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Biomedical subjects

T J Martin

Publications and source records attributed to T J Martin.

At least 361 records · Page 20Linked to original sources
PubMed1985

Biosynthesis of calcitonin by human lung cancer cells.

The ectopic secretion of calcitonin (CT) by a wide variety of nonthyroidal human tumors has been studied by CT RIA, but little information is available concerning the biosynthesis of CT in these tumors. In the present study, a human lung cancer cell line (BEN), secreting high mol wt forms of CT was investigated to characterize the CT gene products synthesized. When conditioned medium from BEN cells was chromatographed through a Bio-Gel P-30 column, larger species of immunoreactive CT were detected with mol wt of approximately 8,000 and 18,000. Little, if any, CT of 3,500 mol wt was detected. To examine CT gene products produced in BEN cells, poly A+ RNA was isolated from BEN cells and subjected to cell-free translation assays and DNA/RNA hybridization assays. In the wheat germ cell-free translation assay, a single BEN cell product which migrated on sodium dodecyl sulfate-polyacrylamide gels with an apparent mol wt of 17,000 could be specifically immunoprecipitated with CT antisera. A similar sized CT-related translation product is produced in wheat germ assays programmed by mRNA prepared from human medullary thyroid carcinomas. In DNA/RNA hybridization assays, a single BEN cell mRNA species of 1,000 base pairs, identical in size to human thyroidal CT mRNA, hybridized to a radiolabeled CT cDNA probe. Hybridization of the CT cDNA probe with BEN cell mRNA was confirmed by RNA dot blot hybridization and cytoplasmic RNA blotting procedures. These results indicate that larger mol wt forms of CT secreted by BEN cells are derived from a translation product and a mRNA which are of similar, if not identical, size as CT gene products produced in human thyroidal tissues. The inability of lung tumor cells to process the CT precursor to calcitonin of 3,500 mol wt may reflect a lack of specific prohormone processing enzymes in these tumor cells or may be due to structural polymorphism in the CT precursor expressed in the lung cells.

Calcitonin↗
PubMed1985

Regulation of plasminogen activator production by bone-resorbing hormones in normal and malignant osteoblasts.

The plasminogen activator (PA) activity of clonal rat osteogenic sarcoma cell (phenotypically osteoblast) and of osteoblast-rich rat calvarial cells is shown to be increased by treatment with the bone-resorbing hormones, PTH, 1,25-dihydroxyvitamin D3, prostaglandin E2, and epidermal growth factor. Dose-dependent increases were observed, after a lag period of 4 to 8 h. Stimulated and control PA activities were inhibited by actinomycin D and cycloheximide but not by cytosine arabinoside. Glucocorticoid hormones prevented the hormone stimulation, but other steroids did not. Calcitonin had no effect either on basal or on hormone-treated PA activity. Isobutyl-methylxanthine alone increased PA activity and enhanced responsiveness to PTH and to prostaglandin E2. These data point to a common pathway in the actions upon osteoblasts of several hormones with diverse initial cellular actions and raise the possibility that the PA/plasmin system may contribute to cellular mechanisms of bone turnover.

Animals↗
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↗
PubMed1985

Sympathetic denervation does not alter the density or properties of alpha-1 adrenergic receptors in rat vas deferens.

Alpha-1 adrenergic receptors in surgically denervated rat vas deferens were studied using radioligand binding assays of [125I] BE 2254 ([125I]BE) and contraction measurements. Scatchard analysis of saturation isotherms of specific [125I]BE binding showed no change in the affinity or density of binding sites 4, 7 or 14 days after denervation of rat vas deferens. The potency of norepinephrine in inhibiting specific [125I]BE binding was also unchanged 7 days after denervation of vas deferens. The potency of phenylephrine in causing contraction in vitro did not change 4, 7 or 14 days after denervation of vas deferens; however, there was a significant increase in the maximum contractile response to phenylephrine at all time points. After partial inactivation of alpha-1 adrenergic receptors in vitro with phenoxybenzamine, there was an equivalent reduction in the number of [125I]BE binding sites in the control and 14-day denervated vas deferens. The equilibrium dissociation constants calculated from contractile measurements for norepinephrine were the same in the control and denervated tissues. However, there was a 2.2-fold increase in contractile sensitivity to norepinephrine 14 days after denervation and a 3.6-fold increase in contractile sensitivity to methacholine 7 days after denervation. These results show that surgical denervation of the rat vas deferens results in an increase in contractile sensitivity to norepinephrine and methacholine and an increase in maximum contraction. However, there is no change in alpha-1 adrenergic receptor density or properties at any time after denervation. Thus, alterations in alpha-1 adrenergic receptors do not contribute to contractile supersensitivity of denervated rat vas deferens.

Animals↗
PubMed1985

Effect of retinoic acid on cellular content and human parathyroid hormone activation of cyclic adenosine 3':5'-monophosphate-dependent protein kinase isoenzymes in clonal rat osteogenic sarcoma cells.

Pretreatment with 10(-8) M retinoic acid for 4 days caused changes in three distinct components of the parathyroid hormone (PTH)-stimulated cyclic adenosine 3':5'-monophosphate response in a clonal rat osteogenic sarcoma cell line, UMR 106-06: the amplitude of the cyclic adenosine 3':5'-monophosphate response to PTH was moderately increased after pretreatment with retinoic acid; while the cellular content of the two isoenzymes of the cyclic adenosine 3':5'-monophosphate-dependent protein kinase was approximately equal in control cells, retinoic acid pretreatment was associated with a marked increase in the ratio of type II to type I holoenzyme activity. This change might be due to a decrease in the type I holoenzyme as suggested by immunofluorescence detection of decreased type I regulatory subunit in fixed cells together with the relative decrease in type I holoenzyme determined biochemically; there was a marked alteration of the pattern of PTH-stimulated protein kinase isoenzyme activation from predominantly type I isoenzyme in control cells to almost exclusively type II isoenzyme in retinoic acid-treated cells. Growth inhibition by submaximal amounts of PTH and retinoic acid when added together was greater than that for either agent alone.

Animals↗
PubMed1985

Effect of retinoids on the growth, ultrastructure, and cytoskeletal structures of malignant rat osteoblasts.

A clonal rat osteogenic sarcoma cell line, UMR 106-06, was used to study the effects of retinoic acid (RA) on its growth and morphology. Retinoic acid caused a reversible, time and dose-dependent inhibition of growth. RA-treated cells were larger, were more adherent to the substratum, and contained fewer mitotic figures. Half-maximal growth inhibition was observed at 10(-8) M. Among the naturally occurring retinoids, RA was clearly the most potent while the arotinoids, Ro 13-7410 and Ro 13-6298, were approximately 50 times more potent than was RA. A similar range of potencies was observed in the cloning efficiencies of the cells in soft agar. Fluorescence microscopy revealed that RA treatment increased the cellular content and organization of F-actin fibers. Ultrastructural changes include decreased chromatin dispersion and increased number of nucleoli per nucleus, decreased rough endoplasmic reticulum, decreased electron density and number of mitochondria, and increased formation of microfilaments and microtubules. These results identify this clonal cell line, which has been extensively characterized as the malignant counterpart of the normal osteoblast, as a target for vitamin A action.

Actins↗
PubMed1984

Characteristics of selective activation of cyclic AMP-dependent protein kinase isoenzymes by calcitonin and prostaglandin E2 in human breast cancer cells.

The characteristics of the cyclic AMP-dependent protein kinase isoenzyme response to calcitonin stimulation have been studied in two human breast cancer cell lines, T47D and MCF 7. Both cell lines possess calcitonin receptors, a calcitonin-responsive adenylate cyclase and the two isoenzymes of the cyclic AMP-dependent protein kinase, types I and II. The adenylate cyclase also responds to prostaglandin E2. Acute activation of the cyclic AMP-dependent protein kinase isoenzymes was determined by using a modification of a multiple small anion exchange column method [Livesey, Kemp, Re, Partridge & Martin (1982) J. Biol. Chem. 257, 14983-14987]. Control experiments showed that post-extraction activation did not influence the data. Calcitonin caused a rapid, selective activation of isoenzyme II in the T 47D cells with half-maximal response at 10(-10)M, and persisting for at least 24h. In MCF 7 cells calcitonin also caused a highly selective activation of isoenzyme II with half-maximal response at 5 X 10(-11) M, but the response was transient with a return to basal isoenzyme activity by 4-6 h. At this time further addition of calcitonin did not restimulate the cyclic AMP-dependent kinase activity. In neither cell line did calcitonin treatment result in activation of isoenzyme I. Prostaglandin E2, on the other hand, the only significant alternative agonist of adenylate cyclase in T 47D cells, activated isoenzymes I and II to an equal extent in these cells, illustrating that two hormones activating adenylate cyclase in the one cell type might exert different effects by their selective actions upon protein kinase isoenzymes.

Breast Neoplasms↗
PubMed1984

Effects of pertussis toxin on adenylate cyclase responses to prostaglandin E2 and calcitonin in human breast cancer cells.

Both calcitonin and prostaglandin E2 (PGE2) stimulate adenylate cyclase activity in the human breast cancer cell line (T 47D). The maximum cyclic AMP response to calcitonin exceeds that of PGE2. When maximal concentrations of the two hormones were added simultaneously to the cells, the amount of cyclic AMP generated was less than that seen with calcitonin alone. When cells were treated with the protein toxin of Bordetella pertussis (islet-activating protein; IAP) which inactivates the inhibitory regulatory component (Ni) of adenylate cyclase, there was no change in basal or calcitonin-responsive adenylate cyclase in intact cells. However, the PGE2 response was augmented at all dose levels, and this effect was dependent on the concentration of IAP. Moreover, in cells pretreated with IAP, simultaneous addition of PGE2 and calcitonin resulted in additivity rather than in inhibition of cyclic AMP production. The additivity of the response to calcitonin and PGE2 after IAP treatment implies activation of separate pools of adenylate cyclase catalytic subunit by the two hormones. These data are consistent with a model in which calcitonin acts on adenylate cyclase in T 47D cells through stimulatory regulatory components alone, whereas PGE2 acts on the same cells through both stimulatory and inhibitory components. The Ni input can limit the maximum effect of PGE2 and is capable of limiting calcitonin effects when the two agonists are used simultaneously.

Adenylate Cyclase Toxin↗
PubMed1984

Selective activation of cyclic AMP dependent protein kinase by calcitonin in a calcitonin secreting lung cancer cell line.

The characteristics of the cyclic AMP-dependent protein kinase isoenzyme response to calcitonin have been studied in a calcitonin-secreting human lung cancer cell line (BEN). These cells secrete a high molecular weight calcitonin-like molecule. They have previously been shown to have calcitonin receptors linked to adenylate cyclase. In this study we demonstrate that the cells contain two cyclic AMP-dependent protein kinase iso-enzymes. Using a recently reported method for studying selective activation of these isoenzymes by hormones in intact cells, it is demonstrated that calcitonin causes selective activation of isoenzyme I, with no significant activation of isoenzyme II. Post-extraction activation was excluded by appropriate controls. The response was rapid (2 min) and persisted for 18 hours. Half maximal response occurred at 3 X 10(-10) M salmon calcitonin.

Calcitonin↗
PubMed1984

Stimulation of plasminogen activator in osteoblast-like cells by bone-resorbing hormones.

Hormonal control of plasminogen activator (PA) was studied in clonal rat osteogenic sarcoma cells which are phenotypically osteoblast, and in osteoblast-rich rat bone cell cultures. The bone-resorbing hormones (parathyroid hormone, prostaglandin E2, epidermal growth factor and 1,25-dihydroxyvitamin D3) stimulated PA activity in both cell types. The relative efficacies of vitamin D metabolites and of prostanoids reflect their relative potencies as stimulators of bone resorption.

Animals↗
PubMed1984

Comparison of substrate specificity of myosin kinase and cyclic AMP-dependent protein kinase.

A series of synthetic peptides corresponding to the amino-terminal region of chicken gizzard myosin light chain (Mr 20 000) have been tested for their capacity to act as substrates for the cAMP-dependent protein kinase. The 18-residue peptide, K6AKTTK11 K12R13PQRATS19NVFS , was stoichiometrically phosphorylated on serine-19 by the cAMP-dependent protein kinase. This is the same residue phosphorylated by the myosin light chain kinase. The cAMP-dependent protein kinase phosphorylated this peptide with an apparent Km of 120 microM and Vmax of 0.29 mumol . min .-1 mg-1. The Km is 17-fold higher and the Vmax 10-fold lower than the corresponding values obtained with this peptide as substrate for the myosin light chain kinase. The kinetics of phosphorylation of shortened peptides corresponding to this 18-residue sequence together with those of another related sequence, RPQRAKAKTTKATSNVFS , indicated that the myosin light chain kinase had a relatively stronger dependence on lysine residues, whereas the cAMP-dependent protein kinase depends more on arginine residues. Although both the cAMP-dependent protein kinase and the myosin light chain kinase phosphorylate the same serine in the myosin light chain peptides, these enzymes are influenced by different nearby basic residues.

Amino Acids↗
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

Tumor-derived growth factor increases bone resorption in a tumor associated with humoral hypercalcemia of malignancy.

Evidence is presented that a tumor-derived transforming growth factor is responsible for stimulating bone resorption and causing hypercalcemia in an animal tumor model of the hypercalcemia of malignancy. Both conditioned medium harvested from cultured tumor cells and tumor extracts of the transplantable rat Leydig cell tumor associated with hypercalcemia contained a macromolecular bone resorbing factor with the chemical characteristics of a tumor-derived transforming growth factor.

Animals↗
PubMed1983

Prostanoids in platelet-vascular interactions.

Prostacyclin, the labile prostanoid product of arachidonic acid metabolism in vascular endothelium, is the most potent known inhibitor of platelet aggregation and is highly effective in relaxing vascular smooth muscle. Its production is probably critically important in the maintenance of an intact vasculature. Although there is some evidence that prostacyclin circulates as a hormone, it is probably most important as a locally active agent in preventing thrombosis and maintaining patent vessels. Several factors can influence prostacyclin production, the most important of which probably act locally at sites of vessel wall injury. The most promising therapeutic approaches toward using prostacyclin's beneficial effects in vascular disease may lie in the use of drugs aimed at increasing prostacyclin production. Among these are thromboxane synthesis inhibitors, which act by diverting prostaglandin endoperoxides through the prostacyclin synthetase pathway, and lipoxygenase inhibitors, which might act chiefly by preventing formation of metabolites capable of inhibiting prostacyclin synthetase.

Blood Platelets↗
PubMed1983

Prostacyclin produced by the pericardium and its influence on coronary vascular tone.

To determine the influence of pericardial fluid prostacyclin on coronary blood flow, the latter was measured in the circumflex artery of anesthetized dogs. Intraaortic infusions of angiotensin II (25 ng . kg-1 . min-1) reduced blood flow and released prostacyclin into pericardial fluid. Epicardial and pericardial superfusion with indomethacin (1 micrograms/ml) abolished prostacyclin release and significantly increased the coronary vasoconstrictor effect of angiotensin II; this treatment did not appear to affect vascular synthesis of prostacyclin. Pericardial prostacyclin may modulate the coronary vasoconstrictor effect of angiotensin, but its general role as a regulator of coronary vascular resistance is probably limited. A more important effect of pericardial prostacyclin may be exerted on the large coronary vessels in the epicardial surface. Release of prostacyclin into pericardial fluid represents a potential mechanism for opposing coronary vasospasm, especially if platelet activation is found to be a contributory factor in vasotonic angina pectoris.

Angiotensin II↗
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

Cyclo-oxygenase products of arachidonic acid metabolism in rat osteoblasts in culture.

The metabolism of arachidonic acid to its cyclo-oxygenase products was studied in monolayer cultures of osteoblast-rich rat calvarial cells and of clonal cell lines from a rat osteogenic sarcoma, enriched in the osteoblast phenotype. Prostanoids were measured by radioimmunoassay after extraction of media and fractionation by high pressure liquid chromatography. In both normal and malignant osteoblasts the major cyclooxygenase product was 6-oxo-prostaglandin F1 alpha, the hydration product of prostacyclin, with lesser amounts of prostaglandin E2 and prostaglandin F2 alpha. No significant thromboxane B2 was detected. Prostaglandins are thought to have a local role in the regulation of bone resorption. These results point to the possible importance of prostacyclin either in bone resorption or in some other local function, e.g., regulation of bone blood flow.

6-Ketoprostaglandin F1 alpha↗