Acute phase reactants ceruloplasmin and haptoglobin and their relationship to plasma prostaglandins in rabbits bearing the VS2 carcinoma.

Results of previous studies have shown that the VX2 carcinoma in rabbits synthesizes large amounts of prostaglandin E2 (PGE2). PGE2 secreted by the tumor is rapidly metabolized and can be measured in plasma as the metabolite 13,14-dihydro-15-keto-PGE2 (PGE2-M). We have previously proposed that the hypercalcemia that occurs in rabbits bearing the VX2 carcinoma is due to excessive secretion of PGE2 by the tumor and its subsequent action on the skeleton as a bone resorption-stimulating factor. In the course of these studies, we noted that the plasma of rabbits bearing the VS2 carcinoma became blue about 1 wk after tumor implantation. The intensity of the color increased markedly thereafter. We therefore measured ceruloplasmin in plasma by both chemical and immunological assay methods. Plasma ceruloplasmin and PGE2-M rose in parallel (within 7-10 days) and preceded by 7-10 days the development of hypercalcemia. 2 wk after tumor implantation, plasma PGE2-M and ceruloplasmin had risen about 20- and 6-fold, respectively, while the rise in plasma calcium was just beginning. Indomethacin, an inhibitor of prostaglandin synthesis, given from the time of tumor implantation prevented completely the hypercalcemia and largely inhibited the rise in ceruloplasmin. When given after hyperprostaglandinemia had developed, indomethacin produced a fall in both PGE2-M and ceruloplasmin. A rise in plasma haptoglobin concentrations similar to that seen for ceruloplasmin was also observed. No changes in plasma albumin concentrations occurred. We conclude that the acute phase reactants ceruloplasmin and haptoglobin rise rapidly in the plasma of rabbits bearing the VX2 carcinoma, and that this increase is related to arachidonic acid metabolism in these animals. It is possible that arachidonic acid metabolites also play a role in the elevations of these two plasma proteins observed in certain patients with malignant tumors.

Evidence that the bone resorption-stimulating factor produced by mouse fibrosarcoma cells is prostaglandin E 2 . A new model for the hypercalcemia of cancer.

A transplantable mouse fibrosarcoma, HSDM(1), produces a potent bone resorption-stimulating factor. The factor can be extracted from the tumor tissue and harvested from the medium of clonal strains of HSDM(1) tumor cells growing in monolayer culture. It has several chemical and biological properties of a prostaglandin. Using radioimmunoassay techniques, we have shown that HSDM(1) cells synthesize and secrete large quantities of prostaglandin E(2) (PGE(2)). The specific bone resorption-stimulating activity of the HSDM(1) factor extracted from the tumor is high and approximately equal to that of PGE(2) as measured in a bone tissue culture system in vitro. Indomethacin, a potent inhibitor of PGE(2) synthesis in HSDM(1) cells, also inhibits production by the cells of the bone resorption-stimulating factor, and has no detectable nonspecific effects on the bone culture assay system. Mice bearing the HSDM(1) tumor have higher levels of both calcium and PGE(2) in serum than control mice. We conclude that PGE(2) is the bone resorption-stimulating factor produced by HSDM(1) tumor cells, and that secretion of PGE(2) by the tumor in vivo accounts for the relative hypercalcemia observed in tumor-bearing animals. The HSDM(1) tumor cell system constitutes a new model for studying the pathogenesis of hypercalcemia associated with certain malignant tumors.

Dietary menhaden oil lowers plasma prostaglandins and calcium in mice bearing the prostaglandin-producing HSDM1 fibrosarcoma.

The omega 3 class of polyunsaturated fatty acids, particularly eicosapentaenoic acid (EPA, 20:5), has been shown to alter the patterns of arachidonic acid (20:4) metabolism in both in vitro and in vivo systems. To examine further the role of arachidonic acid conversion to prostaglandins (PG) in hypercalcemic mice bearing the PG-producing HSDM1 fibrosarcoma, we have performed experiments in which control and tumor-bearing animals were fed diets either low (0.1-0.2% of total fatty acid) or high (17%) in EPA. In all five experiments performed, tumor-bearing mice eating control diets had markedly elevated (average sixfold above control) plasma concentrations of 13,14-dihydro-15-keto-PGE2 (PGE2-M), while in mice bearing HSDM1 tumors and eating the EPA-enriched menhaden oil diet, the elevation was reduced to only twice control values. The increase in plasma calcium concentration (approximately 2.5 mg/dl above control) in tumor-bearing animals was also reduced significantly (P less than 0.05) to only 1.3 mg/dl above control in mice eating the diet enriched in EPA. Plasma immunoreactive hydroxy fatty acids (i12-HETE) and sulfidopeptide leukotrienes (iSRS) were not elevated in tumor-bearing mice and were unaffected by diet. The contents of PGE2, PGF2 alpha, and 6-keto-PGF1 alpha were lower in tumor tissue from animals eating the diet high in EPA, whereas the tissue contents of i12-HETE and iSRS were not altered by diet. Fatty acid analysis of liver and tumor tissue revealed marked increases in certain omega 3 fatty acids (20:5, 22:5, and 22:6) from animals eating the enriched diet. Body weights, tumor weights, and tumor histology were not significantly altered by diet. To determine whether dietary calcium played a role in the elevation of plasma calcium in mice bearing the HSDM1 tumor and the reduction of plasma calcium in animals fed EPA, we compared results in mice fed diets containing 0.80% (normal) and 0.015% (deficient) calcium. The increases in plasma calcium and PGE2-M observed in tumor-bearing mice were the same on both normal and very low calcium intakes. We conclude, in mice of the Swiss albino strain bearing the HSDM1 fibrosarcoma, that consumption of a diet enriched in EPA reduces the production of cyclooxygenase products of arachidonic acid metabolism and thereby reduces the elevation of plasma calcium concentration. Dietary enrichment with EPA did not alter the production of serologically determined lipoxygenase products of arachidonic acid.

Actions of growth factors on plasma calcium. Epidermal growth factor and human transforming growth factor-alpha cause elevation of plasma calcium in mice.

Specific humoral substances produced and secreted by human tumors that cause hypercalcemia have not been identified. Certain growth factors (such as epidermal growth factor, platelet-derived growth factor, and transforming growth factors-alpha and -beta) have been shown to stimulate the resorption of bone in organ culture by both prostaglandin-dependent and prostaglandin-independent pathways. In this report we demonstrate that epidermal growth factor and recombinant human transforming growth factor-alpha induce a significant rise in plasma calcium concentration when administered repeatedly to intact mice for periods ranging from 24 h to 16 d. The elevation of plasma calcium is not dependent on dietary calcium and is not invariably accompanied by an increase in systemic levels of the prostaglandin E2 metabolite 13,14-dihydro-15-keto-prostaglandin E2. The in vivo calcium-mobilizing activity of epidermal growth factor and transforming growth factor-alpha indicate that these or related growth factors need be considered as potential mediators of tumor-induced hypercalcemia.

Cyclooxygenase products of arachidonic acid metabolism by mouse bone in organ culture.

The products of endogenous and exogenous arachidonic acid metabolism via the cyclooxygenase pathway in mouse bone in organ culture were identified and quantitated by the use of high performance liquid chromatography and radioimmunoassay. Production of prostaglandins E2, F2 alpha, and I2 from endogenous substrate was stimulated by incubation of bone with epidermal growth factor and the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate. Addition of arachidonic acid to the culture medium not only resulted in the accumulation of prostaglandins E2, F2 alpha, and I2 but also thromboxane. Bone metabolized prostaglandins E2 and F2 alpha to their respective 13,14-dihydro-15-keto-derivatives. Prostaglandin I2, measured as 6-keto-prostaglandin F1 alpha was synthesized by bone, and metabolic products of prostaglandin I2 or 6-keto-prostaglandin F1 alpha, either 6,15-diketo-prostaglandin F1 alpha or 13,14-dihydro-6,15-diketo-prostaglandin F1 alpha, were also detected in the culture media. Formation of cyclooxygenase products of endogenous and exogenous arachidonic acid metabolism (both basal and stimulated) and bone resorption were inhibited by indomethacin. Bone as a tissue responded biochemically not only to exogenous prostaglandins and agents that enhance endogenous prostaglandin production but also to exogenous arachidonic acid by biosynthesis of prostaglandins, prostacyclin and thromboxane. Furthermore, bone metabolized these cyclooxygenase products to their more stable metabolites.

Protein phosphatase inhibitors and bone resorption: inhibition by okadaic acid and biphasic actions of calyculin-A.

PTH receptors on osteoblasts and calcitonin receptors on osteoclasts are coupled to adenylate cyclase. Despite similar transduction mechanisms, these hormones have opposing physiological actions. We investigated the consequences of persistent protein phosphorylation on bone resorption in neonatal mouse calvariae using okadaic acid (OA) and calyculin-A, two inhibitors of protein phosphatase-1 and -2A. These two inhibitors caused different responses in bone at picomolar and low nanomolar concentrations. OA inhibited, in a dose-dependent manner, bone resorption stimulated by PTH, 1,25-Dihydroxyvitamin D3, phorbol ester, and prostaglandin E2 (PGE2). OA did not inhibit the generation of the second messengers cAMP or PGs and did not have nonspecific toxic effects, as measured by protein and RNA synthesis. Thus, OA appeared to mimic the global inhibitory action of calcitonin on bone resorption. Unlike OA, calyculin-A elicited a biphasic dose response. At concentrations of 3.3 nM and greater, calyculin-A inhibited, in a dose-dependent manner, stimulated bone resorption. However, calyculin-A alone, at 0.625 and 2.5 nM, stimulated bone resorption via a PG-independent pathway. In calvariae, OA and calyculin-A increased phosphorylation of a 58- to 60-kilodalton protein. A protein of similar molecular mass was hyperphosphorylated in OA-treated ROS 17/2.8 osteoblast-like cells. We conclude that in addition to hormonal regulation of protein kinase activity, protein dephosphorylation plays a functionally important role in the modulation of bone resorption.

Evidence for multiple bone resorption-stimulating factors produced by normal human keratinocytes in culture.

Conditioned medium from cultured normal human foreskin keratinocytes enhanced the release of calcium from neonatal mouse calvaria in organ culture. Unfractionated keratinocyte-conditioned medium (KCM) stimulated bone resorption in a dose-dependent manner, but it did not increase the concentration of prostaglandin E2 (PGE2) in the bone culture medium until a maximal dose of KCM for resorption was used. Furthermore, inhibitors of PGE2 synthesis, indomethacin, ibuprofen, and piroxicam, did not inhibit KCM-induced calcium release. High concentrations of KCM increased cAMP production by calvaria in the presence of isobutylmethylxanthine, but the increase was small compared with that produced by a dose of bovine PTH that caused a similar level of bone resorption. The bone resorption-stimulating activity of KCM was not lost after incubation at 56 C for 60 min, but it was lost after heating at 100 C for 10 min. Fractionation of KCM by gel filtration chromatography revealed two distinct peaks of bone resorption-stimulating activity. One peak, KCMI, caused a significant increase in bone resorption at 2 micrograms protein/ml. KCMI did not increase medium PGE2, and inhibition of PGE2 synthesis in bone had no effect on KCMI-induced bone resorption. KCMI failed to increase cAMP production by human osteosarcoma SaOS-2 cells. Another peak, KCMII, caused a dose-dependent increase in bone resorption, and a significant increase in medium calcium was noted at a 20-fold lower concentration (0.1 microgram protein/ml) than with KCMI. In contrast to KCMI, the increase in bone resorption stimulated by KCMII was accompanied by a parallel increase in the production of PGE2, and inhibition of PGE2 synthesis completely inhibited the bone resorption-stimulating activity of KCMII. KCMII also caused an increase in cAMP production by SaOS-2 cells. We conclude that KCM contains at least two distinct bone resorption-stimulating factors, one of which acts via a PG-mediated mechanism and the other by a PG-independent pathway.

Human parathyroid hormone (PTH)-related protein and human PTH: comparative biological activities on human bone cells and bone resorption.

Human PTH-related protein (hPTHrP) has been characterized as a product of tumor cells with sequence homology to the biologically active amino-terminal portion of human PTH (hPTH). We measured the relative activities of synthetic amino-terminal sequences of hPTH-(1-34) and hPTHrP-(1-34) to stimulate production of cAMP in intact human SaOS-2 osteosarcoma cells. Both peptides enhanced cAMP production at concentrations of 2.5-7.5 X 10(-10) M, had parallel dose-response curves, and were of essentially equal potency. Preincubation of SaOS-2 cells with hPTH-(1-34) or hPTHrP-(1-34) for 1 or 4 h induced homologous desensitization to a second challenge with the same peptide as well as heterologous desensitization to the other PTH peptide, but had little or no effect on the action of vasoactive intestinal peptide; the magnitudes of homologous and heterologous desensitization induced by the same doses of hPTHrP-(1-34) or hPTH-(1-34) were similar. Bone resorption-stimulating activity was measured using 40Ca2+ release from neonatal mouse calvariae in organ culture after 72 h of incubation. hPTHrP-(1-34) gave a dose-response between 0.2 and 5 ng/ml (5 X 10(-11) and 1.2 X 10(-9) M), was about 3 times more potent than Lilly bovine PTH standard (assuming a SA of 3000 U/mg; 100 U/ml), gave the same maximum response as hPTH-(1-34), and was 20-30% as potent as hPTH-(1-34). Neither hPTH-(1-34) nor hPTHrP-(1-34) enhanced prostaglandin production in mouse calvariae, and indomethacin did not inhibit the bone resorption-stimulating activities of either peptide. We conclude that hPTHrP-(1-34) and hPTH-(1-34) have similar high specific biological activities to stimulate production of cAMP in human osteoblast-like cells, but that hPTHrP-(1-34) is modestly less potent than hPTH-(1-34) to stimulate bone resorption in mouse calvariae.

Tumor necrosis factor-alpha (cachectin) stimulates bone resorption in mouse calvaria via a prostaglandin-mediated mechanism.

Recombinant human (h) and murine (m) tumor necrosis factor (TNF)-alpha stimulated bone resorption and the production of prostaglandin (PG) E2 in neonatal mouse calvaria in organ culture. In experiments of 72-h duration, the effect on bone resorption was of large magnitude (an average increase in medium calcium of 3.3 mg/dl above control values in 11 separate experiments) and occurred over a concentration range of 0.1-20 ng/ml mTNF and 0.5-50 ng/ml hTNF. Accompanying the TNF-enhanced release of bone calcium there was enhanced accumulation of PGE2 in the culture medium. The increases in medium calcium and PGE2 were both inhibited completely by nontoxic concentrations of 4 different PG cyclooxygenase inhibitors (indomethacin, piroxicam, ibuprofen, and acetylsalicylic acid) but not by the noncyclooxygenase inhibitor salicylic acid. The magnitude of the PGE2 response, but not the calcium release, was less for bones treated with TNF than for those treated with equipotent doses of epidermal growth factor or human transforming growth factors-alpha or -beta, suggesting that the local site of production of PGE2 in bone may be different for TNF than for the other factors. Repeated sc injections of hTNF to intact mice for a 48-h period produced a statistically significant elevation of the plasma calcium concentration. Because TNF is produced by cells of the monocyte/macrophage lineage in response to invasive stimuli such as the presence of tumor, our findings indicate that a host factor produced in response to malignant cells can cause enhanced bone resorption. Thus, the concept of the humoral hypercalcemias of malignancy must be expanded to include mediators not produced by the tumor cells themselves.

Two squamous cell carcinomas not associated with humoral hypercalcemia produce a potent bone resorption-stimulating factor which is interleukin-1 alpha.

Conditioned medium (CM) from two squamous cell carcinoma cell lines, SCC-9 and SCC-13, stimulated bone resorption in neonatal mouse calvariae in organ culture. Enhanced bone resorption induced by CM was associated with an increased production of prostaglandin-E2 (PGE2) by the calvariae. Complete inhibition of stimulated PGE2 synthesis by indomethacin only partially inhibited bone resorption-stimulating activity (BRSA) in the CM. Neither SCC-9 nor SCC-13 CM stimulated cAMP production in rat osteosarcoma cells (ROS 17/2.8). The BRSA in CM was completely inhibited by an antibody to interleukin-1 alpha (IL-1 alpha). Fractionation of SCC-9 CM by gel filtration and HPLC ion exchange chromatography revealed a single peak of BRSA and PGE2 synthesis-stimulating activity at 17-20K (termed SCMII). In mouse calvariae, SCMII increased medium Ca2+ and PGE2 in a dose-dependent manner at concentrations from 20 ng protein/ml to a maximum of 500 ng protein/ml. Preincubation of SCMII with antibody to IL-1 alpha completely inhibited SCMII-induced bone resorption. SCMII also enhanced thymocyte proliferation with activity that was equivalent to 353 U/ml IL-1. Antibodies to IL-1 beta and tumor necrosis factor had no effect on SCMII-induced bone resorption. Using specific enzyme-linked immunosorbent assays for IL-1 alpha and IL-1 beta, IL-1 alpha was measured in high concentrations in both crude and partially purified fractions of SCC-9 and SCC-13 CM. In contrast, IL-1 beta was either undetectable or present in amounts below those that stimulate bone resorption. In addition, SCMII did not enhance cAMP production in bone cells. We conclude that the BRSA produced by the two squamous cell carcinoma cell lines SCC-9 and SCC-13 is IL-1 alpha.

Distribution of calcitonin-containing cells in the normal neonatal human thyroid gland: a correlation of morphology with peptide content.

C-cells have been mapped in the thyroid glands of 6 human neonates by means of immunoperoxidase localization of calcitonin and tissue calcitonin content as measured by radioimmunoassay. The C-cells were concentrated in a zone in the upper two-thirds of the lateral lobes bilaterally, where they were identified individually and in small groups in both an intrafollicular and parafollicular distribution. In contrast to findings in the adult, C-cells were predominantly intrafollicular in the neonate. The relative numbers of C-cells counted per unit area of thyroid tissue correlated strongly with the calcitonin content of immediately adjacent tissue sections. In areas rich in C-cells, as many as 75 immunoperoxidase-stained cells per low-power field were counted, and the concentration of calcitonin was as high as 540 to 2100 mU/g fresh weight, values that were as great as 10 times those observed in the normal adult thyroid gland. The prominence of the C-cell population and increased tissue calcitonin content in the human neonatal thyroid gland may reflect an as yet undefined physiologic role for calcitonin in the newborn.

Hypercalcemia and tumor-prostaglandins: the VX2 carcinoma model in the rabbit.

The VX2 carcinoma produces profound hypercalcemia (17-22 mg/100 ml) in the rabbit about 3-4 wk after transplantation. A bone resorption-stimulation factor (assayed in vitro with mouse calvaria in culture) has been extracted with diethyl ether from the tumor tissue and from the medium of a clonal strain of VX2 cells grown in culture. Serologic methods reveal that the tumors contain 294 plus or minus 51 ng/g fresh weight (mean plus or minus SE, 25 tumors) of prostaglandin E2 (PGE2), a potent bone resorption-stimulating agent. VX2 cells in culture produce 0.5-3.0 mug PGE2 per mg cell protein per 24 hr. The production of bone resorption-stimulating activity and PGE2 by VX2 cells in culture were both inhibited by indomethacin (100 ng/ml). Tumors from normocalcemic, indomethacin-treated rabbits (10-40 mg/rabbit/24 hr) contained little or no bone resorption-stimulating activity nor PGE2. Tumor-bearing rabbits receiving indomethacin continuously did not develop hypercalcemia, however, following cessation of indomethacin administration, hypercalcemia developed rapidly and was again reversed by reinstitution of indomethacin feeding. In untreated, hypercalcemic, tumor-bearing rabbits, initiation of indomethacin treatment was followed by a rapid return of the plasma calcium to the normal range. Systemic venous plasma from hypercalcemic tumor-bearing plasma contained higher concentrations of PGE2 than plasma from normocalcemic control rabbits. Venous drainage of the tumor contained even higher plasma PGE2 concentrations than systemic venous plasma in hypercalcemic animals; plasma PGE2 concentrations locally and in systemic plasma were unmeasurable (less than 70 pg/ml) in normocalcemic, indomethacin-treated, tumor-bearing rabbits. We conclude that PGE2 is a bone resorption-stimulating factor produced by VX2 tumor cells, and that secretion of PGE2 by the tumor in vivo may well be responsible for the hypercalcemia observed in tumor-bearing rabbits.

Pituitary immunoreactive calcitonin-like material: lack of evidence for cross-reactivity with pro-opiomelanocortin.

In mammals, calcitonin (CT) is synthesized, stored, and secreted by intrathyroidal C cells. Several reports have suggested the presence of immunoreactive CT in the pituitary gland. We have studied the rat pituitary gland using a radioimmunoassay for CT and have also found immunoreactive CT-like material. Assay of extracts of whole rat pituitary glands was performed using a radioimmunoassay for human CT, which gave identical dilution curves with synthetic human CT (hCT), synthetic rat CT (rCT), and mouse and rat thyroid extracts, but not with a variety of other pituitary and hypothalamic peptides. Immunoreactive CT (iCT) content of extracts of whole pituitary glands ranged from 6 to 72 pg/mg wet weight of tissue (60-840 pg/whole pituitary gland), whereas iCT was not measureable (less than 5 pg/mg tissue) in similar extracts of hypothalamus and cerebral cortex. Gel filtration studies of pituitary extracts showed a peak of iCT, which eluted with 125I-rCT and diluted in parallel with rCT. To investigate whether the pituitary iCT was related to pro-opiomelanocortin, extracts of ACTH-producing AtT20/D16 cells from mice, which contain the ACTH precursor in large quantities, were examined and no iCT was found. Immunohistochemical studies of rat pituitary glands with peroxidase-antiperoxidase and immunofluorescent techniques showed positive staining for CT in cells in the pars anterior, but not in the pars intermedia of pars nervosa; this staining was not eliminated when the antiserum was absorbed with CT under conditions that completely obliterated staining of rat thyroid glands. Double staining demonstrated essentially two distinct populations of cells, one positive for CT and another positive for ACTH, with less than 1% of the cells positive for both ACTH and CT. Immunoreactive CT-like material was present in the pituitary glands of rats thyroparathyroidectomized 18 days before they were killed, but was diminished. Biosynthetic labeling in vitro of rat pituitary glands with 3H-leucine showed incorporation into prolactin; there was no incorporation into CT. No in vitro secretion of iCT by whole rat pituitary glands either basally or after high K+ stimulation was observed. We conclude that: (1) a substance that has certain immunologic and size characteristics of CT is present in minute amounts in the pituitary gland of rats; (2) this material is not a part of the ACTH precursor; and (3) positive immunohistochemical staining in pituitary glands may not be specific for authentic CT.