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Background/Aims: The recommendations on the time interval between pre-cleaning and reprocessing of endoscopes differ in international guidelines, with a low level of evidence. The aim of this study was to investigate the influence of postponing reprocessing on the reprocessing quality after pre-cleaning the flexible endoscopes.Methods: We reprocessed 124 standardized test tubes simulating endoscope channels after soiling and contamination and determined the reprocessing performance. In addition, we examined contaminated gastroscopes, colonoscopes, and bronchoscopes. The duration of interim storage after pre-cleaning was 16 h for 100 test tubes and up to 24 h for 18 endoscopes. We determined the residual protein content and germ load as markers for cleaning and disinfection performance. In addition, we determined biofilm formation by photometry of crystal violet staining.Results: All test tubes and flexible endoscopes showed residual protein content and germ load significantly below legally prescribed threshold values, independent of the interval between pre-cleaning and reprocessing.Conclusions: Our findings indicate that flexible endoscopes could be stored overnight after pre-cleaning without any influence on the quality of reprocessing. While ensuring patient safety, this could simplify logistical processes and enable cost savings.

The transcription factor signal transducer and activator of transcription 5 (STAT5) is activated conditionally and transiently by external stimuli. Thereupon, STAT5 modulates the transcription of its target genes, promoting cell survival and growth. Constitutive STAT5 activity has been shown to be oncogenic in hematopoietic cells. This correlates with the acquisition of cancer hallmarks, such as ‘cytokine-independent survival’, ‘uncontrolled growth’ and ‘genomic instability’. Chromatin dynamics is of pivotal importance for the regulation of transcriptional activity and DNA damage repair. Accordingly, cancer hallmarks are not only effected by oncogenic ‘driver’ alterations at the DNA level, but also at the chromatin level. Sustained DNA binding of constitutively active STAT5 might have distinct effects on chromatin, which might lead to ‘driver’ chromatin alterations and underlie its oncogenicity. The main goal of the present study was to identify ‘driver’ chromatin alterations and other ‘driver’ events during the oncogenesis process induced by constitutively active STAT5. The constitutively active STAT5 mutant STAT5A-1*6 was previously shown to induce oncogenesis in the IL-3-dependent pro-B cell line Ba/F3 by enabling cytokine-independent survival and growth. Specific aims of this study were to characterize the effects of STAT5A-1*6 expression on (i) cell survival and growth, (ii) expression of selected STAT5 target genes and (iii) chromatin rearrangements. To monitor the oncogenesis process, a stable Ba/F3 cell line – inducibly expressing STAT5A-1*6 upon doxycycline administration (Tet-on expression system) – was generated and validated. Short- and long-term STAT5A-1*6 induction experiments were conducted and STAT5A-1*6 protein levels and activation (Western blot), and cell phenotype in terms of survival, growth and genome stability (cell counting, flow cytometric analysis of cellular DNA content/cell cycle states) were analyzed. Expression of STAT5 target genes including Cis, Osm, Spi2.1, c-Myc, Pim-1, Id-1 and TNFRSF13b was investigated in parallel using RT-qPCR. As expected, STAT5A-1*6 expression enabled cytokine-independent survival and growth of Ba/F3 cells. Cell viability and proliferation rates increased gradually during the initial phase of induction. Interestingly, after 4–5 weeks of induction cell survival and growth no longer depended on STAT5A-1*6 expression. In addition, in one out of four experiments, STAT5A-1*6-expressing cells accumulated chromosomal aberrations. The correlation patterns of STAT5A-1*6 and STAT5 target gene expression suggested dose-dependent STAT5A-1*6-mediated transcriptional activation of STAT5 target genes, at least within the first few weeks of induction. Later on however, sustained expression of the STAT5 target genes c-Myc and Pim-1 became independent of STAT5A-1*6. Altogether, these observations suggest the acquisition of the ‘cytokine-independent survival’, ‘uncontrolled growth’ and ‘genomic instability’ cancer hallmarks, possibly due to continually accumulating ‘driver’ alterations upon sustained expression of STAT5A-1*6. Interestingly, using chromatin immunoprecipitation STAT5 DNA binding to Cis, Osm, Spi2.1, Id-1 and TNFRSF13b was correlated with a strong decrease in histone H3 occupancy, likely reflecting a loss in nucleosomes. This histone H3 loss was particularly prominent at the STAT5 binding sites, regardless of (i) their location within the gene locus, of (ii) transcriptional activation and of (iii) cytokine supplementation. In addition, sustained STAT5A-1*6 DNA binding patterns were associated with broadened histone H3 loss in regions distant from STAT5 binding sites. Taken together, these data strongly suggest that DNA binding of STAT5 causes a local nucleosome loss, and possibly a global nucleosome loss along its target genes. Accordingly, I propose a general STAT5-mediated chromatin decondensation mechanism leading to a nucleosome loss around STAT5 binding sites, at a step preceding transcriptional activation. The broadened STAT5A-1*6-associated histone H3 loss patterns also raise the possibility of distinct STAT5A-1*6-mediated ‘driver’ chromatin alterations, which might misregulate chromatin dynamics and, in turn, promote the acquisition of ‘driver’ DNA alterations (i.e. the ‘genomic instability’ cancer hallmark). Together, these DNA and chromatin alteration events might underlie the oncogenicity of constitutively active STAT5. Further characterization of these events might contribute to a better understanding of the mechanism of STAT5-mediated oncogenesis and possibly to the identification of novel molecular targets for the development of drugs against STAT5-associated cancers., Der Transkriptionsfaktor signal transducer and activator of transcription 5 (STAT5) wird von externen Stimuli zeitlich begrenzt aktiviert. Daraufhin moduliert STAT5 die Transkription seiner Zielgene und vermittelt so eine Zellüberlebens- und Zellwachstumsantwort. Konstitutive STAT5-Aktivität ist onkogen und an der Leukämie- und Lymphomentstehung aus blutbildenden Zellen beteiligt. Diese erwerben dabei Krebsmerkmale wie ‘zytokin-unabhängiges Überleben’, ‘unkontrolliertes Wachstum’ und ‘Genominstabilität’. Die Chromatindynamik ist von zentraler Wichtigkeit für die Regulierung der Transkriptionsaktivität und die Reparatur von DNA-Schäden. Dementsprechend verursachen nicht nur krebsbedingende sogenannte ‘Treiber’-Veränderungen auf DNA-Ebene Krebsmerkmale, sondern auch solche auf Chromatin- Ebene. Daher könnte die dauerhafte DNA-Bindungsaktivität von konstitutiv aktivem STAT5 spezifische Effekte auf das Chromatin haben, die zu ‘Treiber’-Chromatinveränderungen führen und so dessen Onkogenität zugrunde liegen. Das Hauptziel der vorliegenden Arbeit war die Identifizierung von ‘Treiber’-Chromatinveränderungen und anderer ‘Treiber’-Ereignisse während der von konstitutiv aktivem STAT5 induzierten Onkogenese. Es wurde bereits gezeigt, dass die konstitutiv aktive STAT5-Mutante STAT5A-1*6 in der IL-3-abhängigen pro-B-Zelllinie Ba/F3 Onkogenese induziert, indem sie den Zellen zytokin-unabhängiges Überleben und Wachstum ermöglicht. Die vorliegende Arbeit sollte daher im Einzelnen die Effekte der STAT5A-1*6-Expression auf (i) das Zellüberleben und -wachstum, (ii) die Expression ausgewählter STAT5-Zielgene und (iii) Chromatinumstrukturierungen charakterisieren. Um die Onkogenese im Zeitverlauf zu beobachten, wurde eine stabile Ba/F3-Zelllinie entwickelt und validiert, die über Doxycyclin-Gabe induzierbar STAT5A-1*6 exprimiert (Tet-on-Expressionssystem). Nach Induktion der STAT5A-1*6-Expression wurden in Zeitkursexperimenten STAT5A-1*6-Proteinlevel und -Aktivierung (Western blot) sowie der Überlebens- und Wachstumsphänotyp und die Genomstabilität (Zellzählung, durchflusszytometrische Bestimmung des Zell-DNA-Gehalts und der Zellzyklusphase) und die Gesamt-DNA-Menge (Zellzyklusanalyse) analysiert. Zusätzlich wurde mittels RT-qPCR die Expression von STAT5-Zielgenen, u.a. Cis, Osm, Spi2.1, c-Myc, Pim-1, Id-1 und TNFRSF13b, untersucht. Wie erwartet ermöglichte die STAT5A-1*6-Expression Ba/F3-Zellen zytokin-unabhängiges Überleben und Wachstum. Zu Beginn der Zeitkursexperimente nahmen Zellviabiliäts- und Zellproliferationsraten fortlaufend zu. Interessanterweise hingen aber Zellüberleben und -wachstum nach 4–5 Wochen nicht mehr von der STAT5A-1*6-Expression ab. Zudem akkumulierten die Zellen in einem von vier Experimenten Chromosomenaberrationen. Die Expressionsstärke von STAT5A-1*6 korrelierte darüber hinaus mit der von STAT5-Zielgenen – zumindest in den ersten Wochen. Das wies auf eine dosis-abhängige Transkriptionsaktivierung der STAT5-Zielgene durch STAT5A-1*6 hin. Die STAT5-Zielgene c-Myc und Pim-1 wurden hingegen zu späteren Zeitpunkten zunehmend STAT5A-1*6-unabhängig exprimiert. Zusammengenommen legen diese Beobachtungen den Erwerb der Krebsmerkmale ‘zytokin-unabhängiges Überleben’, ‘unkontrolliertes Wachstum’ und ‘Genominstabilität’ nahe – womöglich aufgrund stetig akkumulierender ‘Treiber’-Veränderungen bei langanhaltender STAT5A-1*6-Expression. Chromatin-Immunpräzipitationsexperimente zeigten, dass die STAT5-DNA-Bindung an den Cis, Osm, Spi2.1, Id-1 und TNFRSF13b-Loci mit einer starken Abnahme im Histon-H3-Gehalt korrelierte. Dies spiegelt wahrscheinlich einen Nukleosomenverlust wider. Besonders ausgeprägt war der Histon-H3-Verlust an STAT5-Bindestellen, ungeachtet (i) deren Lage innerhalb des Genlokus, (ii) von Transkriptionsaktivierung und (iii) von Zytokin-Gabe. Zudem korrelierte die anhaltende STAT5A-1*6-DNA-Bindung mit einer Ausdehnung des Histon-H3-Verlusts. Diese Daten lassen den Schluss zu, dass die Bindung von STAT5 an DNA einen Nukelosomenverlust an dessen Bindestelle und möglicherweise auch überall entlang seiner Zielgene verursacht. Daraus könnte sich ein allgemeiner STAT5-vermittelter Chromatindekondensierungs-Mechanismus ableiten, bei dem es zu Nukleosomenverlust in der Umgebung von STAT5-Bindestellen kommt (an einem Schritt vor STAT5-vermittelter Transkriptionsaktivierung). Aus den Ausdehnungsmustern des STAT5A-1*6-assoziierten Histon-H3-Verlusts lässt sich schlussfolgern, dass STAT5A-1*6 spezifische ‘Treiber’-Chromatinveränderungen verursachen könnte. Diese könnten die Chromatindynamik beeinflussen und so wiederum den Erwerb von ‘Treiber’-DNA-Veränderungen (d.h. des Krebsmerkmals ‘Genominstabilität’) begünstigen. Gemeinsam könnten solche DNA- und Chromatinveränderungsereignisse der Onkogenität von konstitutiv aktivem STAT5 zugrunde liegen. Die weitere Charakterisierung dieser Ereignisse könnte dazu beitragen, den Mechanismus hinter STAT5-vermittelter Onkogenese besser zu verstehen. Das könnte neue molekulare Ansatzpunkte für die Entwicklung von Medikamenten gegen STAT5- assoziierte Krebsformen eröffnen.

Additional file 1: Fig. S1. Protein interaction between STAT5A-1*6 and BRD2 cannot be evidenced in co-immunoprecipitation assays. Nuclear lysates from formaldehyde-crosslinked (A-C) or non-crosslinked (D, E) STAT5A-1*6-expressing cells were prepared as described in the Methods section. Nuclear protein enrichment was verified by Western blot using antibodies specific for the nuclear and cytosolic proteins HDAC1 and α-tubulin respectively, and STAT5A-1*6 expression was monitored using the FLAG antibody (A, D). Immunoprecipitations (IP) were performed as described in the “Methods” section using the indicated antibodies. Input (In), immunoprecipitation supernatants (SN) and eluted bead fractions (B) were analysed by immunoblot (IB) using the indicated antibodies (B, E). In panel B, arrow points to BRD2 and (*) indicates a non-specific signal associated with the bead fractions. Bead samples from the IP experiment shown in panel B (crosslinked cells) were further processed for ChIP analysis by qPCR, using the Cis-specific primers depicted in Fig. 2a (C). In panel C, background cut-off (dotted line) was defined as in legend to Fig. 4 (mean IgG background + 2x SD). One-way ANOVA with Dunnett’s multiple comparison test was used to evaluate BRD2 and STAT5 enrichment at the STAT5 binding site (STAT5) and transcription start site (TSS) of the Cis gene, in comparison to the “ORF” region, used as a reference and background control; ***P

Signal transducer and activator of transcription STAT5 is essential for the regulation of proliferation and survival genes. Its activity is tightly regulated through cytokine signaling and is often upregulated in cancer. We showed previously that the deacetylase inhibitor trichostatin A (TSA) inhibits STAT5-mediated transcription by preventing recruitment of the transcriptional machinery at a step following STAT5 binding to DNA. The mechanism and factors involved in this inhibition remain unknown. We now show that deacetylase inhibitors do not target STAT5 acetylation, as we initially hypothesized. Instead, they induce a rapid increase in global histone acetylation apparently resulting in the delocalization of the bromodomain and extra-terminal (BET) protein Brd2 and of the Brd2-associated factor TBP to hyperacetylated chromatin. Treatment with the BET inhibitor (+)-JQ1 inhibited expression of STAT5 target genes, supporting a role of BET proteins in the regulation of STAT5 activity. Accordingly, chromatin immunoprecipitation demonstrated that Brd2 is associated with the transcriptionally active STAT5 target gene Cis and is displaced upon TSA treatment. Our data therefore indicate that Brd2 is required for the proper recruitment of the transcriptional machinery at STAT5 target genes and that deacetylase inhibitors suppress STAT5-mediated transcription by interfering with Brd2 function.

Signal transducer and activator of transcription STAT5 and its upstream activating kinase JAK2 are essential mediators of cytokine signaling. Their activity is normally tightly regulated and transient. However, constitutive activation of STAT5 is found in numerous cancers and a driving force for malignant transformation. We describe here the identification of the synthetic chalcone alpha-Br-2',3,4,4'-tetramethoxychalcone (alpha-Br-TMC) as a novel JAK/STAT inhibitor. Using the non-transformed IL-3-dependent B cell line Ba/F3 and its oncogenic derivative Ba/F3-1*6 expressing constitutively activated STAT5, we show that alpha-Br-TMC targets the JAK/STAT pathway at multiple levels, inhibiting both JAK2 and STAT5 phosphorylation. Moreover, alpha-Br-TMC alters the mobility of STAT5A/B proteins in SDS-PAGE, indicating a change in their post-translational modification state. These alterations correlate with a decreased association of STAT5 and RNA polymerase II with STAT5 target genes in chromatin immunoprecipitation assays. Interestingly, expression of STAT5 target genes such as Cis and c-Myc was differentially regulated by alpha-Br-TMC in normal and cancer cells. While both genes were inhibited in IL-3-stimulated Ba/F3 cells, expression of the oncogene c-Myc was down-regulated and that of the tumor suppressor gene Cis was up-regulated in transformed Ba/F3-1* 6 cells. The synthetic chalcone alpha-Br-TMC might therefore represent a promising novel anticancer agent for therapeutic intervention in STAT5-associated malignancies.

Signal transducer and activator of transcription STAT5 is an essential mediator of cytokine, growth factor and hormone signaling. While its activity is tightly regulated in normal cells, its constitutive activation directly contributes to oncogenesis and is associated to a number of hematological and solid tumor cancers. We previously showed that deacetylase inhibitors can inhibit STAT5 transcriptional activity. We now investigated whether the dietary chemopreventive agent sulforaphane, known for its activity as deacetylase inhibitor, might also inhibit STAT5 activity and thus could act as a chemopreventive agent in STAT5-associated cancers. We describe here sulforaphane (SFN) as a novel STAT5 inhibitor. We showed that SFN, like the deacetylase inhibitor trichostatin A (TSA), can inhibit expression of STAT5 target genes in the B cell line Ba/F3, as well as in its transformed counterpart Ba/F3-1*6 and in the human leukemic cell line K562 both of which express a constitutively active form of STAT5. Similarly to TSA, SFN does not alter STAT5 initial activation by phosphorylation or binding to the promoter of specific target genes, in favor of a downstream transcriptional inhibitory effect. Chromatin immunoprecipitation assays revealed that, in contrast to TSA however, SFN only partially impaired the recruitment of RNA polymerase II at STAT5 target genes and did not alter histone H3 and H4 acetylation, suggesting an inhibitory mechanism distinct from that of TSA. Altogether, our data revealed that the natural compound sulforaphane can inhibit STAT5 downstream activity, and as such represents an attractive cancer chemoprotective agent targeting the STAT5 signaling pathway.

Background: c-Myc has been proposed as a putative target gene of signal transducer and activator of transcription 5 (STAT5). No functional STAT5 binding site has been identified so far within the c-Myc gene locus, therefore a direct transcriptional regulation by STAT5 remains uncertain. c-Myc super-enhancer, located 1.7 Mb downstream of the c-Myc gene locus, was recently reported as essential for the regulation of c-Myc gene expression by hematopoietic transcription factors and bromodomain and extra-terminal (BET) proteins and for leukemia maintenance. c-Myc super-enhancer is composed of five regulatory regions (E1-E5) which recruit transcription and chromatin-associated factors, mediating chromatin looping and interaction with the c-Myc promoter. Results: We now show that STAT5 strongly binds to c-Myc super-enhancer regions E3 and E4, both in normal and transformed Ba/F3 cells. We also found that the BET protein bromodomain-containing protein 2 (BRD2), a co-factor of STAT5, co-localizes with STAT5 at E3/E4 in Ba/F3 cells transformed by the constitutively active STAT5-1*6 mutant, but not in non-transformed Ba/F3 cells. BRD2 binding at E3/E4 coincides with c-Myc transcriptional activation and is lost upon treatment with deacetylase and BET inhibitors, both of which inhibit STAT5 transcriptional activity and c-Myc gene expression. Conclusions: Our data suggest that constitutive STAT5 binding to c-Myc super-enhancer might contribute to BRD2 maintenance and thus allow sustained expression of c-Myc in Ba/F3 cells transformed by STAT5-1*6.