Search PubMed⌕ Search

Biomedical subjects

D Tramontano

Publications and source records attributed to D Tramontano.

At least 55 records · Page 3Linked to original sources
PubMed1986

Thyrotropin-independent mutant clones from FRTL5 rat thyroid cells: hormonal control mechanisms in differentiated cells.

Mutant cells varying in the pathways of their responses to hormonal stimulation are useful in defining the subcellular steps in the mechanisms of hormone action. FRTL5, a strain of normal and differentiated cells originally derived from adult rat thyroids, which depends on TSH for growth in vitro, was used to produce five TSH-independent mutants, after chemical mutagenesis and selection in medium lacking TSH. Their characterization and comparison with wild type cells demonstrated full retention of differentiated thyroid function markers such as thyroglobulin production and active iodide transport, and a slower growth rate. Characterization of cAMP metabolism in mutants revealed levels of basal cAMP and adenylate cyclase and phosphodiesterase activities similar to those of wild type cells kept in a nonproliferative state in medium lacking TSH. Adenylate cyclase responsiveness to very low doses of TSH (10(-12) M) was fully retained in all mutant clones, but the TSH-dependent cAMP elevation, although comparable to that reported in wild type cells, was not followed by significant growth stimulation in mutants. These findings demonstrate that the persistence of functional TSH receptors in these cells and that of growth regulation in them is independent of cAMP elevation.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1986

Properties and regulation of the thyrotropin receptor in the FRTL5 rat thyroid cell line.

Despite extensive use of FRTL5 cells in studies of responses to TSH and anti-TSH receptor antibodies, almost nothing is known of the properties of their TSH receptors, possibly because binding of TSH by these cells is negligible when studied in their usual culture medium. In the present studies, we have demonstrated that specific binding of TSH can readily be demonstrated in confluent monolayers of FRTL5 cells if their culture medium is replaced by Krebs-Ringer bicarbonate (KRB) buffer. In keeping with previous observations concerning the effects of cations on the binding of TSH in other thyroid systems, binding of TSH to FRTL5 was far greater when the medium used was a modified KRB in which an isosmotic substitution of sucrose for NaCl had been made. Kinetic studies of TSH binding in both types of medium suggested the presence of two binding sites, one with a higher affinity and lower maximum binding capacity than the other. The influence of NaCl was to decrease the capacity of both sites, that of the low affinity site to a greater extent than that of the high affinity site, whereas the affinities of the two sites remained unchanged. Correlative studies indicated that physiological responses to TSH were associated mainly with occupancy of the higher affinity sites. Experiments in which TSH binding was studied in cells grown to confluence in the presence of TSH from which TSH was then withdrawn and in cells maintained in the absence of TSH to which TSH was then added demonstrated the occurrence of up-and down-regulation, respectively, of receptor concentrations without a change in their affinities. The reduction in maximum binding capacity induced by TSH was proportionately greater in the case of the high affinity than the low affinity receptor. Down-regulation by TSH was concentration dependent and was demonstrable at a TSH concentration of 10(-11) M, considered to be physiological. Further, maximum down-regulation was induced by 10(-9) M TSH, the approximate concentration at which other responses to TSH in these cells reach their peak. Therefore, down-regulation of TSH receptors can be considered to be one of the physiological responses that TSH elicits.

Aminoisobutyric Acids↗
PubMed1986

Insulin-like growth factor-I stimulates the growth of rat thyroid cells in culture and synergizes the stimulation of DNA synthesis induced by TSH and Graves'-IgG.

The present studies were undertaken to examine the factors that influence the growth of cells of endocrine gland origin, particularly the possible interactions between "nonspecific" growth factors and the trophic hormone for a target endocrine cell. As a model system, we explored the individual and conjoint effects of insulin-like growth factor-I (IGF-I) and TSH on the growth of FRTL5 cells, a nontransformed line of cloned rat thyroid follicular epithelium. In these cells, IGF-I and TSH each produced a dose-dependent enhancement of DNA synthesis and cell proliferation. When added together, IGF-I and TSH were markedly synergistic in stimulating DNA synthesis, producing increases in 3H-thymidine incorporation that were far greater than the sum of the effects of each alone. A similar effect of IGF-I was evident in the case of the stimulation of DNA synthesis produced by immunoglobulin G (IgG) preparations from the blood of patients with Graves' disease. Such IgG bind to the TSH receptor and mimic the actions of TSH therein. It is suggested, therefore, that there exist in the FRTL5 cell line at least two mechanisms for the regulation of growth, one activated at the level of the IGF-I receptor and the other at the level of the TSH receptor. When the two pathways are activated concurrently, a synergistic enhancement of DNA synthesis takes place. The findings indicate that the FRTL5 cell line is an excellent model in which to study these complex interactions and that IGF-I may be a determinant of thyroid cell growth, both normally and in certain thyroid diseases.

Animals↗
PubMed1985

Differential expression of thyroglobulin gene in normal and transformed thyroid cells.

We have measured the synthesis of thyroglobulin in two differentiated cell lines, FRTL-5 and FRTL-424, and two transformed thyroid cell lines, FRA and 1-5G. The untransformed cells actively synthesized and exported thyroglobulin in the medium: however, the FRTL-5 cell line synthesized seven times less thyroglobulin than the FRTL-424 cell line, even though both cell types contained equal amounts of functional thyroglobulin mRNA. In contrast the transformed cells expressed extremely low levels of thyroglobulin mRNA, even though there was no detectable change in gene structure or copy number as determined by Southern blot analysis. On the basis of these data we conclude that (a) the different levels of thyroglobulin synthesis in the two untransformed cell lines are due to stable post-transcriptional alterations in the biosynthesis of thyroglobulin and (b) the transformation of thyroid cells results in a substantial reduction in thyroglobulin gene expression.

Animals↗
PubMed1985

Alteration of erythrocyte membrane lipid fluidity in human obesity.

The lipophilic probe 1,6-diphenyl-1,3,5-hexatriene was incorporated into erythrocyte ghosts of either normal or obese humans, and the polarization of fluorescence was measured between 0 and 40 C. The membrane lipid fluidity, evaluated by fluorescence polarization, was consistently higher in the ghosts from obese subjects. A strong correlation was found between increased 1,6-diphenyl-1,3,5-hexatriene fluorescence polarization and excess body weight. Measurements of cholesterol and phospholipids indicated increased cholesterol and decreased phospholipids in erythrocyte ghosts from obese subjects. These data suggest that alterations in lipid composition in erythrocytes of obese subjects are responsible for abnormal physical properties of plasma membranes, which, in turn, may cause altered enzymatic activities.

Adolescent↗
PubMed1985

Morphological and functional polarity of an epithelial thyroid cell line.

The thyroid epithelial cell line FRT in monolayer culture appeared to be strongly polarized by morphological criteria. Cells were connected by tight junctions, exposed microvilli toward the culture medium and formed domes at confluency. FRT cells were infected with vesicular stomatitis virus (VSV) and Sindbis virus and the budding polarity was examined 8 and 16 h after infection, respectively. VSV budding occurred preferentially from the basolateral domain of plasma membrane, while Sindbis virus budding was mostly apical. The distribution of VSV and Sindbis virus glycoproteins, as determined by the immuno-gold technique, correlated well with the budding polarity. Polarized budding was not observed in isolated cells in suspension.

Animals↗
PubMed1984

The level of thyroglobulin mRNA is regulated by TSH both in vitro and in vivo.

We have studied the effect of thyrotropin on the intracellular concentration of thyroglobulin and of its mRNA both in a differentiated rat thyroid cell line and in the rat thyroid in vivo. Our data demonstrate that the absence of thyrotropin causes a two fold decrease of both thyroglobulin and of its mRNA suggesting that thyroglobulin synthesis in thyroid cells is 50% constitutive.

Animals↗
PubMed1984

Suspension culture reveals a morphogenetic property of a thyroid epithelial cell line.

It is known that freshly dissociated thyroid cell clusters form follicles in suspension culture. Thyroid epithelial cell lines, grown for many generations in vitro, fail to show colloid-containing lumina when cultured as monolayers. Several thyroid cell lines, some transformed, have been tested with respect to their ability to form extracellular lumina when transferred from monolayer to suspension culture. One cell line in particular, the T78 cell line, showed this property when cultured in suspension. Lumina formed within 3 days even in the absence of added thyrotropin (TSH). The ultrastructure of lumina within cell aggregates resembled that of the thyroid follicle in vivo. The ability to undergo morphogenesis may therefore be an intrinsic property of thyroid epithelial cells which is retained for a large number of generations in vitro and is revealed by proper culture conditions. The shift from monolayer to suspension culture may thus lead to the expression of a thyroid differentiated function such as the formation of follicle-like structures.

Animals↗
PubMed1983

Effects of laser irradiation on hematoporphyrin-treated normal and transformed thyroid cells in culture.

Laser irradiation of tissues treated in vivo with the hematoporphyrin derivative (HPD) is known to result in a cytocidal effect, reportedly more pronounced in the tumor than in the surrounding normal tissues. In order to ascertain if this phenomenon had a clear cellular basis, it has been now reproduced in vitro in a model system consisting of normal and transformed cell lines. Epithelial rat thyroid cells were infected and transformed with a RNA oncogenic virus. Both the original (normal) and the viral-transformed (tumorigenic) cells were incubated with HPD and exposed to two types of laser irradiation: 631 nm, continuous wave; and 337.1 nm, pulsed. Under the conditions tested, the percentage survival of the transformed cells was found to be lower (up to approximately 3 times) than that of the normal cells. The cytocidal effect was greater using the pulsed than using the continuous-wave irradiation. The difference between normal and tumor cells was more evident at 30 micrograms than at 50 micrograms of HPD per ml. The HPD not followed by laser irradiation had no effect on the cell growth rate. The findings of a significant difference in the sensitivity to photoactivated HPD between normal and tumor cells under strictly controlled and highly comparable conditions opens new possibilities to the study of the cellular and molecular mechanisms involved in the phototherapy of tumors. Furthermore, studies in vitro on the active components of the photosensitizer and on their selectivity towards the tumor cells, explained at a cellular level, will lead to better approaches to photochemotherapy in vivo.

Animals↗
PubMed1982

Direct visualization of membrane clustering and endocytosis of thyrotropin into cultured thyroid cells.

We prepared a highly fluorescent conjugate of thyrotropin (TSH) which retains approximately 25% of the binding affinity of native TSH towards TSH receptor and approximately 25% of the potency of the native hormone in stimulating the accumulation of cAMP in thyroid cells. Using an image-intensified microscopy system, we observed that our fluorescent TSH bound specifically to diffusely distributed membrane receptors on live rat or bovine embryo thyroid cells grown in culture. At 37 degrees C the fluorescent hormone formed visible patches which were internalized and subsequently degraded. Hence, TSH, like other polypeptide hormones, is internalized by a process of receptor-mediated endocytosis. The addition of bivalent antibodies against the fluorophore rhodamine, to thyroid cells that were previously exposed to sub-optimal concentrations of rhodamine TSH, elicited maximal stimulation of the thyroid adenyl cyclase. Monovalent Fab' fragments did not enhance the response of rhodamine TSH. Therefore we conclude that enhanced surface clustering of TSH molecules increases their capacity to stimulate the adenyl cyclase of thyroid cells.

Animals↗
PubMed1982

Block in the expression of differentiation markers of rat thyroid epithelial cells by transformation with Kirsten murine sarcoma virus.

Well-differentiated epithelial cells, derived from primary cultures of normal rat thyroid glands (T-79 cells), as well as a cloned cell line also derived from normal rat thyroid glands (FRT-L cells) were infected with Kirsten murine sarcoma virus carrying outer coat of the helper Kirsten murine leukemia virus. Infected T-79 and FRT-L cells changed morphologically and began to proliferate rapidly, suggesting malignant transformation by the virus. Both cell lines can support the replication of both transformation-competent and transformation-incompetent viruses such as murine or rat leukemia viruses. Infected T-79 and FRT-L cells had a high colony-forming efficiency (68 and 64%, respectively) when grown in agar and formed tumors when transplanted s.c. into syngeneic rats. These tumors morphologically resemble undifferentiated adenocarcinomas, thus showing that Kirsten sarcoma virus carrying the outer coat of the helper Kirsten murine leukemia virus is able to transform differentiated epithelial cells. Transformed T-79 and FRT-L cells, in contrast to uninfected cells, neither secrete thyroglobulin concentrate iodide, two biochemical markers of differentiated thyroid function. Thus, expression of the differentiated phenotype is blocked as a consequence of cell transformation. The system described may be useful in studying epithelial cell carcinogenesis in terms of regulated expression of differentiated functions.

Animals↗
PubMed1981

Adenosine 3',5'-monophosphate modulates thyrotropin receptor clustering and thyrotropin activity in culture.

A biologically active rhodamine conjugate of thyrotropin binds at 4 degrees C to diffusely distributed membrane thyrotropin receptors which patch and become endocytosed into thyroid cells in a temperature-sensitive process. When the cells are first incubated with 8-bromo-cyclic adenosine monophosphate at 37 degrees C, the conjugate also binds to clustered receptors at 4 degrees C. Furthermore, 8-bromo-cyclic adenosine monophosphate reduces the amount of adenosine 3',5'-monophosphate (cyclic AMP) induced by thyrotropin. Hence, increased intracellular cyclic AMP induces receptor patching and reduces the concentration of cyclic AMP normally induced by thyrotropin. This suggests that cyclic AMP acts both as the second messenger of thyrotropin and also as the regulator of the level of thyrotropin receptors.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1981

Antitumoral action of bovine seminal ribonuclease.

The antitumor action of bovine seminal RNAase is studied as a function of the enzyme concentration and of the number of plated cells. With polyoma transformed hamster kidney cells, a 50% inhibition of cell growth is obtained with a 10 micrograms/ml of enzyme, while at this concentration growth of normal cells is very little affected. On the other hand the higher the number of plated cells, the lesser is the effect. The enzyme is found to be very effective also on tumor cells derived from a spontaneous tumor (neuroblastoma) and on cells derived from a chemically induced tumor (glioma). Amphoterycin B which is known to alter the permeability of eukariotic cells, does not affect the resistance of normal cells to the cytotoxic action of the enzyme.

Animals↗
PubMed1980

In vitro studies on selective inhibition of tumor cell growth by seminal ribonuclease.

The effect on cell growth of bovine seminal RNase has been tested on cells cultured in vitro. A selective inhibition of growth has been observed on tumor cells as compared to normal cells using several virus-transformed cell lines and a neuroblastoma line. The different cellular response of virus-transformed cells does not appear to depend on a differential permeability to the protein of transformed cells with respect to nontransformed ones. The selective cytotoxic action of seminal RNase on tumor cell growth was also compared with the action of other structurally related RNases and of various RNase derivatives prepared by specific chemical modifications. The results indicate that the protein dimeric structure and its enzymic activity are essential requirements for its action.

Animals↗