Molecular complexities of patched signaling in cancer development

Basal Cell Cancer (BCC) is the most common cancer in the Western world. Although BCCs hardly ever metastasize, invasive growth may cause considerable local tissue destruction. BCCs mainly occur as sporadic tumors but can also be found in a hereditary form in the Nevoid Basal Cell Carcinoma Syndrome (NBCCS). The gene underlying the NBCCS is Patched1 (PTCH1), a tumor suppressor gene that is mutated or deleted in these patients. This thesis is focusing in deciphering complexities that are associated with signal tranduction in BCCs and was initiated by the cloning of a second receptor of the Hedgehog (HH) ligand, Patched2 (PTCH2). In similarity to PTCH1, PTCH2 was found to be up-regulated in BCCs, however in contrast to PTCH1 no mutations have been detected in this gene in BCCs. Thus PTCH2 overexpression cannot compensate for a defective PTCH1 allele implying that either the inherent properties of these proteins or the timing and cellular specificity of expression are distinct. An interesting observation during the analysis of PTCH2 expression was that in tissues that included skin and testis a number of PTCH2 splice variants have been detected. Some of these variant mRNA forms appeared to be of potential functional significance as the open reading frame was conserved. Consequently efforts were focused into the analysis of the functional properties of the proteins encoded by the PTCH2 splice variants as well as their relation to PTCH1. Although cellular localization assays or the capacity to internalize Sonic Hedgehog (SHH) did not reveal major differences, the splice variants had distinct properties in influencing signaling as revealed by co-transfection of a reporter gene. In fact the reporter gene used was PTCH2 providing unambiguous evidence that it represents a direct target of HH signaling. These transfection assays established that PTCH1 is a stronger inhibitor of SHH than the PTCH2 splice variants and inclusion of both the internal exon domains and the alternative 3 end is necessary for increased PTCH2 inhibition. Using a Ptch1-/- fibroblast cell line it was also shown that the PTCH2 splice variant with the strongest inhibitory activity is also most effective into reconstituting Desert Hedgehog (DHH) signaling, albeit to a lower extent than that of PTCH1. PTCH1 gene expression is characterized by the presence of three distinct first exons. Each of these is individually spliced with exon two resulting in mRNAs with unique 5 ends. Interestingly in BCCs, overexpression of only one of the three PTCH1 mRNAs is observed implying that signaling dysregulation does not affect PTCH1 expression at large but solely the production of a specific mRNA. By the use of transfection assays it was shown that the PTCH1 protein encoded by the up-regulated mRNA is most effective as an inhibitor of signaling initiated by the PTCH1 interacting protein, Smoothened (SMO). An unexpected finding in the analysis of HH signaling was the observation that gene activation by the transcription factor GLI1 is apparently down-regulated by PTCH1. Although activation by GLI1 is known to be inhibited by the signaling component SU(FU) and the critical amino acids for this interaction have been mapped in GLI1, these are not involved in the observed PTCH1 mediated down-regulation. Deletion mapping of PTCH1 has revealed that the domains encompassed by amino acids 180 to 786 and 1058 to 1210 are of highest significance in inhibiting GLI1 gene activation

The effectiveness of two energy drinks on selected indices of maximal cardiorespiratory fitness and blood lactate levels in male athletes

Background: Consumption of energy drinks has become widespread among athletes. The effectiveness of Red Bull and Hype energy drinks on selected indices of maximal cardiorespiratory fitness and blood lactate levels in male athletes was examined in this study. Methods: Ten male student athletes (age: 22.4 ± 2.1 years, height: 180.8 ± 7.7 cm, weight: 74.2 ± 8.5 kg) performed three randomized maximal oxygen consumption tests on a treadmill. Each test was separated by four days and participants were asked to ingest Red Bull, Hype or placebo drinks 40 minutes before the exercise bout. The VO 2max , time to exhaustion, heart rate and lactate were measured to determine if the caffeine-based beverages influence performance. ANOVA test was used for analyzing data. Results: A greater value was observed in VO 2max and time to exhaustion for the Red Bull and Hype trial compared to the placebo trial (p < 0.05). No significant difference was found in pre- and post-test heart rate for two drinks (p > 0.05). For blood lactate levels no significant changes were observed before and two minute after the test (p > 0.05). Conclusions: Ingestion of Red Bull and Hype prior to exercise testing is effective on some indices of cardiorespira-tory fitness but not on the blood lactate levels

Inhibition of GLI1 gene activation by Patched1

Patched1 (PTCH1) is a human tumour suppressor that acts as an HH (Hedgehog) receptor protein and is important for embryonic patterning. PTCH1 mediates its effects through SMO (Smoothened) and represses the expression of HH target genes such as the transcription factor GLI1 (glioma 1) as well as PTCH1. Up-regulation of these genes has been observed in several cancer forms, including basal cell carcinoma, digestive track tumours and small cell lung cancer. The fact that PTCH1 down-regulates its own expression via ‘negative feedback’ is an important feature in HH signalling, as it keeps the balance between HH and PTCH1 activities that are essential for normal development. In the present study, we provide evidence that a novel mechanism allowing PTCH1 to maintain this balance may also exist. We show that gene activation by GLI1, the transcriptional effector of the pathway, can be down-regulated by PTCH1 without involvement of the canonical cascade of HH signalling events. Specifically, the SMO antagonist cyclopamine has no appreciable effects in blocking this PTCH1-mediated inhibition. Moreover, the negative GLI1 regulator SUFU (Suppressor of Fused) was also found to be dispensable. Additionally, deletion mapping of PTCH1 has revealed that the domains encompassed by amino acids 180–786 and 1058–1210 are of highest significance in inhibiting GLI1 gene activation. This contrasts with the importance of the PTCH1 C-terminal domain for HH signalling

The effect of DNA methylation inhibitor 5-Aza-2'-deoxycytidine on human endometrial stromal cells

Decidualization, the differentiation of endometrial stromal cells is a crucial step for successful implantation of an embryo, development of the placenta and completion of pregnancy to term. Epigenetic mechanisms are thought to be strongly involved in the regulation of processes controlling implantation, placentation, organ formation and foetal growth. Recent studies suggest that decreased DNA methylation facilitates a receptive endometrium. Hence, the aim of this project was to compare the transcriptional profile changes induced by the inhibitor of DNA methylation, 5-Aza-2'-deoxycytidine (AZA) to the transcriptional changes that happen during decidualization

Epigenetic regulation of E-cadherin controls endometrial receptivity

Key to the success of human reproduction is the capacity of an embryo to attach and implant into the endometrial wall after which a nutrient supply is established through placentation. Herein, we have examined the potential epigenetic regulation of uterine receptivity by use of the receptive RL95-2 and nonreceptive AN3-CA endometrial epithelial carcinoma cell lines. Using an in vitro model of embryo implantation, we demonstrate that inhibition of DNA methylation by 5′-aza- 2′-deoxycytidine (AZA), resulted inthe nonreceptive AN3-CA cell line becoming receptiveto BeWo cell spheroid attachment. Examination of components of the adherens junction complex revealed that AZA specifically increased the expression of E-cadherin and plakoglobin at the mRNA and protein levels in AN3-CA cells, and E-cadherin protein expression was found to localize to sites of intercellular contact. Forced expression of E-cadherin in AN3-CA cells significantly enhanced receptivity. Small interfering RNA (siRNA)-mediated depletion of the individual DNA methyltrans-ferase (DNMT) molecules did not induce E-cadherin expression in AN3-CA cells; however, concomitant siRNA-mediated depletion of both DNMT3A and DNMT3B induced the expression of E-cadherin. Furthermore, E-cadherin expression was significantly increased after the concomitant siRNA-mediated depletion of DNMT-1, -3A, and -3B in AN3-CA cells. Therefore, we have provided evidence that E-cadherin plays an important role in uterine receptivity and that E-cadherin expression is epigenetically regulated in AN3-CA cells, suppressed by the combined actions of DNMT-1, -3A, and -3B. Copyright © 2009 by The Endocrine Society

Epigenetic regulation of human trophoblastic cell migration and invasion

Pivotal to successful mammalian reproduction is the ability of a developing embryo to implant to the uterine wall and establish a nutrient supply via placentation. Herein, we have examined the potential epigenetic regulation of human trophoblastic cell migration and invasion by use of the choriocarcinoma cell line, BeWo. Treatment of BeWo cells with a DNA methyltransferase inhibitor, 5'-aza-2'-deoxycytidine (AZA), resulted in conversion of cell morphology to a nonmigratory phenotype. This was exemplified by the ability of AZA to prevent BeWo cell migration in wound healing and transwell migration assays. AZA consequently inhibited BeWo cell invasion through reconstituted basement membrane. Examination of components of the adherens junction complex pivotal for determination of cell phenotype revealed that AZA specifically increased the mRNA level of E-cadherin and plakoglobin (gamma-catenin), but not alpha-catenin and beta-catenin. AZA also increased the gene promoter activity of both plakoglobin and E-cadherin. Protein levels of both plakoglobin and E-cadherin were increased by AZA, and AZA enhanced their localization to sites of intercellular contact. Forced expression of plakoglobin and E-cadherin abrogated BeWo cell migration, indicative that repression of these genes was required for BeWo cell migration. Small interfering RNA-mediated depletion of the individual DNA methyltransferase (DNMT) molecules did not affect plakoglobin and E-cadherin promoter activity or BeWo cell migration. However, increases in plakoglobin and E-cadherin promoter activity and inhibition of BeWo cell migration was achieved with small interfering RNA-mediated depletion of both DNMT-3a and DNMT-3b. Epigenetic regulation of plakoglobin and E-cadherin is therefore pivotal for appropriate trophoblastic invasion in vitro