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D M Findlay

Publications and source records attributed to D M Findlay.

At least 91 records · Page 5Linked to original sources
PubMed1988

Isolation of the osteonectin gene: evidence that a variable region of the osteonectin molecule is encoded within one exon.

A complementary DNA clone for bovine osteonectin was used to isolate the osteonectin gene from two libraries of bovine genomic DNA fragments. Two overlapping clones were obtained whose relationship was determined by restriction mapping and sequence analysis. The two clones contain the entire osteonectin coding region spanning approximately 11 kilobases of genomic DNA. The coding region of the gene was determined, by electron microscopy and DNA sequencing, to reside in nine exons. In addition, there is at least one 5' exon interrupted by an intron in the 5'-nontranslated sequence of the gene. Excluding this 5' exon and the 3'-terminal exon, the exons are small and approximately uniform in size, averaging 130 +/- 17 base pairs. Three of the exons at the 5' end of the gene were sequenced and appear to encode discrete protein domains. For example, the putative exon 2 contains the coding region for the leader peptide of the molecule. The amino-terminal protein sequence was determined for osteonectin extracted from human, rabbit, and chicken bone and compared with those for bovine, mouse, and pig osteonectin. These data suggest that osteonectin is highly conserved between species, interspecies changes being seen primarily at the amino terminus of the protein and specifically in the region encoded by putative exon 3 in the bovine gene.

Amino Acid Sequence↗
PubMed1986

Kinetics of calcitonin receptor internalization in lung cancer (BEN) and osteogenic sarcoma (UMR 106-06) cells.

The fate of 125I-labeled calcitonin and calcitonin receptors in BEN and UMR 106-06 cells was studied after binding of the ligand. At 37 degrees C but not at 4 degrees C, 125I-labeled salmon calcitonin bound to both cell types and was internalized as evidenced by increasing resistance to removal by acid pH. This process was dependent on de novo protein synthesis. Cells were pretreated with salmon calcitonin and washed with acidified buffer to release cell-surface bound hormone and to allow assessment of cell-surface receptor concentration. It was found that initially there was a temperature- and time-dependent calcitonin-induced loss of binding capacity after exposure to salmon calcitonin, suggestive of endocytosis of the calcitonin-receptor complex. In the continued presence of calcitonin, receptors were lost only at the same rate as the normal turnover of calcitonin receptors as assessed in the presence of cycloheximide. These data are consistent with the presence of two functional populations of calcitonin receptors, only one of which is induced to internalize by ligand binding.

Animals↗
PubMed1986

Calcitonin gene-related peptide (CGRP) acts independently of calcitonin on cyclic AMP formation in clonal osteogenic sarcoma cells (UMR 106-01).

In several human cancer cell lines and in a subclone of rat osteogenic sarcoma cells (UMR 106-06) possessing calcitonin receptors and a calcitonin-responsive adenylate cyclase, calcitonin gene-related peptide (CGRP) behaved as a weak calcitonin agonist. In another subclone of the same osteogenic sarcoma (UMR 106-01) with no measurable calcitonin receptors or response, both rat and human CGRP were found to increase cyclic AMP formation in a dose-dependent manner. The data indicate that CGRP is capable of a weak calcitonin-like action in cells with calcitonin receptors, but also that in some cells CGRP activates adenylate cyclase itself, independently of calcitonin receptors.

Animals↗
PubMed1986

Studies on the cytotoxicity of penicillamine in a rat osteogenic sarcoma.

The effect of penicillamine on the growth rate of an osteogenic sarcoma of rats was investigated and compared with cyclophosphamide. Rats were inoculated with a readily transplantable osteogenic sarcoma subcutaneously into the left thigh and treated with penicillamine and cyclophosphamide alone or in combination. Cyclophosphamide inhibited tumour growth. Penicillamine did not delay the appearance or the growth rate of the tumour. Tumour sizes tended to be larger in the penicillamine-treated rats, but there was no evidence that penicillamine interfered with the antitumour effect of cyclophosphamide given in large doses (100 mg/kg).

Animals↗
PubMed1985

Characterization of an osteoblast-like clonal cell line which responds to both parathyroid hormone and calcitonin.

The clonal cell line UMR 106, which was originally derived from a rat transplantable osteogenic sarcoma with an osteoblastic phenotype, was subcloned after the emergence of a calcitonin-responsive adenylate cyclase was noted in late passages. Detailed studies on the stimulation of adenylate cyclase and activation profile of the cyclic AMP-dependent protein kinase isoenzymes in response to parathyroid hormone (PTH) and salmon calcitonin (SCT) were conducted on two subclones (UMR 106-01 and UMR 106-06). Both subclones responded in an identical manner to PTH, which stimulated adenylate cyclase and activated both isoenzyme I and isoenzyme II of cyclic AMP-dependent protein kinase. In contrast, only UMR 106-06 cells responded to calcitonin. At 3 X 10(-8)M SCT, there was a sevenfold stimulation of adenylate cyclase, 84% activation of isoenzyme I, and 44% activation of isoenzyme II. The activation profiles of the isoenzymes to PTH and SCT in UMR 106-06 were similar. Furthermore, their response to SCT correlates with the presence of specific, saturable binding of 125I-labeled SCT. Binding parameters indicate apparent Kd = 0.8 nM and 6,000 receptors/cell. These data point to a significant phenotypic change having taken place in this clonal cell line with prolonged maintenance in culture, with the emergence of a calcitonin receptor linked to adenylate cyclase and protein kinase activation.

Acid Phosphatase↗
PubMed1985

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.

Adenylyl Cyclases↗
PubMed1984

Relationship between internalization and calcitonin-induced receptor loss in T 47D cells.

Exposure of T 47D human breast cancer cells to salmon calcitonin (sCT) resulted in a reduction of binding capacity for [125I]iodo-sCT in washed cells. The reduction was both time and concentration dependent. Recovery of binding capacity in CT-pretreated T 47D cells occurred in the absence of CT, but was prevented by inhibitors of protein synthesis. Studies were carried out to determine the mechanism of CT-induced reduction of binding capacity. When T 47D cells were treated with sCT at 37 C or 4 C and washed with buffer at neutral pH, subsequently measured binding capacity was lost as a function of time of pretreatment. Cells pretreated under the same conditions were washed with isotonic buffer at pH 2.5 to release cell-surface bound sCT and to allow assessment of cell surface receptor concentration. It was found that at 37 C sCT induced a time-dependent loss of cell surface receptors, so that initially the lost binding capacity was largely reclaimable by acid treatment, whereas after longer exposure to sCT, acid treatment was much less effective in regenerating binding capacity. The CT-induced reduction in binding capacity was not observed when cells were pretreated with sCT at 4 C or in the presence of inhibitors of cellular metabolic energy. These results are consistent with the view that initially CT-induced loss of CT receptors in T 47D cells is primarily due to occupancy of cell-surface receptors and later to a reduction in the concentration of cell-surface receptors mediated by an energy requiring internalization process involving the CT-receptor complex; reappearance of receptors requires new protein synthesis.

Breast Neoplasms↗
PubMed1983

The calcitonin receptor on T 47D breast cancer cells. Evidence for glycosylation.

The glycosyl nature of the receptor for the peptide hormone calcitonin has been investigated in a human breast cancer cell line, T 47D. Studies have been carried out to assess the ability of various lectins and of the antibiotic tunicamycin to inhibit specific binding of calcitonin to the cells, to reduce cross-linking of photoactive calcitonin to a macromolecular receptor component and to influence calcitonin stimulation of cyclic AMP. Pre-incubation of cells with low concentrations of tunicamycin for 72 h resulted in a reduction of total specific binding by approx. 80% and a 40% reduction in calcitonin-stimulated adenylate cyclase; formation of the cross-linked receptor component was also inhibited. Wheat-germ lectin showed the most marked inhibition of total specific binding and cyclic AMP production. However, cross-linking of photoactive calcitonin to receptor component was totally inhibited by this lectin. Soya-bean lectin brought about very little reduction in total specific binding but had more profound effects on calcitonin-stimulated cyclic AMP production and cross-linking of photoactive calcitonin. Concanavalin A and lentil lectin showed some inhibition of all parameters. The data indicate that the calcitonin receptor in T 47D cells is associated with glycosyl moieties, the major contributors of which are N-acetyl-D-glucosamine residues, but N-acetyl-D-galactosamine and mannose residues are also associated.

Adenylyl Cyclases↗
PubMed1983

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.

Adenylyl Cyclases↗
PubMed1983

Mechanisms of calcitonin induction of prolonged activation of adenylate cyclase in human cancer cells.

Four human cancer cell lines (MCF 7, T 47D, ZR 75 and BEN), each of which possess calcitonin-responsive adenylate cyclase, have been investigated. Pre-treatment of cells with calcitonin resulted in two effects on their adenylate cyclase. First, the ability of adenylate cyclase to respond to further stimulation by calcitonin was reduced. Secondly, membranes from calcitonin-preincubated cells had increased activity of adenylate cyclase compared with control membranes, the activated state not only surviving washing and cell-disrupting steps but persisting for some hours after the removal of hormone from the incubation medium. This phenomenon has therefore been termed "persistent activation" and may be peculiar to the action of calcitonin. It is dose dependent and is seen with calcitonins of various origins (sCT, hCT, pCT and [Asu1-7] eCT). When pre-bound hormone was removed from the cell surface by acid washing, adenylate cyclase activities in preincubated membranes fell towards control activities and responsiveness of the enzyme to a second challenge with calcitonin returned. Inhibitors of protein synthesis prevented full recovery from desensitization but had no effect on the return towards control of residual enzyme activity in preincubated membranes. These results are consistent with continued receptor occupancy leading to a persistent activation of adenylate cyclase until internalization of the receptor has occurred.

Adenocarcinoma, Scirrhous↗
PubMed1982

Processing of calcitonin and epidermal growth factor after binding to receptors in human breast cancer cells (T 47D).

125I-labelled calcitonin and 125I-labelled epidermal growth factor (EGF) bound to T 47D breast cancer cells at 37 degrees C in a manner that became increasingly resistant to removal by acid pH. Bound 125I-labelled EGF became resistant to acid removal more rapidly than did bound 125I-labelled calcitonin. The shift from acid accessibility to acid inaccessibility was energy-dependent since it was not seen at 4 degrees C and was inhibited in the presence of cell metabolic inhibitors. Radioactivity removed by acid represented intact hormone as assessed by trichloroacetic acid precipitation, whereas radioactivity released spontaneously by the cells was trichloroacetic acid-soluble. Inclusion of 10 mM-NH4Cl in the incubation medium resulted in an accumulation of cell-associated radioactivity without affecting the shift to acid inaccessibility. The accumulated radioactivity was relatively more trichloroacetic acid-precipitable in comparison with that associated with control cells. These data are consistent with internalization of receptor-bound EGF and a similar though slower mechanism of processing for receptor-bound calcitonin. The predominant route of hormone release from cells seems to occur via intracellular degradation rather than dissociation from cell-surface receptors.

Ammonium Chloride↗
PubMed1982

Covalent cross-linking of a photoactive derivative of calcitonin to human breast cancer cell receptors.

A photoaffinity derivative of salmon calcitonin has been produced by transglutaminase-mediated incorporation of N-(beta-aminoethyl)-4-azido-2-nitroaniline into the hormone. The derivative, purified by high pressure liquid chromatography, retained the abilities to bind to cultured T47D breast cancer cells and to stimulate adenylate cyclase in these cells. In both these respects it was equipotent with synthetic salmon calcitonin. Photolysis of the 125I-labeled photoactive salmon calcitonin derivative bound to T47D cells was accompanied by specific labeling of only one component (Mr approximately 85,000) as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Labeling was observed only upon photolysis and was inhibited by unlabeled synthetic salmon calcitonin but not by the inactive calcitonin analogue, 8-glycine human calcitonin. Reduction did not alter the apparent molecular weight of the calcitonin receptor complex. No macromolecular forms of calcitonin were produced by photolysis in the absence of T47D cells.

Affinity Labels↗
PubMed1982

Presence of 1,25-dihydroxyvitamin D3 receptors in established human cancer cell lines in culture.

Receptors for 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] have been described in several human breast cancer cell lines and more recently in human melanoma. The presence of 1,25-(OH)2D3 receptor (1,25-DR) in two cultured breast cancer cell lines was associated with receptors for calcitonin, another hormone thought to have effects on calcium handling. Therefore, it seemed important to examine a range of established human cancer cell lines for the presence of receptors for 1,25-(OH)2D3 and calcitonin. Thirty-three cancer cell lines were examined. 1,25-DR was found to be present in 23 lines, while calcitonin receptors were not detected in any of them. The 1,25-DR from several cell lines sedimented at about 3.5S in sucrose density gradients, had the appropriate specificity for vitamin D metabolites, had Kds of 0.8 to 2.2 x 10(-11) M, and had receptor concentrations of 12 to 99 fmol/mg protein. Ten malignant melanoma and nine colonic carcinoma lines constituted the largest groups of carcinoma cell lines, and seven and eight, respectively, of these were 1,25-DR positive. The high frequency of 1,25-DR positivity in the cultured colonic carcinoma cells is quite different from the low frequency of 1,25-DR in primary colonic carcinomas. It was also interesting that both of two cell lines derived from patients who had had both bone metastases and malignant hypercalcemia were 1,25-DR positive. These various cell lines may provide useful models for the examination of 1,25-(OH)2D3 action in vitro.

Binding, Competitive↗
PubMed1981

Calcitonin binding and degradation by two cultured human breast cancer cell lines (MCF 7 and T 47D).

Two human breast cancer cell lines (MCF 7 and T 47D) possess calcitonin-responsive adenylate cyclase systems. Suspended cells of both lines specifically bound 125I-labelled salmon calcitonin with mean dissociation constants of 1.7 nM (MCF 7) and 1.4 nM (T 47D); mean receptor numbers were 5300 and 24400 per cell respectively. Measurement of specific binding to MCF 7 cells was obscured by rapid and substantial degradation of the labelled hormone. Degradation of 125I-labelled salmon calcitonin: (i) was of high capacity; (ii) lacked the specificity displayed by 125I-labelled salmon calcitonin binding to the same cells; and (iii) was not related to binding since cell incubation supernatants retained full degrading activity. The degrading activity was inhibited by corticotropin (1-24)-tetracosapeptide, insulin and bacitracin. Inclusion of bacitracin in the incubation resulted in apparently fewer numbers of lower affinity receptors on MCF 7 cells, whereas these parameters were identical to T 47D cells incubated in the presence or absence of bacitracin. Eel [2-aminosuberic acid 1,7]-calcitonin was resistant to proteolysis in the presence of either cell line. Analysis of hormone-receptor interactions with calcitonin-responsive cells should take account of potent calcitonin-degrading activities in some cell lines.

Adenylyl Cyclases↗
PubMed1981

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.

Adenylyl Cyclases↗