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G Leclercq

Publications and source records attributed to G Leclercq.

At least 19 recordsLinked to original sources
PubMed1999

Tumor-infiltrating dendritic cells in adenocarcinomas of the breast: a study of 143 neoplasms with a correlation to usual prognostic factors and to clinical outcome.

Dendritic cells (DC) are the most potent antigen-presenting cells, and induce antigen-specific immune responses. Infiltration of tumors by DC is thought to reflect the interaction between the host immune system and tumor cells. Tumor-infiltrating DC (TIDC) are believed to evolve into tumor-antigen pulsed cells and then to migrate to local lymph nodes, where they activate anti-tumor immune responses. Indirect clinical evidence supporting this theory is provided by studies showing that high TIDC densities are associated with favorable prognosis in some tumor types. In the present study, we evaluated 143 primary breast adenocarcinomas for the presence of DC, using immunohistochemistry with the anti-S100 protein antibody. We analyzed the relationship between the degree of infiltration by S100+ TIDC and the usual prognostic factors and clinical outcome. The results show that 42% of breast adenocarcinomas contain S100 TIDC. The number of S100+ TIDC varies according to the grade of tumors as follows: GRIII > GRII > GRI. A relationship is also found between S100+ TIDC and tumor size, lymph-node involvement, estrogen/progesterone receptor status and age. However, the presence of S100+ TIDC, even at the highest density, was not correlated with metastasis-free survival or overall survival.

Adenocarcinoma↗
PubMed1999

Implication of proteasome in estrogen receptor degradation.

In MCF-7 breast cancer cells, estradiol (E2) and pure antiestrogen RU 58668 down-regulate the estrogen receptor (ER). Interestingly, the protein synthesis inhibitor cycloheximide (CHX) abrogated solely the effect of E2 suggesting a selective difference in the degradation of the receptor induced by estrogenic and antiestrogenic stimulations. A panel of lysosome inhibitors (i.e. bafilomycin, chloroquine, NH4Cl, and monensin), calpain inhibitors (calpastatin and PD 150606) and proteasome inhibitors (lactacystin and proteasome inhibitor I) were tested to assess this hypothesis. Among all inhibitors tested, lactacystin and proteasome inhibitor I were the sole inhibitors to abrogate the elimination of the receptor induced by both E2 and RU 58668; this selective effect was also recorded in cells prelabeled with [3H]tamoxifen aziridine before exposure to these ligands. Hence, differential sensitivity to CHX seems to be linked to the different mechanisms which target proteins for proteasome-mediated destruction. Moreover, the two tested proteasome inhibitors produced a slight increase of ER concentration in cells not exposed to any ligand, suggesting also the involvement of proteasome in receptor turnover.

Cysteine Endopeptidases↗
PubMed1999

In vitro intrathymic differentiation kinetics of human fetal liver CD34+CD38- progenitors reveals a phenotypically defined dendritic/T-NK precursor split.

Human CD34+CD38- hematopoietic precursor cells from fetal liver are able to develop into T, NK, and dendritic cells in a hybrid human/mouse fetal thymic organ culture (FTOC). In this report, we pay particular attention to the early events in differentiation of these precursor cells. We show that the CD34+CD38- precursor cells, which are CD4-CD7-cyCD3-HLA-DR-/++ (cy, cytoplasmatic), differentiate into a CD4+ population that remained CD7-cyCD3-HLA-DR++ and a CD4- population that expressed CD7 and cyCD3. The CD4+CD7-cyCD3- cells differentiate into phenotypically and functionally mature dendritic cells, but do not differentiate into T or NK cells. The CD4-CD7+cyCD3+ population later differentiates into a CD4+CD7+cyCD3+HLA-DR- population, which has no potential to differentiate into dendritic cells but is able to differentiate into NK cells and gammadelta and alphabeta T lymphocytes. These findings support the notion that the T/NK split occurs downstream of the NK/dendritic split.

ADP-ribosyl Cyclase↗
PubMed1999

Murine fetal natural killer cells are functionally and structurally distinct from adult natural killer cells.

Natural killer (NK) cell phenotype and activity was studied by analyzing uncultured and short-time-cultured murine NK cells from fetal day 17 spleen and thymus. In contrast to NK cells from adult mice, freshly sorted fetal NK cells did not contain NK receptor transcripts for Ly-49A, B, C/I, D, F, G2, or H. The only NK receptor transcripts that could be detected were Ly-49E and CD94. It is important that Ly-49E was present at a 10- to 30-fold higher level compared with uncultured NK cells from adult mice. After short-time interleukin-2 culture, the level of Ly-49E mRNA was comparable between fetal and adult NK cells. Functionally, fetal NK cells only killed MHC class I-negative tumor cells when activating NK receptors were cross-linked with antibody. We show that fetal NK cells are mature but are different from NK cells in adult mice regarding their NK cell receptor repertoire and function.

Animals↗
PubMed1998

Estrogenic and anti-estrogenic regulation of estrogen receptor in MCF-7 breast-cancer cells: comparison of immunocytochemical data with biochemical measurements.

Data from immunocytochemical assessment of estrogen receptor (ER) regulation in MCF-7 cells under estrogenic and anti-estrogenic stimulation were compared with those obtained by enzyme immunoassay (Abbott ER-EIA). Similar trends were observed, although ER level variations were less marked when assessed immunocytochemically. We confirmed reports of ER disappearance in the presence of estrogens (Es; E2 and DES) and pure anti-estrogens (AEs; RU 58,668 and ICI 164,384) as well as its increase with partial AEs (4-OH-TAM and RU 39,119). E2-induced ER down-regulation was partly blocked by actinomycin D (AMD), okadaic acid (OK) and cycloheximide (CHX) when assessed by these 2 methods. Down-regulation by pure AEs was not impeded by CHX, indicating that they operate differently from Es (i.e., transformation of ER to a form sensitive to constitutive degradation activity). In situ pre-labeling of the cells with [3H]TAZ indicated that all investigated ligands eliminate pre-existing ER through binding to newly synthetized receptors, since [3H]TAZ co-valently associates with ER; E2 and RU 58,668 were more effective than 4-OH-TAM in this regard. CHX blocked ER disappearance even in the presence of pure AEs, which is in contrast to the data established with cells not pre-exposed to [3H]TAZ. Nuclear location of [3H]TAZ-ER complexes may explain this discrepancy, since pure AE-ER complexes were reported to be incapable of nuclear translocation.

Breast Neoplasms↗
PubMed1998

Length increase of the side chain of idoxifene does not improve its antagonistic potency in breast-cancer cell lines.

Linkage of specific residues onto steroidal estrogens through a long aliphatic side chain leads to "pure antiestrogens" devoid of residual estrogenic activity. Therefore, we assessed whether an increase in the length of the side chain of the triphenylethylenic antiestrogen idoxifene might increase its antagonistic potency. Culture of MCF-7 and tamoxifen-resistant variant RTX6 cells in the presence of CB 7675, a (CH2)8 derivative of idoxifene [(CH2)2], ruled out this possibility. This compound partly blocked MCF-7 cell growth only at 10(-6) M to almost the same extent as tamoxifen and failed to inhibit the growth of RTX6 cells, whereas the pure antiestrogen RU 58 668 was effective on both cell lines at much lower concentration. This absence of improvement was reflected in the observation of an efficiency for down-regulating progesterone receptor no better than that of tamoxifen. Pure antiestrogens are known to down-regulate the estrogen receptor, whereas triphenylethylenic antiestrogens up-regulate the receptor; CB 7675 behaves as the latter in agreement with its lack of strong antagonistic activity.

Breast Neoplasms↗
PubMed1998

Exchange of bound estrogens and antiestrogens in MCF-7 cells: evidence for ligand-induced stable configurations of the estrogen receptor.

Estrogens and antiestrogens promote specific conformations of the estrogen receptor (ER). To analyze the influence of such configurations on the stability of the ligand-ER complexes, MCF-7 breast cancer cells were exposed for 1 h to either [3H]E2 or an unlabeled estrogen or antiestrogen (E2, DES, E1, BP; OH-Tam, RU 39,411, ICI 164,384, RU 58,668); mutual exchange rates of bound compounds (i.e., [3H]E2-->ligand; ligand-->[3H]E2) were then analyzed in cell extracts by measuring [3H]E2. Addition of cycloheximide (CHX) to the incubation medium eliminated the potential interference of E2-induced ER loss. Extracts from control untreated cells were labeled with [3H]E2 or one of these various ligands and similarly submitted to exchange. Displacement of bound compounds occurred at moderate temperature (18 degrees C) but not at 4 degrees C. Remarkably, exchange proceeded at a lower rate in extracts from cells preincubated with [3H]E2 or a ligand. Antiestrogens RU 39,411 and RU 58,668 appeared especially refractory to displacement. Such low exchange rates were also recorded in experiments conducted on whole cells although to a higher extent than in extracts from preincubated cells. Enzyme immunoassays demonstrated that absence of major exchange could not be attributed to ER loss. Moreover, displacement of bound ligands appeared independent of their binding affinity for the receptor. These data suggest that estrogen and antiestrogen binding is stabilized by at least one factor (coactivators or corepressors) thus fixing the receptor molecules in a configuration that is relatively resistant to subsequent exchange. FPLC and PgR induction revealed that a significant proportion of ER maintained in a sufficiently flexible status was still able to exchange and transduce the transcriptional message of the displacer ligand.

Chromatography, Gel↗
PubMed1998

4-iodotamoxifen aziridine, a new affinity labeling agent for the rapid detection of estrogen receptor isoforms.

We describe the simple and fast preparation of a new radioiodinated probe for the detection of the estrogen receptor (ER) and its isoforms. Iodotamoxifen aziridine was labeled with iodine 125 ([125I]TAZ) in position 4 of the alpha aromatic ring. The yield was high (>75%), the label was stable and the specific activity was near optimal (1900-2170 Ci/mmol). The apparent relative binding affinity of the probe to a recombinant human ER (hER) was high (RBA = 35 vs estradiol = 100). Electrophoretic studies (SDS-PAGE) with this hER indicated the high potency of [125I]TAZ at very low concentration (<1 nM) to reveal ER bands after a short exposure time (1-4 days). Competition between this probe and various compounds as well as chemical treatments of the ER with SH-reactive chemicals, demonstrated the labeling specificity. Analysis of cytosols from a panel of cell lines and various rat reproductive organs displayed characteristic ER bands (67, 50 and 37 kDa) suppressed by unlabeled E2. Detection in nonreproductive organs of 43 kDa E2-nondisplaceable peptide raised the question upon the presence of altered and/or variant ERs in many tissues. Data concerning human breast cancer cytosols were in complete accordance with those established with [3H]TAZ: high ER polymorphism in most ER-positive samples and peculiar forms (mainly 43 kDa) in ER-negative samples. Hence, [125I]TAZ appears especially useful for the detection of altered ER or related peptides in breast cancers.

Affinity Labels↗
PubMed1998

Evolution towards hormone independence of the MXT mouse mammary tumor is associated with a gradual change in its estrogen receptor molecular polymorphism.

Using a method based on [3H]tamoxifenaziridine ([3H]TAZ) labeling, sequential immunoadsorption with anti-ER monoclonal antibodies, sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and fluorography, we observed a striking change inthe estrogen receptor (ER) electrophoresis pattern of the transplantable MXT mouse mammary tumor. Early, ER "rich" tumors (approximately 100 fmol/mg prot) displayed classical cytosolic 67 and 50 KDa bands. These bands disappeared in favor of a "cytosolic" 35 KDa band during progression towards undifferentiated ER "poor" tumors (approximately 25 fmol/mg prot). Although we can not rule out that this 35 KDa peptide results from in vivo ER proteolysis, it seems unique in view of the following: 1. It is immunoadsorbed not only by an anti-ER monoclonal antibody (H-222) directed to the hormone-binding domain, but also by an anti-ER monoclonal antibody (H-226) which interacts with an epitope in the A/B region close to the DNA-binding domain and is mainly exposed under activation conditions. 2. It does not bind [3H]estradiol([3H]E2) and a tentative to restore its [3H]E2 binding capacity with calmodulin and ATP was unsuccessful. The observation of similar approximately 35 KDa ERs in the nuclear fraction of early tumor transplants and in control uterus suggests that this peptide is already in an activated form. Structural alterations of ER and/or associated "anchorage" nuclear proteins may beat the origin of its cytosolic localization. Moreover, the fact that the addition of calmodulin and ATP to late MXT transplants cytosols fails to increase their [3H]E2 binding capacity indicates that the low ER content of these tumors does not result from a deficiency in the phosphorylation status of the receptor.

Animals↗
PubMed1997

Human thymocytes become lineage committed at an early postselection CD69+ stage, before the onset of functional maturation.

Mature functional CD4 or CD8 single positive (SP) thymocytes differentiate from immature CD4+ 8+ double positive (DP) precursors through a process of positive selection and terminal differentiation. To study CD4/CD8 lineage commitment, human postselection CD69+ thymocytes were separated into distinct subpopulations based on the differential expression of CD27, CD1, and CD45RA/RO. We demonstrate that these CD69+ subpopulations represent transitional stages of a common differentiation pathway during which CD69+ thymocytes that are initially CD27- CD1+ CD45RA- will sequentially up-regulate CD27, down-regulate CD1, and eventually acquire CD45RA upon maturation. Examination of CD4 and CD8 expression on these CD69+ subsets identified an early postselection CD69+ CD27- CD4SP population that gives rise to both CD4SP and CD8SP mature T cells when cultured in mouse thymus organs. In addition, a CD4+ 8+ DP population was identified that is CD69+ and CD27+, which only gives rise to CD8SP progeny upon culture. Although these results suggest that development of CD4SP and CD8SP cells may proceed through distinct intermediates, examination of active biosynthesis of CD4 and CD8 by the various subsets demonstrated that cells that have selectively terminated CD4 synthesis are already present in the CD27- CD4SP and CD27+ DP populations before culture. These data support a model of thymocyte differentiation whereby the decision of thymocytes to differentiate into one or the other lineage occurs concomitantly with, or very soon after, acquisition of CD69 and before the cells acquire CD27, down-regulate CD1, or acquire functional properties.

Animals↗
PubMed1997

Estrogen receptor-negative/progesterone receptor-positive Evsa-T mammary tumor cells: a model for assessing the biological property of this peculiar phenotype of breast cancers.

In 1986 we reported the appearance of a progestin binding protein in the human breast cancer cell line Evsa-T, originally described as lacking both estrogen and progesterone receptors (ER and PR). In this report we show that PR of this cell line displays a binding affinity for [3H]ORG 2058 and a sucrose gradient sedimentation profile similar to those ascribed to PR from MCF-7 or T47D breast cancer cell lines. PR from Evsa-T cells is down-regulated by the progestin R-5020 as well as by the two antiprogestins, ZK 112.993 and ZK 98.299, but does not confer growth sensitivity to these compounds. ER remains undetectable by ligand binding assay, enzyme immunoassay and northern blotting. Our Evsa-T clone could be a valuable model for assessing the mechanisms leading the ER-/PR+ phenotype occurring occasionally in breast cancers and frequently in meningiomas.

Breast Neoplasms↗
PubMed1997

Phenotypic and functional maturation of TCR gammadelta cells in the human thymus.

In contrast to thymic differentiation of TCR alphabeta cells, differentiation stages of TCR gammadelta cells are largely unknown. This report shows that CD1, a known marker of immature TCR alphabeta thymocytes, was expressed on some postnatal TCR gammadelta thymocytes. Only CD1+ TCR gammadelta thymocytes expressed recombination-activating gene-1 mRNA, and they were shown to differentiate into CD1- TCR gammadelta thymocytes. Functionally, sorted CD1- TCR gammadelta thymocytes proliferated in the presence of immobilized anti-CD3 Ab plus exogenous rIL-2 or rIL-15. Interestingly, in contrast to CD1- TCR alphabeta cells, CD1- TCR gammadelta thymocytes also proliferated extensively when cultured with exogenous rIL-2 or rIL-15 alone. FACS analysis as well as reverse transcription-PCR analysis showed that only CD1- TCR gammadelta thymocytes expressed IL-2Rbeta protein and mRNA. The differential expression of maturation markers, such as CD27, CD45RO, and CD45RA, as a function of expression of CD1 was similar in TCR gammadelta and TCR alphabeta thymocytes. An important exception is the expression of CD4 and CD8. Whereas TCR alphabeta thymocytes are mainly CD4-CD8 double positive at the immature CD1+ stage and CD4 or CD8 single positive at the mature CD1- stage, CD1(bright) TCR gammadelta thymocytes all expressed CD4, but only some of them expressed CD8. Some CD1- TCR gammadelta thymocytes also expressed CD8, but were negative for CD4. Collectively, our data clearly show that CD1 is a useful marker to distinguish immature human TCR gammadelta thymocytes from functional mature gammadelta cells based on recombination-activating gene-1 expression, in vitro differentiation, and phenotypic and functional characteristics.

Antigens, CD1↗
PubMed1997

MHC class II molecules are required for initiation of positive selection but not during terminal differentiation of human CD4 single positive thymocytes.

Positive selection of T cell precursors is an MHC dependent, multistep process by which functionally mature CD4+8- helper and CD4-8+ cytotoxic single positive (SP) T cells are generated from immature CD4+8+ double positive (DP) thymocytes. We investigated the requirement for TCR/MHC class II interactions during different stages of positive selection of human CD4 SP thymocytes. We show that sorted CD69- CD4+8+ DP preselection thymocytes cultured in fetal thymus lobes of normal mice were subject to positive selection and differentiated to CD3(high) CD69+, mature CD8 SP, and CD4 SP cells. When cultured in thymus lobes from MHC class II-deficient mice, these precursors failed to develop into mature CD4 SP T cells, indicating that in the hybrid cultures, murine MHC class II molecules are required for the development of mature human CD4 SP T cells. We have previously identified CD4 SP intermediate thymocytes that have received at least some of the signals involved in positive selection, since these cells are CD69+, CD3/TCR(high), and CD8beta- but that are still phenotypically and functionally immature. Here we demonstrate that in contrast to preselection thymocytes, these CD4 SP intermediate thymocytes can give rise to phenotypically mature and functionally CD4 SP progeny both in normal and in MHC class II-deficient thymus lobes. These results suggest that TCR/MHC interactions are required for the initial stages of positive selection, but are not essential during terminal differentiation to functionally mature CD4 SP T cells.

Animals↗
PubMed1997

Antagonism of estrogen receptor and calmodulin association by antiestrogens is not dependent on an interaction with calmodulin.

Previously, two antiestrogens estradiol derivatives (3 and 4) bearing the basic side chain of tamoxifen were shown to impede the binding of the estrogen receptor (ER) to calmodulin (CaM)-Sepharose. In this study, the interaction of these and related compounds with calmodulin was examined using the cyclic AMP phosphodiesterase (cAMP-PDE) assay. Neither of the steroids gave any significant inhibition of the calmodulin dependent cAMP-PDE activity up to a final concentration of 20 microM. For comparison, tamoxifen and nafoxidine produced IC50 values of 6.7 microM +/- 1.0 and 7.4 microM +/- 1.1, respectively. In addition, a comparison was made of the activity of some triphenylethylene derivatives against CaM dependent cAMP-PDE and the ER-CaM Sepharose assays, but no relationship was observed. Overall, these results demonstrate that inhibition of the ER-CaM association by various steroidal and triphenylethylene antiestrogens does not relate to antagonism of calmodulin function or their binding affinity for the estrogen receptor.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1997

Tamoxifen-induced estrogen receptor up-regulation in mammary tumor cells is not related to growth inhibition.

Treatment of estrogen receptor (ER)-positive mammary tumors with tamoxifen produces a dramatic accumulation of ER in the cell nucleus. We investigated whether this phenomenon might be related to the antitumor activity of the drug. Five tamoxifen derivatives for which an influence on MCF-7 cell growth had previously been established were tested for that purpose; two of them inhibited growth, one was growth-stimulatory, and the remaining two were without significant effect. At 1 microM all compounds up-regulated ER in the cell nucleus after 3 days of culture, suggesting that the ER accumulation does not predict the response to tamoxifen treatment. An analysis of a tamoxifen-resistant clone (RT x 6 cells) under similar experimental conditions led to the same conclusion: the ER level significantly increased in the presence of tamoxifen and its 4-hydroxy metabolite.

Antineoplastic Agents, Hormonal↗
PubMed1997

Optimization of glottic exposure during intubation of a patient lying supine on the ground.

Two methods of endotracheal intubation of patients lying on the ground were compared for ease and speed of intubation and minimization of complications in a crossover study of prehospital-oriented emergency physicians. Intubation of a mannequin was attempted by the physicians in either a left lateral decubitus (LLD) position or a kneeling (K) position, followed by the alternate position. The LLD position afforded more rapid intubation, better glottic visualization, and less dental trauma. Eighty-seven percent of physicians completely visualized the glottis in the LLD position, versus 33% of the K position group. Intubation times were 10.5 versus 14.6 seconds in the LLD and K positions, respectively (P < .001). The LLD position is a more effective position (in a mannequin model) than the K position for intubation of patients found lying on the ground, a frequent situation in prehospital care.

Cross-Over Studies↗
PubMed1997

Varying sensitivity of human mammary carcinoma cells to the toxic effect of parvovirus H-1.

We have previously demonstrated lysis of non-established cultures of human mammary carcinoma cells by parvovirus H-1, which has little effect on the proliferation of corresponding normal cultures. In the present study, we examined this effect in a number of breast-tumour specimens and found them to differ as to the amplitude of their response to parvoviral attack. We first investigated whether the differences in cell sensitivity to parvovirus infection reflected the differentiation level of the initial tumour. Among the biochemical and anatomopathological indicators of original tumour differentiation, the presence of oestrogenic receptors (ER) was found to have a predictive value as to the sensitivity of derived cultures to the cytopathic effect of H-1 virus. The ER+ tumour-derived cultures showed an increased sensitivity to the lytic effect of H-1 virus compared with the ER-tumour-derived cultures, in spite of similar average proliferation rates for the two types of cultures. The proliferation rate was more heterogeneous among ER+ tumour-derived cultures and, in this group, the faster growing cultures were also the most sensitive. This observation was corroborated by the study of established cell lines retaining ER expression under in vitro culture conditions. Oestradiol was found to increase the sensitivity of these cells to the parvovirus in parallel with induction of proliferation. This effect appeared to be mediated by ER activation, since it was not observed in the ER-negative cell line MDA-MB-231. These data point to the importance of hormonal influences and cellular parameters, notably differentiation and proliferation, in determining the extent to which human cancer cells can be targets for the cytopathic effect of parvoviruses.

Breast Neoplasms↗