP inhibits hormone-stimulated bone resorption in vitro. Although it is less potent than calcitonin in the rat, CGRP has been shown to have potency comparable to that of calcitonin in other species, and therefore, a role for CGRP as a therapeutic agent in states of increased bone resorption cannot be ruled out.

Evidence is presented that a tumor-derived transforming growth factor is responsible for stimulating bone resorption and causing hypercalcemia in an animal tumor model of the hypercalcemia of malignancy. Both conditioned medium harvested from cultured tumor cells and tumor extracts of the transplantable rat Leydig cell tumor associated with hypercalcemia contained a macromolecular bone resorbing factor with the chemical characteristics of a tumor-derived transforming growth factor.