Biomedical subjects

J C Faye

Publications and source records attributed to J C Faye.

At least 19 recordsLinked to original sources

PubMed1992

Differential initiation of translation of a single estrogen receptor mRNA could explain some estradiol resistance cases.

Cell response to steroid stimulation is generally acknowledged to be mediated by an intracellular protein known as a receptor. Response intensity is related to the affinity of the receptor and to the number of sites occupied by its specific ligand. Although verified in the majority of experimental and clinical studies, certain phenomena of steroid hormone resistance would seem to challenge this assertion. Application of gene molecular biology to determine the action mechanisms of steroid hormones has partially explained cell resistance in terms of genetic modifications. The work presented here shows that in certain cases, estrogen resistance could be explained by regulation of translation of the single messenger RNA coding for the receptor.

Animals↗

PubMed1992

Cytosolic type II estrogen binding site in rat uterus: specific photolabeling with estrone.

A low affinity (Kd = 30 nM), large capacity (Bmax = 2.6 pmol/g tissue) estrogen binding site was photolabeled from estradiol-stimulated rat uterus cytosol. To maximize levels of this binding site and reduce those of the type I binding site, ovariectomized rats were injected with high doses of estradiol (10 micrograms per day) for four days with the last injection two hours before sacrifice. This treatment depleted type I estrogen receptors from the cytosol (by 90%) and raised levels of type II sites in the nucleus without affecting cytosolic type II levels. The type II estradiol binding sites were distinguished from the type I sites on the basis of their dissociation kinetics, pH-sensitivity and their behavior towards potassium chloride, somatostatin, sodium thiocyanate, sulfhydryl reagents and ammonium sulfate precipitation. These type II binding sites could be covalently photolabeled with tritiated estrone. A molecular weight of 43 kDa was found on SDS PAGE.

Affinity Labels↗

PubMed1991

Interactions between trypanocidal drugs and membrane phospholipids. A surface pressure, surface potential and electrophoretic mobility study.

Amphiphilic diphenyl methane derivatives exhibiting both antiproliferative and trypanocidal effects were studied with respect to their interactions with phospholipids, in monolayers and bilayers. These compounds, namely (4-benzyl)-phenoxy-2 trimethylammonium ethane iodide (D1), (4-tertiobutyl)-phenoxy-2 morpholinium ethane chloride (D2), and (4-benzyl)-phenoxy-2 morpholinium ethane chloride (D3), were shown to interact with phosphatidylcholine (PC) and phosphatidylserine (PS) in monolayers, as monitored by surface pressure and surface potential measurements. The film expansion of monolayers, on 10 mM NaCl subphase at pH 7.1, was more pronounced in the presence of D2 and D3 in the subphase before spreading of the lipids than with the injection of the drugs underneath a preformed film. Apparent binding constants of 10(4) M-1 were determined for both drugs from monolayer experiments. With D2 in the presence of PS, results of monolayer compressions and electrophoretic mobility measurements indicate binding of the drug to the lipid molecules only when the molecular area was large. D3 was shown to interact with PS, both in monolayers and bilayers, with a drug-to-lipid binding constant of about 2 x 10(4)M-1, as evaluated from electrophoretic mobility measurements on PS liposomes. These results, which indicate binding of these drugs to phospholipids in the order D2 less than D3, correlate with the biological activity of the drugs, and may account for the discrepancy observed between the drug concentrations required for biological and binding activities.

Animals↗

PubMed1991

Radiation inactivation of estrogen receptor in intact human breast cancer cells (MCF-7). Lack of energy transfer to the estradiol binding domain.

Whole MCF-7 human breast-cancer cells were irradiated at - 78 degrees C in a calibrated Gammacel 60Co irradiator. Freezing or storing conditions induce neither an alteration of the viability of cells nor a change in estradiol binding activity. Hexosaminidase was used as internal marker, and we measured the radiation inactivation size (RIS) of the estrogen receptor in whole cells. After various cell treatments, the estradiol binding unit always presents a molecular mass of 25 kDa. This value, which corresponds to the size of the defined hormone binding domain of the estrogen receptor, suggests that the energy delivered to the protein by the radiation is efficient to inactivate estradiol binding only when the hit occurs directly in the smaller hormone binding domain.

Binding Sites↗

PubMed1990

A potent and selective photoaffinity probe for the anti-estrogen binding site of rat liver.

The anti-estrogen binding site (ABS) is an apparently ubiquitous component of cells that has been shown to be intimately linked with the antiproliferative effects of certain antiestrogenic compounds, like tamoxifen, which is currently used for the treatment of breast cancer. However, the identification and in vitro study of this novel protein has been hampered to date by a lack of convenient probes that will efficiently label the molecule in nonpurified preparations. Thus, using a selective ABS ligand (4-benzylphenoxy-N-ethylmorpholine, MBPE) as starting material, we synthesized a photosensitive azido derivative, [(2-azido-4-benzyl)phenoxy]N-ethylmorpholine (azido-MBPE) that can be prepared in a tritiated form. Azido-MBPE has a high affinity for ABS (Kd = 3 nM), identical to that of tamoxifen, and covalently labels 5 and 12% of membrane-bound and detergent-solubilized ABS, respectively. Its incorporation is selectively and competitively inhibited by other ABS ligands (tamoxifen greater than nitromifen greater than hydroxytamoxifen). [3H]Azido-MBPE potently photolabels either membrane-bound or detergent-solubilized ABS as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under denaturing conditions revealing specific photoincorporation in a protein band of Mr = 40,000. This molecular weight is approximately two times lower than what we observed previously for ABS preparations studied under nondenaturing conditions and postlabeled with [3H]tamoxifen (Mr = 80,000-110,000). In chromatofocusing experiments with photolabeled ABS, a single specifically labeled protein fraction migrating with a pI of 6.4 was found to exhibit a Mr of 40,000 when subsequently electrophoresed on sodium dodecyl sulfate-polyacrylamide gels. These results indicate that [3H]azido-MBPE is a specific high affinity probe of ABS that will prove useful in the ultimate identification of this protein.

Affinity Labels↗

PubMed1990

Target size analysis of estrogen receptor in cultured intact cells: change in receptor structure between subconfluency and superconfluency in culture.

MCF-7 human breast cancer cells were submitted to the tritiated antiestrogen tamoxifen aziridine, frozen at -170 degrees C, stored and irradiated at -78 degrees C in a calibrated Gammacell 60Co irradiator. A three-step protein extraction procedure provided protein samples for the determination of the target size (TS) of the covalently labelled estrogen receptor (ER). From the TS it is shown that ER bound to an antiestrogen was, in whole cells, part of a 265 kDa polypeptide structure if measured in MCF-7 cells at subconfluency, or of a 360 kDa species in superconfluent cells.

Breast Neoplasms↗

PubMed1990

The anti-proliferative properties of 4-benzylphenoxy ethanamine derivatives are mediated by the anti-estrogen binding site (ABS), whereas the anti-estrogenic effects of trifluopromazine are not.

We compared the anti-proliferative properties of 4-benzylphenoxy-N ethyl morpholine (morpho-BPE) and trifluopromazine (TFP) on both the human breast cancer cell lines, MCF7, and its tamoxifen-resistant variant RTx6. We found that the calmodulin antagonist trifluopromazine (TFP) which bound ABS weakly, inhibited MCF7 cell growth but did not follow the relationship observed for diphenylmethane derivatives between MCF7-inhibitory potencies and their Ki. Regarding the tamoxifen-resistant RTx6 cells, TFP but not morpho-BPE induced inhibition of the proliferation. Using a tritiated derivative of morpho-BPE, two distinct binding sites could be demonstrated. Indeed, a low affinity binding site was present in both cell lines whereas a high affinity binding site was mainly found in MCF7 cells although being in lower concentration (less than 10%) in RTx6 cells. Both tamoxifen and TFP displaced morpho-BPE from the two binding sites. The uptake and efflux of the tritiated drug were similar in the two cell lines. The drug did not appear to be metabolized. We concluded that TFP and morpho-BPE belong to distinct classes of molecules and that ABS mediates the anti-proliferative action of diphenylmethane derivatives but not the inhibitory effect of the calmodulin antagonist TFP.

Antineoplastic Agents↗

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

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↗

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

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↗

PubMed1979