KDM6A mutations promote acute cytoplasmic DNA release, DNA damage response and mitosis defects.

BACKGROUND: KDM6A, encoding a histone demethylase, is one of the top ten mutated epigenetic cancer genes. The effect of mutations on its structure and function are however poorly characterized. METHODS: Database search identified nonsense and missense mutations in the N-terminal TPR motifs and the C-terminal, catalytic JmjC domain, but also in the intrinsically disordered region connecting both these two well-structured domains. KDM6A variants with cancer-derived mutations were generated using site directed mutagenesis and fused to eGFP serving as an all-in-one affinity and fluorescence tag to study demethylase activity by an ELISA-based assay in vitro, apoptosis by FACS, complex assembly by Co-immunoprecipitation and localization by microscopy in urothelial cells and apoptosis by FACS. RESULTS: Independent of the mutation and demethylase activity, all KDM6A variants were detectable in the nucleus. Truncated KDM6A variants displayed changes in complex assemblies affecting (1) known interactions with the COMPASS complex component RBBP5 and (2) KDM6A-DNA associated assemblies with the nuclear protein Nucleophosmin. Some KDM6A variants induced a severe cellular phenotype characterized by multiple acute effects on nuclear integrity, namely, release of nuclear DNA into the cytoplasm, increased level of DNA damage indicators RAD51 and p-γH2A.X, and mitosis defects. These damaging effects were correlated with increased cell death. CONCLUSION: These observations reveal novel effects of pathogenic variants pointing at new specific functions of KDM6A variants. The underlying mechanisms and affected pathways have to be investigated in future research to understand how tumor cells cope with and benefit from KDM6A truncations.

[Report from the 9th symposium of the German Association for Bladder Cancer Research]. / Bericht vom 9. Symposium des Deutschen Forschungsverbunds Blasenkarzinom.

The annual symposium of the German Research Association for Bladder Carcinoma (DFBK) was organized on February 7th and 8th, 2020, in Düsseldorf. On the first day, eight international guest speakers invited by the DFBK and the Department of Urology of the Heinrich Heine University Düsseldorf presented the current state of research on bladder cancer (BC). Topics were genomic changes and molecular classification in non-muscle-invasive and muscle-invasive BC, prospects and limits of proteome technology in urine diagnostics, function of chromatin regulators in bladder carcinogenesis, cellular reactions to aneuploidy, organoid technology and biobanking, as well as novel aspects of immunotherapy for BC. The second day was dedicated to new results and ideas of the DFBK members on BC pathomechanisms, diagnostics and therapeutic approaches, and most importantly, discussions on the further development of collaborative projects. Additional information is available at http://www.forschungsverbund-blasenkarzinom.de.

High-soluble CGA levels are associated with poor survival in bladder cancer.

Recently, a neuroendocrine-like molecular subtype has been discovered in muscle-invasive urothelial bladder cancer (BC). Chromogranin A (CGA) is a widely used tissue and serum marker in neuroendocrine tumors. Our aim was to evaluate serum CGA (sCGA) concentrations and their associations with clinical and follow-up data in BC and renal cell carcinoma (RCC). sCGA concentrations were analyzed in the following cohorts: (1) BC training set (n = 188), (2) BC validation set (n = 125), (3) RCC patients (n = 77), (4) healthy controls (n = 97). CGA immunohistochemistry and RT-qPCR analyses were performed in 20 selected FFPE and 29 frozen BC tissue samples. Acquired data were correlated with clinicopathological parameters including comorbidities with known effect on sCGA as well as with patients' follow-up data. sCGA levels were significantly higher in BC but not in RCC patients compared to healthy controls. High sCGA levels were independently associated with poor overall and disease-specific survival both in the BC training (P < 0.001, P = 0.002) and validation set (P = 0.009, P = 0.017). sCGA levels were inversely correlated with glomerulus filtrating rate (GFR) and linearly correlated with creatinine clearance and urea concentrations. These correlations were not related to the prognostic value of sCGA. Tissue CGA levels were low to absent independently of sCGA concentrations. Our results demonstrate elevated levels and an independent prognostic value for sCGA in BC but not in RCC. Despite the significant correlation between sCGA and GFR, the prognostic relevance of sCGA seems not related to impaired renal function or other comorbidities.

[Epigenetics in urothelial cancer: Pathogenesis, improving diagnostics and developing novel treatment options]. / Epigenetik des Urothelkarzinoms : Pathogenese, verbesserte Diagnostik und neue Therapieansätze.

Urothelial carcinoma of the bladder is a common tumor for which improvements in diagnostic markers and new therapy approaches, in addition to or combined with standard chemotherapy, are urgently required. Epigenetic alterations could provide both novel diagnostic markers and therapeutic targets as they are emerging as crucial factors in the development and progression of this tumor type, likely contributing to altered differentiation and metastatic potential. These alterations affect DNA methylation, histone modifications, chromatin remodeling, long noncoding RNAs, and microRNAs. Factors involved in histone modifications and chromatin remodeling appear to be particularly frequently inactivated by mutations. Thus, histone-modifying enzymes may represent good targets for rational new therapeutic approaches, although thorough investigation of their complex functions is a prerequisite. DNA methylation changes and altered miRNA expression provide promising biomarkers for diagnosis and prognosis that need further validation in comprehensive and well-standardized studies.

[Bladder cancer in focus: update 2012 of the German Bladder Cancer Association]. / Das Harnblasenkarzinom im Fokus : Update 2012 des Deutschen Forschungsverbundes Blasenkarzinom e. V.

The German Bladder Cancer Association (DFBK) invited its members to the 3rd annual meeting 2012 in Hannover 4 years after the official founding. The meeting was directed to discuss the progress of ongoing and newly initiated projects and collaborations. In this article we will introduce current research activities and collaborations of the DFBK and would like to invite interested researchers to join this national interdisciplinary research association. The aim of the DFBK is to initiate interdisciplinary collaboration and to support scientific discussions among its members. For further information please visit our website at www.forschungsverbund-blasenkarzinom.de.

In situ x-ray photoelectron spectroscopy of model catalysts: at the edge of the gap.

We present high-pressure x-ray photoelectron spectroscopy (HP-XPS) and first-principles kinetic Monte Carlo study addressing the nature of the active surface in CO oxidation over Pd(100). Simultaneously measuring the chemical composition at the surface and in the near-surface gas phase, we reveal both O-covered pristine Pd(100) and a surface oxide as stable, highly active phases in the near-ambient regime accessible to HP-XPS. Surprisingly, no adsorbed CO can be detected during high CO(2) production rates, which can be explained by a combination of a remarkably short residence time of the CO molecule on the surface and mass-transfer limitations in the present setup.

Minkowski tensor shape analysis of cellular, granular and porous structures.

Predicting physical properties of materials with spatially complex structures is one of the most challenging problems in material science. One key to a better understanding of such materials is the geometric characterization of their spatial structure. Minkowski tensors are tensorial shape indices that allow quantitative characterization of the anisotropy of complex materials and are particularly well suited for developing structure-property relationships for tensor-valued or orientation-dependent physical properties. They are fundamental shape indices, in some sense being the simplest generalization of the concepts of volume, surface and integral curvatures to tensor-valued quantities. Minkowski tensors are based on a solid mathematical foundation provided by integral and stochastic geometry, and are endowed with strong robustness and completeness theorems. The versatile definition of Minkowski tensors applies widely to different types of morphologies, including ordered and disordered structures. Fast linear-time algorithms are available for their computation. This article provides a practical overview of the different uses of Minkowski tensors to extract quantitative physically-relevant spatial structure information from experimental and simulated data, both in 2D and 3D. Applications are presented that quantify (a) alignment of co-polymer films by an electric field imaged by surface force microscopy; (b) local cell anisotropy of spherical bead pack models for granular matter and of closed-cell liquid foam models; (c) surface orientation in open-cell solid foams studied by X-ray tomography; and (d) defect densities and locations in molecular dynamics simulations of crystalline copper.

Characterization of (Fe'Zr,Ti - VO..). defect dipoles in (La,Fe)-codoped PZT 52.5/47.5 piezoelectric ceramics by multifrequency electron paramagnetic resonance spectroscopy.

Ferroelectric 1 mol.% La(3)+ and 0.5 mol.% Fe(3)+ codoped Pb[Zr0(0.54)Ti0(0.46)]O(3) ceramics were studied by means of multifrequency electron paramagnetic resonance (EPR) spectroscopy. The obtained results prove that iron is incorporated at the [Zr,Ti]-site, acting as an acceptor and building a charged Fe'(Zr,Ti) - V(O)..)(.) defect dipole with a directly coordinated oxygen vacancy for partial charge compensation. This feature of the defect associates has hitherto been identified only in hard, exclusively Fe(3)+-doped PZT compounds. The present results show, however, that a similar defect association of the Fe3+ functional center with a V(O)..) also exists in soft, donor-acceptor (La(3)+,Fe(3)+)-codoped PZT. According to models developed by Arlt et al. electric dipoles from defect associates, such as the Fe'(Zr,Ti) - V(O)..)(.) defect associate, which may give rise to an internal bias field that is discussed being responsible for ferroelectric aging.

Using EBSD and TEM-Kikuchi patterns to study local crystallography at the domain boundaries of lead zirconate titanate.

Reliable EBSD mapping of 90 degree domains in a tetragonal ferroelectric perovskite has been achieved for the first time, together with reliable automated orientation determination from TEM-Kikuchi patterns. This has been used to determine misorientation angles at 90 degree domain boundaries and thus local c/a ratios. The sources of orientation noise/error and their effects on the misorientation angle data have been thoroughly analyzed and it is found that this gives a cosine distribution of misorientation angles about the mean with a characteristic width related to the width of the orientation noise distribution. In most cases, a good agreement is found between local c/a ratios and global measurements by X-ray diffraction, but some clear discrepancies have also been found suggesting that real local variations are present, perhaps as a consequence of compositional inhomogeneities.

Concomitant down-regulation of SPRY1 and SPRY2 in prostate carcinoma.

Sprouty proteins encoded by the SPRY genes act as modulators and feedback inhibitors of signalling by epidermal growth factor (EGF) and fibroblast growth factor (FGF). Overactivity of EGF and FGF signalling common in prostate cancer might therefore be exacerbated by Sprouty down-regulation. Indeed, down-regulation of SPRY1 and SPRY2 expression has been independently reported. We found both genes modestly down-regulated by microarray expression analysis of microdissected prostate cancers and by quantitative RT-PCR in macrodissected specimens compared with benign tissues. Importantly, the decreases paralleled each other and expression levels of both genes were significantly lower in cancers that recurred within the average follow-up period of 32 months. In contrast to a previous report, no hypermethylation was found to accompany down-regulation of SPRY2 in cancer tissues and cell lines. We additionally investigated the expression of an SPRY1 alternative transcript presumed to be specific for fetal tissues and found its expression moderately well correlated with expression of the standard transcript through diverse tissues and cell lines. The present study confirms and extends previous reports by demonstrating concomitant down-regulation and a significant association with recurrence of SPRY genes.

Pharmacokinetic studies in Tg.AC and FVB mice administered [14C] benzene either by oral gavage or intradermal injection.

Chronic benzene toxicity has been demonstrated to result in either aplastic anemia or acute myelogenous leukemia, a form of granulocytic leukemia, in exposed people (Snyder and Kalf, Crit. Rev. Toxicol. 24, 177-209, 1994). Aplastic anemia has been demonstrated in animal models following benzene exposure but, heretofore, it has not been possible to replicate benzene-induced granulocytic leukemia in animals. The Tg.AC mouse appears to be the first animal model in which a granulocytic leukemia was produced by treatment with benzene (Tennant et al., The Use of Short- and Medium-Term Tests for Carcinogenic Hazard Evaluation, 1999; French and Saulnier, J. Toxicol. Environ. Health 61, 377-379, 2000). Leukemia was observed in Tg.AC mice to which benzene was administered dermally. Neither orally dosed Tg.AC mice or mice of the parental FVB strain treated by either route of exposure developed leukemia. It is well established that benzene metabolism is required to produce benzene toxicity. To determine whether metabolic differences arising from differences in route of exposure or strain of mouse directed the development of leukemia, the pharmacokinetics of benzene were compared between the two strains and between the two routes of administration. Regardless of the route of exposure or the strain of mouse, seven major metabolites plus unmetabolized benzene were detected in most samples at most time points. Few differences were observed between the two strains following either route of administration. These results suggest that the genetic modification in the Tg.AC mouse, i.e., insertion of the v-Ha-ras construct into the genome, did not disrupt any major pathways involved in determining the pharmacokinetics of benzene. Two significant differences were observed between the two routes of exposure: first, benzene was absorbed more slowly after intradermal injection than after oral gavage, and second, the intradermally dosed mice produced more conjugates of hydroquinone than did the orally dosed mice. These differences in metabolism may be involved in the previously observed differences in hematotoxicity between the two routes of exposure.

Metabolism of [14C]phenol in the isolated perfused mouse liver.

A previous report from this laboratory focused on the metabolism of [14C]benzene (BZ) in the isolated, perfused, mouse liver (C. C. Hedli, et al., 1997, Toxicol. Appl. Pharmacol. 146, 60-68). Whereas administration of BZ to mice results in bone marrow depression (R. Snyder et al., 1993, Res. Commun. Chem. Pathol. Pharmacol. 20, 191-194), administration of phenol (P), the major metabolite of BZ, does not. It was, therefore, of interest to determine whether the metabolic fate of P produced during BZ metabolism differed from that of P metabolized in the absence of BZ. Mouse livers were perfused with a solution of [14C]P in both the orthograde (portal vein to central vein) and retrograde (central vein to portal vein) direction to investigate the metabolic zonation of enzymes involved in P hydroxylation and conjugation. Perfusate samples were collected, separated by HPLC, and tested for radioactivity. Unconjugated metabolites were identified by comparing their retention times with nonradiolabeled standards, which were detected by UV absorption. Conjugated metabolites were identified and collected on the basis of radiochromatogram results, hydrolyzed enzymatically, and identified by co-chromatography with unlabeled BZ metabolites. The objective was to compare and quantify the metabolites formed during the perfusion of P in the orthograde and retrograde directions and to compare the orthograde P-perfusion results with the orthograde BZ results reported previously. Regardless of the direction of P perfusion, the major compounds released from the liver were P. phenylgucuronide, phenylsulfate, hydroquinone (HQ), and HQ glucuronide. A comparison of the results of perfusing P in the orthograde versus the retrograde direction showed that more P was recovered unchanged and more HQ was formed during retrograde perfusion. The results suggest that enzymes involved in P hydroxylation are generally closer to the central vein than those involved in conjugation, and that during retrograde perfusion, P metabolism may be limited by the sub-optimal conditions of perfusion. Comparison of the orthograde perfusion studies of P and BZ revealed that a larger percentage of the radioactivity released from the liver was identified as unconjugated HQ after BZ perfusion than after P perfusion. In addition, the amount of radioactivity covalently bound to liver macromolecules was measured after each perfusion and determined to be proportional to the amount of HQ and HQG detected in the perfusate samples.

Benzene metabolism in the isolated perfused mouse liver.

The hematotoxicity of benzene (BZ) requires its hepatic metabolism, the release of metabolites into the circulation, and the access of metabolites to the bone marrow. Although a range of potentially toxic metabolites produced by the liver was identified using subcellular systems and isolated hepatocytes, these models do not allow identification of the metabolites released from the liver with respect to time and flow through the liver. We developed an isolated perfused mouse liver model to evaluate metabolites released following a single-pass of radiolabeled BZ and after recirculation of single-pass metabolites back through the liver. Reversing the path of flow through the liver changes the orientation of hepatic oxidizing and conjugating enzymes with respect to perfusate flow. Comparison of metabolite production following normal (orthograde, portal vein to hepatic vein) perfusion with reversed (retrograde) perfusion permitted an evaluation of the impact of zonal distributions of these enzymes on BZ metabolism. The major metabolites detected by HPLC, irrespective of the direction of perfusion, were free phenol (P), phenylsulfate (PS), and phenylglucuronide (PG), plus lesser amounts of hydroquinone (HQ) and hydroquinone glucuronide (HQG). Recirculation of the products of single pass orthograde perfusion through the liver yielded P conjugates as well as low levels of free and conjugated HQ. No free P was detected after recirculation. Although no qualitative differences between orthograde and retrograde perfusion were observed, the percentage of free P and P conjugates (PS + PG) found as free P was twice as great following orthograde perfusion as compared to retrograde perfusion. These results suggest that regional differences in the zonation of enzymes involved in oxidation and conjugation may play a critical role in hepatic BZ metabolism.

Membrane function and vascular reactivity.

This communication examines the possibility that nitric oxide (NO) production by endothelial cells results from changes in cell membrane fluidity. Lysophosphatidylcholine (LPC) alters fluidity of the endothelial cell membranes causing vascular relaxation. Through membrane alterations LPC influences function of a number of membrane receptors and modulates enzyme activity. As a result of detergent action, lysophosphatidylcholine (LPC) causes activation of guanylate cyclase, stimulates sialyltransferase and regulates protein kinase C activity. It has already been demonstrated that ionic detergents, such as Triton X-100 also cause vascular relaxation, possibly induced by NO production from endothelial cells. It is postulated that production of nitric oxide results from changes in membrane viscosity; this may represent a mechanism for its regulation in biological systems.