Dagmar-Ulrike Richter

Cathepsin D expression in normal, hyperplastic and malignant endometrial tissue: an immunohistochemical analysis

Cathepsin D (CathD), a lysosomal aspartyl protease secreted by normal and malignant cells, is considered to be involved in breakdown of the extracellular matrix. Aim of the present study was to determine the frequency and tissue distribution of CathD in normal, hyperplastic and malignant endometrium. Paraffin-fixed endometrial tissue was obtained from premenopausal women in the proliferative phase (n = 5), early secretory phase (n = 4) and late secretory phase (n = 4) as well as glandular-cystic hyperplasia (n = 5), endometrial polyps (n = 5), endometrial polyps from the use of tamoxifen (n = 5), adenomatous hyperplasia (AH) grade I (n = 5), grade II (n = 4), grade III (n = 5) and endometroid adenocarcinoma (n = 5). CathD expression was evaluated with the IRS score and ANOVA analysis was used for statistical evaluation. CathD was primarily localised in luminal and glandular epihelium with little staining in stromal cells. The expression of CathD was significantly higher during the late secretory phase than in the proliferative phase. Highest expression of CathD was observed in the late secretory phase and in glandular-cystic hyperplasia, whereas endometroid carcinoma showed no expression. A continuous increase in CathD expression was observed in AH, with a significant difference between AH grade I and III. In conclusion, CathD was found to be expressed in normal and hyperplastic endometrial tissue. CathD immunostaining in normal endometrial glands varied on the basis of the phase of the menstrual cycle, suggesting physiological functions of CathD in endometrial maturation and degradation. Adenocarcinomas did express significant lower amounts of CathD. Therefore, the prognostic value of this parameter remains uncertain. A continuous increase in CathD immunostaining was observed in AH. Since AH grade III can be considered as a precursor of endometrial cancer, CathD could be a possible parameter for assessing malignant transformation.

Immunohistochemical expression of inhibin-alpha in human endometrium and the in vitro secretion of inhibin, estradiol and cortisol in cultured human endometrial glandular cells

BackgroundInhibins are multipotent dimeric glycoproteins, composed of an α-subunit and one of two possible β-subunits (βA or βB). Aims of this study were (a): the immunohistochemical characterisation of normal human endometrium for the inhibin-α subunit; (b) the assessment of the secretion and metabolism of inhibin, E2 and cortisol; (c) the evaluation of any relationship between these three substances in cell culture medium of isolated and cultivated normal human endometrial glandular cells.Materials and methodsSamples of human endometrium were obtained from 34 premenopausal patients. Nineteen endometrial specimen (proliferative [PP] n=8; early secretory [ES] n=7; late secretory phase [LS] n=4) were brought into cell culture. Fifteen endometrial specimen (PP n=5; ES n=5; LS n=5) were paraffin-fixed and used for the immunohistochemical analysis for inhibin-α. Stromal and epithelial cells were separated by collagenase digestions, filtrations, sedimentations and Ficoll-gradient centrifugation. E2 and cortisol were measured with radioimmunoassay (RIA) and inhibin with enzyme-immunoassay (EIA). Statistical analysis was performed with the non-parametric Mann-Whitney rank-sum test and linear regression analysis.ResultsInhibin-α showed a weak (positive) expression during proliferative phase, which increased significantly as the menstrual cycle continued. In secretory glands the mean inhibin concentration was higher than that from proliferative samples. A significant correlation was observed between inhibin and E2 (p<0.001) as well as cortisol and inhibin (p<0.0001) in glands from proliferative phase. Between inhibin and E2 (p<0.05) as well as inhibin and cortisol (p<0.002) a significant correlation in early secretory glands was also noted. In late secretory phase inhibin and E2 (r2=0.78650; p<0.0001), inhibin and cortisol (r2=0.58326; p<0.001) and E2 and cortisol (r2=0.52880; p<0.001) showed a significant correlation.DiscussionIn conclusion, we found a cyclical expression of inhibin-α subunit in the endometrium demonstrated by immunohistochemical means. A higher in vitro secretion of inhibin from secretory glands was also observed. In addition, a significant correlation between inhibin with E2 and cortisol in PP and ES glands and a significant correlation between inhibin, E2 and cortisol in LS glands could also be demonstrated. We conclude that inhibin can be associated with E2 and cortisol metabolism, playing an important role in paracrine/autocrine mechanisms in the endometrium and possibly exerting its function through cortisol and E2. The cortisol concentration also correlates with E2, suggesting a link between these steroids in the endometrial function. The correlation of inhibin, E2 and cortisol suggest complex autocrine/ paracrine mechanisms in human endometrial glands, modulated and controlled by all these three substances.

Absolute quantification of human chorionic gonadotropin-β mRNA with TaqMan™ detection

We describe a reverse transcriptase-polymerase chain reaction (RT-PCR) for determination of human chorionic gonadotropin-β (HCGβ) mRNA copies using the TaqMan™ system. To evaluate our quantitative assay, we analyzed HCGβ transcripts of all protein coding genes (HCGβ 5, 3, 8, and 7) in human RNA panels of different normal tissues and in glycodelin-A-stimulated trophoblast cell cultures. Absolute quantification using HCGβ TaqMan probe was found to be highly reproducible. Our study of RNA panels confirms recently published results that expression of HCGβ transcripts is a common feature of a great variety of different normal tissues. High levels of HCGβ mRNAs (>1.000 molecules per 200 ng RNA) were detected in placenta, uterus, and testis. An increase of HCGβ mRNA expression (1.7-fold) was detected at 150 µg/mL glycodelin-A treatment in trophoblast cell culture. Time-dependence study showed that the increase in HCGβ mRNA level was evident at 60 min after glycodelin-A treatment. In summary, we have developed a highly sensitive one-tube, one-enzyme quantitative RT-PCR system that is time-saving and avoids postamplification procedures.

Influence of phytoestrogens on the proliferation and expression of adhesion receptors in human mammary epithelial cells in vitro.

Tumor metastasis is associated with integrin-mediated adhesion and hyaluronan receptor expression. Accumulating evidence suggests that phytoestrogens, which are naturally occurring, plant-derived phytochemicals, could inhibit tumorigenesis during the development of breast cancer. Less is known, however, about the regulation of adhesion receptors by phytoestrogens and, particularly, their potency to influence proliferation of primary human breast cells in comparison with the steroid hormone 17&bgr;-estradiol. Throughout the proliferation experiments, we used primary human mammary epithelial cells from normal tissue that was derived from plastic surgery. For receptor expression (&bgr;1, &agr;2, &agr;3, CD44), we used the cell line MCF-7. Both investigations were carried out by flow cytometry. The phenotype of primary human mammary epithelial cells was microscopically characterized by analyzing the distribution of ZO-1, cytokeratin and the estrogen receptors &agr; and &bgr;. The integrins and the hyaluronan receptor were significantly up-regulated with 17&bgr;-estradiol in human MCF-7 cells. In contrast, genistein and daidzein did not affect the expression at a concentration of 100 μmol/l. In all proliferation experiments with a significant stimulation of the primary human mammary epithelial cell growth due to 17&bgr;-estradiol, in general, genistein and daidzein did not influence S-phase and G2/M-phase cells. Additionally, the stimulative effect of 17&bgr;-estradiol could be inhibited. As the phytoestrogens do not up-regulate adhesion receptors in human breast cells and, regarding proliferation, are able to abolish the stimulatory effect of 17&bgr;-estradiol, we suggest that phytoestrogens could have beneficial effects for the prevention or inhibition of carcinogenesis in hormone-dependent malignancies.

Stimulation of hCG protein and mRNA in first trimester villous cytotrophoblast cells in vitro by glycodelin A

Abstract Aim: Human chorionic gonadotropin (hCG) is produced by fetal trophoblast cells and secreted into maternal circulation mainly in the first trimester of pregnancy. Another glycoprotein, glycodelin A, is one of the main products of the maternal decidua during this period. The purpose of this study was to investigate the effect of glycodelin A on hCG release by isolated cytotrophoblast cells in vitro. Methods: Cytotrophoblast cells were prepared from human first trimester placenta and incubated with varying concentrations of glycodelin A. Supernatants were assayed for hCG protein concentrations, and quantification of beta hCG mRNA was carried out by RT-PCR. Expression of hCG was analysed in stimulated trophoblast cells and in unstimulated controls by immunocytochemistry. Results: Glycodelin A induces a dose-dependent increase of hCG production. An increase of hCG expression was measured at 100 and 200 μg/mL glycodelin-A treatment in trophoblast cell culture by TaqMan assay on mRNA level. We found a moderate staining of hCG in control trophoblast cells, whereas a strong hCG staining was seen in glycodelin A-treated trophoblast cells. Conclusions: HCG is a marker for the differentiation process of trophoblast cells. Our results suggest that glycodelin A secreted by the decidualized endometrium is involved in the regulation of hormones produced by the trophoblast.

Angiogenic factors and acute-phase proteins in serum samples of preeclampsia and HELLP patients: a matched-pair analysis

Objectives: To investigate the serum level distribution of angiogenic markers (PlGF, endoglin, sFlt-1) and acute-phase proteins (SAA, CRP) in patients with HELLP syndrome or preeclampsia (PE) including matched controls. Methods: The matching procedure revealed 46 controls for 23 HELLP cases, and 81 controls for 42 preeclamptic patients. Maternal serum concentrations were determined by immunoassays. Results: SAA and CRP levels were significantly higher in HELLP patients compared with controls. This finding was not observed in preeclamptic subgroup. Pro-angiogenic PlGF is significantly lower in PE and HELLP syndrome. Anti-angiogenic endoglin is significantly higher in PE and HELLP syndrome. The sFlt-1 analysis supports the anti-angiogenic shift in HELLP and preeclamptic patients, but with smaller differences between subgroups. The SAA/PlGF ratio showed the highest ROC value of all tested parameters in discrimination between HELLP and HELLP controls. Conclusions: These findings support the concept that patients with HELLP syndrome have both an anti-angiogenic state and a pronounced inflammatory response, while patients with PE are characterized only by an anti-angiogenic shift.

Regulation of progesterone production in human term trophoblasts in vitro by CRH, ACTH and cortisol (prednisolone)

Background: In most mammals, onset of labor is accompanied with progesterone withdrawal. In humans, cortisol blockade of progesterone is a possible mechanism involved in the initiation of labor. Therefore, aim of the study was to clarify the effect of CRH, ACTH and cortisol (prednisolone) on the release of progesterone by term trophoblast cells in vitro. Methods: Cytotrophoblast cells were prepared from human term placentas by standard dispersion of villous tissue followed by a percoll gradient centrifugation step. Trophoblasts were incubated with CRH, ACTH as well as with prednisolone Results: The release of progesterone is decreased in CRH- and ACTH-treated trophoblast cell cultures compared to untreated trophoblast cells. Addition of prednisolone in varying concentrations leads to an increase of trophoblast progesterone production. Conclusions: The results suggest that CRH and ACTH directly modulate the endocrine function of trophoblasts in culture by downregulating progesterone production. Prednisolone on the other hand showed a stimulating effect on progesterone production in term trophoblast cells in vitro. Because blockade of progesterone is a possible mechanism involved in initiation of labor, we may speculate that CRH and ACTH are directly involved in the auto- or paracrine regulation of this procedure.

Development and characterization of monoclonal antibodies for the immunohistochemical detection of glycodelin A in decidual, endometrial and gynaecological tumour tissues

Aims : Glycodelin is a glycoprotein with a molecular weight of 28 kDa. Unusual LacdiNAc structures have been identified on glycodelin A, isolated from amniotic fluid. Three major functions of this glycoprotein have been identified. Glycodelin is an immunosuppressive molecule, a marker of morphological differentiation, and a contraceptive. Because no monoclonal antibodies for glycodelin A are commercially available, our aim was to develop and characterize three monoclonal antibodies against this glycoprotein.

Effects of phytoestrogens genistein and daidzein on production of human chorionic gonadotropin in term trophoblast cells in vitro

Background. Phytoestrogens are a diverse group of non-steroidal compounds that occur naturally in many plants. Because they possess a ring system similar to estrogens they are able to bind to estrogen receptors in humans. In the present study we tested the effects of the phytoestrogens genistein and daidzein on the production of human chorionic gondaotropin (hCG) in isolated trophoblast cells of term placentas in vitro. Methods. Genistein and daidzein were incubated at different concentrations with trophoblast cells. Untreated cells were used as controls. At designated times aliquots were removed and tested for hCG production. Results. Production of the protein hormone hCG was influenced by the phytoestrogens genistein and daidzein in trophoblast cells. We found a significant decrease of hCG production in genistein- and daidzein-treated trophoblast cells that was concentration-dependent. Compared with daidzein, genistein seems to be a more efficient inhibitor of the production of hCG. Conclusion. The phytoestrogens genistein and daidzein can reduce hCG production in human term trophoblasts. Both phytoestrogens belong to the group of isoflavones, which are enriched in soy-containing foods and are widely consumed by humans for putative beneficial health effects. Because both phytoestrogens have inhibitory effects on hCG production during pregnancy, exposure to these estrogen-like compounds during sensitive periods of development may have the capacity to alter the function of the reproductive system and thereby influence fertility.

Antiproliferative activity of lignans against the breast carcinoma cell lines MCF 7 and BT 20

PurposePhytoestrogens are plant-derived, non-steroidal phytochemicals with anticarcinogenic potential. The major structural classes are the isoflavones and lignans. The aim of this study was to compare the effect of the plant-derived lignans secoisolariciresinol and matairesinol with the human lignans enterodiol and enterolactone as well as with 17β estradiol and tamoxifen on cell proliferation of breast carcinoma cell lines.MethodsThe influence of the lignans, 17β estradiol and tamoxifen on cell proliferation was determined using the BrdU test in MCF 7 and BT 20 cell lines.ResultsEnterodiol and enterolactone induced a stronger inhibition of cell growth in MCF 7 and BT 20 cells than secoisolariciresinol and matairesinol. The inhibition effects were less expressed in the BT 20 than in the MCF 7 cells.ConclusionsThe human lignans enterodiol and enterolactone are more biologically active than their precursors secoisolariciresinol and matairesinol, and may be defined as the real drugs in cancer prevention.