Claudia A. Krusche

Histone deacetylase-1 and -3 protein expression in human breast cancer: a tissue microarray analysis

SummaryImpaired histone acetylation was recognized to be involved in carcinogenesis. Furthermore, histone deacetylase (HDAC) inhibitors induce differentiation of breast cancer cells and inhibit tumour growth. These results prompted us to study HDAC-1 and -3 expression in breast tumours to establish their potential therapeutic and prognostic significance.HDAC-1 und HDAC-3 protein expression was analyzed immunohistochemically on a tissue microarray (TMA) containing 600 core biopsies from 200 patients. HDAC-1 and -3 expression was correlated to steroid hormone receptor-, Her2/neu- and proliferation status of tumours as well as to overall and disease free survival.Moderate or strong nuclear immunoreactivity for HDAC-1 was observed in 39.8% and for HDAC-3 in 43.9% of breast carcinomas. HDAC-1 and -3 expression correlated significantly with oestrogen and progesterone receptor expression (both p< 0.001). HDAC-1 expression predicted significantly better disease free survival (DFS: p=0.044), in particular, in patients with small tumours of all differentiation types (DFS: p=0.016). Multivariate analysis demonstrated that HDAC-1 is an independent prognostic marker.Our data suggest that evaluation of HDAC-1 protein expression enables a more precise assessment of the prognosis of breast cancer patients. Thus, HDAC-1 expression analysis might be clinically useful to facilitate an individual, risk-directed, adjuvant systemic therapy in breast cancer patients.

Progestins, progesterone receptor modulators, and progesterone antagonists change VEGF release of endometrial cells in culture.

The influences of the synthetic progestin, medroxyprogesterone acetate (MPA), the progesterone receptor modulator J867, and the antagonist ZK137316 were studied in vitro on isolated endometrial epithelial cells, as well as endometrial fibroblasts. We evaluated the expression of estrogen receptor alpha (ER) and the progesterone receptor (PR) by RT-PCR. ER and PR were strongly expressed in the fibroblasts and epithelial cells under treatment with 10(-8) M 17beta-estradiol (E(2)). Treatment with 10(-6) M J867 or ZK137316 upregulated the PR expression as did E(2), in contrast to treatment with 10(-6) M MPA, which caused a downregulation of PR in epithelial cells, but not in fibroblasts. In addition, the vascular endothelial growth factor (VEGF) release into the cell culture medium was analyzed by a VEGF-ELISA. VEGF which plays an important role in angiogenesis, is regulated by steroid hormones as well as hypoxia. E(2) stimulates VEGF release into the medium in both cell types. MPA reduces VEGF release significantly in the fibroblast cell culture, but increases it in the epithelial cell culture. ZK137316, in the presence or absence of E(2), reduces VEGF release in fibroblast cell culture. J867 increases the VEGF production in fibroblasts only in the presence of E(2). Both compounds show no significant effects, compared to E(2), in epithelial cell culture. The different results for the epithelial cells and fibroblasts indicate that the pharmacological effects of PR modulators (PRMs) and progesterone antagonists (PAs) may be cell specific and depend on the presence or absence of partial progestagenic agonistic activities. This observation opens up new perspectives for various clinical applications.

Apoptosis of Extravillous Trophoblast Cells Limits the Trophoblast Invasion in Uterine but not in Tubal Pregnancy During FirstTrimester

During the first trimester of pregnancy extravillous trophoblast cells (EVT) invade the maternal decidua. Invasion normally is reduced from the second trimester onwards and stops in the inner third of the myometrium. By contrast, in extrauterine tubal pregnancy, trophoblast invasion may even penetrate the tubal wall, which ultimately leads to the rupture of the fallopian tube. Induction of apoptosis of EVT cells, by maternal immune competent cells, may be an important mechanism to limit EVT invasion in uterine pregnancy. Tissue specimens from first and second trimester uterine pregnancy and first trimester tubal pregnancy were analyzed for apoptosis by TUNEL- and M30-staining. By immunohistochemical double labelling, maternal leukocyte subtypes were co-localized to apoptotic cells and in this context, the number of CD56(+)NK cells was analyzed. Our data show that apoptosis is confined to the decidua basalis. Most apoptotic cells are single cytokeratin-positive epithelial cells residing in the stromal compartment. Consequently these cells can only be EVT cells. Maternal leukocytes are not apoptotic. They are located in close contact to apoptotic cells. The number of apoptotic cells in the second trimester (1.8+/-0.7 per cent) is reduced compared to first trimester (5.6+/-0.7 per cent) of uterine pregnancy. In parallel, the number of NK cells declines from first (24.4+/-2.9) to second (12.4+/-1.8) trimester. Furthermore, apoptosis is significantly reduced in ectopic (0.9+/-0.3 per cent) compared to eutopic first trimester pregnancies. Consequently, we suggest that in first trimester uterine pregnancy, induction of EVT cell apoptosis by the maternal immune system is one mechanism to limit EVT invasion. During the second trimester, in parallel to declining numbers of NK cells, the mechanism changes. However, in tubal pregnancy due to differing immunological microenvironments at the ectopic implantation site, apoptosis induction fails, which deleteriously may result in uncontrolled invasion and penetration of the tubal wall.

Full-Length Complementary DNA and the Derived Amino Acid Sequence of Horse Uteroglobin

Abstract After its original description as a steroid-dependent protein in the rabbit uterus, uteroglobin became one of the best characterized proteins. However, detailed knowledge of its physiological role remains an enigma. In this study we investigate how its structure is phylogenetically conserved in the horse compared to other mammalian species. Northern blot analysis showed that in horses, the main expression of uteroglobin appears in lung, uterus, and prostate tissues. Western blot analysis demonstrated that the dimeric form of uteroglobin is found predominantly in biological compartments. Using a RACE-PCR technique, we cloned and sequenced the full-length cDNA (473 base pairs) that encodes equine uteroglobin. The nucleotide sequence was shown to characterize the primary structure of this protein. This enabled us to add equine uteroglobin to a comparative amino acid alignment of 8 other uteroglobin molecules, and finally, to unravel 14 evolutionary completely conserved amino acids. We summarize these results with a computer-based 3-D model of horse uteroglobin, and discuss new concepts on the physiological role of uteroglobin, in particular as a specific binding protein.

Cyclooxygenase-2 Supports Tumor Proliferation in Vestibular Schwannomas

BACKGROUND:Recent studies have shown that cyclooxygenase-2 (COX-2) plays an important role in tumor growth and neovascularization. However, COX-2 expression in vestibular schwannomas (VSs) has not been investigated. OBJECTIVE:To analyze the pattern of COX-2 expression in sporadic and neurofibromatosis type 2 (NF2)–associated VSs and its relationship with tumor proliferation and microvessel density. METHODS:Fifteen sporadic and 15 NF2-associated VSs were examined for COX-2 expression, microvessel density, and proliferation rate by immunohistochemical methods. Immunohistochemical scores were used to interpret the extent and intensity of COX-2 staining. Microvessel density (MVD) was determined using von Willebrand factor (vWf). Proliferation rate was quantified using Ki-67. The relationship among COX-2 expression, MVD, and proliferation rate was statistically analyzed. RESULTS:COX-2 expression was detected in 29 (96.67%) of 30 VSs, with no significant difference between sporadic and NF2-associated VSs (P = .722). In 6 (20%) VSs, COX-2 expression was graded as strong, in 12 (40%) as moderate, and in 11 (36.7%) as weak. VSs with high proliferation showed significantly higher COX-2 expression (P = .015) than VSs with low proliferation. COX-2 expression and MVD did not show specific biological correlations (P = .035). CONCLUSION:Our data demonstrate that COX-2 is expressed in VSs. High COX-2 expression in VSs with high proliferation rates suggests that the COX-2 pathway may be involved in the development and growth of VSs.

Estradiol and medroxyprogesterone acetate regulated genes in T47D breast cancer cells.

Many mammary tumors express estrogen receptors (ER) and progesterone receptors (PR), and there is increasing evidence that progestins influence gene expression of breast tumor cells. To analyse the impact of progestins on breast cancer cells, we compared (a) the expression of two cytokines, involved in tumor progression, and searched (b) for differentially regulated genes by a microarray, containing 2400 genes, on T47D breast cancer cells cultured for 6 days with 17beta-estradiol (E2) or E2+medroxyprogesterone acetate (E2+MPA). Lower amounts of PDGF and TNFalpha were found in culture supernatants of E2+MPA treated T47D cells. MPA addition induced a 2.8-3.5-fold increase of the mRNA expression of (a) tristetraprolin, which is involved in the posttranscriptional regulation of cytokine biosynthesis, and (b) zinc-alpha2-glycoprotein and Na, K-ATPase alpha1-subunit, which both resemble differentiation markers of breast epithelium. In contrast, the mRNA expression of lipocalin 2, which promotes matrixmetalloproteinase-9 activity, was decreased five-fold in E2+MPA treated cells. Our data show that the expression of genes from various functional gene families is regulated differentially by E2 and E2+MPA treatment in T47D cells. This suggests that exogenous progestins applied for therapy and endogenous changes of the progesterone levels during the menstrual cycle both influence breast cancer pathophysiology.

Desmoglein 2-Dependent Arrhythmogenic Cardiomyopathy Is Caused by a Loss of Adhesive Function

Background—The desmosomal cadherin desmoglein 2 (Dsg2) localizes to the intercalated disc coupling adjacent cardiomyocytes. Desmoglein 2 gene (DSG2) mutations cause arrhythmogenic cardiomyopathy (AC) in human and transgenic mice. AC is characterized by arrhythmia, cardiodilation, cardiomyocyte necrosis with replacement fibrosis, interstitial fibrosis, and intercalated disc dissociation. The genetic DSG2 constellations encountered are compatible with loss of adhesion and altered signaling. To further elucidate pathomechanisms, we examined whether heart-specific Dsg2 depletion triggers cardiomyopathy. Methods and Results—Because DSG2 knockouts die during early embryogenesis, mice were prepared with cardiomyocyte-specific DSG2 ablation. Healthy transgenic animals were born with a functional heart presenting intercalated discs with incorporated desmosomal proteins. Dsg2 protein expression was reduced below 3% in the heart. All animals developed AC during postnatal growth with pronounced chamber dilation, calcifying cardiomyocyte necrosis, aseptic inflammation, interstitial and focal replacement fibrosis, and conduction defects with altered connexin 43 distribution. Electron microscopy revealed absence of desmosome-like structures and regional loss of intercalated disc adhesion. Mice carrying 2 mutant DSG2 alleles coding for Dsg2 lacking part of the adhesive EC1-EC2 domains present an indistinguishable phenotype, which is similar to that observed in human AC patients. Conclusions—The observations show that the presence of Dsg2 is not essential for late heart morphogenesis and for cardiac contractility to support postnatal life. On increasing mechanical demands, heart function is severely compromised as evidenced by the onset of cardiomyopathy with pronounced morphological alterations. We propose that loss of Dsg2 compromises adhesion, and that this is a major pathogenic mechanism in DSG2-related and probably other desmosome-related ACs.

Mammaglobin 1: not only a breast‐specific and tumour‐specific marker, but also a hormone‐responsive endometrial protein

Classen‐Linke I, Moss S, Gröting K, Beier H M, Alfer J & Krusche C A 
(2012) Histopathology 61, 955–965

The progesterone antagonist onapristone retards the advanced endometrial transformation after gonadotropin stimulation in rabbits.

Ovarian stimulation with gonadotropins (GN) during human in vitro fertilization and embryo transfer (IVF/ET) therapy alters the ovarian steroid output, especially that of progesterone. As a consequence, endometrial transformation is advanced, and embryo implantation is hampered. This study used the rabbit model to determine if the application of the progesterone antagonist (PA) onapristone (ONA) could retard endometrial development after GN-stimulation. Rabbits were GN-stimulated twice daily with 5 IU FSH and 5 IU LH on 3 consecutive days with a) hMG (n = 10) or b) with a mixture of recombinant FSH and LH (n = 10). The animals were then mated, and hCG was injected i.v. to ensure ovulation. This day is designated as day 0 post coitum (d 0 p.c.). On day 2 p.c., five animals of each group were treated with 20 mg ONA/kg body weight and five with vehicle for control. On d 5 p.c. endometrial transformation was analyzed by morphology, uteroglobin (Ugl)-mRNA expression, and proliferation. Embryos were flushed from the uteri. Their number and morphology was evaluated. The endometrium of GN-stimulated control animals demonstrated very long endometrial glands and narrow stromal septa. Ugl-mRNA expression was restricted to the cells at the bottom of the gland. 17.0 +/- 4.6% (mean +/- SD) of glandular cells and 6.0 +/- 5.3% of luminal epithelial cells proliferated. In ONA-treated animals, endometrial glands were significantly shorter, and the pattern of arborization was less pronounced. Endometrial gland cells and luminal epithelial cells expressed Ugl-mRNA. Furthermore, glandular and luminal cells proliferated with high intensity (38.6 +/- 6.8% and 36.4 +/- 9.3%, respectively). These results indicate that the status of endometrial differentiation was retarded after ONA-treatment. Nevertheless, the embryos of these ONA-treated animals were well developed. In conclusion, after GN-stimulation, ONA treatment retarded the advanced endometrial transformation in rabbits. Therefore, postovulatory administration of a PA might be a possible strategy to modulate the advanced endometrial development in IVF-cycles.

Expression and regulation of 17β-hydroxysteroid dehydrogenase 7 in the rabbit ☆

We cloned partial cDNA sequences of rabbit 17beta-hydroxysteroid dehydrogenase 1 (17beta-HSD1) and 17beta-HSD7. We analyzed the tissue distribution of 17beta-HSD7 as well as the expression in corpus luteum and endometrium during pseudopregnancy. The obtained cDNA sequence of 17beta-HSD7 coded for all functional regions of the protein and showed 86 and 81% similarity to human and rodent sequences, respectively. The partial sequence of rabbit 17beta-HSD1 was 76 and 82% similar to rodent and human sequences. By Northern analysis 17beta-HSD7 expression was predominantly found in reproductive organs like ovary, oviduct, endometrium, placenta and mammary gland. Substantial expression was also apparent in the heart, stomach and cerebellum. The 17beta-HSD1 could be detected in placenta by reverse transcriptase-polymerase chain reaction (RT-PCR), so far. During rabbit pseudopregnancy 17beta-HSD7 expression was found to be regulated in corpus luteum as well as in endometrium. In the corpus luteum, strongest expression occurred from d10 to d14 of pseudopregnancy (p. hCG) and was downregulated on d16 p. hCG. In endometrium strongest expression of 17beta-HSD7 was found on d6 p. hCG, when the endometrium was differentiated to its implantation permissive status.