Biomedical subjects

F Bayard

Publications and source records attributed to F Bayard.

At least 91 records · Page 5Linked to original sources

PubMed1990

Modulation of the estrogen receptor structure, evidence of a heterogeneity.

In order to analyse the molecular weight polymorphism of the estrogen receptor (ER) in MCF-7 cells, we have developed a procedure which allowed in situ linkage of ER by (3H) tamoxifen aziridine and provided labelled proteins in conditions which minimized protease activities. After labelling, cell lysis was performed in SDS buffer containing various concentrations of [symbol: see text]-mercaptoethanol. Proteins extracted with phenolic solution and precipitated by cold acetone were analysed by SDS PAGE. It appears that beside the form of 67 kDa already described, binding entities of tamoxifen aziridine were also present at a molecular mass of 110 kDa and 45 kDa. On the other hand, investigations on the effect of 12-0-Tetradecanoyl Phorbol 13-Acetate (TPA) showed that TPA induces a decrease of the 67 kDa entity.

Breast Neoplasms↗

PubMed1989

Tumor necrosis factor-alpha inhibits follicle-stimulating hormone-induced differentiation in cultured rat granulosa cells.

We have investigated the effects of TNF-alpha on FSH-induced LH receptor expression, cAMP and progesterone production in cultured rat granulosa cells. TNF-alpha (0.5-100 ng/ml) inhibits the stimulating action of FSH on LH receptor formation in a dose-dependent manner with an IC50 of 1 ng/ml and an almost complete suppression of LH receptor induction for 50-100 ng/ml TNF-alpha. The inhibitory effect of TNF-alpha is not due to variations in cell number or viability but rather to a reduction of the LH receptor content per cell with no change in binding affinity (KD = 0.8 x 10(-10)M). TNF-alpha also inhibits the FSH-induced cAMP production but at a lower extent, with a maximum reduction of 60% for 100 ng/ml TNF-alpha. Moreover, TNF-alpha impairs the LH receptor formation induced by forskolin, cholera toxin or 8-Bromo-cAMP, indicating that the cytokine also acts at a step distal to FSH receptor and to cAMP formation. Finally, TNF-alpha decreases dramatically the progesterone synthesis that is stimulated by FSH, with a reduction to undetectable levels on and after 10 ng/ml TNF-alpha. These results suggest that TNF-alpha may drastically reduce the capacity of granulosa cells to differentiate upon FSH stimulation and to respond to LH during the physiological ovarian follicular maturation. Such anti-gonadotropic action of TNF-alpha on granulosa cell differentiation may be also relevant to the alteration of ovarian function during physiopathological processes like inflammatory or infection diseases.

Animals↗

PubMed1989

Thyroid function tests in ageing and their relation to associated nonthyroidal disease.

Thyroid function tests of 179 euthyroid geriatric inpatients (83 +/- 6 yr) unaffected by acute diseases or malnutrition were investigated and compared with those of 76 ambulatory healthy younger subjects (42 +/- 13 yr). Elderly population was divided in three groups, respectively: group G I (n = 37, 65-78 yr), group G II (n = 64, 79-85 yr) and group G III (n = 78, over 85 yr). Severity-of-illness index of the patients was evaluated at entry in the study protocol. While total thyroxine (TT4), free triiodothyronine (FT3) and TSH levels remained unchanged, circulating total triiodothyronine (TT3) was significantly lower (113 +/- 32 vs 150 +/- 31 ng/dl, p less than 0.05) and free thyroxine (FT4) was significantly higher (12.4 +/- 2.7 vs 10.3 +/- 2.3 pg/ml, p less than 0.05) in aged people. Furthermore, TT3 decreased significantly from 130 +/- 36 in G I to 110 +/- 33 in G II and to 108 +/- 25 in G III (p less than 0.01), and FT4 increased progressively although not significantly in the same groups. A close correlation was found between TT3 and severity index in male observations only (r = -0.43, p less than 0.01), as well as between FT4 and severity index in both sexes (r = 0.51, p less than 0.001 for men, r = 0.21, p less than 0.01 for women). These data suggest that thyroid function tests have to be cautiously interpreted in a geriatric population, particularly in relation to the severity of the clinical state, and reference values should be determined for TT3 and FT4 in the ageing process.

Age Factors↗

PubMed1989

Opposing effects of basic fibroblast growth factor and transforming growth factor-beta on the proliferation of cultured bovine retinal capillary endothelial (BREC) cells.

Transforming Growth Factor-beta (TGF-beta) inhibits the serum and basic fibroblast growth factor (bFGF)-induced proliferation of cultured bovine retinal endothelial capillary (BREC) cells in a dose-dependent manner. The concentration of TGF-beta required to get half-maximal inhibition (ED50) are 10 pg ml-1 in serum and 17 pg ml-1 in the presence of additional bFGF (1 ng ml-1). These TGF-beta ED50 values are greatly increased when BREC cells were seeded at high density: 610 pg ml-1 in serum and 1 ng ml-1 in the presence of additional bFGF. At low initial cell density BREC cells are more sensitive to TGF-beta than aortic bovine arch endothelial (ABAE) cells for which TGF-beta ED50 values are respectively 40 pg ml-1 and 100 pg ml-1 in serum and in the presence of additional bFGF. In contrast, at high cell density BREC cells appeared to be more resistant to TGF-beta inhibition than ABAE cells for which TFG-beta ED50 values are 210 and 300 pg ml-1. Moreover bFGF added at increasing concentrations neutralize totally TGF-beta inhibition of BREC cell proliferation but only partially that of ABAE cell proliferation. Our results suggest a key role of equilibrium TFG-beta bFGF on the proliferation of BREC cells.

Animals↗

PubMed1989

Autocrine regulation of bovine retinal capillary endothelial cell (BREC) proliferation by BREC-derived basic fibroblast growth factor.

Fibroblast growth factors (FGFs) are mitogenic for bovine retinal capillary endothelial cells (BREC) seeded at a low density. Seeding BREC cells at a high density greatly reduces their requirement for basic FGF (bFGF) in order to proliferate actively. We show here that monolayers of BREC cells synthesize and release into the culture medium a growth factor, which on the basis of biological activity, heparin affinity, immuno-cross reactivity with anti-bFGF antibodies and mRNA analysis, has been identified as basic fibroblast growth factor. These data indicate that BREC cells are able to synthesize and release bFGF, which can act as a promoting-growth factor for these cells by a para- and/or autocrine mechanism. We suggest thus, that this para- and/or autocrine mechanism involving bFGF may play a key role in preretinal neovascularization, particularly in diabetic patients presenting a proliferative retinopathy.

Animals↗

PubMed1989

Long term effects of continuous subcutaneous infusion of the somatostatin analog octreotide in the treatment of acromegaly.

The marked pituitary tumor shrinkage achieved by continuous sc infusion (CSI) of the long-acting somatostatin analog octreotide in one acromegalic patient led us to treat 16 other acromegalic patients for up to 24 months by CSI. This therapy, given in doses ranging from 100-600 micrograms/day, resulted in normalization of the mean daily serum GH (mGH) and insulin-like growth factor I levels in 9 of the 17 patients (53%). In 7 patients, mean daily serum GH decreased but not to normal; 3 of these patients had hyperprolactinemia which was not influenced by octreotide. One patient was completely unresponsive. In contrast to the biochemical results, 80% of the patients had marked clinical improvement. Side-effects consisted of slightly impaired carbohydrate tolerance in 2 patients and cholelithiasis in 2 patients. Pituitary tumor size decreased in only 3 patients; in 1 of them visual field defects disappeared rapidly. These results suggest that octreotide treatment may prove beneficial before surgery in patients with macroadenomas, although its efficacy varies widely. Potential responsivity can usually be determined by a short course (24 h) of CSI of octreotide.

Acromegaly↗

PubMed1989

Differential estrogenic responsiveness of MCF-7 cells. Relationship to the presence of two different estrogen receptors.

Estradiol stimulation of thymidine incorporation and progesterone receptor synthesis is at a maximum in exponentially growing cells. These activities are found to disappear in confluent MCF-7 cells. Since no significant differences in the binding of estradiol to its receptor site (Kd = 10(-10) M, Bmax = 150 fm/mg protein) are observed in these two conditions, receptor structure was analyzed in both cell populations. Various methods demonstrated that receptor size is related to the state of confluence. The hydrodynamic properties of estradiol receptors complexed with 3H-estradiol from cells in the two different growth phases are similar in low ionic strength but different in high ionic strength media. Moreover, when the cell extracts are analyzed in denaturing conditions, cells which are sensitive to estradiol are found to contain a monomeric binding entity of 62 kD, whereas the unresponsive cells have a 47 kD binding entity.

Breast Neoplasms↗

PubMed1989

[Culture of retinal capillary endothelial cells].

The culture of retinal capillary endothelial cells involves certain problems concerning contamination by pericytes, the maintenance of differentiation and the duration of culture viability. A procedure for the isolation and culture of capillary endothelial cells from bovine retina which overcomes these difficulties, is described. Microvessel fragments isolated by mechanical dispersion and filtration techniques adhere strongly to dishes coated with extracellular matrix produced by bovine corneal endothelial cells. The first migrating cells emerge from the original microvessel fragments two days after plating. This technique and subsequent cloning provides migrating and proliferating cells derived only from the retinal capillaries and uncontaminated by other cell types such as pericytes. Endothelial cells were grown on gelatin coated dishes in a serum supplemented medium (10% calf serum). Cell proliferation was significantly enhanced by the addition of basic fibroblast growth factor (1 ng/ml) to the culture medium. In these culture conditions, retinal capillary endothelial cells can be repeatedly passing without the loss of their principal morphological characteristics and some of the differentiated properties of endothelial cells. Primary cultures and subcultures, at least up to the 8th passage, formed a monolayer of small, elongated, tightly-packed, contact inhibited cells which expressed Factor VIII-related antigen. Ultrastructural examination by transmission electron microscopy of confluent bovine retinal capillary endothelial cells showed many tight junctions and Webel Palade granules. These studies provide new means for the isolation and culture of retinal capillary endothelial cells and presents evidence for growth factor requirements for the ability of cells to be repeatedly passing.

Animals↗

PubMed1988

Inhibition of MCF-7 cell growth by 12-O-tetradecanoylphorbol-13-acetate and 1,2-dioctanoyl-sn-glycerol: distinct effects on protein kinase C activity.

We have investigated the effects of phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and permeant diacylglycerol 1,2-dioctanoyl-sn-glycerol (DiC8) on MCF-7 cell proliferation and protein kinase C activity. DiC8 mimics the effects of TPA on both cell morphology and proliferation, with an ED50 value of 11 micrograms/ml for cell growth inhibition. As with TPA and phorbol 12,13-dibutyrate, DiC8 enhances the degree of phosphorylation of an endogenous Mr 28,000 protein in a time- and dose-dependent manner. The effect is measurable upon 5 min of cell treatment with each protein kinase C activator and reaches a maximum at 30 min. The ED50s observed are 5 ng/ml and 20 micrograms/ml, respectively, for phorbol esters and DiC8. The Mr 28,000 protein is found in the cytosolic fraction and is phosphorylated on serine residues by both TPA and DiC8. Further characterization of the phosphorylated proteins using a highly resolutive two-dimensional electrophoresis demonstrates that the two-protein kinase C activators lead to slightly distinct protein phosphorylation patterns with an extra set of proteins phosphorylated under TPA but not DiC8 stimulation. Contrary to TPA, DiC8 induces only a partial and transient translocation of protein kinase C activity from the cytosolic to the particulate compartment. Moreover, no down-regulation of protein kinase C is observed after prolonged treatment of MCF-7 cells with DiC8, while only 10% of the initial protein kinase C level remains present in cells treated with TPA for 48 h. However, this remainder enzymatic activity is sufficient to induce the phosphorylation of the Mr 28,000 protein at its maximal level. In conclusion, our results reinforce the hypothesis of a negative modulatory role of protein kinase C in MCF-7 cell proliferation but suggest that the two activators TPA and DiC8 could induce distinct molecular events with regard to the enzyme recruitment and activity as well as to its further processing.

Breast Neoplasms↗

PubMed1988

Solubilization of a tamoxifen-binding protein. Assessment of its molecular mass.

Recent findings point to a role of Antioestrogen-Binding Site (ABS) in some of the growth-modulatory effects of antioestrogens. In the present study, a method for the solubilization of ABS from rat uterus microsomal fractions by using 3-(3-cholamidopropyl)dimethylammonio-1-propanesulphonate (CHAPS; 20 mM) and KCl (0.4 M) is described. Decreasing the CHAPS concentration below the critical micelle concentration led to long-term stabilization of the protein. All of the membrane-bound ABS was recovered in the extract, and only one class of binding site, with a high affinity for [3H]tamoxifen (KA = 5 x 10(8) M-1) was detectable. This binding was time-dependent and reversible: at 4 degrees C, the association rate constant was ka = 7.2 x 10(4) M-1.s-1, and the reverse rate constant was kd = 1.0 x 10(-4) s-1. Solubilized ABS exhibited an affinity and specificity similar to those of the membrane-bound sites. Under disaggregating conditions, solubilized ABS had an apparent sedimentation coefficient, s20,w, of 5.2 S and a Stokes radius of 6.4 nm. From these two values, molecular masses of 160,000 Da for the detergent-ABS complex, and 110,000 for the protein moiety, were estimated. Assessment of the size of the membrane-bound ABS by a radiation inactivation technique is also described. The 'radiation inactivation size', corresponding to the mass of 1 mol of protein structure(s) whose associated tamoxifen-binding activity is abolished after a single hit by ionizing radiation, was estimated to be 80,000 Da.

Animals↗

PubMed1988

Further evidence for a biological role of anti-estrogen-binding sites in mediating the growth inhibitory action of diphenylmethane derivatives.

Several diphenylmethane derivatives have been synthesized with variable affinities for Anti-estrogen Binding Sites (ABS) but not for the estrogen receptor. Using these molecules as probes it is shown that their binding affinities for ABS correlate with their abilities to inhibit the growth of MCF-7 human breast cancer cells. In contrast they have no influence on the proliferation of tamoxifen-resistant variant cells (RTx6) in which ABS are undetectable. These data support the conclusion that ABS has a functional role in the anti-proliferative effect of triphenylethylene anti-estrogens and structurally related compounds.

Benzhydryl Compounds↗

PubMed1988

Presence of low affinity estrogen binding sites in guinea-pig Kurloff cells.

Estrogen treatment of guinea-pig leads to an increase in the number of lymphoid cells containing Kurloff inclusions. The presence of estradiol binding sites in cytosolic extracts of Kurloff cells was investigated. We confirm here our previous inability to measure the typical type I estrogen receptor by using the classical dextran-charcoal procedure to separate bound and free ligand. We report now that low affinity estrogen binding sites (Kd approximately equal to 11 nM) can be detected when Kurloff cell extracts were fractionated on hydroxylapatite or Sephadex LH-20 after binding assay. Although the real function of these binding sites remains to be defined, the question arises again whether Kurloff cell represents a target cell for estrogens.

Animals↗

PubMed1988

Modulation of estrogen receptors by phorbol diesters in human breast MCF-7 cell line.

Treatment of MCF-7 cells with 12-O-tetradecanoylphorbol 13-acetate (TPA) results in an inhibition of cell proliferation and a reduction in the number of estrogen receptors (ER), shown by binding studies and immunoassay. The decrease in ER concentration induced by phorbol ester derivatives parallels their growth inhibitory effect. Moreover, the estrogen receptor of TPA-resistant RPh4 cells (which are insensitive to the antiproliferative and morphological effects of TPA) is not affected by TPA treatment. The reduction in ER concentration appear to be a specific phenomenon since it contrasted with the 2-fold increase in total cell protein content which included an increase in progesterone receptor (PgR). We also found that addition of TPA does not affect estrogen induction of PgR.

Binding, Competitive↗

PubMed1987

TPA induces subcellular translocation and subsequent down-regulation of both phorbol ester binding and protein kinase C activities in MCF-7 cells.

Phorbol ester TPA has been previously shown to induce a rapid translocation, followed by a progressive decline of protein kinase C activity in MCF-7 cells (J.M. Darbon et al, 1986, Biochem. Biophys. Res. Comm. 137: 1159-1166). We show now a parallel TPA-induced movement of phorbol ester binding sites from the cytosolic to the particulate fraction with no change in the binding affinities for the (3H) PDBu probe (KD congruent to 2 nM). The subcellular redistribution process is followed by a rapid decrease of the phorbol ester binding capacity at the membrane level. The concomitant decline in both phorbol ester binding and protein kinase C activities that we observed during the course of TPA treatment strongly argues for a real down-regulation of the enzyme in phorbol ester-treated MCF-7 cells. The molecular mechanisms of these events and their relations to the inhibition of cell growth remain to be clarified.

Animals↗

PubMed1987

Influence of 12-O-tetradecanoylphorbol-13-acetate on proliferation and maturation of human breast carcinoma cells (MCF-7): relationship to cell cycle events.

Exposure of MCF-7 human mammary carcinoma cells to 12-O-tetradecanoylphorbol-13-acetate (TPA) results in changes in cell morphology and arrest of cell growth. The inhibition of cell proliferation and the increase in cell volume are concentration dependent; these effects are reversible upon removal of the tumor promoting agent. Electron microscopic studies reveal that TPA increases endoplasmic reticulum and induces the appearance of secretory granules. MCF-7 cells treated by TPA therefore present morphological characteristics of secretory cells. These effects of TPA on MCF-7 cells are accompanied by specific disruption of cell cycle events, a block of cells in G1 at the expense of S base, and a delayed passage through G2. Studies in which a cell cycle lock in G1 is produced by tamoxifen show that exposure of such cells to PA produces cell morphological changes and an inability to progress through the cell cycle when estradiol is added.

Breast Neoplasms↗

PubMed1987

Antiestrogens, different sites of action than the estrogen receptor?

Nonsteroidal antiestrogens display antagonistic as well as agonistic properties when compared with estrogens. These observations and studies of their molecular interactions suggest that part of their activity is mediated through estrogen receptor (ER)-mediated pathways. However, other data, concerning mainly effects on growth and cellular proliferation, cannot be explained on this basis. Recent investigations have shown binding of these molecules to a number of other intracellular binding sites beside ER. Using different cell variants of the human breast cancer cell line MCF-7 we could confirm the peculiar role of one of these sites, the specific antiestrogen-binding site (ABS). Published data on ABS are critically reviewed.

Animals↗

PubMed1987

Shrinking of a growth hormone-producing pituitary tumor by continuous subcutaneous infusion of the somatostatin analog SMS 201-995.

SMS 201-995, a long-acting somatostatin analog, was given as the initial treatment to an acromegalic patient. SMS 201-995 (200 micrograms, sc, three times daily) reduced, but did not normalize, serum GH levels. Complete and prolonged control of GH secretion was obtained with a 600-micrograms daily continuous sc infusion (CSI), and the patient was treated in this way for 6 months. Rapid improvement of clinical signs and symptoms of acromegaly occurred, as did major tumor shrinkage. The other pituitary functions did not change. After 6 months, the daily SMS 201-995 dose was progressively reduced; GH secretion remained suppressed. After 12 months of treatment, GH secretion was controlled with a CSI of 100 micrograms SMS 201-995 daily, but not with two daily sc 100-micrograms injections. Further significant reduction in tumor size occurred. We conclude that CSI of SMS 201-995 resulted in constant GH normalization and marked clinical and morphological improvement. This form of treatment should be considered as an alternative to ablative treatment of acromegaly.

Adult↗

PubMed1986

Phorbol esters inhibit the proliferation of MCF-7 cells. Possible implication of protein kinase C.

The effect of tumor promoter phorbol esters on cell proliferation was investigated in human breast cancer cell line MCF-7. During a 4-day culture period, the various phorbol ester derivatives TPA, PDD, PDBu, PDBz and PDA inhibited the proliferation of MCF-7 cells in a dose-dependent manner, with respective IC50 of 0.06, 0.75, 2.4, 3.6 and 15 X 10(-9) M. The 4-O-met-TPA, alpha PDD and alph PHR were ineffective at 2 X 10(-7) M, the highest concentration tested. Using a 3H-PDBu probe, we demonstrated the presence of specific, high affinity binding sites in intact cultured cells, with a Kd of about 9 X 10(-9) M. Unlabelled TPA, PDD, PDBU and PDBz competed with 3H-PDBu with respective IC50 of 35, 12.5, 150 and 220 X 10(-9) M. High concentrations of PDA, 4-O-met-TPA and alpha PDD slightly inhibited the 3H PDBu binding, whereas alpha PHR did not until 10(-5) M. The correlation that we observed between the relative potencies of the various phorbol derivatives for inhibiting both PDBu binding and cell proliferation, suggests that tumor promoter phorbol esters may induce growth arrest in MCF-7 cells by the mediation of protein kinase C.

Breast Neoplasms↗