Retinoblastoma binding protein 4 maintains cycling neural stem cells and prevents DNA damage and Tp53-dependent apoptosis in rb1 mutant neural progenitors

Retinoblastoma-binding protein 4 (Rbbp4) is a WDR adaptor protein for multiple chromatin remodelers implicated in human oncogenesis. Here we show Rbbp4 is overexpressed in zebrafish rb1-embryonal brain tumors and is upregulated across the spectrum of human embryonal and glial brain cancers. We demonstrate in vivo Rbbp4 is essential for zebrafish neurogenesis and has distinct roles in neural stem and progenitor cells. rbbp4 mutant neural stem cells show delayed cell cycle progression and become hypertrophic. In contrast, rbbp4 mutant neural precursors accumulate extensive DNA damage and undergo programmed cell death that is dependent on Tp53 signaling. Loss of Rbbp4 and disruption of genome integrity correlates with failure of neural precursors to initiate quiescence and transition to differentiation. rbbp4; rb1 double mutants show that survival of neural precursors after disruption of Rb1 is dependent on Rbbp4. Elevated Rbbp4 in Rb1-deficient brain tumors might drive proliferation and circumvent DNA damage and Tp53-dependent apoptosis, lending support to current interest in Rbbp4 as a potential druggable target

Prognostic value and functional consequences of cell cycle inhibitor p27Kip1 loss in medulloblastoma

BACKGROUND: The cyclin-dependent kinase inhibitor p27(Kip1) functions during normal cerebellar development and has demonstrated tumor suppressor functions in mouse models of medulloblastoma. Because P27 loss is associated with increased proliferation, we assessed whether P27 absence in surgical medulloblastoma specimens correlated with response to therapy in pediatric patients enrolled in two large studies. Additionally, we examined the functional consequence of p27(Kip1) loss in the SmoA1 medulloblastoma model to distinguish whether p27(Kip1) reduces tumor initiation or slows tumor progression. FINDINGS: Analysis of 87 well-characterized patient samples identified a threshold of P27 staining at which significant P27 loss correlated with poor patient outcome. The same criteria, applied to a second test set of tissues from 141 patients showed no difference in survival between patients with minimal P27 staining and others, suggesting that P27 levels alone are not a sufficient prognostic indicator for identifying standard-risk patients that may fail standard therapy. These findings were in contrast to prior experiments completed using a mouse medulloblastoma model. Analysis of cerebellar tumor incidence in compound mutant mice carrying the activated Smoothened (SmoA1) allele that were heterozygous or nullizygous for p27(Kip1) revealed that p27(Kip1) loss did not alter the frequency of tumor initiation. Tumors haploinsufficient or nullizygous for p27(Kip1) were, however, more invasive and displayed a higher proliferative index, suggesting p27(Kip1) loss may contribute to SmoA1 medulloblastoma progression. CONCLUSIONS: These studies revealed P27 loss affects medulloblastoma progression rather than initiation and that this putative biomarker should not be used for stratifying children with medulloblastoma to risk-based therapeutic regimens

Cytoskeleton’s Role in KIR2.1 Trafficking

Alteration of the inward rectifier current IK1, carried by KIR2.1 channels, affects action potential duration, impacts resting membrane stability and associates with cardiac arrhythmias. Congenital and acquired KIR2.1 malfunction frequently associates with aberrant ion channel trafficking. Cellular processes underlying trafficking are intertwined with cytoskeletal function. The extent to which the cytoskeleton is involved in KIR2.1 trafficking processes is unknown. We aimed to quantify the dependence of KIR2.1 trafficking on cytoskeleton function. GFP or photoconvertible Dendra2 tagged KIR2.1 constructs were transfected in HEK293 or HeLa cells. Photoconversion of the Dendra2 probe at the plasma membrane and subsequent live imaging of trafficking processes was performed by confocal laser-scanning microscopy. Time constant of green fluorescent recovery (τg,s) represented recruitment of new KIR2.1 at the plasma membrane. Red fluorescent decay (τr,s) represented internalization of photoconverted KIR2.1. Patch clamp electrophysiology was used to quantify IKIR2.1. Biochemical methods were used for cytoskeleton isolation and detection of KIR2.1 cytoskeleton interactions. Cytochalasin B (20 μM), Nocodazole (30 μM) and Dyngo-4a (10 nM) were used to modify the cytoskeleton. Chloroquine (10 μM, 24 h) was used to impair KIR2.1 breakdown. Cytochalasin B and Nocodazole, inhibitors of actin and tubulin filament formation respectively, strongly inhibited the recovery of green fluorescence at the plasma membrane suggestive for inhibition of KIR2.1 forward trafficking [τg,s 13 ± 2 vs. 131 ± 31* and 160 ± 40* min, for control, Cytochalasin B and Nocodazole, respectively (*p < 0.05 vs. control)]. Dyngo-4a, an inhibitor of dynamin motor proteins, strongly slowed the rate of photoconverted channel internalization, whereas Nocodazole and Cytochalasin B had less effect [τr,s 20 ± 2 vs. 87 ± 14*, 60 ± 16 and 64 ± 20 min (*p < 0.05 vs. control)]. Cytochalasin B treatment (20 μM, 24 h) inhibited IKIR2.1. Chloroquine treatment (10 μM, 24 h) induced intracellular aggregation of KIR2.1 channels and enhanced interaction with the actin/intermediate filament system (103 ± 90 fold; p < 0.05 vs. control). Functional actin and tubulin cytoskeleton systems are essential for forward trafficking of KIR2.1 channels, whereas initial backward trafficking relies on a functional dynamin system. Chronic disturbance of the actin system inhibits KIR2.1 currents. Internalized KIR2.1 channels become recruited to the cytoskeleton, presumably in lysosomes

Evaluation of the similarity of gene expression data estimated with SAGE and Affymetrix GeneChips

BACKGROUND: Serial Analysis of Gene Expression (SAGE) and microarrays have found awidespread application, but much ambiguity exists regarding the evaluation of these technologies. Cross-platform utilization of gene expression data from the SAGE and microarray technology could reduce the need for duplicate experiments and facilitate a more extensive exchange of data within the research community. This requires a measure for the correspondence of the different gene expression platforms. To date, a number of cross-platform evaluations (including a few studies using SAGE and Affymetrix GeneChips) have been conducted showing a variable, but overall low, concordance. This study evaluates these overall measures and introduces the between-ratio difference as a concordance measure pergene. RESULTS: In this study, gene expression measurements of Unigene clusters represented by both Affymetrix GeneChips HG-U133A and SAGE were compared using two independent RNA samples. After matching of the data sets the final comparison contains a small data set of 1094 unique Unigene clusters, which is unbiased with respect to expression level. Different overall correlation approaches, like Up/Down classification, contingency tables and correlation coefficients were used to compare both platforms. In addition, we introduce a novel approach to compare two platforms based on the calculation of differences between expression ratios observed in each platform for each individual transcript. This approach results in a concordance measure per gene (with statistical probability value), as opposed to the commonly used overall concordance measures between platforms. CONCLUSION: We can conclude that intra-platform correlations are generally good, but that overall agreement between the two platforms is modest. This might be due to the binomially distributed sampling variation in SAGE tag counts, SAGE annotation errors and the intensity variation between probe sets of a single gene in Affymetrix GeneChips. We cannot identify or advice which platform performs better since both have their (dis)-advantages. Therefore it is strongly recommended to perform follow-up studies of interesting genes using additional techniques. The newly introduced between-ratio difference is a filtering-independent measure for between-platform concordance. Moreover, the between-ratio difference per gene can be used to detect transcripts with similar regulation on both platforms

Association of potentially inappropriate medications with outcomes of inpatient geriatric rehabilitation : A prospective cohort study.

BACKGROUND Higher age is associated with multimorbidity, which may lead to polypharmacy and potentially inappropriate medication (PIM). OBJECTIVE To evaluate whether PIM on admission for geriatric inpatient rehabilitation is associated with rehabilitation outcome regarding mobility and quality of life. MATERIAL AND METHODS A total of 210 patients were included. Medications at hospital admission were analyzed with the Screening Tool of Older Persons' potentially inappropriate Prescriptions (STOPP) and the number of PIMs individual patients were taking was determined. The study population was then divided into two groups, one with and one without PIM. The main rehabilitation outcomes, quality of life and mobility, were assessed on admission and discharge. Associations between PIM and the main outcomes were analyzed using the two-tailed Student's t-test and Spearman correlations. RESULTS In total 131 PIMs were identified by STOPP. Of the patients 91 (43%) were taking at least 1 PIM, and 119 patients (57%) were not taking any PIM. Patients with no PIM had a significantly better quality of life on admission (p < 0.05) and discharge (p < 0.005). The number of PIMs was not associated with the rehabilitation outcomes mobility and quality of life (Spearman's ρ = -0.01, p = 0.89 and ρ = -0.02, p = 0.7, respectively). The quality of life and mobility increased identically in both groups from admission to discharge but the group with PIM did not reach the levels of those without PIM. CONCLUSION The use of PIM may have a negative impact on the quality of life of elderly people but patients with and without PIM achieved comparable improvements in quality of life and mobility. Further studies are required to assess the long-term outcomes of patients taking PIM following inpatient rehabilitation

Enhanced Survival of High-Risk Medulloblastoma-Bearing Mice after Multimodal Treatment with Radiotherapy, Decitabine, and Abacavir

Children with high-risk SHH/TP53-mut and Group 3 medulloblastoma (MB) have a 5-year overall survival of only 40%. Innovative approaches to enhance survival while preventing adverse effects are urgently needed. We investigated an innovative therapy approach combining irradia- tion (RT), decitabine (DEC), and abacavir (ABC) in a patient-derived orthotopic SHH/TP53-mut and Group 3 MB mouse model. MB-bearing mice were treated with DEC, ABC and RT. Mouse survival, tumor growth (BLI, MRT) tumor histology (H/E), proliferation (Ki-67), and endothelial (CD31) staining were analyzed. Gene expression was examined by microarray and RT-PCR (Ki-67, VEGF, CD31, CD15, CD133, nestin, CD68, IBA). The RT/DEC/ABC therapy inhibited tumor growth and enhanced mouse survival. Ki-67 decreased in SHH/TP53-mut MBs after RT, DEC, RT/ABC, and RT/DEC/ABC therapy. CD31 was higher in SHH/TP53-mut compared to Group 3 MBs and decreased after RT/DEC/ABC. Microarray analyses showed a therapy-induced downregulation of cell cycle genes. By RT-PCR, no therapy-induced effect on stem cell fraction or immune cell inva- sion/activation could be shown. We showed for the first time that RT/DEC/ABC therapy improves survival of orthotopic SHH/TP53-mut and Group 3 MB-bearing mice without inducing adverse effects suggesting the potential for an adjuvant application of this multimodal therapy approach in the human clinic

Replication protein A safeguards genome integrity by controlling NER incision events

Continued association of RPA with sites of incomplete nucleotide excision repair averts further incision events until repair is completed

Diffuse infiltrating tumour with the molecular profile of an atypical teratoid rhabdoid tumour (AT/RT SHH-1B) in an adult patient

We describe a 46-year-old patient with an IDH-wildtype diffusely infiltrating atypical teratoid/rhabdoid tumour (AT/RT), SHH-1B molecular subtype. The unusual histology and subsequent diagnosis in an adult patient will be discussed.</p

Chronic Propafenone Application Increases Functional K IR2.1 Expression In Vitro.

Expression and activity of inwardly rectifying potassium (K IR) channels within the heart are strictly regulated. K IR channels have an important role in shaping cardiac action potentials, having a limited conductance at depolarized potentials but contributing to the final stage of repolarization and resting membrane stability. Impaired K IR2.1 function causes Andersen-Tawil Syndrome (ATS) and is associated with heart failure. Restoring K IR2.1 function by agonists of K IR2.1 (AgoKirs) would be beneficial. The class 1c antiarrhythmic drug propafenone is identified as an AgoKir; however, its long-term effects on K IR2.1 protein expression, subcellular localization, and function are unknown. Propafenone's long-term effect on K IR2.1 expression and its underlying mechanisms in vitro were investigated. K IR2.1-carried currents were measured by single-cell patch-clamp electrophysiology. K IR2.1 protein expression levels were determined by Western blot analysis, whereas conventional immunofluorescence and advanced live-imaging microscopy were used to assess the subcellular localization of K IR2.1 proteins. Acute propafenone treatment at low concentrations supports the ability of propafenone to function as an AgoKir without disturbing K IR2.1 protein handling. Chronic propafenone treatment (at 25-100 times higher concentrations than in the acute treatment) increases K IR2.1 protein expression and K IR2.1 current densities in vitro, which are potentially associated with pre-lysosomal trafficking inhibition

Itch/β-arrestin2-dependent non-proteolytic ubiquitylation of SuFu controls Hedgehog signalling and medulloblastoma tumorigenesis

Suppressor of Fused (SuFu), a tumour suppressor mutated in medulloblastoma, is a central player of Hh signalling, a pathway crucial for development and deregulated in cancer. Although the control of Gli transcription factors by SuFu is critical in Hh signalling, our understanding of the mechanism regulating this key event remains limited. Here, we show that the Itch/β-arrestin2 complex binds SuFu and induces its Lys63-linked polyubiquitylation without affecting its stability. This process increases the association of SuFu with Gli3, promoting the conversion of Gli3 into a repressor, which keeps Hh signalling off. Activation of Hh signalling antagonises the Itch-dependent polyubiquitylation of SuFu. Notably, different SuFu mutations occurring in medulloblastoma patients are insensitive to Itch activity, thus leading to deregulated Hh signalling and enhancing medulloblastoma cell growth. Our findings uncover mechanisms controlling the tumour suppressive functions of SuFu and reveal that their alterations are implicated in medulloblastoma tumorigenesis