Faculty Bibliography

Hormonal activation of cAMP-dependent protein kinase has been studied in cultured cells derived from a rat osteogenic sarcoma and in osteoblast-rich cells grown from newborn rat calvaria. Both cell strains contain adenylate cyclase activities which respond to parathyroid hormone (PTH) and a variety of prostanoids. PTH, prostaglandin E2 (PGE2), and prostacyclin (PGI2) were all capable of activating cAMP-dependent protein kinase(s) in suspensions of the two cell types. Activation was very rapid in all cases, being detectable at 10 sec and maximal between 30-60 sec. Using saturating concentrations of hormones, the protein kinase activity ratio remained elevated (between 0.6-0.9) for up to 35 min after the start of PGE2 stimulation, but declined toward basal activity ratio 5-10 min after stimulation with PTH or PGI2. Each of the hormones caused a dose-dependent increase in activation of cAMP-dependent protein kinase in both cell types. Half-maximal activation of the enzyme occurred at 2 X 10(-9) M bovine PTH for calvarial cells, at 10(-8) M bPTH for osteogenic sarcoma cells, and at 2-4 X 10(-8) M PGE2 and 1-3 X 10(-7) M PGI2 for both cell types. Maximal activation of protein kinase occurred before maximal cAMP accumulated, implying that only a fraction of cAMP is biologically significant. These two cell strains provide a useful means of analyzing postreceptor events in the hormonal regulation of bone cells

Certain metabolic properties of hormonally responsive osteogenic sarcoma cells derived from a transplantable rat tumor have been compared with those of related normal rat bone cells. All studies were carried out on cells grown in monolayer culture. Normal rat bone cells derived by repeated collagenase/trypsin digestion of newborn rat calvaria. Bone cells selected for comparison were thought to be osteoblast-like, as judged by enrichment of alkaline phosphatase and adenylate cyclase responsiveness to parathyroid hormone and prostaglandin E2. The adenylate cyclases of the two cell strains were similarly stimulated by a range of prostanoids and their metabolites and analogs. Morphology showed the two cell strains to be similar; the only obvious difference was a multilayering of cells in the sarcoma cultures, while the normal cultures showed abundant extracellular fibril formation which was not seen in the tumor cells. Investigation of the cAMP-dependent protein kinase isoenzymes showed the presence of two forms in both cell types, one eluting at a low salt concentration and the other at a high salt concentration. There was approximately twice the amount of the first isoenzyme compared to the second isoenzyme. The results indicate the usefulness of the two cell strains to elucidate further the molecular mechanisms of action of parathyroid hormone and prostaglandins

Protein degradation has been measured in confluent monolayers of eleven lines of contact-inhibited cells and ten transformed lines as the rate of release of trichloroacetic acid-soluble radioactivity after prelabeling cell protein with [3H]leucine. Insulin, at contrations from 10(-12) M to 10(-6) M, has been added at the beginning of the 4-hour degradation period to detect selective effects of this hormone as an inhibitor of the inducible proteolysis occurring in serum-free medium. In addition insulin binding measurements have been performed on selected cell lines in an attempt to relate receptor properties to insulin action. Substantial effects of insulin are found in most cells with a selective inhibition at low insulin concentrations noted in several of the transformed lines. The difference in insulin sensitivity is not entirely definitive because temperature-sensitive transformation mutants of NRK cells are not more sensitive to insulin at a temperature where they show the transformed phenotype. Although insulin receptors on different cell lines have similar binding properties, two of the hepatomas used, H35 and MH1C1, show inhibition of protein degradation at insulin concentrations where receptor occupancy is extremely low. Calvarial osteoblast-like cells have a high rate of protein degradation which can be reduced by growth factors but not by insulin. The lack of an insulin response is a consequence of poor insulin binding to the cells. Insulin binds to the osteogenic sarcoma cells in substantial amounts. However, its normal action to inhibit the induced proteolysis is restricted because with these cells no increase of proteolysis occurs in serum-free medium. Generally higher rates of protein degradation are observed in the contact-inhibited lines than the transformed cells. We suggest that this difference may provide a selective growth advantage to transformed cells

Prostaglandins are potent bone resorbing agents, the most potent of those tested being prostaglandin E2. The labile prostanofd, prostacylin, is also capable of resorbing bone, but its potency is not known. The responsiveness of rat bone and bone tumour cell adenylate cyclase has been used to assess the relative efficacies of a wide range of stable and labile prostaglandins, their metabolites and analogues upon bone. Prostaglandins may be important local regulators in bone, and it will be critical, therefore, to define the precise action of prostaglandin-like molecules on bone cells. There is ample evidence for a role for prostaglandins in the pathogenesis of malignant hypercalcaemia in a number of animal tumour models. Evidence is also accumulating in human cancers, suggesting that prostaglandin production may be important in the establishment of osseous metastases, and in generating excessive resorption adjacent to metastatic deposits in bone, particularly in breast and renal cortical carcinomas. The nature of the responsible proslaglandin-like molecules is undetermined, and this needs to be investigated by defining the pathways of arachidonic acid metabolism in tumours. When this has been achieved, drug therapy can be directed at the appropriate enzyme activities. The current application of cyclo-oxygenase inhibitors in these clinical syndromes is of little benefit. © 1980.