Genetic and epigenetic changes in the mitogen activated protein kinase (MAPK) signaling render urothelial cancer a potential target for tyrosine kinase inhibitor (TKI) treatment. However, clinical trials of several TKIs failed to prove efficacy. In this context, we investigated changes in MAPK signaling activity, downstream apoptotic regulators and changes in cell cycle distribution in different urothelial cancer cell lines (UCCs) upon treatment with the multikinase inhibitor sorafenib. None of the classical sorafenib targets (vascular endothelial growth factor receptor 1/-receptor 2, VEGFR1/-R2; platelet-derived growth factor receptor ?/-receptor ?, PDGFR-?/-?; c-KIT) was expressed at significant levels leaving RAF proteins as its likely molecular target. Low sorafenib concentrations paradoxically increased cell viability, whereas higher concentrations induced G1 arrest and eventually apoptosis. MAPK signaling remained partly active after sorafenib treatment, especially in T24 cells with an oncogenic HRAS mutation. AKT phosphorylation was increased, suggesting compensatory activation of the phosphatidylinositol-3-kinase (PI3K) pathway. Sorafenib regularly down regulated the anti-apoptotic myeloid cell leukemia 1 (Mcl-1) protein, but combinatorial treatment with ABT-737 targeting other B-cell lymphoma 2 (Bcl-2) family proteins did not result in synergistic effects. In summary, efficacy of sorafenib in urothelial cancer cell lines appears hampered by limited effects on MAPK signaling, crosstalk with further cancer pathways and an anti-apoptotic state of UCCs. These observations may account for the lack of efficacy of sorafenib in clinical trials and should be considered more broadly in the development of signaling pathway inhibitors for drug therapy in urothelial carcinoma.
Notch signalling regulates cell fate in most tissues, promoting precursor cell proliferation in some, but differentiation in others. Accordingly, downregulation or overactivity variously contributes to cancer development. So far, little is known about Notch pathway activity and function in the normal urothelium and in urothelial carcinoma (UC). We have therefore investigated expression of Notch pathway components in UC tissues and cell lines and studied the function of one receptor, NOTCH1, in detail.
With rapidly increasing numbers of publications, assessments of study quality, reporting quality, and classification of studies according to their level of evidence or developmental stage have become key issues in weighing the relevance of new information reported. Diagnostic marker studies are often criticized for yielding highly discrepant and even controversial results. Much of this discrepancy has been attributed to differences in study quality. So far, numerous tools for measuring study quality have been developed, but few of them have been used for systematic reviews and meta-analysis. This is owing to the fact that most tools are complicated and time consuming, suffer from poor reproducibility, and do not permit quantitative scoring.
The aim of this study was to develop valid and reliable disease-specific questionnaires for adult patients with cystic fibrosis and for parents of minors with cystic fibrosis for assessing patient experience with cystic fibrosis care.
Urothelial carcinoma (UC) is characterized by multiple recurrent chromosomal changes on a background of increasing genomic instability. To define target genes of recurrent deletions and amplifications, we explored which gene alterations are common in UC, in two recently established cell lines, BC44 and BC61.
Several novel therapeutic agents have demonstrated ability to improve overall survival in metastatic castration-resistant prostate cancer (mCRPC) in recent years. With as many as 5 new agents approved within the last 5 years and an ongoing lack of comparative and prospective data, strategies for patient selection and sequencing of drug treatments are urgently needed. This review will summarize current clinical evidence and relevant molecular mechanisms in mCRPC. The understanding of these mechanisms may provide valuable assistance in making therapeutic decisions, especially while robust clinical data remain sparse.
Previous studies have shown that class-I histone deacetylase (HDAC) 8 mRNA is upregulated in urothelial cancer tissues and urothelial cancer cell lines compared to benign controls. Using urothelial cancer cell lines we evaluated whether specific targeting of HDAC8 might be a therapeutic option in bladder cancer treatment.
Antimicrobial photodynamic therapy (aPDT) is a promising method to treat local bacterial infections. The therapy is painless and does not cause bacterial resistances. However, there are gaps in understanding the dynamics of the processes, especially in periodontal treatment. This work describes the advances in fundamental physical and mathematical modeling of aPDT used for interpretation of experimental evidence. The result is a two-dimensional model of aPDT in a dental pocket phantom model. In this model, the propagation of laser light and the kinetics of the chemical reactions are described as coupled processes. The laser light induces the chemical processes depending on its intensity. As a consequence of the chemical processes, the local optical properties and distribution of laser light change as well as the reaction rates. The mathematical description of these coupled processes will help to develop treatment protocols and is the first step toward an inline feedback system for aPDT users.
Epigenetic modifiers such as histone deacetylases (HDACs) have come into focus as novel drug targets for cancer therapy due to their functional role in tumor progression. Since common pan-HDAC inhibitors have adverse side effects and minor anti-cancer activity against solid tumors, enzyme-specific inhibitors were developed. HDAC6 is especially well-suited for specific inhibition due to its unique domain structure and mode of action and has been suggested to provide an exceptionally suitable target for cancer therapy. However, expression and function of HDACs have been insufficiently studied in urothelial cancers (UC), a disease urgently requiring new therapeutic approaches. The present study sought to evaluate HDAC6 as a target for treatment of urothelial cancers with enzyme-specific inhibitors. We observed moderate HDAC6 overexpression in urothelial cancer tissues and a broad range of expression in urothelial cancer cell lines. In the cell lines Tubacin was the most potent inhibitor, compared with Tubastatin and ST-80, but still active only at high micromolar concentrations. HDAC6 expression levels correlated poorly with sensitivity to enzyme inhibition. Combined treatments with heat shock, HSP90 inhibition by 17-AAG, proteasome inhibition by bortezomib, or DNA-damaging agents did not result in significant synergistic effects. Experiments with siRNA-mediated knockdown further underlined that urothelial cancer cells do not critically depend on HDAC6 expression for survival.
Multiple epigenetic alterations contribute to prostate cancer progression by deregulating gene expression. Epigenetic mechanisms, especially differential DNA methylation at imprinting control regions (termed DMRs), normally ensure the exclusive expression of imprinted genes from one specific parental allele. We therefore wondered to which extent imprinted genes become deregulated in prostate cancer and, if so, whether deregulation is due to altered DNA methylation at DMRs. Therefore, we selected presumptive deregulated imprinted genes from a previously conducted in silico analysis and from the literature and analyzed their expression in prostate cancer tissues by qRT-PCR. We found significantly diminished expression of PLAGL1/ZAC1, MEG3, NDN, CDKN1C, IGF2, and H19, while LIT1 was significantly overexpressed. The PPP1R9A gene, which is imprinted in selected tissues only, was strongly overexpressed, but was expressed biallelically in benign and cancerous prostatic tissues. Expression of many of these genes was strongly correlated, suggesting co-regulation, as in an imprinted gene network (IGN) reported in mice. Deregulation of the network genes also correlated with EZH2 and HOXC6 overexpression. Pyrosequencing analysis of all relevant DMRs revealed generally stable DNA methylation between benign and cancerous prostatic tissues, but frequent hypo- and hyper-methylation was observed at the H19 DMR in both benign and cancerous tissues. Re-expression of the ZAC1 transcription factor induced H19, CDKN1C and IGF2, supporting its function as a nodal regulator of the IGN. Our results indicate that a group of imprinted genes are coordinately deregulated in prostate cancers, independently of DNA methylation changes.
Carcinoma of the bladder is the fifth most common cancer whose incidence continues to rise. MTDH/AEG-1 is associated with the initiation and progression of many cancers including breast, hepatocellular, ovarian, and colorectal carcinomas. However, the expression and functional importance of MTDH/AEG-1 in bladder cancer remains unknown. The present study was aimed at exploring the functional role of MTDH/AEG-1 in selected bladder cancer cell lines.
Rosacea is associated with mental distress and depression, yet no studies have looked at the mediating effect of stigmatization and other factors on psychological sequelae. Using a survey of affected individuals, the objective of this study was to explore relationships between self-reported symptom severity, discomfort, stress reactivity, quality of life, and symptoms of depression and anxiety. Participants were recruited from the mailing list of a German rosacea organization. The survey instrument included the Dermatology Life Quality Index (DLQI), the rejection scale of the Questionnaire on Experience with Skin Complaints (QES), and the German version of the Hospital Anxiety and Depression Scale (HADS-D). A total of 168 persons with rosacea were participated. The sample returned a HADS anxiety score of 7.2?±?4.60, a HADS depression score of 5.1?±?4.09, a DLQI of 4.05?±?4.67, and a QES rejection score of 2.,61?±?3.,37. Path analysis suggested that symptoms of anxiety and depression are linked with somatic symptoms indirectly, mediated through quality of life and stigmatization. Men are more negatively affected. Physicians treating rosacea patients should consider recommending psychological co-treatment for patients who have experienced stigmatization or who report low life quality.
Diagnostic and prognostic tools for prostate cancer (PC) are suboptimal, causing overtreatment of indolent PC and risk of delayed treatment of aggressive PC. Here, we identify six novel candidate DNA methylation markers for PC with promising diagnostic and prognostic potential.
This study presents a novel numerical model for laser ablation and laser damage in glass including beam propagation and nonlinear absorption of multiple incident ultrashort laser pulses. The laser ablation and damage in the glass cutting process with a picosecond pulsed laser was studied. The numerical results were in good agreement with our experimental observations, thereby revealing the damage mechanism induced by laser ablation. Beam propagation effects such as interference, diffraction and refraction, play a major role in the evolution of the crater structure and the damage region. There are three different damage regions, a thin layer and two different kinds of spikes. Moreover, the electronic damage mechanism was verified and distinguished from heat modification using the experimental results with different pulse spatial overlaps.
Changes in DNA methylation frequently accompany cancer development. One prominent change is an apparently genome-wide decrease in methylcytosine that is often ascribed to DNA hypomethylation at retroelements comprising nearly half the genome. DNA hypomethylation may allow reactivation of retroelements, enabling retrotransposition, and causing gene expression disturbances favoring tumor development. However, neither the extent of hypomethylation nor of retroelement reactivation are precisely known. We therefore assessed DNA methylation and expression of three major classes of retroelements (LINE-1, HERV-K, and AluY) in human urinary bladder cancer tissues and cell lines by pyrosequencing and quantitative reverse transcription-polymerase chain reaction, respectively. We found substantial global LINE-1 DNA hypomethylation in bladder cancer going along with a shift toward full-length LINE-1 expression. Thus, pronounced differences in LINE-1 expression were observed, which may be promoted, among others, by LINE-1 hypomethylation. Significant DNA hypomethylation was found at the HERV-K_22q11.23 proviral long terminal repeat (LTR) in bladder cancer tissues but without reactivation of its expression. DNA methylation of HERVK17, essentially absent from normal urothelial cells, was elevated in cell lines from invasive bladder cancers. Accordingly, the faint expression of HERVK17 in normal urothelial cells disappeared in such cancer cell lines. Of 16 additional HERV-Ks, expression of 7 could be detected in the bladder, albeit generally at low levels. Unlike in prostate cancers, none of these showed significant expression changes in bladder cancer. In contrast, expression of the AluYb8 but not of the AluYa5 family was significantly increased in bladder cancer tissues. Collectively, our findings demonstrate a remarkable specificity of changes in expression and DNA methylation of retroelements in bladder cancer with a significantly different pattern from that in prostate cancer.
During the treatment of surface water to drinking water, ozonation is often used for disinfection and to remove organic trace substances, whereby oxidation by-products can be formed. Here we use the example of tolyltriazole to describe an approach for identifying relevant oxidation by-products in the laboratory and subsequently detecting them in an industrial-scale process. The identification process involves ozonation experiments with pure substances at laboratory level (concentration range mg L(-1)). The reaction solutions from different ozone contact times were analyzed by high performance liquid chromatography - quadrupole time-of-flight mass spectrometry (HPLC-QTOF-MS) in full scan mode. Various approaches were used to detect the oxidation by-products: (i) target searches of postulated oxidation by-products, (ii) comparisons of chromatograms (e.g., UV/VIS) of the different samples, and (iii) color-coded abundance time courses (kinetic) of all detected compounds were illustrated in a kind of a heat map. MS/MS, H/D exchange, and derivatization experiments were used for structure elucidation for the detected by-product. Due to the low contaminant concentrations (ng L(-1)-range) of contaminants in the untreated water, the conversion of results from laboratory experiments to an industrial-scale required the use of HPLC-MS/MS with sample enrichment (e.g., solid phase extraction.) In cases where reference substances were not available or oxidation by-products without clear structures were detected, reaction solutions from laboratory experiments were used to optimize the analytical method to detect ng L(-1) in the samples of the industrial processes. We exemplarily demonstrated the effectiveness of the methodology with the industrial chemicals 4- and 5-methyl-1H-benzotriazole (4- and 5-MBT) as an example. Moreover, not only did we identify several oxidation by-products in the laboratory experiments tentatively, but also detected three of the eleven reaction products in the outlet of the full-scale ozonation unit.
Retroelements constitute a large part of the human genome. These sequences are mostly silenced in normal cells, but genome-wide DNA hypomethylation in cancers might lead to their re-expression. Whether this re-expression really occurs in human cancers is largely unkown. We therefore investigated expression and DNA methylation of several classes of retroelements in human prostate cancer tissues and cell lines by quantitative reverse transcription-polymerase chain reaction and pyrosequencing, respectively. The most striking finding was strong and generalized increased expression of the HERV-K_22q11.23 provirus in cancers, including de novo expression of a spliced accessory Np9 transcript in some tumors. In parallel, DNA methylation in the long terminal repeat (LTR) decreased. Conversely, HERVK17 expression was significantly diminished in cancer tissues, but this decrease was unrelated to LTR methylation. Expression of both proviruses was restricted to androgen-responsive prostate cancer cell lines and LTRs sequences containing steroid hormone-responsive elements bound the androgen receptor and conferred androgen responsiveness to reporter constructs. Expression of LINE-1 5-untranslated region (UTR) and 3-UTR sequences in prostate cancers rather decreased, despite significant hypomethylation of the internal LINE-1 promoter. Increased expression of the young AluYa5 and AluYb8 families was restricted to individual tumors. Our findings demonstrate a surprising specificity of changes in expression and DNA methylation of retroelements in prostate cancer. In particular, LINE-1 hypomethylation does not lead to generalized overexpression, but specific human endogenous retrovirus-K proviruses display conspicuous changes in their expression hinting at significant functions during prostate carcinogenesis.
Overexpression of the classical homeobox transcription factor HOXC6 is frequent in prostate cancers and correlates with adverse clinical parameters. Since surprisingly many HOXC6 target genes are downregulated in prostate cancer, it has been posited that oncogenic effects of HOXC6 in prostate cancer may be unmasked by concurrent epigenetic downregulation of target genes exerting tumor suppressive effects. To test this hypothesis, we have studied the expression of three HOXC6 target genes, CNTN1 (encoding a cell adhesion protein), DKK3 and WIF1 (encoding WNT growth factor antagonists) as well as DNA methylation of DKK3 and WIF1. HOXC6 upregulation and association with poor prognosis were confirmed in our tissue series. The three target genes were each significantly downregulated in cancer tissues and expression of each one correlated inversely with that of HOXC6. Cases with lower WIF1 expression showed significantly earlier recurrence (p = 0.021), whereas no statistical significance was reached for CNTN1 and DKK3. Hypermethylation of DKK3 or WIF1 gene promoters was observed in a subset of cancers with downregulated expression, but was often weak. Our data support the hypothesis that HOXC6 target genes exerting tumor-suppressive effects are epigenetically downregulated in prostate cancer, but DNA methylation appears to follow or bolster rather than to cause their transcriptional inactivation.
Aberrant activation and mutation status of proteins in the phosphatidylinositol-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) and the mitogen activated protein kinase (MAPK) signaling pathways have been linked to tumorigenesis in various tumors including urothelial carcinoma (UC). However, anti-tumor therapy with small molecule inhibitors against mTOR turned out to be less successful than expected. We characterized the molecular mechanism of this pathway in urothelial carcinoma by interfering with different molecular components using small chemical inhibitors and siRNA technology and analyzed effects on the molecular activation status, cell growth, proliferation and apoptosis. In a majority of tested cell lines constitutive activation of the PI3K was observed. Manipulation of mTOR or Akt expression or activity only regulated phosphorylation of S6K1 but not 4E-BP1. Instead, we provide evidence for an alternative mTOR independent but PI3K dependent regulation of 4E-BP1. Only the simultaneous inhibition of both S6K1 and 4E-BP1 suppressed cell growth efficiently. Crosstalk between PI3K and the MAPK signaling pathway is mediated via PI3K and indirect by S6K1 activity. Inhibition of MEK1/2 results in activation of Akt but not mTOR/S6K1 or 4E-BP1. Our data suggest that 4E-BP1 is a potential new target molecule and stratification marker for anti cancer therapy in UC and support the consideration of a multi-targeting approach against PI3K, mTORC1/2 and MAPK.
The use of complementary and alternative medicine (CAM) is increasing in Europe as well as in the USA, but CAM courses are infrequently integrated into medical curricula. In Europe, but also especially in the USA and in Canada, the attitudes of medical students and health science professionals in various disciplines towards CAM have been the subject of investigation. Most studies report positive attitudes. The main aim of this study was to compare the attitudes towards CAM of medical and psychology students in Germany. An additional set of questions concerned how CAM utilisation and emotional and physical condition affect CAM-related attitudes. Two hundred thirty-three medical students and 55 psychology students were questioned concerning their attitudes towards CAM using the Questionnaire on Attitudes Towards Complementary Medical Treatment (QACAM). Both medical students and psychology students were sceptical about the diagnostic and the therapeutic proficiency of doctors and practitioners of CAM. Students attitudes towards CAM correlated neither with their experiences as CAM patients nor with their emotional and physical condition. It can be assumed that German medical and psychology students will be reluctant to use or recommend CAM in their professional careers. Further studies should examine more closely the correlation between attitudes towards CAM and the students worldview as well as their existing knowledge of the effectiveness of CAM.
Urinary tract infections are common, obnoxious and in some cases even life endangering. They are most often caused by uropathogenic Escherichia coli. Persistent and seemingly recurrent infections may be caused by bacteria establishing intracellular reservoirs in the urothelial epithelium. A better understanding of the mechanisms involved in the complex interplay between host and pathogens is required for preventing and controlling these infections. A paper in this issue of Laboratory Investigation reports changes in gene expression in urothelial carcinoma cells following infection by uropathogenic bacteria which go along with the activation of DNA methyltransferase 1 and increased methylation at the CDKN2A gene. These pioneer findings should stimulate the further development of in vitro models for studying urothelial infections and prompt more extensive analyses of epigenetic alterations elicited by bacterial infections in the urinary tract.
Analyses of DNA methylation in human cancers have identified hypermethylation of individual genes and diminished methylation at repeat elements as common alterations, and have thereby provided important mechanistic insights into cancer biology as well as biomarkers for cancer detection, prognosis and prediction of therapy responses. The techniques available in the past were best suited for investigations of individual candidate genes and sequences, whereas recently developed high-throughput techniques promise to generate unbiased and comprehensive surveys of DNA methylation states across entire genomes. In this minireview we give a short overview of established and novel techniques and outline some major questions that can now be addressed to develop further cancer biomarkers and therapies based on DNA methylation.
Molecular mechanisms driving cancer development and progression are rarely unique to one cancer type. Rather, recent genomic studies of urological cancers suggest that common mechanisms recur with variations. Examples include alterations in hypoxia response regulation, epigenetic regulator proteins, and signal transduction pathways in renal, prostatic and urothelial carcinomas. Consideration of these variations alongside the common basic cancer mechanisms might be important for the successful development of targeted therapies.
The products of transcription by the multisubunit enzyme RNA polymerase III (Pol III), such as 5S rRNA, tRNAs, U6 snRNA, are important for cell growth, proliferation and differentiation. The known range of the Pol III transcriptome has expanded over recent years, and novel functions of the newly discovered and already well known transcripts have been identified, including regulation of stress responses and apoptosis. Furthermore, transcription by Pol III has turned out to be strongly regulated, differing between diverse class III genes, among cell types and under stress conditions. The mechanisms involved in regulation of Pol III transcription are being elucidated and disturbances in that regulation have been implicated in various diseases, including cancer. This review summarizes the novel data on the regulation of RNA polymerase III transcription, including epigenetic and gene specific mechanisms and outlines recent insights into the cellular functions of the Pol III transcriptome, in particular of SINE RNAs.
Non-target screening via high performance liquid chromatography-mass spectrometry (HPLC-MS) has gained increasingly in importance for monitoring organic trace substances in water resources targeted for the production of drinking water. In this article a new approach for evaluating the data from non-target HPLC-MS screening in water is introduced and its advantages are demonstrated using the supply of drinking water as an example. The crucial difference between this and other approaches is the comparison of samples based on compounds (features) determined by their full scan data. In so doing, we take advantage of the temporal, spatial, or process-based relationships among the samples by applying the set operators, UNION, INTERSECT, and COMPLEMENT to the features of each sample. This approach regards all compounds, detectable by the used analytical method. That is the fundamental meaning of non-target screening, which includes all analytical information from the applied technique for further data evaluation. In the given example, in just one step, all detected features (1729) of a landfill leachate sample could be examined for their relevant influences on water purification respectively drinking water. This study shows that 1721 out of 1729 features were not relevant for the water purification. Only eight features could be determined in the untreated water and three of them were found in the final drinking water after ozonation. In so doing, it was possible to identify 1-adamantylamine as contamination of the landfill in the drinking water at a concentration in the range of 20 ng L(-1). To support the identification of relevant compounds and their transformation products, the DAIOS database (Database-Assisted Identification of Organic Substances) was used. This database concept includes some functions such as product ion search to increase the efficiency of the database query after the screening. To identify related transformation products the database function "transformation tree" was used.
Hypermethylation of the PITX2 (paired-like homeodomain transcription factor 2) gene promoter is strongly associated with recurrence after radical prostatectomy. We hypothesized that PITX2 hypermethylation leads to PITX2 silencing and that decreased PITX2 expression is likewise associated with poor prognosis in prostate cancers. Moreover, it is unknown so far how PITX2 hypermethylation relates to other molecular changes in prostate cancer, such as ERG oncogenic activation in about half of all cases.
Using focused ion beam etching techniques, micropillar cavities were fabricated from a high reflective AlAs/AlGaAs distributed Bragg reflector planar cavity containing self-assembled InP quantum dots in (Al(0.20)Ga(0.80))(0.51)In(0.49)P barrier layers. The mode spectra of pillars with different diameters were investigated using micro-photoluminescence, showing excellent agreement with theory. Quality factors of the pillar cavities up to 3650 were observed. Furthermore, for a microcavity pillar with 1.26 mum diameter, single-photon emission is demonstrated by performing photon correlation measurements under pulsed excitation.
The RNA-binding protein Musashi1 (MSI1) is a marker of progenitor cells in the nervous system functioning as a translational repressor. We detected?MSI1?mRNA in several bladder carcinoma cell lines, but not in cultured normal uroepithelial cells, whereas the paralogous MSI2 gene was broadly expressed. Knockdown of?MSI1?expression by siRNA induced apoptosis and a severe decline in cell numbers in 5637 bladder carcinoma cells. Microarray analysis of gene expression changes after?MSI1?knockdown significantly up-regulated 735 genes, but down-regulated only 31. Up-regulated mRNAs contained a highly significantly greater number and density of Musashi binding sites. Therefore, a much larger set of mRNAs may be regulated by Musashi1, which may affect not only their translation, but also their turnover. The study confirmed p21(CIP1) and Numb proteins as targets of Musashi1, suggesting additionally p27(KIP1) in cell-cycle regulation and Jagged-1 in Notch signalling. A significant number of up-regulated genes encoded components of stress granules (SGs), an organelle involved in translational regulation and mRNA turnover, and impacting on apoptosis. Accordingly, heat shock induced SG formation was augmented by Musashi1 down-regulation. Our data show that ectopic?MSI1?expression may contribute to tumorigenesis in selected bladder cancers through multiple mechanisms and reveal a previously unrecognized function of Musashi1 in the regulation of SG formation.
The cortical cytoskeleton network connects the actin cytoskeleton to various membrane proteins, influencing cell adhesion, polarity, migration and response to extracellular signals. Previous studies have suggested changes in the expression of specific components in prostate cancer, especially of 4.1 proteins (encoded by EPB41 genes) which form nodes in this network.
Many metastatic cancers recapitulate the epithelial-to-mesenchymal transition (EMT) resulting in enhanced cell motility and invasiveness. The EMT is regulated by several transcription factors, including the zinc finger protein SNAI2, also named Slug, which appears to exert additional functions during development and cancer progression. We have studied the function of SNAI2 in prostate cancer cells. Quantitative RT-PCR analysis showed strong SNAI2 expression particularly in the PC-3 and PC3-16 prostate carcinoma cell lines. Knockdown of SNAI2 by specific siRNA induced changes in EMT markers and inhibited invasion of both cell lines into a matrigel matrix. SNAI2 siRNA-treated cells did not tolerate detachment from the culture plates, likely at least in part due to downregulation of integrin alpha6beta4. SNAI2 knockdown disturbed the microtubular and actin cytoskeletons, especially severely in PC-3 cells, resulting in grossly enlarged, flattened, and sometimes multinuclear cells. Knockdown also decreased cell proliferation, with a prominent G0/G1 arrest in PC3-16. Together, our data imply that SNAI2 exerts strong effects on the cytoskeleton and adhesion of those prostate cancer cells that express it and is necessary for their proliferation and invasiveness.
Imprinted genes are often arranged in clusters epigenetically controlled by differentially methylated regions (DMR) containing bivalent histone modifications. Both DNA hypermethylation and hypomethylation in cancer can therefore disturb imprinted gene expression. We have studied expression, DNA methylation and histone modifications of TFPI2, a presumed tumor suppressor, and that of other genes in the 7q21 imprinted gene cluster in prostate cancer.
The epithelial-mesenchymal transition (EMT) is regarded as an important step in cancer metastasis. Snail, a master regulator of EMT, has been recently proposed to act additionally as a cell survival factor and inducer of motility. We have investigated the function of Snail (SNAI1) in prostate cancer cells by downregulating its expression via short (21-mer) interfering RNA (siRNA) and measuring the consequences on EMT markers, cell viability, death, cell cycle, senescence, attachment, and invasivity. Of eight carcinoma cell lines, the prostate carcinoma cell lines LNCaP and PC-3 showed the highest and moderate expression of SNAI1 mRNA, respectively, as measured by quantitative RT-PCR. Long-term knockdown of Snail induced a severe decline in cell numbers in LNCaP and PC-3 and caspase activity was accordingly enhanced in both cell lines. In addition, suppression of Snail expression induced senescence in LNCaP cells. SNAI1-siRNA-treated cells did not tolerate detachment from the extracellular matrix, probably due to downregulation of integrin ?6. Expression of E-cadherin, vimentin, and fibronectin was also affected. Invasiveness of PC-3 cells was not significantly diminished by Snail knockdown. Our data suggest that Snail acts primarily as a survival factor and inhibitor of cellular senescence in prostate cancer cell lines. We therefore propose that Snail can act as early driver of prostate cancer progression.
Although donorship issues and the integration of the new organs are the most distinguishing aspects of lung transplantation (LTx) compared to other kinds of high-tech medicine, there is a paucity of papers on that matter. Therefore, we aimed to evaluate these aspects in young adult LTx recipients with at least 1-year survival.
Low density (approximately 10(7) cm(-2)), small sized InGaAs quantum dots were grown on a GaAs substrate by metal-organic vapor-phase epitaxy and a special annealing technique. The structural quantum dot properties and the influence of the annealing technique was investigated by atomic force microscope measurements. High-resolution micro-photoluminescence spectra reveal narrow photoluminescence lines, with linewidths down to 11 microeV and fine structure splittings of 25 microeV. High signal to noise ratios (approximately 140) and a nearly background free autocorrelation measurement indicate an excellent optical quality and single photon emission behavior. Furthermore, time resolved measurements reveal excitonic decay times typically in the range between 800 and 2300 ps and biexcitonic decay times around 300 ps.
Rhodamine B and its five de-ethylated transformation products could be identified in a groundwater sample. Using high-performance thin-layer chromatography (HPTLC) six fluorescent zones were detected in the sample. In order to identify the compounds in the zones by exact mass mass spectrometry (MS) measurements and tandem mass spectrometry (MS/MS), they were extracted from the HPTLC plate for subsequent analysis by nano-chip high-performance liquid chromatography quadrupole-time-of-flight mass spectrometry (nano-chip HPLC/QTOFMS). In addition, chemical derivatisation experiments on HPTLC plates were applied to detect the presence of a primary amino group in the transformation products. From the combined analytical results it was possible to allocate rhodamine B and its five de-ethylated transformation products to the six different HPTLC zones. The quantification of rhodamine B in different groundwater samples was carried out by a high-performance liquid chromatography/triple quadrupole mass spectrometry (HPLC/MS/MS). The maximum detected concentration of rhodamine B was 83 microg L(-1).
The cellular FLICE-inhibitory protein (c-FLIP) is a modulator of death receptor-mediated apoptosis and plays a major role in T- and B-cell homeostasis. Three different isoforms have been described on the protein level, including the long form c-FLIP(L) as well as 2 short forms, c-FLIP(S) and the recently identified c-FLIP(R). The mechanisms controlling c-FLIP isoform production are largely unknown. Here, we identified by sequence comparison in several mammals that c-FLIP(R) and not the widely studied c-FLIP(S) is the evolutionary ancestral short c-FLIP protein. Unexpectedly, the decision for production of either c-FLIP(S) or c-FLIP(R) in humans is defined by a single nucleotide polymorphism in a 3 splice site of the c-FLIP gene (rs10190751A/G). Whereas an intact splice site directs production of c-FLIP(S), the splice-dead variant causes production of c-FLIP(R). Interestingly, due to differences in protein translation rates, higher amounts of c-FLIP(S) protein compared with c-FLIP(R) are produced. Investigation of diverse human cell lines points to an increased frequency of c-FLIP(R) in transformed B-cell lines. A comparison of 183 patients with follicular lymphoma and 233 population controls revealed an increased lymphoma risk associated with the rs10190751 A genotype causing c-FLIP(R) expression.
PIWIL2, a member of Argonaute family of proteins, is exclusively expressed in testis and functions in development and maintenance of germline stem cells. Recently, ectopic expression of PIWIL2 has been reported in a variety of human and mouse tumors. To investigate a potential involvement of PIWIL2 in human bladder cancer, we examined its expression in several human bladder cancer cell lines, normal uroepithelial cell cultures, and some bladder tissues.
Prostate cancer is one of the most frequent cancers in males in Western industrialized countries. Its course is highly variable, from indolent to highly lethal. Genetic changes vary accordingly, with chromosomal losses, gains and translocations, although often recurrent, differing between individual cases of the disease. In contrast, certain epigenetic changes are highly consistent, in particular hypermethylation of a specific set of genes, and others regularly associated with progression, such as global DNA hypomethylation, certain chromatin modifications and altered levels and composition of polycomb complexes. Although changes in polycombs and DNA methylation appear to both accompany the progression of prostate cancer, recent studies do not suggest that they cause one another. However, they may contribute to establishing and maintaining an aberrant differentiation potential of prostate cancer initiating cells. Global DNA hypomethylation in prostate cancer may relate to adaptative changes in several signaling pathways typical of this cancer type, including innate immunity responses. Similarly, adaptative changes in the expression and function of chromatin regulators required to diminish the dependency of prostate cancer cells on androgens may shape the epigenome, beyond individual genes regulated by the androgen receptor. Because of their crucial role, epigenetic alterations may become highly useful for diagnostics and therapy of prostate cancer.
Defects in apoptotic pathway contribute to uncontrolled proliferation of cancer cells and confer resistance to chemotherapy. Growth arrest and DNA damage inducible, alpha (GADD45alpha) is up-regulated on docetaxel treatment and may contribute to docetaxel-mediated cytotoxicity. We examined the mechanism of regulation of GADD45alpha in prostate cancer cells and the effect of its up-regulation on sensitivity to docetaxel chemotherapy. Expression of GADD45alpha in PC3 cells was higher than that in Du145 and LNCaP cells (17- and 12-fold, respectively; P < 0.05). Although the proximal promoter region was unmethylated in all three cell lines, methylation of a 4 CpG region upstream of the proximal promoter correlated inversely with gene expression levels. Methylation was reversed by treatment of Du145 and LNCaP cells with DNA methyltransferase inhibitors, leading to reactivation of GADD45alpha expression in these cells. The 5 4 CpG region was also frequently methylated in prostate cancer tissues. Methylation of this region correlated inversely with gene expression in prostate cancer and benign prostate tissues. The methyl binding protein MeCP2 was associated with the methylated 4 CpGs in Du145 cells, and knockdown of MeCP2 in these cells (Du145 MeCP2(-)) led to a significantly increased expression of GADD45alpha (3-fold; P = 0.035) without affecting the methylation status of the gene. Enhanced sensitivity to docetaxel was observed by up-regulation of GADD45alpha in Du145 cells by recombinant expression of GADD45alpha or pretreatment with 5-azacytidine. Our results show that GADD45alpha is epigenetically repressed and is a potential target for treatment of prostate cancer.
We demonstrate efficient (>30%) quantum frequency conversion of visible single photons (711 nm) emitted by a quantum dot to a telecom wavelength (1313 nm). Analysis of the first- and second-order coherence before and after wavelength conversion clearly proves that pivotal properties, such as the coherence time and photon antibunching, are fully conserved during the frequency translation process. Our findings underline the great potential of single photon sources on demand in combination with quantum frequency conversion as a promising technique that may pave the way for a number of new applications in quantum technology.
To determine histone deacetylase (HDAC) isoenzyme expression patterns in urothelial cancer tissues and cell lines and investigate their potential to predict the efficacy of the HDAC inhibitor vorinostat.
The role of Hedgehog (HH) signaling in bladder cancer remains controversial. The gene encoding the HH receptor and negative regulator PATCHED1 (PTCH1) resides on a region of chromosome 9q, one copy of which is frequently lost in bladder cancer. Inconsistent with PTCH1 functioning as a classic tumor suppressor gene, loss-of-function mutations in the remaining copy of PTCH1 are not commonly found. Here, we provide direct evidence for a critical role of HH signaling in bladder carcinogenesis. We show that transformed human urothelial cells and many urothelial carcinoma cell lines exhibit constitutive HH signaling, which is required for their growth and tumorigenic properties. Surprisingly, rather than originating from loss of PTCH1, the constitutive HH activity observed in urothelial carcinoma cell lines was HH ligand dependent. Consistent with this finding, increased levels of HH and the HH target gene product GLI1 were found in resected human primary bladder tumors. Furthermore, on the basis of the difference in intrinsic HH dependence of urothelial carcinoma cell lines, a gene expression signature was identified that correlated with bladder cancer progression. Our findings therefore indicate that therapeutic targeting of the HH signaling pathway may be beneficial in the clinical management of bladder cancer.
Epigenetic aberrations are frequent in prostate cancer and could be useful for detection and prognostication. However, the underlying mechanisms and the sequence of these changes remain to be fully elucidated. The tumor suppressor gene RARRES1 (TIG1) is frequently hypermethylated in several cancers. Having noted changes in the expression of its paralogous neighbor gene LXN at 3q25.32, we used pyrosequencing to quantify DNA methylation at both genes and determine its relationship with clinicopathological parameters in 86 prostate cancer tissues from radical prostatectomies. Methylation at LXN and RARRES1 was highly correlated. Increasing methylation was associated with worse clinical features, including biochemical recurrence, and decreased expression of both genes. However, expression of three neighboring genes was unaffected. Intriguingly, RARRES1 methylation was influenced by the genotype of the rs6441224 single-nucleotide polymorphism (SNP) in its promoter. We found that this SNP is located within an ETS-family-response element and that the more strongly methylated allele confers lower activity in reporter assays. Concomitant methylation of RARRES1 and LXN in cancerous tissues was also detected in prostate cancer cell lines and was shown to be associated with repressive histone modifications and transcriptional downregulation. In conclusion, we found that genotype-associated hypermethylation of the ETS-family target gene RARRES1 influences methylation at its neighbor gene LXN and could be useful as a prognostic biomarker.
Non-aqueous capillary electrophoresis (NACE) background electrolytes are most often composed of a mixture of methanol and acetonitrile (ACN) with soluble ammonium salts added as electrolyte. In this study on NACE-MS, we used a mixture of glacial acetic acid and ACN giving rise to an acidic background electrolyte (BGE) with a very low dielectric constant. Impressive changes in selectivity and resolution were observed for structurally closely related indole alkaloids including diastereomers upon addition of ammonium formate as electrolyte and upon variation of the solvent ratio. In order to obtain best separation and MS detection conditions and to reveal the influence of the parameters of the BGE on separation and detection and vice versa of the MS parameters on separation, an optimization strategy was employed using a design of experiments in a central composite design with response surface methodology. It was proven that at high electroosmotic flow conditions capillary electrophoretic separations and thus optimization can be realized without interference from the coupling to an MS system. Several significantly interacting parameters were revealed, which are not accessible with classical univariate optimization approaches. With this optimization, alkaloid mixtures from a plant extract of Mitragyna speciosa, containing a large number of diastereomeric compounds were successfully separated.
Epigenetic dysregulation comprising DNA hypermethylation and hypomethylation, enhancer of zeste homologue 2 (EZH2) overexpression and altered patterns of histone modifications is associated with the progression of prostate cancer. DNA methylation, EZH2 and histone modifications also ensure the parental-specific monoallelic expression of at least 62 imprinted genes. Although it is therefore tempting to speculate that epigenetic dysregulation may extend to imprinted genes, expression changes in cancerous prostates are only well documented for insulin-like growth factor 2 (IGF2). A literature and database survey on imprinted genes in prostate cancer suggests that the expression of most imprinted genes remains unchanged despite global disturbances in epigenetic mechanisms. Instead, selective genetic and epigenetic changes appear to lead to the inactivation of a sub-network of imprinted genes, which might function in the prostate to limit cell growth induced via the PI3K/Akt pathway, modulate androgen responses and regulate differentiation. Whereas dysregulation of IGF2 may constitute an early change in prostate carcinogenesis, inactivation of this imprinted gene network is rather associated with cancer progression.
Epigenetic alterations contribute significantly to the development and progression of prostate cancer, the most prevalent malignant tumor in males of Western industrialized countries. Here, we review recent research on DNA methylation alterations in this cancer type. Hypermethylation of several genes including GSTP1 is well known to occur in a consistent and apparently coordinate fashion during the transition from intraepithelial neoplasia to frank carcinoma. These hypermethylation events have shown promise as biomarkers for detection of prostate carcinoma. Many other individual genes have been shown to undergo hypermethylation, which is typically associated with diminished expression. These investigations indicate additional candidates for biomarkers; in particular, hypermethylation events associated with progression can be employed to identify more aggressive cases. In addition, some of genes silenced by aberrant methylation in prostate have been shown to exhibit properties of tumor suppressors, revealing insights into mechanisms of carcinogenesis. Whereas most studies in the past have used candidate gene approaches, new techniques allowing genome-wide screening for altered methylation are increasingly employed in prostate cancer research and have already yielded encouraging results.
Related JoVE Video
Journal of Visualized Experiments
What is Visualize?
JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.
How does it work?
We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
Video X seems to be unrelated to Abstract Y...
In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.