Estrone and dehydroepiandrosterone sulfatase activities and plasma estrone sulfate levels in human breast carcinoma.

The activity of the two membrane-bound sulfatases, estrone and dehydroepiandrosterone sulfatases, are reported in human breast carcinoma tissues. In 21 tested tumors (12 from post-menopausal women and 9 from nonmenopausal women), the two sulfatases were consistently present. The apparent Km values for estrone and dehydroepiandrosterone sulfatases were, respectively, 6.8 and 14.9 microM. In terms of maximal velocity, the sulfatase activities are not correlated to the estrogen or progesterone receptor status of the tumors or to the hormonal status of the donors. It may be concluded that these two activities are not hormone dependent. Estrone sulfate, the substrate of estrone sulfatase, has been measured in plasma of postmenopausal women. The mean levels (nmol/liter) of plasma estrone sulfate were compared in post-menopausal women with (n = 51) or without (n = 39) breast cancer. For the first age group (48 to 55 years old), no statistically significant difference in these levels was observed [1.91 +/- 1.06 versus 1.50 +/- 1.04 (mean +/- t0.95 (Formula: see text) S.E.)]. For the two other age groups (56 to 65 and 66 to 80 years of age), the differences were statistically significant [1.46 +/- 0.43 versus 0.77 +/- 0.21 (p less than 0.02) and 1.77 +/- 0.53 versus 0.81 +/- 0.22 (p less than 0.01)]. The usefulness of plasma estrone 3-sulfate levels as an indicator of the real estrogen status of postmenopausal women is discussed.

Effect of progesterone on hydrophobic cell-associated proteoglycans bound to cholesterol sulfate in glandular epithelial cells of guinea-pig endometrium.

Sulfate incorporation was studied in subcultured glandular epithelial cells of guinea-pig endometrium untreated or treated with 10(-8) M 17 beta-estradiol alone or associated with various concentrations of progesterone. In the cells treated with progesterone in association with 17 beta-estradiol, the maximum of the 35S-labelled cell-associated macromolecules failed to bind with an anion-exchange resin (53% of total radioactivity) and had a hydrophobic character. This fraction was separated as an aggregate when the cells were extracted with 4 M guanidine-HCl, and separated as a single component in the presence of Triton X-100, suggesting that it aggregates with cellular lipid. The guanidine-extracted material contained 23.5% proteoglycans. However, the bulk of the radioactivity was in the sulfated lipids (68-75%), essentially represented by cholesterol sulfate. In the progesterone-treated cells, the amount of cholesterol sulfate was significantly higher than in 17 beta-estradiol-treated or untreated cells (1.35-1.5-fold). Thus, the effect of progesterone is located on a lipophilic proteoglycan associated with cholesterol sulfate. These results are discussed in relation to the preparation of the endometrium for embryo implantation.

Free fatty acids and fatty acids of triacylglycerols in normal and hyperkeratotic human stratum corneum.

The content of the free fatty acids and the fatty acids of triacylglycerols has been measured in human plantar stratum corneum from normal and hyperkeratotic subjects with palmoplantar keratoderma. Fatty acids of triacylglycerols in normal tissues showed a characteristic pattern with a relative abundance of short-chain length and unsaturated fatty acids. Free fatty acid fraction was characterized by the predominance of saturated compounds. The relative amount of short-chain and monoene fatty acids in the hyperkeratotic stratum corneum was increased. These results seem to show a defect in the maturation of fatty acids in the living epidermis and present new evidence that the abnormality of lipid metabolism can influence the process of desquamation in stratum corneum.

Adenosine 3',5'-monophosphate mediates progesterone effect on sulfate uptake in endometrial epithelial cells.

We have previously shown that progesterone increased sulfate uptake in glandular epithelial cells of guinea pig endometrium. To investigate whether cAMP might be the cause of the progesterone effect on sulfate uptake, cAMP accumulation and the effect of cAMP on sulfate uptake were evaluated in cells treated with 17 beta-estradiol alone or with progesterone. Progesterone provoked an increase in the intracellular cAMP accumulation in cells treated with 17 beta-estradiol. Moreover, cAMP or forskolin elicited the same marked increase in sulfate uptake as that observed with progesterone. The effect of progesterone on sulfate uptake was abolished by blocking either the cAMP pathway or the genomic action of progesterone and was independent of the cAMP-activatable apical chloride channel. This study is the first evidence of cAMP activation of sulfate uptake and suggests a genomic effect of progesterone on the production of cAMP which activates the sulfate transport system in a short term activation and a long term activation independent of transcriptional or translational events. The endometrium is a unique tissue that undergoes profound highly regulated modifications during the secretory phase in providing a suitable environment for embryo implantation. The regulation of sulfate uptake could participate in this process.

Progesterone control of fibronectin secretion in guinea pig endometrium.

Immunohistochemistry with a polyclonal antibody raised against human plasma fibronectin (Fn) was used to determine the localization of Fn in endometrial sections of guinea pig uteri isolated at the first, fourth, sixth, or tenth day of the estrous cycle. Immunoreactive Fn was constantly visualized in the endometrial stroma but absent from the epithelial layer. Fn was detected in the uterine lumen on the first or fourth day of the estrous cycle and was absent from the other sections. To determine the origin of this luminal Fn the ability of subcultured endometrial cells to produce Fn was tested, and the hormonal regulation of Fn secretion was studied. Cells were treated by estradiol alone or in association with progesterone, progesterone alone, or untreated. Whatever the hormonal treatment, stromal cells constantly secreted immunoreactive Fn into the culture medium. In the same way, the amount of Fn synthesized and basally secreted by epithelial cells was not affected by any hormonal treatments. However, Fn was found in the apical secretions of the untreated or estradiol-treated epithelial cells but was undetectable in the apical compartment when the epithelial cells were treated by progesterone alone or in association with estradiol. These results indicate that Fn is constitutively secreted by stromal cells and that subcultured epithelial cells of guinea pig endometrium secrete Fn from both their basal and apical membrane domains. However, the apical secretion of Fn is specifically suppressed by progesterone.

Estrone and dehydroepiandrosterone sulfatase activities in normal and pathological human endometrium biopsies.

The properties of estrone (E1) and dehydroepiandrosterone (DHEA) sulfatase activities are reported. Endometrial biopsy specimens were obtained using a Novak curette. Cycle stage was assessed from histological dating of endometrium, plasma estradiol and progesterone levels, and patient history. Both sulfatases are membrane-bound enzymes. The optimum pHs in Tris-HCl buffer were 6.5 for E1 sulfatase and 7.3 for DHEA sulfatase. Lowest activities and different optimum pHs were obtained with imidazole, maleate, or acetate buffers. DHEA sulfatase is more sensitive to thermal inactivation than E1 sulfatase. From kinetic studies, apparent Km values of 3.1 microM for E1 sulfatase and 5.7 microM for DHEA sulfatase were calculated. Noncompetitive inhibition of E1 sulfatase by DHEA sulfate and of DHEA sulfatase by E1 sulfate were demonstrated. The effects of inorganic ions and unconjugated steroids were also tested. These results are consistent with two different activities hydrolyzing E1 or DHEA sulfates. Neither activity varies during normal menstrual cycles nor is not correlated to plasma progesterone or 17 beta-estradiol levels. An isolated increase in E1 sulfatase occurred in the proliferative phase of irregular menstrual cycles, postantibiotic-treated salpingitis, or hyperplastic endometrium.

Insulin up-regulates vascular endothelial growth factor and stabilizes its messengers in endometrial adenocarcinoma cells.

Angiogenesis is crucial for tumor growth and dissemination. Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that promotes vascular growth and therefore tumoral growth and metastasis. Overweight, frequently associated with hyperinsulinemia, constitutes the major risk factor for endometrial carcinoma. Thus, elevated insulin levels may partly explain the increased risk of endometrial cancer observed in obese postmenopausal women. The aim of the present work was to test the role of insulin in the control of VEGF expression in endometrial carcinoma cells (HEC-1A). We have shown that insulin induced a biphasic expression of VEGF messenger ribonucleic acid, with an early, but low, induction (4 h of stimulation) and a delayed, but high, induction (24 h). The delayed effect of insulin on VEGF expression involved transcriptional and posttranscriptional regulation, as evidenced by the increased rate of VEGF transcription and the prolonged half-life of VEGF messenger ribonucleic acid. Simultaneously we observed higher levels of VEGF protein in the conditioned medium of stimulated cells compared with unstimulated ones. Therefore, insulin could contribute to the increased risk of endometrial carcinoma due to its ability to induce VEGF expression and thus participate in the maintenance of an angiogenic phenotype.

Gene transfer into subcultured endometrial cells using lipofection.

Lipofection using the Lipofectin reagent was optimized to transiently transfect subcultured guinea pig endometrial stromal cells with a beta-galactosidase gene driven by a simian virus 40 promoter. Efficient transfection was obtained in the following conditions: a value of six for the ratio of lipofectin to DNA, a low cellular density (10(5) cells per 35-mm well) at the time of subculture (48 h before lipofection) and a lipofection duration of 12 hours. Lipofection was compared to calcium phosphate precipitation previously optimized in the same culture model. At a low cellular density, the lipofection method was found to be more efficient than the calcium phosphate precipitation. This result gives a great relevance to lipofection since the cultured cells available in an experiment are often limited. Then, using cells at low density and a plasmid containing the chloramphenicol acetyltransferase (cat) gene linked to an estrogen response element, it was shown that the lipofection procedure is a suitable tool for the evaluation of gene regulation by estrogen.

Ultrastructural changes induced by oestradiol-17 beta, progesterone and oestrone-3-sulphate in guinea-pig endometrial glandular cells grown in primary culture.

The effects of oestradiol-17 beta, progesterone and oestrone-3-sulphate were studied in primary cultures of guinea-pig endometrial glandular epithelial cells. Comparative ultrastructural studies were performed by means of transmission electron microscopy on cells grown either without hormones or with oestradiol-17 beta (2 nmol/l), oestradiol-17 beta (2 nmol/l) plus progesterone (50 nmol/l), or oestrone sulphate (0.1 mumol/l). In the control medium, without steroid hormones, the majority of epithelial cells were poorly differentiated, although numerous small mitochondria were present and abundant lipid droplets could be observed. Oestradiol-17 beta stimulated metabolic activity in the cells. Progesterone added to oestradiol-17 beta-primed cells stimulated the development of the endoplasmic reticulum-Golgi system. Oestrone sulphate induced a higher level of differentiation characterized by large clear mitochondria, well-developed Golgi complexes, and active nuclei, suggesting secretory activity. In all cases, the cultured cells displayed deep invaginations of the nuclear membrane associated with nuclear pores, known as nucleolar channels. After treatment with oestrone sulphate these channels were associated with a characteristic reticular nucleolus. We conclude that cultured endometrial epithelial cells display secretory activity in response to treatment with oestradiol-17 beta plus progesterone, or with oestrone sulphate.

Specific effect of oestrone sulphate on protein synthesis and secretion by cultured epithelial cells from guinea-pig endometrium.

Patterns of induced protein synthesis and secretion in guinea-pig endometrial epithelial cell cultures in response to oestrone sulphate alone and oestrone sulphate plus progesterone were investigated. Epithelial cells were cultured for 3 days in growth medium, then washed three times in a steroid-free medium. For each experiment, anticytokeratin immunostaining was used to discriminate the epithelial cells from the stromal cells. Only experiments in which the control dishes displayed more than 80% of anticytokeratin-immunostained cells were further processed. After this period oestradiol-17 beta (20 nmol/l; control), oestradiol-17 beta (20 nmol/l) plus progesterone (0.5 mumol/l), oestrone sulphate (1 mumol/l) or oestrone sulphate (1 mumol/l) plus progesterone (0.5 mumol/l) were added to the medium for 48 h. An immunocytochemical progesterone receptor assay showed that oestradiol-17 beta increased the progesterone receptor content of cells, and progesterone added to cultured cells in the presence of oestradiol-17 beta induced a significant increase in oestrogen sulphotransferase activity assessing the hormone responsiveness of the cultured cells. In these culture conditions and after 16 h of incubation, oestradiol-17 beta induced a 1.7-fold increase in [3H]thymidine incorporation into DNA, and [35S]methionine incorporation into cellular proteins was linearly increased up to 8 h. Biochemical changes induced by the different hormone treatments were studied by labelling the proteins with a 6-h pulse of [35S]methionine. The proteins present in the medium and in cells were analysed by two-dimensional polyacrylamide gel electrophoresis, followed by fluorography.(ABSTRACT TRUNCATED AT 250 WORDS)

Progesterone effect on intracellular inorganic sulphate in uterine epithelial cells.

The effect of progesterone on the available intracellular sulphate pool in subcultured glandular epithelial cells from guinea-pig endometrium is reported. Progesterone in concert with 17 beta-estradiol was shown to cause an increase in the available intracellular sulphate pool. The maximum effect was obtained for 10(-8) M and 10(-7) M progesterone. This effect of progesterone on the available intracellular sulphate pool essentially concerned the intracellular inorganic sulphate and was inhibited by the antiprogesterone steroid RU 486 (5 x 10(-7) M). Sulphate incorporation into the endometrial epithelial cells was suppressed by the inhibitor of anion transport diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and the protein synthesis inhibitor, cycloheximide. These results would suggest that a sulphate transport system may be involved in the accumulation of the intracellular sulphate, stimulated by progesterone. This phenomenon could be an early process in the preparation of the endometrium for implantation.

Progesterone stimulates sulfate uptake in subcultured endometrial epithelial cells.

The effect of progesterone was studied on the sulfate entry in glandular epithelial cells of guinea-pig endometrium subcultured in bicameral chambers on matrix-coated filters in a chemically defined medium. At post-confluency (8 days of subculture), cells were treated with 10 nM estradiol alone or in association with various concentrations of progesterone. Optimal progesterone action was at a 16 h incubation time and a 10 nM hormonal concentration. Progesterone increased in a dose-dependent fashion the sulfate uptake specifically in glandular epithelial cells, preferentially from the basal surface. Progesterone effect on the sulfate uptake occurred only in estradiol-primed epithelial cells and was inhibited by the antiprogestin steroid RU-486. The progesterone-dependent increase in sulfate uptake was inhibited by the inhibitor of anion exchange, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). At physiological sulfate concentrations, progesterone essentially induces a high-affinity DIDS-sensitive transport system.

Transfected endometrial cultured cells: a system to study gene-regulation by estrogens.

Glandular epithelial (GE) and stromal cells were isolated from guinea-pig endometrium, cultured and subcultured separately. At the end of subculture, the purity of each cell population was higher than 95% and cells displayed a high level of estrogen receptors. Calcium phosphate transfection conditions were defined using a control plasmid containing the bacterial CAT gene driven by viral promoter and enhancer sequences. Transfection experiments were performed with other plasmids in which CAT gene was linked to different estrogen response elements (EREs) derived from those of vitellogenin genes. CAT activity was significantly increased by estradiol-17 beta treatment only when GE or stromal cells were transfected with plasmids containing EREs previously reported as functional EREs in other cell types. This induction was abolished by ICI 164,384 diethylstilbestrol was as effective as estradiol-17 beta for CAT induction and estradiol-17 alpha was ineffective. Transiently transfected endometrial cells in subculture are a suitable system to study the estrogen effect on gene regulatory elements.

Effects of 17 beta-estradiol and growth factors on c-fos gene expression in endometrial epithelial cells in primary culture.

Primary culture of guinea-pig endometrial cells was made quiescent by serum depletion. When added to quiescent cells, 17 beta-estradiol (E2) alone affected neither c-fos and c-myc gene expression, nor DNA synthesis and cell proliferation. Insulin or epidermal growth factor (EGF) only induced DNA synthesis. An association of both growth factors allowed significant cell proliferation without inducing c-fos or c-myc expression. A response including c-fos induction (maximal expression at 75 min), but not c-myc expression, DNA synthesis and a marked cell proliferation was only obtained when 17 beta-estradiol was associated with insulin plus EGF. In this case, cycloheximide raised the c-fos gene expression. These data suggest that in endometrial epithelial cells, E2 is mitogenic only when it acts in association with EGF plus insulin and the c-fos gene expression may not be correlated with cell proliferation.

ERE environment- and cell type-specific transcriptional effects of estrogen in normal endometrial cells.

Our previous results have suggested a repression of E2 (17beta-estradiol) effect on the c-fos gene of cultured guinea-pig endometrial cells. To investigate this repression, the expression of three human c-fos gene recombinants, pFC1-BL (-2250/+41), pFC2-BL (-1400/+41) and pFC2E (-1300/-1050 and -230/+41), known to be E2-responsive in Hela cells, was studied in stromal (SC) and glandular epithelial cells (GEC). In both cellular types, pFC1-BL was not induced by E2, even in the presence of growth factors or co-transfected estrogen receptor. The pattern of pFC2-BL and pFC2E expression was strikingly different and depended on the cellular type: pFC2-BL and pFC2E induction was restricted to the glandular epithelial cells and did not occur in the SCs. We argue for a repression of E2 action which is dependent on the estrogen-responsive cis-acting element (ERE) environment and also cell type-specific involving DNA/protein and/or protein/protein interactions with cellular type-specific factors.

Identification and characterization of an early estrogen-regulated RNA in cultured guinea-pig endometrial cells.

A cDNA library was prepared from quiescent guinea-pig endometrial glandular epithelial cells stimulated for 2 h with estradiol-17 beta (E2) in the presence of cycloheximide. It was screened by differential hybridization for estrogen-regulated sequences. Six recombinants containing E2-regulated sequences were identified. One of them, called gec1 was then characterized by Northern blot hybridization. The gec1 mRNA was 1,800 bases in size. A 2-fold increase in the gec1 mRNA level was achieved at 120 min after E2 treatment. The E2 action on gec1 gene required the presence of cycloheximide. The cloned gec1 cDNA was 1 kb in size. The sequence so far determined did not show similarity with well characterized genes. This is the first report on a cloned cDNA probe of early estrogen-induced mRNA in a primary culture of endometrial epithelial cells.

In vitro effect of synthetic progestogens on estrone sulfatase activity in human breast carcinoma.

The effect of progesterone and nine synthetic progestogens on the activity rate of microsome estrone sulfatase obtained from human breast carcinoma tissues was studied. The progestogens were classified into three groups: group I with a strict inhibitor effect: demegestone and chlormadinone acetate; group II with a strict activator effect: medroxyprogesterone acetate, quingestanol acetate, lynestrenol and progesterone and group III with a nonsignificant effect: dydrogesterone, promegestone, norgestrel and danazol. Demegestone was the most potent inhibitor and medroxyprogesterone acetate and quingestanol acetate had the highest activator effect. The effect of Triton X-100, a nonionic detergent, was also tested. This detergent consistently increased the microsome estrone sulfatase activity. A comparison was made between the effects of demegestone, medroxyprogesterone acetate and danazol on estrone sulfatase activity measured with or without Triton X-100 in the incubation medium. The presence of the detergent modified the progestogen action. Our results suggest that synthetic progestogens can influence the estrone sulfatase activity measured in human breast carcinoma tissues. However, the effect of progestogens was dependent on experimental conditions. Progestogens such as demegestone and chlormadinone acetate which inhibited estrone sulfatase activity in intact preparations, can reduce the intracellular production of biological active estrogen via the sulfatase pathway.

Proliferation of guinea-pig uterine epithelial cells in serum-free culture conditions: effect of 17 beta-estradiol, epidermal growth factor and insulin.

The effects of 17 beta-estradiol (E2), epidermal growth factor (EGF) and insulin, alone or in association on guinea-pig uterine epithelial cell proliferation were examined in serum-free culture conditions. Primary cultures of epithelial cells were made quiescent by serum depletion, then incubated in a chemically defined medium. In this medium, insulin increased DNA synthesis but not in a dose-dependent manner for concentrations ranging from 0.2 to 10 micrograms/ml. A significant effect of EGF was found only for the highest concentration tested (100 ng/ml). E2 alone or in the presence of insulin (1 microgram/ml) had no effect whatsoever on the concentration tested (10(-10)-10(-5)M). Insulin (10 micrograms/ml) plus EGF (100 ng/ml) exerted on DNA synthesis and cell proliferation a significant additive effect which was identical to the growth stimulation induced by 10% fetal calf serum. The effects of insulin plus EGF were not modified by the addition of E2. These findings suggest that E2 is not directly mitogenic for uterine epithelial cells in defined culture conditions and that the mitogenic response to optimal concentration of insulin plus EGF is independent of E2.

17beta-estradiol inhibits forskolin-induced vascular endothelial growth factor promoter in MCF-7 breast adenocarcinoma cells.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor whose expression is induced by the cAMP-dependent signalling pathway in several cell types, and by estrogens in some human breast cancer cells. Here, we investigated the cross-talk between estrogens and cAMP/PKA-dependent signalling pathway in human breast cancer MCF-7 cells. The results show that, in the absence of any CRE and ERE, forskolin induces whereas estrogens have no effect on VEGF promoter. Moreover, estrogens, through estrogen receptors, partly inhibit the forskolin-induced VEGF promoter in MCF-7 human breast cancer cells. Therefore, in breast cancers, estrogens could partly inhibit the effect of ligand-activated G protein-coupled receptors on VEGF expression.

Relationship between expansion of the CAG repeat in exon 1 of the androgen receptor gene and idiopathic male infertility.

OBJECTIVE: To determine whether expansion of CAG repeats in exon 1 of the androgen receptor is correlated with impaired spermatogenesis in patients with male idiopathic infertility. DESIGN: A retrospective study. SETTING: Medical school in Besançon, France. PARTICIPANT(S): Thirty-seven infertile patients with azoospermia or oligospermia and 50 fertile controls. INTERVENTION(S): History, physical, hormonal assays, semen analysis, and collection of blood samples in order to study the androgen receptor's gene. MAIN OUTCOME MEASURE(S): Blood samples were collected from each infertile patient and control. The length of the CAG repeat segment was evaluated by using polymerase chain reaction (PCR) electrophoresis in exon 1 and PCR single-strand conformation polymorphism in exons 2-8. RESULT(S): The mean length of the CAG repeats was significantly different between infertile and fertile patients (23.91 +/- 0.5 vs. 22.20 +/- 0.4). No mutation was detected in exons 2-8 of the androgen receptor gene in infertile patients. CONCLUSION(S): Expansion of the CAG repeat segment of the androgen receptor is correlated with male idiopathic infertility. The number of CAG repeats may therefore have a modulatory effect on normal androgen receptor function.