Hervé Mittre

Estradiol membrane binding sites on human breast cancer cell lines. Use of a fluorescent estradiol conjugate to demonstrate plasma membrane binding systems.

A fluorescent estradiol macromolecular complex was used to study and to characterize steroid binding to membranes of living target cells. Ligand binding to plasma membranes was quantitated with a sensitivity of 0.1 nM. In this way, we found two types of estradiol-binding sites on hormone sensitive MCF-7 cells. Type A sites (8000-16000 sites per cell) were rapidly saturated at low concentrations of the estradiol-bovine serum albumin-fluorescein isothiocyanate macromolecular complex (E2-BSA-FITC). They had a greater affinity for the complex than did the type B sites for which a phenomenon of cooperative fixation was shown. The complex binding was displaced by estrogenic molecules, but not by non-estrogenic compounds, such as cortisol or progesterone. We also studied complex binding on another breast cancer cell line, MDA-MB-231 (MDA), without intracellular estrogen receptors. These cells showed a specific plasma membrane binding system for estrogen, but lacked the high affinity type A binding site. Then, we report the effects of enzyme treatments (trypsin, phospholipase A2 and neuraminidase) on E2-BSA-FITC binding to MCF-7 cell membranes. The quantity of complex bound to membranes decreased after phospholipase and neuraminidase treatments and increased after trypsin. But, in the three cases, the binding was no longer specific because it could not be displaced by E2-BSA or by estradiol. The enzymatic effects were reversible and specific binding was totally restored within 24 h. However, in the presence of the protein synthesis inhibitor, cycloheximide, no restoration of specific binding occurred on trypsin-treated cells. Estrogen binding to MCF-7 and MDA cell plasma membranes thus possesses the three characteristics of all mediated transport processes across biological membranes: saturability, substrate specificity, and specific inhibition. However, the high affinity type A binding site was found only on the estrogen-sensitive cell line, MCF-7.

Array CGH analysis in high‐risk pregnancies: comparing DNA from cultured cells and cell‐free fetal DNA

To compare results of array comparative genomic hybridization (CGH) on cell‐free fetal (cff) DNA from amniotic fluid supernatant and DNA from cultured amniocytes in high‐risk pregnancies.

Estrogen response of MCF-7 cells grown on diverse substrates and in suspension culture: Promotion of morphological heterogeneity, modulation of progestin receptor induction; cell-substrate interactions on collagen gels☆

In this study we observed the incidence of hormone sensitivity in the response of MCF-7 cells to estrogen stimulation when the cells were cultured in different contact environments (hydrophilic plastic, bovine corneal extracellular matrix, type I collagen and in suspension culture). The major purpose was to describe the influence of cell to cell and cell to substrate contacts on the morphological response to estrogen treatment. However, other parameters including growth and induction of progestin receptor were also explored, keeping in mind that the MCF-7 cell line, although representative of normal mammary epithelium in that it contains a similar hormone receptivity, was selected in vitro from a metastatic population in a pleural effusion. Although substrate conditions did not modify growth enhancement by estrogens, progestin receptor levels were significantly higher in three-dimensional spheroid cultures in which cell to cell contacts were optimal due to elimination of basal contact. A careful morphological survey of large surfaces lead to an objective opinion of the overall effect of the hormone treatment on the non-cloned cell line in which a marked heterogeneity in the response of individual cells was observed. In terms of morphofunctional differentiation, the edification of acini with dense microvillus coating was best in suspension culture. When sections were made perpendicular to the plane of cultures on collagen gel rafts two other phenomena were noted: decrease in intercellular junctions, resulting in reduced cell to cell cohesion, and accumulation biodegradation products in the collagen lattice. This suggested a hormone-mediated interaction between the metastatic cells and the fibrillar substrate, collagen I, one of the major constituents of tissue stroma. This estrogen response might be related to the metastatic phenotype and must be distinct from their hormone sensitivity in terms of growth and differentiation since hormone receptivity is generally considered to be a favorable prognosis for breast cancer.

Supernumerary marker chromosomes management in prenatal diagnosis.

To assess the practical usefulness of array‐comparative genomic hybridization (a‐CGH) when supernumerary marker chromosomes (SMCs) are detected during prenatal diagnosis, we retrospectively studied SMC management in our laboratory before a‐CGH availability. In this 11‐year study, SMCs were observed in 20/16,810 routine karyotypes (0.12%). Their chromosomal origin, ascertained in 13 cases, remained elusive in seven using conventional cytogenetics and FISH. In the literature, most of SMCs (2/3) are easily identified through conventional cytogenetics and targeted FISH, and in these cases a‐CGH would have been unneeded. This technique would have been less helpful in nine cases, that is, bisatellited SMC, isochromosomes and translocation derivatives. On the other hand, a‐CGH would have been helpful for the 11 remaining cases. It would have improved diagnostic accuracy of six SMC whom chromosomal origin was ascertained by cytogenetics and FISH and for which prognosis was only based on literature and ultrasonographic data. Among five unidentified SMCs, a‐CGH would have been more reassuring for four heterochromatic SMCs than normal ultrasonography alone and would have characterized the unidentified case associated with malformations that was interrupted. However potential pitfalls should be outlined. Using high level resolution chip expose to polymorphism detection and misinterpretation, a very sensitive problem in prenatal diagnosis. Moreover, low grade mosaicism could remain undetectable with this technique, leading to erroneous conclusions. Wisest use of a‐CGH should be a complementary approach in prenatal management of SMC. It is specifically appropriate when SMC interpretation remains equivocal and only indirectly based on mode of inheritance, literature data and ultrasonography.

Aberrant adrenal sensitivity to vasopressin in adrenal tumours associated with subclinical or overt autonomous hypercortisolism: is this explained by an overexpression of vasopressin receptors?

Objective  Abnormal responsiveness to arginine vasopressin (AVP) was previously observed in cortisol‐producing adrenocortical tumours but the mechanism remains unclear. The aim of this study was to characterize the effect of AVP on cortisol secretion from adrenocortical tumours compared to normal human adrenal gland.

Cushing's syndrome associated with a nested stromal epithelial tumor of the liver: hormonal, immunohistochemical, and molecular studies

CONTEXT Ectopic ACTH syndrome (EAS) is principally associated with aggressive malignant tumors but also with neuroendocrine tumors of good prognosis. Recently, rare nonhepatocytic nested stromal and epithelial tumors (NSET) were characterized by their possible association with Cushings syndrome of which biochemical and physiopathological features were still incompletely studied. OBJECTIVE To describe the clinical and hormonal characteristics of an EAS originating from a liver NSET and further understand the mechanism of cortisol overproduction. DESIGN AND SETTING This is a clinical case report from the Endocrinology Department of Caen University Hospital, France. PATIENT AND INTERVENTION A 17-year-old female patient was found to have a large liver NSET with mild Cushingoid clinical features and intense biological hypercortisolism but moderate ACTH secretion. Resection of the tumor was curative with a 30-month follow-up. RESULTS The epithelial component of the tumor coexpressed ACTH mildly, corticotropin-releasing hormone (CRH) strongly, and 11beta-hydroxysteroid dehydrogenase at a level comparable with normal human hepatocytes. CONCLUSIONS Liver NSET is a new cause of EAS, which may evoke hypercortisolism by multiple biochemical pathways.

Involvement of the phospholipase C second messenger system in the regulation of steroidogenesis in small bovine luteal cells

We have previously suggested that the interaction between luteinizing hormone (LH) and its receptor, in addition to stimulating adenylate cyclase, is able to trigger a negative regulatory signal at a step beyond cAMP synthesis (Benhaim et al. (1987) FEBS Lett. 223, 321-326). The present study was conducted to determine whether the phospholipase C system is involved in this phenomenon. Small bovine luteal cells from pregnant cows were incubated with phospholipase C, A23187, an ionophore of calcium and/or phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), in the presence or absence of bovine luteinizing hormone or dibutyryl cyclic AMP (dbcAMP). A23187 associated with PMA was able to mimic the stimulatory effect of phospholipase C on basal progesterone production, whereas neither A23187 nor PMA alone had any effect. In the presence of high doses of LH, phospholipase C inhibited progesterone and cAMP production in a dose-dependent manner. A23187 and PMA were able to mimic the inhibition of progesterone synthesis but stimulated LH-induced cAMP accumulation. When cells were stimulated by high doses of dbcAMP, phospholipase C and A23187 but not PMA inhibited progesterone synthesis. These observations suggest that (1) phospholipase C can mimic the post-cAMP negative regulatory signal induced in vitro by high doses of LH, in the presence of an activation of PKC; (2) phospholipase C is also able to mimic in vitro the luteolytic properties of prostaglandin F2 alpha that we previously described (Benhaim et al. (1987) Prostaglandins 33, 227-239); and (3) under basal conditions or in the presence of low doses of LH, the phospholipase C system slightly stimulates steroidogenesis.

Aromatase expression in the normal human adult adrenal and in adrenocortical tumors: biochemical, immunohistochemical and molecular studies.

OBJECTIVE The aromatase enzyme catalyzes the final stage of estrogen biosynthesis pathway from androgens. Its expression in the adrenal is poorly studied except for the rare estrogen-producing adrenocortical tumors. In order to further characterize aromatase expression in the adrenal, we evaluated the aromatase enzyme activity, Cyp19a1 gene expression level, and promoter utilization in normal adrenal tissues and in adrenocortical secreting tumors. DESIGN AND METHODS Six normal adult adrenals (NA), 2 feminizing adrenal tumors (FT), 10 cortisol-producing adenomas with overt (CS, n=4) or sub-clinical Cushing syndrome (SCS, n=6) and 3 aldosterone-producing adenomas (APA) were studied. Tissue aromatase activity was determined by the tritiated ((3)H)-water method. Total aromatase mRNA were measured by a competitive RT-PCR. Promoter regions PII and PI.4-derived transcripts were also studied in NA, FT, and other steroid-producing tumors by a semi-quantitative comparative RT-PCR. Immunofluorescence analysis was performed in normal human adrenal tissues. RESULTS Aromatase activity was detected in NA tissues and in all tumor subtypes, at high levels in both FT. In NA, aromatase immunofluorescence was detected in the cytoplasm of steroidogenic cells, mainly from zona reticularis. Compared with NA, aromatase transcript levels were similar in CS and APA, lower in SCS and similar or higher in FT. Promoter analysis suggested predominant PII utilization in NA, APA, and SCS, but similar PII and PI.4 utilization in CS tumors. CONCLUSION Aromatase is expressed at similar levels in normal adrenal and in adrenocortical tumors, but at variably high levels in FT. Different promoter utilization patterns are found among tumor subtypes.

De novo 15q13.3 microdeletion with cryptogenic west syndrome

West syndrome is a well‐recognized form of epilepsy, defined by a triad of infantile spasms, hypsarrhythmia and developmental arrest. West syndrome is heterogenous, caused by mutations of genes ARX, STXBP1, KCNT1 among others; 16p13.11 and 17q21.31 microdeletions are less frequent, usually associated with intellectual disability and facial dysmorphism. So‐called “idiopathic” West syndrome is of better prognostic, without prior intellectual deficiency and usually responsive to anti‐epileptic treatment. We report on a boy falling within the scope of idiopathic West syndrome, with no dysmorphic features and normal development before the beginning of West syndrome, with a good resolution after treatment, bearing a de novo 15q13.3 microdeletion. Six genes are located in the deleted region, including CHRNA7, which encodes a subunit of a nicotinic acetylcholine receptor, and is frequently associated with epilepsy. Exploration of the 15q13.3 region should be proposed in idiopathic West syndrome.

Effects of phorbol esters on steroidogenesis in small bovine luteal cells

The possible influence of an activator of protein kinase C, the tumor‐promoting phorbol ester, PMA (phorbol‐12‐myristate‐13‐acetate), upon small bovine luteal cell steroidogenesis was investigated in vitro. PMA had no significant effect on basal and dibutyryl cyclic AMP (dbcAMP)‐stimulated progesterone production but markedly modulated the LH‐stimulated progesterone and cAMP productions. PMA potentiated the LH‐stimulated cAMP accumulation whatever the dose of LH used. It also potentiated the LH‐induced progesterone production in the presence of low doses of LH. Paradoxically, in the presence of maximal or submaximal effective doses of LH, PMA exerted a time‐ and dose‐dependent inhibition of progesterone synthesis. Diacylglycerol was able to mimic the effects of PMA on LH‐induced steroidogenesis. These observations suggest that the Ca2+‐ and phospholipid‐dependent protein kinase C can modulate the regulation by LH of small bovine luteal cell steroidogenesis at a step before the synthesis of cAMP. They also suggest that the interaction between LH and its receptor is able to trigger a negative regulatory signal which would be only expressed for high doses of LH and in the presence of an activator of PKC.