Parathyroid hormone-related protein of malignancy: active synthetic fragments.

Peptides corresponding to the amino-terminal region of the parathyroid hormone-related protein (PTHrP) of humoral hypercalcemia of malignancy were synthesized. A 34-amino acid peptide, PTHrP(1-34), was two to four times more potent than bovine or human PTH(1-34) in bioassays promoting the formation of adenosine 3',5'-monophosphate (cAMP) and plasminogen activator activity in osteogenic sarcoma cells and adenylate cyclase activity in chick kidney membranes. Like parathyroid hormone itself, in which the activity resides in the first 34 residues, PTHrP peptides of less than 30 residues from the amino terminus showed substantially reduced activity. PTHrP(1-34) had only 6% of the potency of bovine PTH(1-34) in promoting bone resorption in vitro. PTHrP(1-34) strongly promoted the excretion of cAMP and phosphorus and reduced the excretion of calcium in the isolated, perfused rat kidney consistent with the symptoms seen in malignant hypercalcemia.

Properties of a calcitonin receptor and adenylate cyclase in BEN cells, a human cancer cell line.

A human lung cancer cell line (BEN cells) was found to have a calcitonin-responsive adenylate cyclase. Various calcitonins and synthetic analogs stimulated adenylate cyclase activity withe same relative potency as they show in lowering blood calcium in the rat. Preincubation of the cells with calcitonin, followed by washing, led to loss of subsequent adenylate cyclase response to hormone. This was a dose-dependent phenomenon. The binding of [125I]salmon calcitonin to freshly subcultured cells was studied. The ability of calcitonins and analogs to compete for binding paralleled their efficacies in stimulating adenylate cyclase. Binding was saturable, reversible, and consisted of a single class of noninteracting sites with a mean Kd of 10.75 X 10(-10) M, K of 0.93 X 10(9)/M, and mean receptor number of 2.71 X 10(4)/cell. It is not known whether the calcitonin receptor is inappropriate to the cell of origin of the tumor. The BEN cells provide a means of isolating and studying the properties of the calcitonin receptor and of evaluating the significance for the tumor of a hormone-responsive adenylate cyclase.

Morphological and biochemical characterization of four clonal osteogenic sarcoma cell lines of rat origin.

The ultrastructural and biochemical properties of four clonal osteogenic sarcoma lines, UMR 104, 105, 106, and 108, have been compared with uncloned osteogenic sarcoma cells and normal osteoblast-rich cells derived from newborn rat calvaria. High alkaline phosphatase activity and activation of adenylate cyclase by parathyroid hormone were used as biochemical markers of osteoblastic cells. Cloning enriched both of these parameters above those of the parent tumor and far higher than that seen in normal cells, suggesting enrichment of the osteoblast phenotype. Both of these properties have been retained through many passages in culture. Morphologically, the clonal lines have also retained the "blast"-like appearance of the uncloned osteogenic sarcoma cells and consist mainly of flat, relatively featureless cells. Many cells with mitotic figures were observed, indicating continuous cell division taking place in the malignant cells. Each clonal line gave rise to characteristic tumors when reinjected into rats. It is concluded that the clonal osteogenic sarcoma lines are highly differentiated tumor lines which have conserved the differentiated properties of the mature osteoblast, making them a suitable model for the study of the effects of hormones on the growth of a differentiated tumor, as well as for the study of hormonal regulation of the osteoblast.

Calcitonin and 1,25-dihydroxyvitamin D3 receptors in human breast cancer cell lines.

Five human breast cancer cell lines (MCF 7, T 47D, BT 20, MDA 157, and MDA 231) and a human breast epithelial cell line (HBL 100) have been found to contain specific high-affinity receptors for 1,25-dihydroxyvitamin D3, Kd values ranged from 0.6 to 2.0 X 10(-11) M and receptor concentration from 31 to 150 fmol/mg cytosol protein. Two of the breast cancer lines (MCF 7 and T 47D) contain specific high-affinity receptors for calcitonin and a calcitonin-responsive adenylate cyclase, which have been characterized with the aid of salmon, eel, and human calcitonins and in several substituted analogues of human calcitonin. The 1,25-dihydroxyvitamin D3 receptor may reflect a normal property of the breast cell. Breast cancer cell lines provide a useful source of 1,25-dihydroxyvitamin D3 receptors. Their coexistence with a calcitonin receptor and biological response in some breast cancers offers the opportunity to investigate new aspects of breast cancer endocrinology.

Interactive regulation of signalling pathways in bone cells: possible modulation of PGE2-stimulated adenylyl cyclase activity by protein kinase C.

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.

Effects of calcitonin gene-related peptide on cyclic AMP formation in chicken, rat, and mouse bone cells.

Mixed bone cell cultures obtained by sequential collagenase-trypsin digestion of newborn chick, rat, and mouse calvaria responded to calcitonin gene-related peptide (CGRP) with a dose-dependent increase in cyclic AMP formation. The amplitude of response to CGRP in each species was less than that to parathyroid hormone (PTH). The CGRP effect was not the result of an action as a weak calcitonin agonist, since in most instances a calcitonin effect was not observed. Only in early digests of mouse calvarial cells were consistent stimulatory effects of calcitonin on cyclic AMP noted, and these were always considerably less in amplitude than those to CGRP. It is concluded that chick, rat, and mouse bones contain cells in osteoblast-rich populations that respond specifically to CGRP with a rise in cyclic AMP.

Regulation of alkaline phosphatase expression in a neonatal rat clonal calvarial cell strain by retinoic acid.

A clonal cell strain, UMR 201, was established from a culture of rat calvarial cells by the process of limiting dilution on a collagen substratum. One-day-old neonatal rat calvaria stripped of periosteum were placed on collagen in alpha-MEM with 10% fetal bovine serum (FBS). Cells that grew out from the calvaria were passaged eight times to select cells with the ability to proliferate in culture before cloning was attempted. Cells from the clonal strain were homogeneous in appearance with a doubling time in culture of about 24 hours. The UMR 201 cells formed predominantly type 1 collagen. When treated with retinoic acid (RA), all cells showed an intense staining for alkaline phosphatase (ALP). This effect of RA on the expression of ALP activity was reversible and was time and dose dependent. The earliest change was observed within 6 hours. In contrast, single and isolated clumps of untreated cells stained positively for ALP only when they were confluent. Coincubation with dactinomycin up to 3 hours after the addition of RA completely prevented the expression of ALP, whereas dactinomycin became progressively less effective when added at later times. This is interpreted as indicating a regulatory role of RA on the gene expression of ALP. Other hormones acting on bone, such as 1,25(OH)2 vitamin D3 and dexamethasone, also modulate ALP activity. The cells showed morphologic evidence of senescence after passage 12. Our preliminary studies showed that the UMR 201 cells had the characteristics of relatively undifferentiated mesenchymal cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein kinase-C-induced down-regulation of calcitonin receptors and calcitonin-activated adenylate cyclase in T47D and BEN cells.

T47D human breast cancer cells and BEN human lung cancer cells were preincubated with the tumor-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA). In both cell lines there was a decrease in the binding of 125I-labeled salmon calcitonin ([125I]sCT) which was dependent on the dose and time of exposure to PMA. The effect on binding comprised at least two components: the apparent affinity for binding of [125I]sCT was decreased by PMA, and the rate of internalization of bound [125I]sCT was increased more than 2-fold in the presence of PMA. By using dinitrophenol to inhibit cellular metabolic energy and, therefore, receptor internalization, the PMA effects on receptor affinity were dissociated from those on endocytosis. The effects on binding were reflected in a decreased stimulation by sCT of adenylate cyclase activity. This was specific for the calcitonin receptor system, since PMA had no effect on prostaglandin-E2-stimulated adenylate cyclase in the T47D cell. Protein kinase-C (PKC) was implicated in the inhibitory effects of PMA on both binding and adenylate cyclase activation, since inhibition was reduced by simultaneous incubation with the PKC inhibitors H7 and H8. These results suggest that PKC is capable of mediating down-regulation of the CT receptor, and this is most likely by phosphorylation of the receptor itself or an associated protein.

Biological activities and receptor interactions of des-Leu16 salmon and des-Phe16 human calcitonin.

Analogs of salmon (des-Leu16 sCT) and human (des-Phe16 hCT) calcitonin were prepared in which the amino acid from position 16 was omitted. The biological activities were assessed in vivo in the rat hypocalcemic assay and in vitro by studying competition for binding of [125I]sCT and adenylate cyclase stimulation in human breast cancer cells (T 47D). Deletion from position 16 resulted in substantial loss of biological activity in each system, indicating the importance for a hydrophobic residue at position 16 in the intact calcitonin molecule.

Conformational requirements for activity of salmon calcitonin.

A series of deletion-substitution analogs of salmon calcitonin (SCT) have been prepared containing combinations of a glycine substitution in position 8 and deletions of serine-2 and tyrosine-22. Biological activity of the analogs with respect to native SCT were determined in the rat hypocalcemic assay and by studying stimulation of cAMP formation and competition for binding of 125I-labeled SCT in T47 D human breast cancer cells. It was found that each of the analogs retained full potency, irrespective of the means of assessment. It is suggested that conservation of the alpha-helical region of SCT, along with the overall tertiary structure, are more important for peptide potency than chain length per se.

Functional properties of hormonally responsive cultured normal and malignant rat osteoblastic 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.

Glucocorticoid treatment facilitates cyclic adenosine 3',5'-monophosphate-dependent protein kinase response in parathyroid hormone-responsive osteogenic sarcoma cells.

Late passage cultures of a clonal osteogenic sarcoma line (ROS 17/2.8) failed to respond to PTH with activation of cAMP-dependent protein kinase isoenzymes despite showing a sensitive and dose-dependent increase in cAMP after treatment with the hormone. When cells were treated with hydrocortisone or dexamethasone, protein kinase responsiveness to PTH was readily demonstrated; such treatment also resulted in enhanced cAMP production. Forskolin preincubation resulted in a cAMP response to PTH of similar magnitude to that seen with hydrocortisone but no activation of cAMP-dependent protein kinase occurred. Thus, the effect of glucocorticoid cannot be explained merely by the increased amplitude and sensitivity of the cAMP response which developed with glucocorticoid treatment in these cells. The data indicate that cellular activation of cAMP-dependent protein kinase does not automatically follow cAMP generation and that information transfer can be restored by pharmacological means.

Transforming growth factor-beta modulates receptor binding of calciotropic hormones and G protein-mediated adenylate cyclase responses in osteoblast-like cells.

Transforming growth factor-beta (TGF beta) produced by osteoblasts is present in high levels in bone and influences bone formation, replication of bone cells, and expression of osteoblast protein products. Interactions between bone active hormones and locally released and activated TGF beta were studied by examining the influence of TGF beta preincubation on PTH, calcitonin (CT), and vitamin D receptors in an osteoblastic cell line (UMR 106-06). Preincubation of UMR 106-06 cells with 1 ng/ml TGF beta for 3 days increased specific binding of [125I]PTH-related protein (PTHrP)(1-84) to 140% of that in control cells, but [125I]salmon CT binding decreased to 50% of controls. Binding isotherms indicated that the changes in binding were due to altered receptor numbers since affinities for 125I-labeled PTH and CT remained unchanged. The effect on receptor levels was time dependent, requiring 24 h preincubation with TGF beta for measurable changes, and dose dependent, with maximal effects seen with 1 ng/ml TGF beta. Binding of [3H]1,25(OH)2 vitamin D3 was increased to 130% of control in cytosolic extracts of UMR 106-06 cells pretreated for 3 days with 1 ng/ml TGF beta. Scatchard plots suggested an increase in receptor number without change in affinity. The adenylate cyclase response to PTH increased to 150% of control cells after 3 days of treatment with 1 ng/ml TGF beta; however, the adenylate cyclase response to CT was little changed. Forskolin- and cholera toxin-stimulated adenylate cyclase responses were increased by TGF beta treatment to 130-160% of control, indicating an increase in the stimulatory subunit of the G protein. Increased abundance of both Gs and Gi proteins were indicated by increased cholera toxin- or pertussis toxin-dependent [32P] NAD ribosylation of 47-kilodalton (kDa) and 42-kDa or 40-kDa proteins, respectively, in TGF beta-treated cells. Our data support a complex regulatory effect of TGF beta on UMR 106-06 cells with increases in PTH receptors, vitamin D receptors, and G proteins, whereas there is an apparent down-regulation of CT receptors. TGF beta might induce a more differentiated osteoblast phenotype of these cells, which already express differentiated features such as high alkaline phosphatase activity, PTH and vitamin D receptors, and collagenase production. Since low doses of PTH stimulate bone formation in vivo, TGF beta released or activated at sites of new bone formation might locally modulate PTH activity be allowing increased PTH receptor and postreceptor effectiveness.

Local secretion of parathyroid hormone-related protein by an osteoblastic osteosarcoma (UMR 106-01) cell line results in growth inhibition.

Parathyroid hormone-related protein (PTHrP) has been implicated as being important in the growth of tumor cells responsive to the peptide. We utilized a rat osteoblastic osteosarcoma cell line, UMR 106-01, which has PTHrP receptors and a PTHrP-responsive adenylate cyclase/cAMP messenger system, to produce a modified cell line that overexpresses PTHrP. The human PTHrP cDNA sequence was transfected by electroporation into UMR 106-01 cells and the stable cell lines UMR-36 and UMR-34 were established. The modified cell line, UMR-36, had increased levels of PTHrP mRNA compared with control cell lines and secreted PTHrP into the culture medium at levels of 0.01-0.1 pmol/10(7) cells in 12 h. The secreted peptide was biologically active as indicated by its ability to activate adenylate cyclase. The number of UMR-36 cells following 9 days in culture was reduced by up to 80% compared with control lines, which was associated with decreased (3)H-thymidine incorporation into genomic DNA. Addition of 1000-fold excess of the PTHrP antagonist, PTHrP(7-34), to UMR-36 cells resulted in the escape of growth inhibition and increased rate of growth. In vivo, tumors derived from UMR-36 cells were smaller in size compared with tumors derived from control cells. In conclusion, increased autocrine secretion of, and responsiveness to, PTHrP results in inhibited growth kinetics of an osteoblast-like bone tumor cell line in vitro and in vivo.

Induction of calcitonin receptor expression by glucocorticoids in T47D human breast cancer cells.

When T47D cells were maintained long term in medium containing 0.1 mumol cortisol/l, calcitonin receptor (CTR) expression was stimulated compared with the very low levels of binding in untreated cells grown from frozen stocks. The time-course of the appearance of CTR following treatment with cortisol was slow, requiring up to 3 weeks of continuous exposure of the cells to the steroid. Binding capacity of control cells also increased slowly with time in culture, but after 3 months was only 20-30% of that in cells continuously treated with cortisol. Removal of cortisol resulted in rapid loss of CTR so that binding was reduced to approximately 50% of treated cell levels within 1 week of removal. Scatchard analysis of the binding data showed that the increased binding capacity induced by cortisol was due solely to a change in average receptor number per cell, with no change in receptor affinity. That this induction of CTR was due to a glucocorticoid effect was shown by the more rapid (less than 96 h) and more potent (less than 1 nmol/l) action of dexamethasone than of cortisol. In addition, induction was inhibited by the glucocorticoid inhibitor RU486. The induced receptors were shown to be functional, since salmon calcitonin-stimulated adenylate cyclase was induced in parallel with CTR. These results indicate that glucocorticoids are potential regulators of the CTR.

Independent down-regulation of insulin and calcitonin receptors on a human tumour cell line.

A human lung cancer cell line (BEN cells) with a calcitonin receptor and calcitonin-responsive adenylate cyclase also possesses an insulin receptor. This has been characterized and found to have properties similar to those of other mammalian cell insulin receptors. A receptor number of 58 000 per cell was calculated from curvilinear Scatchard plots, and dissociation of bound labelled insulin by dilution was facilitated by the addition of unlabelled insulin, consistent with negatively co-operative interactions among binding sites. Preincubation of cells with either calcitonin or insulin led to loss of hormone binding in washed cells. In the case of calcitonin this was associated with loss of adenylate cyclase response. For each hormone the state of down-regulation was characterized by a decrease in receptor number, and for calcitonin there was also a low in sensitivity of adenylate cyclase. Down-regulation to calcitonin was more rapid than that to insulin and in each case recovery had occurred by 16 h after removal of the hormone. Induction of down-regulation was specific, in that preincubation with one hormone did not influence the subsequent binding or response of the other. Such data are consistent with independent modulation of peptide receptors in the same cell.

Membranes from a transplantable osteogenic sarcoma responsive to parathyroid hormone and prostaglandins: regulation of adenylate cyclase and of hormone metabolism.

Adenylate cyclase activity in particulate fractions from a transplantable rat osteogenic sarcoma was stimulated in a dose-dependent manner by prostaglandins E1 and E2 (PGE1 and PGE2) and parathyroid hormone (PTH). Prostaglandin F2alpha was active at a high concentration (3 x 10(-4) mol/l). Pretreatment of membranes with collagenase plus hyaluronidase reduced the magnitude of the PTH effect but did not affect the size of the PGE1 effect. Guanosine 5'-triphosphate and its synthetic analogue 5'-guanylylimidodiphosphate (Gpp(NH)p) activated adenylate cyclase in particulate preparations from the osteogenic sarcoma. The latter agent produced much larger effects, although the concentrations required for half-maximal enzyme activation were the same for both agonists (approximately 2 x 10(-6) mol/l). The effects of PTH and Gpp(NH)p were supra-additive at some concentrations of hormone. The effects of PGE1 and Gpp(NH)p were supra-additive at all hormone concentrations tested. Pre-incubation of membrane particles for 6 min with PTH produced an enzyme activation which was not reversed by dilution through washing; pre-incubation with PGE1 did not produce this effect. The response of membrane adenylate cyclase to Gpp(NH)p (10(-4) mol/l) was 75% greater in preparations pre-incubated with PTH than in membranes pre-incubated in buffer alone or in buffer containing PGE1. The basal rate of cyclic AMP production in the adenylate cyclase assay system decreased over a 35 min incubation period. This decrease was prevented by addition of PTH or PGE1. Addition of NaF or Gpp(NH)p produced a steady increase in the rate of production of cyclic AMP with time. Membrane preparations did not reduce the biological activity of PTH and did not degrade 125I-labelled PTH. The results demonstrate that the PTH- and PGE-responsive adenylate cyclases of the osteogenic sarcoma have distinctly different properties and that particulate preparations of the tumour do not metabolize PTH.

Insulin release from a cloned precursor beta cell line.

This paper describes the establishment in long-term tissue culture of a functional, clonal beta (B) cell line UMR 407/3 derived from neonatal rat pancreas. Immunofluorescence demonstrated specific and uniform staining for insulin. Transmission electron microscopy showed the presence of microvilli and cytoplasmic granules. The doubling time in culture was approximately 60 h in 2% (v/v) fetal calf serum with inhibition of growth at confluence. Biochemical studies demonstrated the incorporation of [3H]leucine into proinsulin and insulin, with insulin comprising 43.6% of the total radioactivity incorporated into immune complexes. When incubated at 37 degrees C for 30 min with Krebs-Ringer bicarbonate buffer (pH 7.4), the amount of insulin released on stimulation by 16.7 mmol glucose/l, 20 mmol DL-glyceraldehyde/l or 20 mmol alpha-ketoisocaproate/l was significantly higher compared with 5.6 mmol glucose/l. The mean insulin content was equivalent to 99 +/- 0.4 fmol (S.E.M.)/5 X 10(5) cells. Regulated insulin release was maintained through at least 15 passages in culture. The cells showed morphological evidence of senescence after passage 26 and this was associated with significant reduction in stimulated insulin release as well as insulin content. The ability of the cells of this clonal line to grow in soft agar suggests that it is a precursor cell line. The clonal B cell lines isolated so far may thus represent variably committed rather than fully differentiated B cells in culture. These clonal non-neoplastic cell lines will be useful models with which to study the regulation of maturation/differentiation of B cells and insulin gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of guanyl nucleotides on parathyroid hormone-responsive adenylate cyclase in chick kidney.

Both guanosine 5'-triphosphate (GTP) AND 5'-guanylylimidodiphosphate (Gpp(NH)p) activated adenylate cyclase (EC4.6.1.1) in chick kidney plasma membranes. Half-maximal stimulation occurred at 3-1 X 10(-6)M for both agents. The maximum increases in adenylate cyclase activity produced by GTP and Gpp(NH)p were respectively 130 and 720% over basal activity. At the end of a 12 min incubation period GTP concentration was 85% of that originally added in the presence of an ATP-regenerating system but less than 20% in its absence. GTP and guanosine 5'-diphosphate inhibited the activation of adenylate cyclase by Gpp(NH)p, suggesting that they all acted at a common site. Gpp(NH)p facilitated the stimulation of adenylate cyclase activity by bovine parathyroid hormone (BPTH) and by the synthetic amino terminal fragment BPTH (1-34), decreasing the concentrations required for half-maximal enzyme activation by a factor of approximately eight in both cases. This property was not shared by the native nucleotide GTP. Gpp(NH)p rendered active (at certain concentrations) a synthetic parathyroid hormone peptide fragment, BPTH (2-34), which was incapable of Activating adenylate cyclase in the absence of the nucleotide analogue. This suggested that the GTP analogue, in addition to a direct effect upon adenylate cyclase activity, was capable of influencing hormone interaction with the enzyme complex.

Production of parathyroid hormone-related protein by a rat parathyroid cell line.

Parathyroid hormone-related protein (PTHrP), the peptide associated with humoral hypercalcemia of malignancy, has been identified in fetal and adult parathyroid glands. We here report a sub-clone of a rat parathyroid cell line which secretes a single peptide species corresponding in size to PTHrP(1-84). Biological activity of the secretion product was blocked by a specific antiserum against PTHrP, but not by parathyroid hormone (PTH) antiserum. Secretion of PTHrP by these cells was regulated by extracellular calcium in the physiological range. A single messenger RNA species for PTHrP was identified, though PTH mRNA could not be shown in these cells. Hybrid CAT genes containing 700-1000 bp of 5'-flanking DNA from the human PTH or PTHrP genes were transfected into these cells, and the PTHrP gene was expressed at 10-fold higher levels than the PTH gene. These cells thus provide a valuable model system for investigation expression of PTHrP in a non-transformed cell line.