calcitonin and Osteosarcoma

Although calcitonin gene-related peptide (CGRP) may act as a local factor in bone, its mechanisms of action on osteoblasts are not well understood. We previously showed the presence of CGRP transcripts and peptide in human OHS-4 osteoblastic cells. The authors investigated the expression of CGRP receptor (CGRP-R) and its intracellular signalling properties in OHS-4 cells. Semi-quantitative RT-PCR analysis showed that OHS-4 cells express much more CGRP-R than calcitonin (CT)-R transcripts. After amplification of CGRP-R by RT-PCR and cloning of amplified fragments, the predicted CGRP-R sequence in OHS-4 cells was found to share 100% identity with human lung CGRP-R. Biochemical analysis showed that hCGRP did not increase intracellular cAMP levels in synchronized OHS-4 cells whatever was the cell cycle position. However, adenylate cyclase activity was functional, as human parathyroid hormone increased cAMP levels. In contrast, hCGRP induced a rapid, transient and dose-dependent increase in free cytosolic calcium levels. The data show that CGRP increases intracellular free Ca2+concentration but is not coupled to adenylate cyclase in CGRP receptor-positive OHS-4 osteosarcoma cells, suggesting that CGRP induces downstream events driven by phospholipase C in these cells.

Topics: Animals; Base Sequence; Calcitonin; Calcitonin Gene-Related Peptide; Calcium; Cyclic AMP; DNA Primers; Humans; Intracellular Fluid; Osteosarcoma; Receptors, Calcitonin; Receptors, Calcitonin Gene-Related Peptide; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Tumor Cells, Cultured

In order to test the hypothesis that salmon calcitonin has direct effects to modulate tyrosyl-protein phosphorylation in human osteosarcoma cells, SaOS-2 cells (with very high steady-state levels of skeletal alkaline phosphatase) were exposed to calcitonin, in duplicate serum-free cultures, at concentrations ranging from 10(-13) to 10(-9) mol/liter, for 0-60 minutes at 37 degrees C. Phospho-tyrosyl proteins were identified by autoradiography of Western blots after incubation with 125I-labeled antiphosphotyrosine antibodies (or with unlabeled antibodies and 125I-labeled protein A) and quantitated by laser densitometry. The results of these studies revealed (1) time-dependent effects of salmon calcitonin (sCt) (at 3 x 10(-12) mol/liter) to increase the level of tyrosylphosphorylation of at least six proteins, with apparent molecular weights of 20, 25, 27, 41, 48, and 135 kD (P < 0.05 for each); and (2) dose-dependent effects of sCt (during 15 minutes of exposure) to increase the level of tyrosyl-phosphorylation of at least 10 proteins with apparent molecular weights of 19, 20, 27, 35, 41, 102, 135, 195, 220, and 244 kD (P < 0.05 for each). A supplementary study of calcitonin effects on tyrosyl-protein phosphorylation in a subpopulation of SaOS-2 cells with very low steady-state levels of skeletal alkaline activity revealed similar responses--time and dose-dependent increases in the tyrosyl-phosphorylation of at least seven proteins with apparent molecular weights of 44, 48, 57, 62, 101, 244, and 280 kD (P < 0.05 for each).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Alkaline Phosphatase; Calcitonin; Cell Differentiation; Dose-Response Relationship, Drug; Humans; Osteoblasts; Osteosarcoma; Phosphorylation; Proteins; Time Factors; Tumor Cells, Cultured; Tyrosine

Structural requirements for binding to the bone calcitonin (CT) receptor and for CT bioactivity both in vitro and in vivo were assessed for a series of N-terminally truncated, N alpha-acetylated, fragments of salmon calcitonin (sCT). Sequential deletion of amino acid residues from the amino-terminus of [Ala7]sCT-(2-32) peptide amide first led to partial agonists and, upon deletion of residues 1 to 7, to a high affinity antagonist, N alpha-acetyl-sCT-(8-32)-NH2. The presence of two separate domains within the sCT sequence is proposed: (I) a binding domain comprising residues 9-32 and (II) an activation domain requiring residues 3 to 6. N alpha-acetyl-sCT-(8-32)-NH2, in several bioassays including plasminogen activator release from LLC-PK1 cells (pA2 = 7.31), cAMP production in UMR-106-06 cells (pA2 = 7.81) and in the fetal rat long bone resorption assay showed potent antagonistic properties.

Topics: Acetylation; Animals; Bone Resorption; Calcitonin; Cell Line; Cyclic AMP; Kidney; Molecular Structure; Osteosarcoma; Peptide Fragments; Plasminogen Activators; Radioligand Assay; Rats; Tumor Cells, Cultured

We have previously shown that both parathyroid hormone (PTH) and prostaglandin E2 (PGE2) stimulate the activity of creatine kinase BB (CKBB) in rat bone cells in culture. Therefore, morphologically distinct rat osteogenic sarcoma cells in culture were tested for stimulation of CKBB activity by hormones that regulate skeletal tissues. PTH stimulated CKBB in the osteoblast-like clone ROS 17/2; 1 alpha,25(OH)2D3 inhibited this activity while PGE2, CT and 24R,25(OH)2D3 had no significant effect. PGE2 stimulated CKBB activity in the fibroblast-like clone ROS 24/1, which was unresponsive to PTH, CT and Vitamin D metabolites. 24R,25(OH)2D3 as well as PGE2 (but not PTH, CT or 1 alpha 25(OH)2D3) stimulated CKBB in clone ROS 25/1, suggesting that this fibroblast-like clone has some chondroblast-like character. Both PTH and PGE2 stimulated the brain type isoenzyme of CK (CKBB), although the osteogenic sarcoma cell clones contain a significant proportion of the muscle type of CK (CKMM). Thus, increased CKBB activity can serve as an additional characteristic marker for the action of steroid and polypeptide hormones and for prostaglandins.

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Calcitonin; Calcitriol; Clone Cells; Creatine Kinase; Dihydroxycholecalciferols; Dinoprostone; Fibroblasts; Hormones; Isoenzymes; Osteoblasts; Osteosarcoma; Parathyroid Hormone; Prostaglandins E; Rats