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G Smets

Publications and source records attributed to G Smets.

At least 19 recordsLinked to original sources
PubMed1998

The antiproliferative activity of all-trans-retinoic acid catabolites and isomers is differentially modulated by liarozole-fumarate in MCF-7 human breast cancer cells.

The clinical use of all-trans-retinoic acid (ATRA) in the treatment of cancer is significantly hampered by the prompt emergence of resistance, believed to be caused by increased ATRA catabolism. Inhibitors of ATRA catabolism may therefore prove valuable for cancer therapy. Liarozole-fumarate is an anti-tumour drug that inhibits the cytochrome P450-dependent catabolism of ATRA. ATRA, but also its naturally occurring catabolites, 4-oxo-ATRA and 5,6-epoxy-ATRA, as well as its stereoisomers, 9-cis-RA and 13-cis-RA, show significant antiproliferative activity in MCF-7 human breast cancer cells. To further elucidate its mechanism of action, we investigated whether liarozole-fumarate was able to enhance the antiproliferative activity of ATRA catabolites and isomers. Liarozole-fumarate alone up to a concentration of 10(-6) M had no effect on MCF-7 cell proliferation. However, in combination with ATRA or the ATRA catabolites, liarozole-fumarate (10(-6) M) significantly enhanced their antiproliferative activity. On the contrary, liarozole-fumarate (10(-6) M) was not able to potentiate the antiproliferative activity of the ATRA stereoisomers, most probably because of the absence of cytochrome P450-dependent catabolism. Together, these findings show that liarozole-fumarate acts as a versatile inhibitor of retinoid catabolism in that it not only blocks the breakdown of ATRA, but also inhibits the catabolic pathway of 4-oxo-ATRA and 5,6-epoxy-ATRA, thereby enhancing their antiproliferative activity.

Antineoplastic Agents↗
PubMed1998

All-trans-retinoic acid metabolites significantly inhibit the proliferation of MCF-7 human breast cancer cells in vitro.

All-trans-retinoic acid (ATRA) is well known to inhibit the proliferation of human breast cancer cells. Much less is known about the antiproliferative activity of the naturally occurring metabolites and isomers of ATRA. In the present study, we investigated the antiproliferative activity of ATRA, its physiological catabolites 4-oxo-ATRA and 5,6-epoxy-ATRA and isomers 9-cis-RA and 13-cis-RA in MCF-7 human breast cancer cells by bromodeoxyuridine incorporation. MCF-7 cells were grown in steroid- and retinoid-free medium supplemented with growth factors. Under these culture conditions, ATRA and its naturally occurring catabolites and isomers showed significant antiproliferative activity in MCF-7 cells in a concentration-dependent manner (10[-11] M to 10[-6] M). The antiproliferative activity of ATRA catabolites and isomers was equal to that of the parent compound ATRA at concentrations of 10(-8) M and 10(-7) M. Only at 10(-6) M were the catabolites and the stereoisomer 13-cis-RA less potent. The stereoisomer 9-cis-RA was as potent as ATRA at all concentrations tested (10[-11] M to 10[-6] M). In addition, we show that the catabolites and isomers were formed from ATRA to only a limited extent. Together, our findings suggest that in spite of their high antiproliferative activity the catabolites and isomers of ATRA cannot be responsible for the observed growth inhibition induced by ATRA.

Breast Neoplasms↗
PubMed1997

125I-Tyr0-hCRH labelling characteristics of corticotropin-releasing hormone receptors: differences between normal and adenomatous corticotrophs.

The presence of corticotropin-releasing hormone (CRH) receptors has been previously demonstrated in corticotrophs from normal pituitaries using a method combining immunocytochemistry and liquid emulsion autoradiography. The aim of this study was to compare the characteristics of the 125I-Tyr0-hCRH binding in corticotrophs from normal pituitaries (three obtained at autopsy and one obtained at surgery) with corticotrophs from pituitary adenomas (six corticotroph adenomas responsible for Cushing's disease and two silent corticotroph adenomas secreting a biologically inactive ACTH molecule). In normal corticotrophs, the larger part of the 125I-Tyr0-hCRH binding was localised in patchy conglomerates at the centre of the cell and, to a much lesser degree, in a diffuse pattern at the cell periphery. In adenomatous corticotrophs, CRH receptor expression is disturbed both quantitatively and qualitatively. Except for a minority of cells in one adenoma, all adenomatous corticotrophs showed only peripherally bound 125I-Tyr0-hCRH and no centrally localised binding. Furthermore, adenomatous corticotrophs revealed a statistically significant lower signal intensity when compared to normal corticotrophs and a strongly negative correlation was found between the labelling area in adenomatous corticotrophs and both the basal and CRH-stimulated plasma ACTH levels. These findings suggest defective processing of CRH receptors and could be relevant to the sustained ACTH secretion by adenomatous corticotrophs in Cushing's disease and, more generally, provide an explanation to its pathology. The silent corticotrophs secreting a biologically inactive ACTH molecule were characterised by a very faint signal intensity, although present on almost every cell.

Adenoma↗
PubMed1997

Fluorescein-labeled tyramide strongly enhances the detection of low bromodeoxyuridine incorporation levels.

Immunocytochemical detection of bromodeoxyuridine (BrdU) labeling can be hampered by low BrdU incorporation levels. We describe here an amplification method for weak BrdU immunosignals. The tyramide signal amplification method based on catalyzed reporter deposition (CARD) uses fluorescein-labeled tyramide as a substrate for horseradish peroxidase. The enzyme catalyzes the formation of highly reactive tyramide radicals with a very short half-life, resulting in the binding of fluorescein-conjugated tyramide only at the site of the enzymatic reaction. MCF-7 cells were grown in vitro in medium containing charcoal-stripped fetal bovine serum supplemented by growth factors. Under these culture conditions, the BrdU immunosignal was hard to detect but could be enhanced specifically by the tyramide signal amplification system, resulting in clear-cut differences between BrdU-negative and BrdU-positive cells. This enabled rapid and objective quantification of the BrdU labeling index without the risk of underestimating the number of cells in S-phase. Therefore, this amplification of BrdU immunosignals might also prove valuable for in vivo cancer prognosis, cell kinetics studies, and computer-assisted image analyses.

Breast Neoplasms↗
PubMed1996

Liarozole potentiates the all-trans-retinoic acid-induced structural remodelling in human breast carcinoma MCF-7 cells in vitro.

Liarozole inhibits cytochrome P-450-dependent enzymes that play a key role in all-trans-retinoic acid (ATRA) catabolism. In MCF-7 cells, liarozole potentiates the antiproliferative effects of ATRA. The present study demonstrates this synergistic effect on cell differentiation of MCF-7 cell cultures as measured by immunocytochemistry for cytokeratins 8, 18, and 19, actin, E-cadherin, desmoglein and desmoplakins I & II. ATRA concentration-dependently (10(-8) M-10(-6) M) induced changes in actin stress fibers and cytokeratin intermediate filaments. These changes were accompanied by a more obvious interaction of these filaments with junctional complexes. Surface area and volume of the MCF-7 cells increased markedly after ATRA exposure, with extensive filopodia formation. Liarozole (10(-6) M) alone had no effect on cell morphology, cytokeratin or actin organization, or on cellular junctions. In combination with ATRA (10(-9) M and 10(-8) M), liarozole potentiated the ATRA-induced effects. The MCF-7 cell cultures used showed morphological heterogeneity, consisting of at least two cellular subpopulations. This was reflected in the staining for E-cadherin, desmoglein and desmoplakins I & II. ATRA increased E-cadherin staining at cell-cell contact sites, but had no influence on the staining patterns of desmoglein and desmoplakins I & II. Similar to what has been observed for the cytoskeletal differentiation parameters, liarozole alone had no influence on E-cadherin, desmoglein or desmoplakins I & II expression, but in combination with ATRA again intensified the effects on E-cadherin distribution. These effects on MCF-7 cells agree with previously obtained observations concerning the inhibition of ATRA catabolism by liarozole. Furthermore, our data support the hypothesis that the antiproliferative properties of the drug are accompanied by induction of differentiation.

Antineoplastic Agents, Hormonal↗
PubMed1995

Liarozole, an antitumor drug, modulates cytokeratin expression in the Dunning AT-6sq prostatic carcinoma through in situ accumulation of all-trans-retinoic acid.

Liarozole showed antitumoral activity in the Dunning AT-6sq, an androgen-independent rat prostate carcinoma. To investigate its potential mechanism of action, the effects of the drug doses (ranging from 3.75 to 80 mg/kg b.i.d.) on endogenous plasma and tissue all-trans-retinoic acid levels and on the differentiation status of the tumor cells were evaluated. To follow modulation of differentiation, cytokeratins were localized in the (un)treated tumors by immunocytochemistry and quantitatively determined by immunoblotting. Results showed that liarozole statistically significantly reduced tumor weight from 30 mg/kg upwards and induced accumulation of all-trans-retinoic acid both in plasma and tumors. In the tumors, a statistically significant accumulation was already noted from 7.5 mg liarozole/kg upwards. Concomitantly, the differentiation status shifted from a keratinizing towards a non-keratinizing squamous carcinoma, which was further confirmed by the cytokeratin profile of the carcinoma (presence of CK 8, 10, 13, 14, 18, 19). Immunoblotting revealed an overall decrease in cytokeratin content, except for CK 8. These findings suggest that the antitumoral properties of liarozole might be related to an increase in the degree of tumor differentiation through accumulation of all-trans-retinoic acid.

Animals↗
PubMed1995

The contribution of stimulus attributes of three-dimensional solid forms and level of discrimination to the visual and haptic percept of balance.

Design-trained subjects sorted fourteen solid forms under either visual or haptic conditions into groups on the basis of perceived similarity of balance or along a balance continuum. After these tasks of coarse and fine discrimination, each form was rated on ten bipolar stimulus attributes. Multidimensional scaling and property-fitting analyses were performed on the results of four experiments to determine the combined contribution of stimulus attributes and level of discrimination on the visual and haptic percept of balance. A balance dimension emerged for both modalities, but only when subjects were inclined to attend globally to the structure of a form by the coarse-discrimination task. Results indicate that visual balance is a holistic property of forms which derives from the synthesis of physical stimulus information. For touch, subjects appear to have equated balance with a symmetric distribution of weight/shape about the central axis of a form. Findings are related to theoretical notions of balance.

Adult↗
PubMed1991

Identification of a D1 dopamine receptor, not linked to adenylate cyclase, on lactotroph cells.

1. We studied the lactotroph cells of the rat by both in vivo and in vitro pharmacological techniques for the presence of D1-receptors. Both approaches revealed the presence of D2-receptor, stimulated by quinpirole (resulting in an inhibition of prolactin secretion) and blocked by domperidone. 2. Administration of fenoldopam, the most selective D1-receptor agonist currently available, resulted in a dose-dependent decrease of prolactin secretion in vivo (after pretreatment with alpha-methyl-p-tyrosine) and in vitro (cultured pituitary cells). This increase was dose-dependently blocked by the selective D1-receptor antagonist, SCH 23390, and although the effect of fenoldopam was less than that obtained by D2-receptor stimulation, these data suggest that a D1-receptor also controls prolactin secretion. 3. In order to detect the location of these dopamine receptors, autoradiographic studies were performed by use of [3H]-SCH 23390 and [3H]-spiperone as markers for D1- and D2-receptors, respectively. Specific binding sites for [3H]-SCH 23390 were demonstrated. Fenoldopam dose-dependently reduced [3H]-SCH 23390 binding, but had no effect on [3H]-spiperone binding. Immunocytochemical labelling of prolactin cells after incubation with [3H]-SCH 23390 revealed that the granulae and hence, D1 binding sites were present on the lactotroph cells. 4. Radioligand binding studies performed on membranes from anterior pituitary cells revealed the presence of the D2-receptor (54 fmol mg-1 protein) with a Kd of 0.58 nM for [3H]-spiperone, but failed to detect D1-receptors. 5. Finally, we studied the effect of dopamine and of fenoldopam on the adenosine 3':5'-cyclic monophosphate (cyclic AMP) content of anterior pituitary cells. Although cyclic AMP increased upon prostacyclin administration, indicating an intact adenylate cyclase system, fenoldopam failed to increase the cyclic AMP production. 6. It is tempting to speculate that fenoldopam reduces prolactin secretion through interaction with a non-cyclase-linked D1-receptor on the lactotroph cells.

Adenylyl Cyclases↗
PubMed1991

Receptors for corticotropin-releasing hormone in human pituitary: binding characteristics and autoradiographic localization to immunocytochemically defined proopiomelanocortin cells.

Using autoradiography combined with immunocytochemistry, we demonstrated that the target cells of CRH in the human pituitary were proopiomelanocortin cells. Scatchard analysis of [125I]Tyr0-oCRH saturation binding revealed the presence of one class of saturable, high affinity sites on pituitary tissue homogenate. The equilibrium dissociation constant (Kd) for [125I]Tyr0-oCRH ranged from 1.1-1.6 nM, and the receptor density was between 200-350 fmol/mg protein. Fixation of cryostat sections with 4% paraformaldehyde before tracer incubation improved both tissue preservation and localization of the CRH receptor at the cellular level. Additional postfixation with 1% glutaraldehyde inhibited tracer diffusion during subsequent immunocytochemistry and autoradiography. [125I]Tyr0-oCRH was found in cytoplasmic inclusions or at the cell periphery of ACTH/beta-endorphin cells in the anterior pituitary. Single cells of the posterior pituitary were also CRH receptor positive. Cells staining for PRL or GH were CRH receptor negative. We conclude that CRH binds only to high affinity receptors on ACTH/beta-endorphin cells in the human pituitary.

Adrenocorticotropic Hormone↗
PubMed1990

Metastasizing neuroendocrine carcinoma of the larynx with calcitonin and somatostatin secretion and CEA production, resembling medullary thyroid carcinoma.

A 55-year-old man presented with a metastasizing moderately differentiated neuroendocrine carcinoma of the larynx (atypical carcinoid). Immunocytochemical demonstration of neuroendocrine markers (neuron-specific enolase and chromogranin-A) and presence of membrane-bound neurosecretory granules in the cells established the neuroendocrine nature of the tumour. In addition, the tumour was found to produce calcitonin, somatostatin and carcino-embryonic antigen (CEA). Calcitonin and somatostatin were also secreted. On the basis of this particular marker constellation the tumour closely resembles medullary thyroid carcinoma. Review of the recent literature on carcinoids of the larynx reveals immunoreactivity for calcitonin and CEA in a high percentage of cases.

Brain Neoplasms↗
PubMed1989

Ontogeny of hormone-secreting cells of the rat pituitary gland: an immunocytochemical study on dissociated cells.

An immunocytochemical study was undertaken in foetal, prepubertal and mature rats to determine the time of differentiation of various types of adenohypophyseal cells during development. Freshly dissociated pituitary cells from foetal (18-21 days postconception), neonatal (from birth up to 30 days) and adult rats (more than 8 weeks) were characterized using immunocytochemical methods. All types of hormone-producing cells were present at day 18 postconception, although only 20% of the cells were immunolabelled. Adrenocorticotropin (ACTH)-secreting cells accounted for the highest number of hormone-positive cells. Growth hormone-secreting cells increased remarkably from day 18 postconception onwards. Prolactin-secreting cells were not seen in the foetal adenohypophysis and did not start to increase until 10 days after birth, whereas by that time the number of ACTH, thyrotropin, follicle-stimulating and luteinizing hormone-secreting cells had stopped increasing. By day 30 after birth, 80-95% of the cells were immunoreactive.

Adrenocorticotropic Hormone↗
PubMed1989

Transcriptional pattern of 21-hydroxylase gene (P-450C21) during embryonic development, before, and after birth in mice as determined by in situ hybridization.

21-Hydroxylase is a member of the P-450 superfamily of genes involved in the biosynthesis of cortisol and aldosterone in the adrenal cortex. Congenital adrenal hyperplasia, a well-characterized disease, originates from a lack of this enzyme. We present in this report an in situ hybridization study aimed at detecting 21-hydroxylase activity during murine development, from mid gestation to adulthood. Our results demonstrate that even during the embryonic period the adrenal cortex is the only major site of transcription of this enzyme, which is detectable beginning at embryonic day 14. In addition, a peculiar topographical pattern of transcriptional activity, characteristic of the stage of differentiation of the gland, could be drawn. Using a computer-assisted method, we were able to quantitate the relative transcription level at each stage of development. A steady increase in the level of transcription was demonstrated throughout embryonic life to birth, with a drop during the prepubertal period and a final rise at adult age. The possible physiological significance of our findings is discussed.

Adrenal Cortex↗
PubMed1989

Fully automated measurements by light microscopy of tissue sections using a cellular array computer.

Software was developed for the acquisition, segmentation and analysis of microscopic OD-images on a VICOM digital image processor, extended with a VISIOMORPH morphoprocessor board. The delineation algorithms for peroxisomes, lysosomes, and nuclei in liver, kidney, and adrenal gland sections start by thresholding the difference between the original image and a low pass filtered version. The resulting binary mask is then processed by morphological operations in order to produce an object overlay. The efficiency of the programs is evaluated by comparing delineated objects at different OD-levels, created by varying the stain or by multiplying the original pixel values with constant factors. Manual delineation on some images is also used as a reference. More complex algorithms are used for the delineation of muscle fibres in ATP-ase-stained sections and immunocytochemically labelled cells in monolayer preparations. Muscle images from parallel sections with different stainings are matched with a coordinate transform, enabling the transfer of the object mask from a single delineated image to the unprocessed images and thus obtain all necessary information for fibre classification. After segmentation, the OD-images and their object overlays are fed into a data extraction program, measuring for each delineated object user-selected features. Data are sent to a VAX for statistical interpretation.

Adrenal Cortex↗
PubMed1988

Prolactin cell subpopulations separated on discontinuous Percoll gradient: an immunocytochemical, biochemical, and physiological characterization.

A single-step procedure was devised to separate PRL cells from the rat anterior pituitary gland. After dissociation, cells were centrifuged on a Percoll gradient. Three layers were recovered. The composition of the different layers was evaluated using immunocytochemistry (with antisera to the six pituitary hormones), and in situ hybridization [with DNA complementary to PRL or to GH messenger RNA (mRNA)]. Both methods yielded identical values. PRL cells were recovered in the lower density layer (layer 1) with a good yield (that is 81% of the total PRL cells of the initial cell suspension) and in addition, markedly enriched (indeed 85% of the cells in layer 1 stained for PRL). A second layer (layer 2: intermediate density) contained most of the remaining PRL cells which were, however, heavily contaminated mainly by GH cells and cells that did not stain for any of the known pituitary hormones. A third layer (layer 3: higher density) was enriched in GH cells to 93% (representing, however, only 10% of the initial pituitary GH cells). In addition, PRL and GH were measured by RIA in culture medium and in cell lysates. Hormone biosynthesis was monitored by polyacrylamide gel electrophoresis and autoradiography after culture in the presence of [35S]methionine. These experiments confirmed that layer 1 was enriched in cells containing, and producing, PRL and depleted from GH cells. Cells in layer 2 contained and produced more GH than PRL. PRL cells from layer 1 responded to dopamine and to vasoactive intestinal polypeptide in the same way as PRL cells in the unseparated pituitary cell population. In contrast PRL cells in layer 2 had a lower basal secretion rate but a higher response to vasoactive intestinal polypeptide. Unless this represents a paracrine effect of non-PRL cells, PRL cells in layer 2 exhibit different properties and may therefore form a distinct subpopulation of PRL cells.

Animals↗
PubMed1988

Discrepancy between prolactin (PRL) messenger ribonucleic acid and PRL content in rat fetal pituitary cells: possible role of dopamine.

Data are controversial concerning the time when PRL-synthesizing cells are detected for the first time in the rat pituitary. Using a very sensitive immunocytochemical technique, we could visualize only a few PRL cells before day 10 after birth. At that time, pituitary PRL was still 200 times less abundant than in the adult (on a tissue weight basis) whereas PRL mRNA per mg total RNA was only 80 times lower than in the adult. However, by in situ hybridization, we could demonstrate the presence of PRL mRNA in cells from fetal day 18 on. We have also followed the expression of GH gene in rat pituitary cells during development. In contrast to results obtained with PRL cells, quantitative analysis of cDNA probe hybridization to GH mRNA correlated well with measurements of immunostained cells. We found that PRL was released in the blood from fetal day 19 onwards. Thus, at that time PRL is synthesized and secreted but not stored. We therefore measured brain dopamine levels, and the data support the idea that the rise in dopamine levels after birth contributes to PRL storage. We confirmed in vitro that newborn pituitary cells can store PRL when cultured in the presence of dopamine.

Animals↗
PubMed1987

Linear Percoll gradient centrifugation of rat anterior pituitary cells. A simple method for prolactin cell enrichment.

Prolactin (PRL) cells were purified from nulliparous normal female adult Wistar rat pituitary cell suspensions by linear Percoll density gradient centrifugation, a procedure yielding single cells. Lactotrophs were found in two different layers, the first containing 70% PRL cells in the density range 1.055 to 1.065 g/ml, the second with 28% PRL cells in the range 1.070 to 1.080 g/ml. Both cell fractions contained more than 90% viable cells with an intact ultrastructure. The physiological integrity of the 70% enriched PRL cells was assessed by their basal PRL secretion, their secretory response to TRH and dopamine, and their cAMP production in a basal situation and after incubation with dopamine.

Animals↗
PubMed1987

Immunocytochemical and ultrastructural findings in a mature retroperitoneal teratoma.

Report is made of a mature retroperitoneal teratoma in a 32-year-old man. Investigation of the tumor revealed cells immunoreactive for ACTH, Met-enkephalin, beta-LPH, serotonin, FSH, BPP, S100, Neuron-specific-enolase. These cells were mainly present in the glandular epithelium, lining the cysts of the tumor. Ultrastructurally, neuro-secretory granules were demonstrated in the cytoplasm of the tumoral endocrine cells. At no time did the patient display endocrine symptoms.

Adrenocorticotropic Hormone↗