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G Leclercq

Publications and source records attributed to G Leclercq.

At least 91 records · Page 5Linked to original sources
PubMed1995

Major molecular weight heterogeneity of estrogen receptor from breast cancer is not related to neoplasia.

Recent investigation from our laboratory revealed that the estrogen receptor (ER) from breast cancer is characterized by a high molecular weight polymorphism: SDS-polyacrylamide gel electrophoresis of [3H]-tamoxifen aziridine ([3H]-TAZ) labeled cytosols usually display several bands corresponding to the native receptor (67 KDa) and lower molecular cleavage products. High frequency of such altered receptors was confirmed here by size exclusion FPLC of [125I]-E2 labeled cytosols from a series of 98 breast cancers: on the average, 60% of the ER molecules were strongly degraded (Mr < or = 37 KDa). The absence of transcriptional activating domains (ABC domains) in such receptors was further demonstrated by assessing their ability to bind to hydroxylapatite (HAP). Thus, in presence of 500 mM KCI, 55% of ERs from another series of 54 cytosols failed to strongly adsorb to this phosphocalcic matrix, a characteristic property of receptors without exposed ABC domains. Finally, [3H]-TAZ labeled cytosols from normal uterine tissue and MCF-7 human breast cancer cells growing in nude mice displayed identical multibands electrophoretic patterns revealing in both cases native and cleaved receptors. Since latter receptor forms were never detected in MCF-7 cells growing in monolayer culture, we put forward the hypothesis that they were produced under the action of proteolytic enzymes acting at the time of tissue processing. Hence, most of the truncated receptors detected in human breast cancer cytosols should not be markers of malignancy.

Animals↗
PubMed1994

Evaluation of estrogen receptor, antiestrogen binding sites and calmodulin for antiestrogen resistance of two clones derived from the MCF-7 breast cancer cell line.

Estrogen receptor (ER), antiestrogen binding sites (AEBS) and calmodulin (CaM) are potential targets of antiestrogen (AE) action. To analyse further which of these targets are primarily involved in the antiproliferative activity of these drugs against human breast cancers, two cell clones, namely the RTx6 and LY-2 variants, selected from MCF-7 cells for their resistance to high doses of tamoxifen (TAM) and the Keoxifen (KEO) analog LY 117018, respectively, were studied for their sensitivity to hydroxytamoxifen (OH-TAM) and KEO as well as the strong calmodulin antagonist calmidazolium. The effects of these drugs on both cell growth and progesterone receptor (PgR) concentration were assessed. Binding properties for ER, AEBS and CaM of each compound were also measured. Our results confirmed that basal growth of RTx6 and LY-2 cells was more resistant to OH-TAM and KEO than parent MCF-7 cells, although both displayed a significant inhibition at the highest doses assessed. In regard to calmidazolium inhibition, each variant behaved as did the MCF-7 line indicating that a modification at the CaM level was not responsible for their lower sensitivity to AEs. Nor could the association of CaM to ER which did not differ among all cell lines. Resistance of these variants was not related to AEBS in view of the total lack of such sites in RTx6 cells. However, under estrogenic growth stimulation such sites may play some role, since LY-2 cells in the presence of estradiol displayed a real antiestrogen-resistant pattern while RTx6 cells were more sensitive than MCF-7 cells to OH-TAM. This property was not found in the antagonism against estradiol-induced PgR synthesis which was observed with each variant. Thus the PgR concentration of RTx6 cells was strongly down-regulated by OH-TAM and KEO and reduced in LY-2 cells to the same extent as in MCF-7 cells. All these observations show that AE resistance is not entirely related to ER mediated events and that alterations at the ER and CaM levels are unlikely to account for the lower AE sensitivity of the variants investigated.

Binding Sites↗
PubMed1994

Developmentally ordered appearance of a functional Fc gamma receptor on TCR V gamma 3 thymocytes. Evidence for stimulation-induced expression.

In the present report, we show that the Fc gamma receptor (Fc gamma R) becomes expressed on mature TCR V gamma 3 thymocytes, but it is absent on immature TCR V gamma 3 cells. Both Fc gamma RIII are Fc gamma RIII are expressed. The in vivo expression of the Fc gamma R on mature TCR V gamma 3 thymocytes coincides with an activated phenotype of the cells. In vitro, anti-CD3 stimulation of Fc gamma R-negative, immature TCR V gamma 3 thymocytes results in the expression of the Fc gamma R. The Fc gamma R of short-term IL-2-cultured, mature TCR V gamma 3 thymocytes is functional in an Ab-mediated cytotoxicity assay. In addition, it is shown that cross-linking of the Fc gamma R induces an increase in cytoplasmic Ca2+. Collectively, our findings show that mature TCR V gamma 3 thymocytes express a functional Fc gamma R; evidence is presented that this could be a result of in vivo activation of these cells.

Animals↗
PubMed1994

Human CD34+ fetal liver stem cells differentiate to T cells in a mouse thymic microenvironment.

Hematopoietic stem cells differentiate in the thymus to T cells along precisely defined intermediates. This process is thymic epithelium dependent and involves cytokines and cell-cell interactions between thymic stroma and T-cell precursors. Here we report that highly purified human CD34++ fetal liver stem cells differentiate to mature T cells, when seeded into isolated fetal thymic lobes of severe combined immunodeficient mice, and subsequently cultured in vitro. The human stem cells differentiate sequentially into CD4+CD8-CD3-, CD4+CD8+CD3-, CD4+CD8+CD3+, and finally, CD4+CD8-CD3+4 and CD4-CD8+CD3++ cells. Phenotypic analysis for additional maturation markers showed that these CD4 and CD8 single-positive thymocytes are fully maturate cells. By immunochemistry, human HLA-DR+ cells with a dendritic morphology could be detected. This novel chimeric human-mouse fetal thymus organ culture offers a tool to study human T-cell ontogeny in vitro and is a rapid and reliable test method for T-cell precursor activity of cultured or transfected human stem cells.

Animals↗
PubMed1994

Estradiol derivatives bearing the side-chain of tamoxifen antagonize the association between the estrogen receptor and calmodulin.

Calmodulin (CaM) is known to associate with the estrogen receptor (ER). The antiestrogen tamoxifen impedes this association suggesting that the latter would play an important role in CaM-dependent enzymatic catalyses. The ethoxyaminoalkyl side-chain of tamoxifen which confers antiestrogenicity appears to be involved in this antagonism. Antiestrogenic estradiol derivatives bearing the side-chain of tamoxifen in position 11 beta (RU 39,411) or 7 alpha (RU 45,144) were tested for their potential antagonism towards the association between CaM and ER. According to molecular modelling studies, such graftings position the chain in an orientation corresponding to that found in tamoxifen. Both compounds impeded the binding of ER to CaM-Sepharose at the same concentrations as found with tamoxifen indicating similar effectiveness. Steroidal analogs with or without a side-chain in a non-appropriate orientation failed to show this property. On the contrary, a non-conjugated side-chain analog antagonized the binding of the receptor indicating that the steroidal backbone of RU 39,411 and RU 45,144 did not play a major role in this regard. Since this free side-chain had been reported to be totally devoid of antiestrogenicity, one may consider that the steroidal backbone of these two antiestrogens participate to their antiproliferative activity. One may speculate that within the cell, ER should convey such compounds to CaM leading to a blockade of CaM-dependent catalyses. This hypothesis would also be relevant to the stilbene backbone of tamoxifen.

Animals↗
PubMed1994

Estradiol-induced down-regulation of estrogen receptor. Effect of various modulators of protein synthesis and expression.

Incubation of MCF-7 cells with estradiol (E2) down-regulates estrogen receptor (ER) resulting in a progressive reduction of the capacity of cells to concentrate selectively [3H]E2. Scatchard plot analysis failed to detect any transformation of residual receptors into peptides of lower binding affinity. [3H]Estrone gave an identical ER disappearance pattern with an ER half-life comprised between 2 and 3 h. A similar value was established by incubating the cells with [3H]tamoxifenaziridine ([3H]TAZ) for 1 h before the addition of excessive unlabeled E2 which induced ER-down regulation and impeded any further labeling of the residual receptors. Submission of the [3H]TAZ labeled cell extracts to SDS-PAGE revealed no progressive emergence of low molecular weight cleavage products of the receptor (< 67 kDa). Two inhibitors of protein kinases, H-7 at 40 microM and H-89 at 20 microM, failed to block the E2-induced ER down-regulation. On the contrary, the protein phosphatases 1 and 2A inhibitor, okadaic acid, was effective with concentrations higher than 0.1 microM indicating that a dephosphorylation mechanism was involved in this phenomenon. Cycloheximide (CHX) also significantly reduced the receptor decrease at concentrations higher than 1 microM. G-C specific intercalating agents [actinomycin D (AMD) and chromomycin A3 at 1 microM] also prevented ER disappearance; ethidium bromide (EB) and quinacrine were ineffective. AMD and CHX operated immediately after their addition to the medium indicating an inhibitory action on the synthesis of an RNA and/or a peptide with high turnover rate involved in ER decline. Moreover, AMD produced its suppressive effects under conditions impeding any labeling of newly synthetized receptors (i.e. [3H]TAZ with an excess of unlabeled E2) rejecting the possibility of an increasing ER production which may partially hamper its disappearance. Finally, E2-induced ER mRNA down-regulation was similarly abolished by AMD while EB and CHX were devoid of effect.

Cycloheximide↗
PubMed1994

Importance of A/B and C domains of the estrogen receptor for its adsorption to hydroxylapatite.

Regulatory properties of estrogen receptor (ER) result from the existence of functional domains within its primary structure. Thus, A/B and C domains which are rich in tyrosyl residues control gene expression while the E domain confers estrogen binding capacity. Hydroxylapatite (HAP) is known to adsorb ER. Scatchard plot analysis of [3H]estradiol binding patterns of HAP batches to which cytosolic ER had been adsorbed revealed that AB and/or C domains are mainly responsible for this property. Thus, treatment of these batches with the tyrosine reagent tetranitromethane (TNM) led to a dramatic release of adsorbed receptors. This did not occur with ER preparations devoid of exposed ABC domains obtained by selective immunoextraction with H-226 anti-ER monoclonal antibody prior to HAP assay. KC1 treatment (500 mM) of HAP batches also led to a release of bound receptors especially those devoid of exposed ABC domains. Such binding characteristics were also found with full length and truncated ERs produced in yeast: the full length receptor strongly interacted with HAP while the truncated receptor devoid of AB and C domains displayed only a weak adsorption. Additional investigation revealed that estradiol binding to cytosolic ER does not modify its reactivity towards TNM.

Adsorption↗
PubMed1993

Intrathymic differentiation of V gamma 3 T cells.

Whereas there is considerable information on the phenotypic and functional maturation of T cell receptor (TCR) alpha/beta thymocytes, comparatively little is known of the maturational processes that affect development of TCR-gamma/delta thymocytes. One class of gamma/delta T cells, those bearing the V gamma 3 gene product, are generated only during the early fetal stages of thymic development, and then migrate to the skin. Here we examine the intrathymic differentiation of these V gamma 3+ cells. The earliest V gamma 3 cells to appear in the thymus expressed low levels of TCR (V gamma 3low) and high levels of heat stable antigen (HSA). Over the next few days, V gamma 3+ thymocytes appeared which expressed high levels of TCR (V gamma 3high) and very low levels of HSA. The antigens CD5, CD45RB, and MEL14 were also differentially expressed on V gamma 3low versus V gamma 3high thymocytes, but the shift in expression was the opposite as compared with immature and mature TCR-alpha/beta thymocytes. Transfer experiments of sorted V gamma 3low/HSAhigh thymocytes to SCID thymic lobes showed that these cells were indeed the precursors of V gamma 3high/HSAlow thymocytes. The phenotype of the V gamma 3high thymocytes was similar to that of the postthymic V gamma 3+ cells found in the skin of adult mice. The differentiation of V gamma 3low in V gamma 3high thymocytes was also observed in fetal thymic organ culture. Addition of cyclosporin A (CsA) to these cultures had little effect on the appearance of V gamma 3low/HSAhigh cells, but blocked the appearance of V gamma 3high/HSAlow cells. These results show that, like alpha/beta T cells, V gamma 3+ thymocytes differentiate from TCRlow precursors to cells with a mature phenotype and that CsA inhibits this transition.

Animals↗
PubMed1993

Exogenous IL-7 promotes the growth of CD3-CD4-CD8-CD44+CD25+/- precursor cells and blocks the differentiation pathway of TCR-alpha beta cells in fetal thymus organ culture.

Addition of human rIL-7 to fetal thymic organ culture started at day 13, 14, or 15 did not influence the number of cells generated during a 12-day culture period. However, the IL-7 treatment resulted in a preferential expansion of cells with a phenotype characteristic for cells at an early step of differentiation. The cells were CD4-CD8-CD3-CD2- and SCA-1+. Analysis of the coordinate expression of CD44 and CD25 on these cells showed that the majority of the cells were either CD44+CD25- or CD44+CD25intermediate. TCR-alpha beta cells were present but in a significantly lower number as compared to the control cultures. The cell number of TCR-gamma delta cells was increased. All these effects were moderate after 6 days, but unequivocal after 12 days of culture. Treatment of the fetal organ culture with mAb-neutralizing murine IL-7 resulted in an inhibition of the proliferation of the fetal thymocytes. No particular subset studied was preferentially inhibited. By using a model of reconstitution of 14-day embryonic thymuses depleted of thymocytes by deoxyguanosine and reconstituted with fetal day 13 liver cells and set up in organ culture with or without IL-7, it was shown in a clear cut way that IL-7 indeed promotes expansion of the early precursor cells and TCR-gamma delta cells, but prevents the generation of TCR-alpha beta cells. In addition, reconstitution experiments were set up in the presence of mAb-neutralizing murine IL-7. This treatment resulted in the inhibition of the growth of the fetal thymocytes without inhibiting preferentially a particular subset. These data indicate that IL-7 acts at an early step of T cell differentiation and plays a role to expand precursor cells, but prevents this population from additional differentiation towards the TCR-alpha beta pathways, whereas the differentiation towards TCR-gamma delta cells is not influenced or even enhanced.

Animals↗
PubMed1993

Receptors for oestrogen, progesterone and epidermal growth factor in normal and tumorous canine mammary tissues.

Receptors for oestrogen (ER), progesterone (PR) and epidermal growth factor (EGF-R) are found in normal mammary tissue (NMT) and/or mammary tumours (MT) from all species studied, including dogs. The aims of this study were to define the possible influences of mammary histology, age, location in the mammary chain and of hormonal status and cycle stage in the expression of ER, PR and EGF-R in mammary tissues from healthy dogs and from dogs with MT. Carcinomas that had lost their glandular structure had significantly lower amounts of receptors. NMT either from healthy or affected dogs had significantly higher amounts of ER than MT. PR levels were significantly higher in benign lesions than in NMT. Steroid receptors in NMT from healthy dogs varied significantly with age (older dogs having more ER), location (posterior glands having higher ER concentrations) and cycle stage (the highest ER concentrations being found in the mid-luteal phase and the lowest PR concentrations in the early luteal phase). In NMT from affected dogs, higher steroid receptor concentrations were found in posterior glands; as in healthy dogs, ER concentrations were low in the follicular phase and high in the luteal phase and PR were high in anoestrus. Steroid receptor content in MT did not vary significantly with age, location or cycle stage or with hormonal status, but tended to vary with cycle stage in a manner similar to that found in NMT from the same dogs. In dogs affected with MT and treated with medroxyprogesterone acetate (MPA), NMT had low concentrations of PR but MT from the same dogs had high PR concentrations. EGF-R were found in the majority of the samples (+/- 65% of MT and +/- 85% of NMT) but there was no significant relation between the concentrations and the parameters studied. Nevertheless, EGF-R content was higher in NMT in the proliferative stages (oestrus, early and mid-luteal phase) than in the non-proliferative stages (early pro-oestrus and anoestrus). EGF-R and ER were significantly and positively correlated only in malignant tumours. There is no apparent difference between affected and healthy dogs in the regulation of ER, PR and EGF-R expression in NMT; on the other hand, some differences between NMT and MT are observed in the regulation of PR (for example under the influence of MPA) and in the correlation between EGF-R and ER expression.

Animals↗
PubMed1992

Antagonistic effect of triphenylethylenic antiestrogens on the association of estrogen receptor to calmodulin.

Binding of (3H)-estradiol labeled estrogen receptor from uterine cytosol to calmodulin was demonstrated by both affinity chromatography and sucrose gradient sedimentation. Triphenylethylene antiestrogens (tamoxifen family) with strong antagonistic activity against the calmodulin-dependent c-AMP phosphodiesterase largely reduced the binding of the receptor. Relevance of this observation with regard to the major antiproliferative activity (cytotoxicity) of these drugs is discussed.

Animals↗
PubMed1992

Presence of CD8 alpha-CD8 beta-positive TcR gamma/delta thymocytes in the fetal murine thymus and their in vitro expansion with interleukin-7.

Several groups have described that a low percentage of in vitro cultured T cell receptor (TcR) gamma/delta cells express CD8. Contrary to TcR alpha/beta cells, however, CD8 on these TcR gamma/delta cells was shown to be a CD8 alpha homodimer. We describe here that addition of interleukin-7 (IL-7) to a short-term in vitro culture of fetal day 14 thymic lobes in an organ culture system or of fetal day 18 fetal thymocytes in cell suspension yields CD8 beta-positive TcR gamma/delta cells. This is not the result of IL-7-induced expression of CD8 beta on previously CD8 beta-negative cells. It is due to IL-7-induced expansion of CD8 alpha-CD8 beta-positive TcR gamma/delta cells which are shown to be present in the starting fetal thymocyte cell population.

Animals↗
PubMed1992

Accumulation of a non-binding form of estrogen receptor in MCF-7 cells under hydroxytamoxifen treatment.

It is well known that MCF-7 cells, when incubated with hydroxytamoxifen (OH-Tam) loose their capacity to bind [3H]estradiol. By using Western blotting and [3H]tamoxifen aziridine labeling of KCl extracts from these cells we found that this loss in binding capacity was not associated with a disappearance of the estrogen receptor (ER) protein, an event known to occur after incubation with estradiol. Attempts to label under exchange conditions these ER molecules, which, on the basis of enzyme immunoassays appear to accumulate under OH-Tam treatment, were unsuccessful. Cell fractionation suggested that their origin is nuclear. Assessment of a few triphenylethylenic antiestrogens, as far as their inhibitory potency towards the in vitro MCF-7 cell growth is concerned, indicated a correlation between accumulation of these non-binding ER molecules and the antiestrogen antiproliferative action. However, we were unable to demonstrate absence of such an ER accumulation in two tamoxifen-resistant variants. Impaired folding of the ER protein or impaired phosphorylation of its hormone-binding domain are attractive hypotheses to account for these non-binding ER molecules. Whether these ER molecules have any physiological role, such as competition with the "normal" receptor molecules for the estrogen responsive elements on the DNA is unknown and deserves further study.

Blotting, Western↗
PubMed1992

Expression of c-erbB2, TGF-beta 1 and pS2 genes in primary human breast cancers.

The presence of c-erbB2, TGF-beta 1 and pS2 mRNAs was examined in primary breast tumours. The c-erbB2 mRNA was overexpressed in 34% of the tumours. There was a positive, statistically significant correlation between c-erbB2 gene overexpression and nodal status. TGF-beta 1 mRNA was detected in 84% of the tumours, regardless of their clinical status. When possible, the c-erbB2 and TGF-beta 1 proteins were identified immunohistochemically on frozen sections from the same tumours. For TGF-beta 1, the mRNA and immunohistochemical results were divergent in 6 cases, 5 of which did contain clearly detectable mRNA but did not stain with the antibody. The pS2 mRNA was detected in 22% of the tumours and in the BT474 cell line. There was a significant correlation between the presence of pS2 mRNA and of oestrogen receptors. No statistically significant correlation was observed between pS2 and TGF-beta 1 genes expression and the clinical parameters of the tumours.

Aged↗
PubMed1992

Cytokine production and responsiveness of fetal T-cell receptor V gamma 3 thymocytes.

The aim of this study was to examine the cytokine production and cytokine responsiveness of the first T-cell receptor (TcR) positive cells that appear in the murine fetal thymus, namely TcR V gamma 3 cells. It is shown that IL-2-cultured fetal TcR V gamma 3 thymocytes were capable of producing IL-3, GM-CSF, TNF-alpha and IFN-gamma upon TcR triggering. IL-2, IL-4, IL-5 and IL-6 could not be detected. With regard to cytokine responsiveness, TcR V gamma 3 cells proliferated to a high extent when high concentrations of rIL-2 were added. rIL-4 or rIL-7 alone, but not rIL-1 alone, were capable of inducing a modest proliferation of TcR V gamma 3 thymocytes. When combined with low concentrations of IL-2, a synergistic effect could be observed with IL-1, IL-4 or IL-7. It is shown that the synergistic effect of IL-2 with IL-4 was mainly due to induction of IL-2 receptor expression. The synergistic effect of IL-2 and IL-7 on the proliferation of TcR V gamma 3 cells could only be partially inhibited by anti-IL-2 receptor MoAb, and this antibody had no effect on the IL-2 + IL-1 cultures. These observations can explain the extensive proliferation of TcR V gamma 3 thymocytes during fetal life and they indicate that TcR V gamma 3 thymocytes have the potential to play a functional role during fetal thymus development.

Animals↗
PubMed1992

Modulatory effect of nonesterified fatty acids on structure and binding characteristics of estrogen receptor from MCF-7 human breast cancer cells.

Arachidonic, docosahexaenoic and oleic acids were found to produce a dose-dependent cytotoxic effect on MCF-7 cells; palmitic and stearic acids were totally ineffective in this regard suggesting that solely unsaturated fatty acids were able to arrest mammary tumor cell growth. Similarly, only former acids were able to decrease the binding capacity and affinity (increase Kd value) of the cells for 3H-E2 in a dose-dependent manner. In the case of arachidonic acid (the reference fatty acid), this decrease was associated with a slight cleavage of the native 67 KDa estrogen receptor (ER) into 50 and 25-30 KDa peptides as demonstrated by sequential labeling of high-salt cell extracts with 3H-tamoxifen aziridine, specific immunoadsorption with H-222 anti-ER monoclonal antibody, SDS-PAGE and fluorography. Both, modifications in binding characteristics of ER and cleavage of the native 67 KDa receptor were found to be extremely marked when unsaturated fatty acids were directly added to the high-salt cell extracts. This clear influence on the ER structure was reflected on enzyme immunological assay (EIA) by a reduction of ER immunoreactivity of approximately 50% in presence of arachidonic acid. Our observations are discussed in terms of possible interference of unsaturated fatty acids either through transmembrane modulation of phosphokinases and/or phospholipases implicated in ER mechanism of action, or through an intracellular interaction between ER and these acids acting as second messengers in regulation of cellular functions.

Breast Neoplasms↗
PubMed1992

[Turner's syndrome and pregnancy in donor oocytes and in vitro fertilization. Three case reports].

It is extremely rare for pregnancy to occur spontaneously in a woman with a mosaic or non-mosaic karyotype 45 x 0. (Till now only 13 patients with a homogeneous x 0 karyotype have been reported out of 62 patients who between them have had 138 pregnancies). Furthermore these pregnancies have been plagued by a number of important complications or fetal malformations (21% have had chromosome anomalies). It is therefore clear that in vitro fertilization with donor oocytes is a tempting solution for these women. The authors having three personal cases try and point out the conditions under which this technique can be used. It is important to realise that from the age of 10 or 12 years these patients have to have hormone treatment in order to avoid hypoplasia and hypovascularization of the uterus. Those conditions explain why so many these women had premature labours and also suffer from pre-eclampsia. Caesarean section was often found to be necessary because of feto-pelvic disproportion.

Adult↗