Alan Šerman

Epigenetic alterations of the Wnt signaling pathway in cancer: a mini review

Epigenetic mechanisms play a crucial role in cellular proliferation, migration and differentiation in both normal and neoplastic development. One of the key signaling pathways whose components are altered through the epigenetic mechanisms is the Wnt signaling pathway. In this review, we briefly discuss the key concepts of epigenetics and focus on the recent advances in the Wnt signaling pathway research and its potential diagnostic and therapeutic implications.

Epigenetic control of cell invasion – the trophoblast model

Abstract Trophoblast implantation and placentation allow the survival of the young embryo and its normal development inside the uterus. In order for these processes to function properly, the trophoblast has to undergo a series of characteristic changes that lead to its adhesion and invasion of the uterus. This is achieved, among other mechanisms, by inactivation of specific tumor suppressor genes, commonly by methylation of their promoters. Cell adhesion and tissue invasion are also characteristics of malignant tumors and patterns of methylation similar to that seen in trophoblast are found in various tumor types. Another important mechanism that aids trophoblast cells invasion is their transition from epithelial to mesenchymal phenotype. Such a transition is also a common characteristic of invading malignant cells. Thus, studying tissue invasion and its control mechanisms can benefit the understanding of both the trophoblast and malignant cells behavior.

An in vivo rat model to study epigenetic control of cell invasion

Due to it ability to invade deep into endometrium, as well as into other tissues at ectopic sites (testis, kidney capsule), throphoblast plays an important role in shaping the future placenta. To accomplish this task, it is necessary for throphoblast cells to differentiate into highly invasive throphoblast giant cells (TGC). The behaviour of TGC during implantation resembles that of cancer cells during metastasis. In both cases, the invasive phenotype is to a large degree controlled epigenetically, by DNA methylation, with resulting gene expression silencing. DNA demethylating agents, such as 5-azacitidine (5azaC), reverse the gene expression and change cell behaviour; already used in cancer therapy, 5azaC is also useful experimentally to elucidate epigenetic pathways in normal and malignant cells. In this paper we describe an in vivo rat model of throphoblast cell invasion, in which cells are exposed to 5azaC and transplanted ectopically under kidney capsule. We conclude that temporal variation in exposure to 5azaC, such as the gestation day, affects the throphoblast cells differentiation, and thus changes their invasive properties. We suggest that this in vivo model could be useful to study steps in epigenetic control of both the placental development and cancer cell spread.

Glycosylation pattern and axin expression in normal and IUGR placentae

Abstract Objective: The aim of this study was to investigate the protein glycosylation pattern and AXIN1 protein expression in human placentae of normal pregnancies and compare them with placentae of pregnancies complicated with intrauterine growth restriction (IUGR). Methods: A total of 38 placentae (17 placentae of IUGR fetuses from singleton pregnancies and gestational age-matched 21 control placentae from normal singleton pregnancies) were collected from the Clinical Hospital Sveti Duh, Department of Gynecology and Obstetrics, Zagreb, Croatia. Gestational age was determined according to the last menstrual period (LMP) and by ultrasound measurements. Expression of glycoproteins was measured by Western blotting with SNA, UEA-I, PHA-E and DBA lectins as probes whereas expression of AXIN1 was determined by immunohistochemistry. Results: Comparison of detected sugars revealed differences in protein glycosylation between normal and IUGR placentae. Higher expression of AXIN1 protein located mostly in the cytoplasm of syncytiotrophoblast and to a lesser extent in its nuclei was found in IUGR placentae. Conclusion: Results of our study suggest that changes in glycoprotein content may contribute to restricted placenta growth and development. Higher expression of AXIN1 protein in IUGR placentae indicates a role of Wnt/β-catenin signaling pathway in pathology of placental development.

ELF5 transcription factor expression during gestation in humans and rats - an immunohistochemical analysis.

Abstract Objective: The purpose of this study was to measure immunohistochemically the expression of ELF5 protein in term human and rat placentas and in human placentas associated with gestational diabetes (GD) and intrauterine growth restriction (IUGR). Methods: The results were quantitated stereologically using the stereological variable of volume density. A semiquantitative analysis was performed independently by a certified pathologist. Results: Total expression of ELF5 protein was higher in pathological pregnancies than in corresponding control term placentas, with both methods of quantifications showing similar results. In addition, ELF5 expression was also higher in connective tissue and blood vessels in chorionic villi in IUGR placentas (but not in GD placentas) compared to healthy controls. ELF5 is higher in placenta as a whole and in most of its components in both pathologies. The two exceptions are chorionic plates in IUGR and decidua in GD, where its expression is lower than in healthy controls. Conclusions: We have shown that IUGR and GD are associated with significantly increased levels of ELF5 protein in placentas, which suggests that ELF5 may play an important role in normal placentation. However, these are term placentas and to study ELF5 in premature births would give better insight into human placentation in health and disease.

Structural changes in the rat placenta during the last third of gestation discovered by stereology

Structural changes in the rat placenta during the last third of gestation were for the first time assessed by stereology. Fischer female rats were euthanized on the day 16 or day 19 of gestation, and 35 placentas were collected. Three randomly selected placentas from each group were stereologically analyzed for the absolute volume. The proportion of the glycogenic cells and the trophoblast giant cells (TGC) in the basal part of the placenta was calculated using volume density. The absolute volume of the rat placenta on the day 16 of gestation was determined as 0.0638 cm3. The labyrinth comprised 0.0274 cm3, the basal plate 0.0271 cm3 and the decidua 0.0093 cm3. On the day 19 of gestation, the absolute volume of the placenta was 0.1627 cm3, the labyrinth occupied 0.0922 cm3, the basal plate 0.0596 cm3 and the decidua 0.0109 cm3. The volume density of trophoblast giant cells was 0.174 cm0 on the day 16 and 0.107 cm0 on the day 19 of gestation. The glycogenic cells comprised 0.379 percentage of the basal plate on the day 16 and 0.236 on the day 19 of gestation. We conclude that the absolute volume of the whole placenta and the labyrinth has increased from day 16 to the day 19 of gestation. In contrast, the volume density of glycogenic cells and trophoblast giant cells was higher on the day 16 than on the day 19 of gestation, probably due to the intensive trophoblast invasion during that time.

Negative regulators of Wnt signaling pathway SFRP1 and SFRP3 expression in preterm and term pathologic placentas

Abstract Objective: Since Wnt signaling pathway plays a pivotal role in the placental development, we explored the expression of its negative regulators, SFRP1 and SFRP3 proteins in placentas from pathological pregnancies and compared their levels with those in healthy placentas. Methods: Placentas (n = 79) were stained for SFRP1, and SFRP3 proteins by immunohistochemistry and their expression levels were quantified by stereological variable of volume density (Vv, mm°). Results: Significantly higher expressions of SFRP1 and SFRP3 were found in all investigated groups of term and preterm pathologic placentas as well as in preterm control placentas in comparison with normal-term placentas. Conclusions: Our findings indicate the active involvement of negative Wnt regulators SFRP1/SFRP3 in placental development and important role in pathology of pregnancy.

Reply to comment on "Structural changes in the rat placenta during the last third of gestation discovered by stereology"

Dear Editor, We are thankful to our colleagues for their interest in our paper and comments that they have provided. We could not agree more with the part of the letter regarding the method of sampling. On the basis of our 16-year-long experience in research on rat placentas, we underline sampling as one of the most important concepts in stereology, especially in the light of biological variability [1]. This variability is the reason why we approach sampling with the greatest care, as it is evident from all our published papers in the field of stereology. In addition, we consider it as one of the best methods for quantification of biological materials. Systematic random sampling, which combines both the unbiasedness of random sampling and the efficiency of a systematic sampling, was used in our research. It is based on the selection of the final sample systematically, while the first sample was selected randomly within the first sampling interval [2]. Sections were sampled at a ratio 1:10 (Section Sampling Fraction-SSF), with a random section (RS) selection from the first ten sections (in this case, the 3rd section) and then every 10th section after this initial selection. Preliminary measurements were performed for each of the experimental groups. In addition, due to its small size, it is possible to analyze the entire section of the rat placenta as a single field, which we did, as seen in Figure 2 [3]. However, this is not possible for human placenta, which, due to its larger size, cannot fit into a single section or single field for stereological analysis.

Impact of 5-azacytidine on rat decidual cell proliferation

The DNA demethylating agent 5‐azacytidine (5‐azaC) has a teratogenic influence during rat development influencing both the embryo and the placenta. Our aim was to investigate its impact on early decidual cell proliferation before the formation of placenta. Thus, female Fischer rats received 5‐azaC (5 mg/kg, i.p.) on the 2nd, 5th or 8th day of gestation and the decidual tissues were harvested on gestation day 9. They were then analysed immunohistochemically for expression of cell proliferation marker proliferating cell nuclear antigen (PCNA) in decidual cells and for global DNA methylation using the coupled restriction enzyme digestion, random amplification and pyrosequencing assays. We found that 5‐azaC administered on the 5th and 8th (but not on 2nd) day of gestation led to increased PCNA expression in decidual cells compared with untreated controls. No significant changes in DNA methylation were detected, with either method, in any of the treated rat groups compared with untreated controls. Thus, we conclude that 5‐azaC can stimulate decidual cell proliferation without simultaneously changing global DNA methylation level in treated cells.