vasoactive-intestinal-peptide and Osteosarcoma

Vasoactive intestinal peptide (VIP) was modified at the C terminus with a spacer and four amino acids to serve as a chelating moiety. The modified peptide, TP 3654, was labeled with Tc-99m and evaluated in normal volunteers, as well as in patients with a history of cancer. Renal clearance (67%) was the primary route of excretion, with approximately 20% of the radioactivity clearing through the hepatobiliary system. No adverse reaction was noted in any of the subjects and all, except one small, of the known lesions as seen by CT, MRI, Tc-99m-MIBI, or mammography were correctly identified within a few minutes of an i.v. injection of approximately 10 mCi of Tc-99m-TP 3654 (specific activity 11.3 x 10(3) Ci/m mol). The scans were in concordance in nine patients. In the remaining two, one with a visible mass in the neck from high grade spindle cell sarcoma and the other with a palpable mass in a breast from ductal epithelial hyperplasia, were localized only with Tc-99m-TP 3654, but not with Tc-99m-MIBI. Both malignancies are known to express VIP receptors. The VIP analog promises to be a nontoxic and reliable agent for imaging cancers in humans that express VIP receptors.

Topics: Adenocarcinoma; Adult; Amino Acid Sequence; Autoradiography; Bone Neoplasms; Breast Neoplasms; Female; Humans; Male; Middle Aged; Molecular Sequence Data; Neoplasms; Organotechnetium Compounds; Osteosarcoma; Radionuclide Imaging; Radiopharmaceuticals; Receptors, Vasoactive Intestinal Peptide; Technetium Tc 99m Sestamibi; Vasoactive Intestinal Peptide

A number of studies have demonstrated the pivotal role of collagen molecules in modulating cell growth and differentiation. In order to analyze the direct effects of collagen type I on the osteoblastic phenotype, we have devised an in vitro culture system for studying the interactions between bovine collagen type I and Saos-2 cells, a human osteoblastic cell line. Saos-2 cells were cultured both on top of collagen-coated culture dishes as well as inside a three-dimensional collagen network. Plating on dishes treated with collagen induced maximal adhesion of Saos-2 cells after 24-hour incubation. Cells cultured on collagen gel matrix expressed about 2.5-fold more alkaline phosphatase when compared with untreated plastic dishes. On collagen-coated dishes the responsiveness of Saos-2 cells to parathyroid hormone was decreased, whereas no modifications were observed in the effect of vasoactive intestinal peptide on these cells. Using a microfluorimetric measurement of DNA, an increase of proliferation was observed in Saos-2 cells cultured on collagen gel. Saos-2 cells were also able to colonize collagen sponges and in this three-dimensional network they were able to synthesize osteocalcin, as assessed both by immunocytochemistry and radioimmunoassay. In this study we have demonstrated that bovine collagen type I exhibits favorable effects on attachment and functional and growth activities of a human osteoblastic cell line, encouraging its use as a bone graft material.

Topics: Alkaline Phosphatase; Bone Neoplasms; Cell Adhesion; Cell Differentiation; Cell Division; Cell Transformation, Neoplastic; Collagen; Cyclic AMP; Cytophotometry; DNA, Neoplasm; Extracellular Matrix; Fluorescent Antibody Technique; Gels; Humans; Immunohistochemistry; Microscopy, Electron; Osteoblasts; Osteocalcin; Osteosarcoma; Parathyroid Hormone; Phenotype; Radioimmunoassay; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

The effect of four different neuropeptides and norepinephrine (NE) on cyclic AMP formation in four different osteoblastic cell lines and in isolated neonatal mouse calvarial bone cells has been examined. In the rat osteosarcoma cell line UMR-106-01, vasoactive intestinal polypeptide (VIP, 0.001-1 microM), calcitonin gene-related peptide (CGRP, 0.3-30 nM), and NE (0.1-300 microM), but not neuropeptide Y (NPY, 0.001-1 microM) or substance P (SP, 0.1-10 microM), caused a dose-dependent stimulation of cyclic AMP formation. The stimulatory effects were synergistically potentiated by forskolin (0.1-3 microM). The effects of NE and VIP were time dependent, with an optimal effect seen at 5 minutes. The amount of cyclic AMP accumulated in cells stimulated with NE and VIP was in the same range. The amplitude of the cyclic AMP response induced by CGRP was smaller than that caused by VIP and NE. In the human osteosarcoma cell line Saos-2, NE (0.1 microM) and VIP (0.3 microM) stimulated cyclic AMP formation, and the effect was synergistically potentiated by forskolin. In the absence of forskolin, no effect of CGRP (30 nM) could be seen in the Saos-2 cells, but in the presence of forskolin (3 microM) a stimulatory effect was observed. SP and NPY did not change basal cyclic AMP levels in Saos-2 cells. In the osteoblastic osteosarcoma cell line of rat, ROS 17/2.8, NE (0.1 microM) caused a significant stimulatory action on cyclic AMP formation that was synergistically potentiated by forskolin (3 microM), VIP, CGRP, and SP did not affect the cellular content of cyclic AMP in ROS 17/2.8.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Calcitonin Gene-Related Peptide; Cell Line; Cells, Cultured; Colforsin; Cyclic AMP; Mice; Neuropeptide Y; Neuropeptides; Norepinephrine; Osteoblasts; Osteosarcoma; Substance P; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

We have reported previously that tumour-promoting phorbol esters modulate both basal and vasoactive intestinal polypeptide (VIP)-stimulated adenylyl cyclase activity in GH3 (an established pituitary cell line). Here, we probe the receptor and cell specificity of this response. Experiments were performed in the presence of isobutylmethylxanthine. Unlike the response in GH3 cells, the tumour-promoting phorbol ester (tetradecanoyl phorbol acetate (TPA] did not affect either basal adenylyl cyclase activity nor VIP-stimulated activity in the rat osteosarcoma subclones UMR 106-01 and UMR 106-06. In addition, the cyclase responses to parathyroid hormone (PTH), and, in the case of UMR 106-06, to calcitonin were unaffected by tumour-promoting phorbol ester. However, prostaglandin E2-stimulated cyclase activity in both of these subclones was attenuated in a dose-dependent manner.

Topics: Adenylyl Cyclases; Animals; Cattle; Dinoprostone; Osteosarcoma; Protein Kinase C; Rats; Salmon; Signal Transduction; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

We have investigated the actions of 17 beta-estradiol (E2) on the production of cAMP stimulated by synthetic human PTH [hPTH-(1-34)], synthetic hPTH-related protein [hPTHrP-(1-34)], and vasoactive intestinal peptide (VIP) in human (SaOS-2) and rat (ROS 17/2.8) osteoblast-like osteosarcoma cells. In SaOS-2 cells, hPTH-(1-34) (2.5 nM), hPTHrP-(1-34) (2.5 nM), and VIP (10-100 nM) stimulated the accumulation of cAMP markedly (greater than 20- to 30-fold in 1 h). Cells were preincubated in serum-free medium for 4-24 h, then in the absence or presence of E2 for 4 h before a 1-h stimulation with peptide hormone in the absence of E2. In SaOS-2 cells, pretreatment with E2 (10(-12)-10(-8) M) for 4 h inhibited by up to 50% the accumulation of cAMP stimulated by hPTH-(1-34) or hPTHrP-(1-34), but E2 had no inhibitory effect on VIP action. 17 alpha-Estradiol had no inhibitory action on hPTH- or hPTHrP-stimulated accumulation of cAMP at concentrations as high as 10(-8) M. Additional evidence against a nonspecific effect of E2 was the total lack of inhibition of cAMP accumulation stimulated by hPTH-(1-34) or hPTHrP-(1-34) in ROS 17/2.8 cells at concentrations of E2 up to 10(-6) M. We conclude that E2 can act directly and rapidly in human osteoblast-like cells to modulate selectively the ability of hPTH and hPTHrP to enhance the production of cAMP.

Topics: Animals; Dose-Response Relationship, Drug; Drug Interactions; Estradiol; Humans; Neoplasm Proteins; Osteoblasts; Osteosarcoma; Parathyroid Hormone; Parathyroid Hormone-Related Protein; Peptide Fragments; Proteins; Rats; Teriparatide; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

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.

Topics: Animals; Animals, Newborn; Bone and Bones; Bone Resorption; Calcium; Cyclic AMP; Dinoprostone; Humans; Mice; Neoplasm Proteins; Organ Culture Techniques; Osteoblasts; Osteosarcoma; Parathyroid Hormone; Parathyroid Hormone-Related Protein; Peptide Fragments; Proteins; Teriparatide; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Influx of extracellular Ca++ into bone cells has been postulated as an early action of PTH and other bone resorption-stimulating factors. To test this hypothesis directly, we measured the cytosolic free Ca2+ concentration ([Ca2+]i) in two hormone-responsive human (SaOS-2 and G-292) and two rat osteosarcoma cell lines (Ros 25/1 and Ros 17/2.8) and in primary cultures of bone cells from neonatal mouse calvaria using the fluorescent Ca2+ indicator Quin 2. Actions of bovine PTH-(1-34), vasoactive intestinal peptide, epidermal growth factor, prostaglandin E2, and ionomycin were studied. Medium cAMP (20 min; 37 C; 25 microM 3-isobutyl-1-methylxanthine) was quantitated by RIA. Basal [Ca2+]i was: SaOS-2, 126 +/- 8 nM; G-292, 61 +/- 6 nM; Ros 25/1, 109 +/- 15 nM; Ros 17/2.8, 363 +/- 42 nM; and primary cultures, 266 +/- 39 nM (mean +/- SE; n = 3-14). In each cell type, no acute (1 sec to 20 min) spike in [Ca2+]i was observed in response to PTH (24-120 nM), vasoactive intestinal peptide (100 nM), epidermal growth factor (17 nM), or prostaglandin E2 (2.8 microM). However, in SaOS-2 cells only, PTH reproducibly increased [Ca2+]i 10-15% above basal values beginning about 3 min after hormone addition, and this small increase returned to baseline at 15-20 min. Ionomycin (100 nM) elicited an immediate spike in [Ca2+]i to levels 2- to 4-fold above basal in all cells; the peak [Ca2+]i decayed rapidly (within 4-5 min) to baseline in G-292, Ros 25/1, and Ros 17/2.8 cells. The decay of peak [Ca2+]i in SaOS-2 was prolonged. To test for intact hormone responses in Quin 2-loaded cells, cAMP accumulation was measured. In SaOS-2 and Ros 17/2.8, both control and Quin 2-loaded cells showed similar increases in cAMP in response to PTH. Considering the limitations of the Quin 2 technique, we conclude that in the four hormone-responsive bone cell lines and primary cultures of bone cells tested, acute elevation of [Ca2+]i is not an inevitable consequence of receptor occupancy and/or adenylate cyclase activation by bone resorption-stimulating hormones.

Topics: Aminoquinolines; Animals; Bone Resorption; Calcium; Cell Line; Cells, Cultured; Cyclic AMP; Cytosol; Dinoprostone; Epidermal Growth Factor; Ethers; Humans; Ionomycin; Membrane Potentials; Mice; Osteosarcoma; Parathyroid Hormone; Prostaglandins E; Rats; Vasoactive Intestinal Peptide

We have previously reported that vasoactive intestinal peptide (VIP) stimulates bone resorption in organ culture via a cAMP-dependent mechanism. Here we describe functional receptors for VIP on a clonal line of human osteosarcoma cells, SaOs-2. SaOs-2 cells respond to VIP with an increase in cAMP. The effect was rapid (2 min) and dose dependent from 0.15-15 nM VIP, with half-maximal stimulation at 1.4 nM. SaOs-2 cells produce prostaglandin E2 (PGE2) and respond to exogenous PGE2 with increases in cAMP approximately one third as great as those induced by VIP. However, the VIP-stimulated increases in cAMP occurred without detectable increases in PGE2 production, and increases in cAMP were unaffected by the cyclooxygenase inhibitor indomethacin. SaOs-2 cells pretreated with VIP for 24 h were significantly less responsive to a second acute challenge with VIP, but retained their ability to respond to PGE2. Similarly, pretreatment with PGE2 induced homologous desensitization to PGE2, but had no effect on the VIP-stimulated increase in cAMP. These patterns of response paralleled those previously described in whole bone in organ culture. Binding studies with [125I]VIP demonstrated specific, saturable, high affinity receptors for VIP on SaOs-2 cells. Scatchard analysis of [125I]VIP binding at 37 C resulted in a curvilinear plot. Analysis based upon the assumption of two independent binding sites gave Kd values of 0.44 and 17 nM for high and low affinity binding sites, respectively. The numbers of high and low affinity sites per cell were determined to be 8,500 and 57,000, respectively. Binding of [125I]VIP was partially inhibited by two related peptides, secretin and PHI-27, but not by PTH, calcitonin or a variety of unrelated peptides. We conclude that the action of VIP on human SaOs-2 cells is similar to that observed in intact mouse calvaria, and that these cells provide a good model for the study of the initial steps of VIP action in bone.

Topics: Binding, Competitive; Cell Line; Cyclic AMP; Humans; Indomethacin; Kinetics; Osteosarcoma; Receptors, Cell Surface; Receptors, Vasoactive Intestinal Peptide; Vasoactive Intestinal Peptide