Extended Prophylactic Effect of N-tert-Butyl-α-phenylnitron against Oxidative/Nitrosative Damage Caused by the DNA-Hypomethylating Drug 5-Azacytidine in the Rat Placenta.

Antioxidant N-tert-Butyl-α-phenylnitron (PBN) partly protected embryos from the negative effects of a DNA demethylating drug 5-azacytidine during pregnancy. Our aim was to investigate PBN's impact on the placenta. Fischer rat dams were treated on gestation days (GD) 12 and 13 by PBN (40 mg/kg), followed by 5azaC (5 mg/kg) after one hour. Global methylation was assessed by pyrosequencing. Numerical density was calculated from immunohistochemical expression in single cells for proliferating (PCNA), oxidative (oxoguanosine) and nitrosative (nitrotyrosine) activity. Results were compared with the PBN-treated and control rats. PBN-pretreatment significantly increased placental weight at GD15 and GD20, diminished by 5azaC, and diminished apoptosis in GD 20 placentas caused by 5azaC. Oxoguanosine expression in placentas of 5azaC-treated dams was especially high in the placental labyrinth on GD 15, while PBN-pretreatment lowered its expression on GD 15 and GD 20 in both the labyrinth and basal layer. 5azaC enhanced nitrotyrosine level in the labyrinth of both gestational stages, while PBN-pretreatment lowered it. We conclude that PBN exerted its prophylactic activity against DNA hypomethylating agent 5azaC in the placenta through free radical scavenging, especially in the labyrinthine part of the placenta until the last day of pregnancy.

Defining health-related quality of life in localized and advanced stages of breast cancer - the first step towards hereditary cancer genetic counseling.

The important goal in breast cancer treatment is to improve patient quality of life. Due to the huge economic burden, it is necessary to estimate the health state utility values for different breast cancer stages accurately. A group of 114 women filled out the EuroQol-5D-3L questionnaire at two time points. The participants were divided into three groups, as follows: group 1 including healthy high-risk individuals; group 2 including patients with localized stage breast cancer; and group 3 including patients with advanced stage breast cancer. Results were expressed either as summary health state utility score or summary visual-analog score. The EuroQol utility index score and EuroQol visual-analog score were statistically significantly higher in the group of healthy high-risk individuals. The EuroQol visual-analog score was mostly correlated with the anxiety/depression and pain/discomfort quality of life dimensions. Health state utility values for different breast cancer stages are a necessary tool to perform economic analyses in breast cancer management decision making, due to its huge economic burden. Special attention should be paid to assessment of the psychosocial aspects of the disease, as well as pain management.

Influence of hyperthermal regimes on experimental teratoma development in vitro.

We screened for the impact of hyperthermal regimes varying in the cumulative equivalent minutes at 43°C (CEM43°C) and media composition on tumour development using an original teratoma in vitro model. Rat embryos (three germ layers) were microsurgically isolated and cultivated at the air-liquid interface. During a two week period, ectodermal, mesodermal and endodermal derivatives developed within trilaminar teratomas. Controls were grown at 37°C. Overall growth was measured, and teratoma survival and differentiation were histologically assessed. Cell proliferation was stereologically quantified by the volume density of Proliferating Cell Nuclear Antigen. Hyperthermia of 42°C, applied for 15 minutes after plating (CEM43°C 3.75 minutes), diminished cell proliferation (P Ë‚ .0001) and enhanced differentiation of both myotubes (P Ë‚ .01) and cylindrical epithelium (P Ë‚ .05). Hyperthermia of 43°C applied each day for 30 minutes during the first week (CEM43°C 210 minutes) impaired overall growth (P Ë‚ .01) and diminished cell proliferation (P Ë‚ .0001). Long-term hyperthermia of 40.5°C applied for two weeks (CEM43°C 630 minutes) significantly impaired survival (P Ë‚ .005). Long-term hyperthermia of 40.5°C applied from the second day when differentiation of tissues begins (CEM43°C 585 minutes) impaired survival (P Ë‚ .0001), overall growth (P Ë‚ .01) and cartilage differentiation (P Ë‚ .05). No teratomas survived extreme regimes: 43°C for 24 hours (CEM43°C 1440 minutes), hyperthermia in the scant serum-free medium (CEM43°C 630 minutes) or treatment with an anti-HSP70 antibody before long-term hyperthermia 40.5°C from the second day (CEM43°C 585 minutes). This in vitro research provided novel insights into the impact of hyperthermia on the development of experimental teratomas from their undifferentiated sources and are thus of potential interest for future therapeutic strategies in corresponding in vivo models.

Hypermethylation of Secreted Frizzled Related Protein 1 gene promoter in different astrocytoma grades.

AIM: To identify the involvement of Secreted Frizzled Related Protein 1 (SFRP1) promoter hypermethylation in different malignancy grades of astrocytoma and assess its association with beta-catenin, lymphoid-enhancer factor 1, and T-cell factor 1. METHODS: Twenty-six astrocytoma samples were collected from 2008-2015. Promoter hypermethylation was evaluated by methylation-specific polymerase-chain-reaction and protein expression by immunohistochemistry and stereological analysis. The staining intensity was scored by comparing immunoreactivity with normal tissue and by using 10% and 50% cut-offs. RESULTS: SFRP1 promoter methylation was found in 32% of astrocytomas. The number of hypermethylated samples increased in higher astrocytoma grades and was the highest in glioblastoma (P=0.042 compared to other astrocytoma grades). There was 45.8% of samples with the lack of or weak expression of SFRP1 protein and 29.2% with strong expression. Samples with methylated promoter expressed significantly less SFRP1 than samples with unmethylated promoter (P=0.031). Beta-catenin expression levels were elevated. Yet, glioblastomas with unmethylated SFRP1 promoter had significantly less beta-catenin (P=0.033). Strong expression of lymphoid-enhancer factor 1 was associated to higher astrocytoma grades (P=0.006). CONCLUSION: SFRP1 gene was epigenetically silenced in glioblastomas when compared to low astrocytoma grades, which may suggest that the lack of its protein is involved in astrocytoma progression.

Methylation-associated silencing of SFRP1 gene in high-grade serous ovarian carcinomas.

Wnt is a highly conserved signaling pathway responsible for tissue regeneration, maintenance and differentiation of stem cells in adults. Its aberrant activation through reduced expression of Wnt signaling pathway inhibitors, such as proteins from the SFRP family, is commonly seen in many tumors. In the present study we explored SFRP1 protein expression using immunohistochemistry in 11 low-grade serous ovarian carcinomas (LGSC), 42 high-grade serous ovarian carcinomas (HGSC), and 5 normal ovarian tissues (controls). SFRP1 gene methylation was analyzed by methylation-specific PCR in 8 LGSCs, 13 HGSCs and control samples. SFRP1 gene was unmethylated and SFRP1 protein expression was strong in normal ovaries (n=5). Although SFRP1 gene was unmethylated in almost all of the LGSC cases (7/8, 88%), SFRP1 protein expression was significantly lower than in normal ovaries (p<0.05). Seven out of 13 HGSCs (54%) showed SFRP1 gene hypermethylation and protein expression level was also significantly lower than in normal ovaries (p<0.001). Our preliminary data show loss of SFRP1 protein expression caused by the SFRP1 promoter hypermethylation in a subset of HGSCs. SFRP1 protein expression was also lost in LGSCs but different regulatory mechanisms may be involved. Further studies should elucidate the clinical and therapeutic relevance of the observed molecular alterations.

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.

Epigenetic drug 5-azacytidine impairs proliferation of rat limb buds in an organotypic model-system in vitro.

AIM: To establish an organotypic in vitro model of limb bud development to verify whether epigenetic drug and teratogen 5-azacytidine (5azaC) has an effect on limb buds independent of its effects on the placenta. METHODS: Fischer strain rat fore- and hindlimb buds were microsurgically isolated from 13 days old embryos and cultivated in vitro for two weeks at the air-liquid interface in Eagle's minimum essential medium (MEM) with 50% rat serum. 30 µmol of 5azaC was added to the fresh medium. Overall growth was measured by an ocular micrometer. Routine histology, immunohistochemical detection of the proliferating cell nuclear antigen (PCNA), and stereological quantification of PCNA expression were performed. RESULTS: At four time points, significantly lower overall growth was detected for fore- and hindlimb bud explants cultivated with 5azaC in comparison to controls. After the culture period, numerical density of the PCNA signal for both types of limb buds was lower than for controls (P<0.001). Limb buds were initially covered by immature epithelium and contained mesenchyme, myotubes, single hemangioblasts, hemangioblast aggregates, blood islands, and capillaries. Regardless of the treatment, cartilage and epidermis differentiated, but cells and structures typical for vasculogenesis disappeared. CONCLUSION: Our findings, obtained outside of the maternal organism, stress the importance of compromised cell proliferation for 5azaC impact on limb buds. This investigation points to the necessity to establish alternatives to in vivo research on animals using teratogenic agents.

WNT5A, ß­catenin and SUFU expression patterns, and the significance of microRNA deregulation in placentas with intrauterine growth restriction.

Placental insufficiency is a common cause of intrauterine growth restriction (IUGR). It affects ~10% of pregnancies and increases fetal and neonatal morbidity and mortality. Although Wnt and Hh pathways are crucial for embryonic development and placentation, their role in the pathology of IUGR is still not sufficiently explored. The present study analyzed the expression of positive regulators of the Wnt pathway, WNT5A and ß­catenin, and the expression of the Hh pathway negative regulator suppressor of fused (SUFU). Immunohistochemical and reverse transcription­quantitative PCR (RT­qPCR) assays were performed on 34 IUGR and 18 placental tissue samples from physiologic singleton­term pregnancies. Epigenetic mechanisms of SUFU gene regulation were also investigated by methylation­specific PCR analysis of its promoter and RT­qPCR analysis of miR­214­3p and miR­378a­5p expression. WNT5A protein expression was higher in endothelial cells of placental villi from IUGR compared with control tissues. That was also the case for ß­catenin protein expression in trophoblasts and endothelial cells and SUFU protein expression in trophoblasts from IUGR placentas. The SUFU gene promoter remained unmethylated in all tissue samples, while miR­214­3p and miR­378a­5p were downregulated in IUGR. The present results suggested altered Wnt and Hh signaling in IUGR. DNA methylation did not appear to be a mechanism of SUFU regulation in the pathogenesis of IUGR, but its expression could be regulated by miRNA targeting.

Genetic risk factors in melanoma etiopathogenesis and the role of genetic counseling: A concise review.

Melanoma is a highly aggressive cancer originating from melanocytes. Its etiopathogenesis is strongly related to genetic, epigenetic, and environmental factors. Melanomas encountered in clinical practice are predominantly sporadic, whereas hereditary melanomas account for approximately 10% of the cases. Hereditary melanomas mainly develop due to mutations in the CDKN2A gene, which encodes two tumor suppressor proteins involved in the cell cycle regulation. CDKN2A, along with CDK4, TERT, and POT1 genes, is a high-risk gene for melanoma. Among the genes that carry a moderate risk are MC1R and MITF, whose protein products are involved in melanin synthesis. The environment also contributes to the development of melanoma. Patients at risk of melanoma should be offered genetic counseling to discuss genetic testing options and the importance of skin UV protection, avoidance of sun exposure, and regular preventive dermatological examinations. Although cancer screening cannot prevent the development of the disease, it allows for early diagnosis when the survival rate is the highest.

New insight into the role of PTCH1 protein in serous ovarian carcinomas.

The Hedgehog (Hh) signaling pathway is essential for normal embryonic development, while its hyperactivation in the adult organism is associated with the development of various cancers. The role of the Hh signaling pathway in ovarian cancer has not been sufficiently investigated. Therefore, the present study investigated the role of protein patched homolog 1 (PTCH1), a component of the Hh signaling pathway, and changes in the promoter methylation status of the corresponding gene in a cohort of low­(LGSC) and high­grade serous ovarian carcinomas (HGSC) and HGSC cell lines (OVCAR8 and OVSAHO). PTCH1 protein expression level was analyzed using immunohistochemistry in tissue samples and immunofluorescence and western blotting in cell lines. DNA methylation patterns of the PTCH1 gene were analyzed using methylation­specific PCR. PTCH1 protein expression was significantly higher in HGSCs and LGSCs compared with controls (healthy ovaries and fallopian tubes). Similarly, ovarian cancer cell lines exhibited significantly higher PTCH1 protein expression compared with a normal fallopian tube non­ciliated epithelial cell line (FNE1). PTCH1 protein fragments of different molecular weights were detected in all cell lines, indicating possible proteolytic cleavage of this protein, resulting in the generation of soluble N­terminal fragments that are translocated to the nucleus. DNA methylation of the PTCH1 gene promoter was exclusively detected in a proportion of HGSC (13.5%) but did not correlate with protein expression. PTCH1 protein was highly expressed in serous ovarian carcinoma tissues and cell lines, while PTCH1 promoter methylation was only detected in HGSC. Further investigation is required to elucidate the possible mechanisms of PTCH1 activation in serous ovarian carcinomas.

Endometrial Glucose Transporters in Health and Disease.

Pregnancy loss is a frequent occurrence during the peri-implantation period, when there is high glucose demand for embryonic development and endometrial decidualization. Glucose is among the most essential uterine fluid components required for those processes. Numerous studies associate abnormal glucose metabolism in the endometrium with a higher risk of adverse pregnancy outcomes. The endometrium is incapable of synthesizing glucose, which thus must be delivered into the uterine lumen by glucose transporters (GLUTs) and/or the sodium-dependent glucose transporter 1 (SGLT1). Among the 26 glucose transporters (14 GLUTs and 12 SGLTs) described, 10 (9 GLUTs and SGLT1) are expressed in rodents and 8 (7 GLUTs and SGLT1) in the human uterus. This review summarizes present knowledge on the most studied glucose transporters in the uterine endometrium (GLUT1, GLUT3, GLUT4, and GLUT8), whose data regarding function and regulation are still lacking. We present the recently discovered SGLT1 in the mouse and human endometrium, responsible for controlling glycogen accumulation essential for embryo implantation. Moreover, we describe the epigenetic regulation of endometrial GLUTs, as well as signaling pathways included in uterine GLUT's expression. Further investigation of the GLUTs function in different endometrial cells is of high importance, as numerous glucose transporters are associated with infertility, polycystic ovary syndrome, and gestational diabetes.

Aberrant expression of SFRP1, SFRP3, DVL2 and DVL3 Wnt signaling pathway components in diffuse gastric carcinoma.

Diffuse gastric carcinoma (DGC) is characterized by poorly cohesive cells, highly invasive growth patterns, poor prognosis and resistance to the majority of available systemic therapeutic strategies. It has been previously reported that the Wnt/ß-catenin signaling pathway serves a prominent role in the tumorigenesis of gastric carcinoma. However, the mechanism underlying the dysregulation of this pathway in DGC has not been fully elucidated. Therefore, the present study aimed to investigate the expression profiles of Wnt antagonists, secreted frizzled-related protein 1 (SFRP1) and secreted frizzled-related protein 3 (SFRP3), and dishevelled protein family members, dishevelled segment polarity protein 2 (DVL2) and dishevelled segment polarity protein 3 (DVL3), in DGC tissues. The association between the expression levels of these factors and the clinicopathological parameters of the patients was determined. Protein and mRNA expression levels in 62 DGC tumor tissues and 62 normal gastric mucosal tissues obtained from patients with non-malignant disease were measured using immunohistochemical and reverse transcription-quantitative PCR (RT-qPCR) analysis. Significantly lower protein expression levels of SFRP1 (P<0.001) and SFRP3 (P<0.001), but significantly higher protein expression levels of DVL2 (P<0.001) and DVL3 (P<0.001) were observed in DGC tissues compared with in control tissues by immunohistochemistry. In addition, significantly lower expression levels of SFRP1 (P<0.05) and higher expression levels of DVL3 (P<0.05) were found in in DGC tissues compared with those in normal gastric mucosal tissues using RT-qPCR. According to correlation analysis between the SFRP1, SFRP3, DVL2 and DVL3 protein expression levels and the clinicopathological characteristics of patients with DGC, a statistically significant correlation was found between the SFRP3 volume density and T stage (r=0.304; P=0.017) and between the SFRP3 volume density and clinical stage (r=0.336; P=0.008). In conclusion, the findings of the present study suggested that the Wnt signaling pathway components SFRP1, SFRP3, DVL2 and DVL3 may be aberrantly expressed in DGC tissues, implicating their possible role in the development of this malignant disease. The present data also revealed a positive relationship between SFRP3 protein expression and the clinical and T stage of DGC.

Dishevelled family proteins (DVL1-3) expression in intrauterine growth restriction (IUGR) placentas.

Dishevelled family proteins (DVL1, DVL2, and DVL3) are cytoplasmic proteins that are involved in canonical and non-canonical Wnt signaling pathway during embryonic development. The role of DVL proteins in the placental tissue remains mostly unknown. In the current study, we explored the role of Dishevelled proteins in naturally invasive tissue, trophoblast. Formalin-fixed paraffin-embedded samples of 15 term placentas from physiologic term pregnancies and 15 term placentas from pregnancies complicated with intrauterine growth restrictions (IUGR) were used for the study. Expression levels of mRNA for DVL1, DVL2, and DVL3 in placentas were analyzed by quantitative real-time PCR (qRTPCR). DVL1, DVL2, and DVL3 protein expression were semi-quantitatively analyzed using immunohistochemistry. The expression of DVL2 and DVL3 proteins was significantly higher in trophoblasts in placental villi from IUGR pregnancies compared with the control group of term placentas. In contrast, DVL3 protein expression was significantly higher in endothelial cells in placental villi from IUGR pregnancies compared with normal term placentas. The observed differences at protein levels between normal and IUGR placentas were not confirmed at the mRNA levels of DVL genes. Our data indicate the active involvement of DVL proteins in IUGR-related placentas. No significant changes were observed in DVL mRNA levels between the two groups of placentas. Further studies are required to explore the clinical relevance of these observations.

The role of glycogen synthase kinase 3 (GSK3) in cancer with emphasis on ovarian cancer development and progression: A comprehensive review.

Glycogen synthase kinase 3 (GSK3) is a monomeric serine-threonine kinase discovered in 1980 in a rat skeletal muscle. It has been involved in various cellular processes including embryogenesis, immune response, inflammation, apoptosis, autophagy, wound healing, neurodegeneration, and carcinogenesis. GSK3 exists in two different isoforms, GSK3α and GSK3ß, both containing seven antiparallel beta-plates, a short linking part and an alpha helix, but coded by different genes and variously expressed in human tissues. In the current review, we comprehensively appraise the current literature on the role of GSK3 in various cancers with emphasis on ovarian carcinoma. Our findings indicate that the role of GSK3 in ovarian cancer development cannot be decisively determined as the currently available data support both prooncogenic and tumor-suppressive effects. Likewise, the clinical impact of GSK3 expression on ovarian cancer patients and its potential therapeutic implications are also limited. Further studies are needed to fully elucidate the pathophysiological and clinical implications of GSK3 activity in ovarian cancer.

Dishevelled family proteins in serous ovarian carcinomas: a clinicopathologic and molecular study.

Dishevelled family proteins (DVL1, DVL2, and DVL3) are cytoplasmic mediators involved in canonical and non-canonical Wnt signaling that are important for embryonic development. Since Wnt signaling promotes cell proliferation and invasion, its increased activation is associated with cancer development as well. To get deeper insight into the behavior of Dishevelled proteins in cancer, we studied their expression in serous ovarian carcinomas [both low- (LGSC) and high-grade (HGSC)], and HGSC cell lines OVCAR5, OVCAR8, and OVSAHO. DVL protein expression in serous ovarian carcinomas tissues was analyzed using immunohistochemistry, while DVL protein and mRNA expressions in HGSC cell lines were analyzed using Western blot and quantitative real-time PCR. DVL1 protein expression was significantly higher in LGSC compared with normal ovarian tissue, while DVL3 was overexpressed in both LGSC and HGSC. DVL2 and DVL3 protein expression was higher in HGSC cell lines when compared with normal control cell line FNE1, while DVL1, DVL2, and DVL3 mRNA expression was significantly increased only in OVSAHO cell line. Survival analysis revealed no significant impact of DVL proteins on patients' outcome. Our data show an active involvement of Dishevelled family proteins in serous ovarian carcinomas. Further studies should confirm the clinical relevance of these observations.

Novel Epigenetic Biomarkers in Pregnancy-Related Disorders and Cancers.

As the majority of cancers and gestational diseases are prognostically stage- and grade-dependent, the ultimate goal of ongoing studies in precision medicine is to provide early and timely diagnosis of such disorders. These studies have enabled the development of various new diagnostic biomarkers, such as free circulating nucleic acids, and detection of their epigenetic changes. Recently, extracellular vesicles including exosomes, microvesicles, oncosomes, and apoptotic bodies have been recognized as powerful diagnostic tools. Extracellular vesicles carry specific proteins, lipids, DNAs, mRNAs, and miRNAs of the cells that produced them, thus reflecting the function of these cells. It is believed that exosomes, in particular, may be the optimal biomarkers of pathological pregnancies and cancers, especially those that are frequently diagnosed at an advanced stage, such as ovarian cancer. In the present review, we survey and critically appraise novel epigenetic biomarkers related to free circulating nucleic acids and extracellular vesicles, focusing especially on their status in trophoblasts (pregnancy) and neoplastic cells (cancers).

A Free Radical Scavenger Ameliorates Teratogenic Activity of a DNA Hypomethylating Hematological Therapeutic.

The spin-trap free radical scavenger N-tert-butyl-α-phenylnitron (PBN) ameliorated effects of several teratogens involving reactive oxygen species (ROS). We investigated for the first time whether PBN could ameliorate teratogenesis induced by a DNA hypomethylating hematological therapeutic 5-azacytidine (5azaC). At days 12 and 13 of gestation, Fisher rat dams were pretreated by an i.v. injection of PBN (40 mg/kg) and 1 h later by an i.p. injection of 5azaC (5mg/kg). Development was analyzed at gestation day 15 in embryos and day 20 in fetuses. PBN alone did not significantly affect development. PBN pretreatment restored survival of 5azaC-treated dams' embryos to the control level, restored weight of embryos and partially of fetuses, and partially restored crown-rump lengths. PBN pretreatment converted limb adactyly to less severe oligodactyly. PBN pretreatment restored global DNA methylation level in the limb buds to the control level. Cell proliferation in limb buds of all 5azaC-treated dams remained significantly lower than in controls. In the embryonic liver, PBN pretreatment normalized proliferation diminished significantly by 5azaC; whereas in embryonic vertebral cartilage, proliferation of all 5azaC-treated dams was significantly higher than in PBN-treated dams or controls. Apoptotic indices significantly enhanced by 5azaC in liver and cartilage were not influenced by PBN pretreatment. However, PBN significantly diminished ROS or reactive nitrogen species markers nitrotyrosine and 8-hydroxy-2'deoxyguanosine elevated by 5azaC in embryonic tissues, and, therefore, activity of this DNA hypomethylating agent was associated to the activation of free radicals. That pretreatment with PBN enhanced proliferation in the liver and not in immature tissue is interesting for the treatment of 5azaC-induced hepatotoxicity and liver regeneration.

Epigenetic deregulation through DNA demethylation seems not to interfere with the differentiation of epithelia from pre-gastrulating rat embryos in vitro.

One of the epigenetic mechanisms controlling differentiation during mammalian development is the process of DNA methylation. The differentiation of tissues in pre-gastrulating rat embryos cultivated in vitro under the influence of the demethylating agent 5-azacytidine (5azaC) was investigated. Eight-day-old Fisher rat embryos consisting of epiblast and hypoblast (primitive ectoderm and primitive endoderm) were isolated and cultivated in serum-supplemented medium by air-lifting method in vitro. A single dose of 5azaC (30 microM) was added to the culture medium on day 5 of cultivation. After 14 days, teratoma-like structures developed and were processed by routine histology. When compared to controls, the explants treated with 5azaC showed a statistically significant higher incidence of neuroblasts, myotubes, cartilage, and blood islands. On the other hand, the incidence of stratified squamous, columnar and glandular epithelium was not statistically different from controls. It seems that differentiation of epithelia was not sensitive to DNA demethylation caused by 5azaC like differentiation of other tissues, especially mesodermal derivatives.

Development of epithelia in the ectopic transplant of the fetal rat epiglottis.

Embryonic in situ development is strictly regulated within the specific microenvironment of developing tissues. However, for regenerative medicine purposes (supplementation of damaged tissues/organs), transplantation to ectopic sites has been considered. To investigate developmental potential of fetal epiglottic epithelia at an ectopic site, fetal epiglottis was transplanted under the kidney capsule and its development compared to fetal and adult epiglottis. Seventeen-day-old Fischer rat epiglottides were microsurgically isolated under a dissecting microscope and transplanted under the kidney capsule of adult males. After 14 days, classic histology and immunohistochemical detection of the Proliferating Cell Nuclear Antigen (PCNA) were done in isolated and accordingly fixed transplants. The 17-day-old fetal epiglottis and adult epiglottis were processed in the same way. The 17-day-old fetal epiglottides were covered with immature epithelium expressing PCNA in almost all cells. Adult epiglottis was covered with two types of epithelia (stratified squamous epithelium and ciliated pseudostratified epithelium). In the stratified squamous epithelium PCNA was abundantly expressed in the basal cell layer and absent from more superficial and more differentiated cells. Transplants survived well during the experimental period. On their surface ciliated pseudostratified epithelium could be easily recognized, but squamous epithelium was almost absent. PCNA was expressed in basal cells of the ciliated pseudostratified epithelium and was absent from the more differentiated superficial cells. It seems that at this ectopic site further differentiation of the epiglottic epithelia can proceed but differentiation of squamous epithelium seems not to be favored. It seems that this ectopic site is optimal for further differentiation of the epiglottic epithelium towards ciliated pseudostratified epithelium.