Hypomethylation and expression of BEX2, IGSF4 and TIMP3 indicative of MLL translocations in Acute Myeloid Leukemia

Background Translocations of the Mixed Lineage Leukemia (MLL) gene occur in a subset (5%) of acute myeloid leukemias (AML), and in mixed phenotype acute leukemias in infancy - a disease with extremely poor prognosis. Animal model systems show that MLL gain of function mutations may contribute to leukemogenesis. Wild-type (wt) MLL possesses histone methyltransferase activity and functions at the level of chromatin organization by affecting the expression of specific target genes. While numerous MLL fusion proteins exert a diverse array of functions, they ultimately serve to induce transcription of specific genes. Hence, acute lymphoblastic leukemias (ALL) with MLL mutations (MLLmu) exhibit characteristic gene expression profiles including high-level expression of HOXA cluster genes. Here, we aimed to relate MLL mutational status and tumor suppressor gene (TSG) methylation/expression in acute leukemia cell lines. Results Using MS-MLPA (methylation-specific multiplex ligation-dependent probe amplification assay), methylation of 24 different TSG was analyzed in 28 MLLmu and MLLwt acute leukemia cell lines. On average, 1.8/24 TSG were methylated in MLLmu AML cells, while 6.2/24 TSG were methylated in MLLwt AML cells. Hypomethylation and expression of the TSG BEX2, IGSF4 and TIMP3 turned out to be characteristic of MLLmu AML cell lines. MLLwt AML cell lines displayed hypermethylated TSG promoters resulting in transcriptional silencing. Demethylating agents and inhibitors of histone deacetylases restored expression of BEX2, IGSF4 and TIMP3, confirming epigenetic silencing of these genes in MLLwt cells. The positive correlation between MLL translocation, TSG hypomethylation and expression suggested that MLL fusion proteins were responsible for dysregulation of TSG expression in MLLmu cells. This concept was supported by our observation that Bex2 mRNA levels in MLL-ENL transgenic mouse cell lines required expression of the MLL fusion gene. Conclusion These results suggest that the conspicuous expression of the TSG BEX2, IGSF4 and TIMP3 in MLLmu AML cell lines is the consequence of altered epigenetic properties of MLL fusion proteins

SET-NUP214 fusion in acute myeloid leukemia- and T-cell acute lymphoblastic leukemia-derived cell lines

<p>Abstract</p> <p>Background</p> <p><it>SET-NUP214 </it>fusion resulting from a recurrent cryptic deletion, del(9)(q34.11q34.13) has recently been described in T-cell acute lymphoblastic leukemia (T-ALL) and in one case of acute myeloid leukemia (AML). The fusion protein appears to promote elevated expression of <it>HOXA </it>cluster genes in T-ALL and may contribute to the pathogenesis of the disease. We screened a panel of ALL and AML cell lines for <it>SET-NUP214 </it>expression to find model systems that might help to elucidate the cellular function of this fusion gene.</p> <p>Results</p> <p>Of 141 human leukemia/lymphoma cell lines tested, only the T-ALL cell line LOUCY and the AML cell line MEGAL expressed the <it>SET(TAF-</it>Iβ)-<it>NUP214 </it>fusion gene transcript. RT-PCR analysis specifically recognizing the alternative first exons of the two <it>TAF-</it>I isoforms revealed that the cell lines also expressed <it>TAF-</it>Iα-<it>NUP214 </it>mRNA. Results of fluorescence in situ hybridization (FISH) and array-based copy number analysis were both consistent with del(9)(q34.11q34.13) as described. Quantitative genomic PCR also confirmed loss of genomic material between <it>SET </it>and <it>NUP214 </it>in both cell lines. Genomic sequencing localized the breakpoints of the <it>SET </it>gene to regions downstream of the stop codon and to <it>NUP214 </it>intron 17/18 in both LOUCY and MEGAL cells. Both cell lines expressed the 140 kDa SET-NUP214 fusion protein.</p> <p>Conclusion</p> <p>Cell lines LOUCY and MEGAL express the recently described <it>SET-NUP214 </it>fusion gene. Of special note is that the formation of the <it>SET </it>exon 7/<it>NUP214 </it>exon 18 gene transcript requires alternative splicing as the <it>SET </it>breakpoint is located downstream of the stop codon in exon 8. The cell lines are promising model systems for <it>SET-NUP214 </it>studies and should facilitate investigating cellular functions of the the SET-NUP214 protein.</p

Chronologie der Wnt-Zielgenexpression und Visualisierung des nukleären β\beta-Catenin/TCF-Komplexes

Der Wnt-Signalweg spielt in der Embryogenese und in der Homöostase adulter Stammzellsysteme eine wichtige Rolle. Über die Kontrolle der Genexpression reguliert der Wnt-Signalweg die Proliferation, Differenzierung und Zelltypfestlegung. Hier erfolgte erstmalig eine zeitaufgelöste Analyse der Wnt-Zielgenexpression. Beispielhaft wurde der zeitliche Verlauf der Expression von $\it c-Myc$, $\it CCND1$, $\it CDKN1A$, $\it Ramp3$, $\it Plf/Mrp$ und $\it TfR$ nach Aktivierung des Wnt-Signalwegs in der Brustdrüsenepithelzelllinie C57MG untersucht. Dabei zeigte sich, dass die Art der Signalweg-Aktivierung Auswirkungen auf Richtung, Stärke und den chronologischen Verlauf der Expression hat, und direkt sowie indirekt regulierte Gene unterschieden werden können. Zudem konnte die Wnt-3a-induzierte Interaktion von endogenem $\beta$-Catenin und TCF-4 in C57MG-Zellen und in kolorektalen Tumorzelllinien mit konstitutiv aktivem Wnt-Signalweg fluoreszenzmikroskopisch durch immunzytochemische Markierung in hoher Auflösung dargestellt werden

Chronologie der Wnt-Zielgenexpression und Visualisierung des nukleären β\beta-Catenin/TCF-Komplexes

Der Wnt-Signalweg spielt in der Embryogenese und in der Homöostase adulter Stammzellsysteme eine wichtige Rolle. Über die Kontrolle der Genexpression reguliert der Wnt-Signalweg die Proliferation, Differenzierung und Zelltypfestlegung. Hier erfolgte erstmalig eine zeitaufgelöste Analyse der Wnt-Zielgenexpression. Beispielhaft wurde der zeitliche Verlauf der Expression von $\it c-Myc$, $\it CCND1$, $\it CDKN1A$, $\it Ramp3$, $\it Plf/Mrp$ und $\it TfR$ nach Aktivierung des Wnt-Signalwegs in der Brustdrüsenepithelzelllinie C57MG untersucht. Dabei zeigte sich, dass die Art der Signalweg-Aktivierung Auswirkungen auf Richtung, Stärke und den chronologischen Verlauf der Expression hat, und direkt sowie indirekt regulierte Gene unterschieden werden können. Zudem konnte die Wnt-3a-induzierte Interaktion von endogenem $\beta$-Catenin und TCF-4 in C57MG-Zellen und in kolorektalen Tumorzelllinien mit konstitutiv aktivem Wnt-Signalweg fluoreszenzmikroskopisch durch immunzytochemische Markierung in hoher Auflösung dargestellt werden

<it>CD7 </it>in acute myeloid leukemia: correlation with loss of wild-type <it>CEBPA</it>, consequence of epigenetic regulation

Abstract Background CD7 is a negative prognostic marker in myeloid malignancies. In acute myeloid leukemia (AML), an inverse correlation exists between expression of wild-type CEBPA and CD7. Aim of this study was to find out whether C/EBPα is a negative regulator of CD7 and which other regulatory mechanisms might be involved. Results As already described for primary AML cells, the majority of AML cell lines tested were either C/EBPα+/CD7- or C/EBPα-/CD7+. However, the existence of isolated CD7+ cell lines expressing wild-type C/EBPα challenges the notion that C/EBPα acts as a unique repressor of CD7. Furthermore, ectopic expression of CEBPA did not reduce CD7 in CD7+ cells and knock-down of C/EBPα failed to induce CD7 in CD7- cells. In contrast, the DNA demethylating agent Aza-2'deoxycytidine triggered CD7 expression in CD7- AML and in T-cell lines suggesting epigenetic regulation of CD7. Bisulfite sequencing data confirmed that CpGs in the CD7 exon1 region are methylated in CD7- cell lines, and unmethylated in CD7+ cell lines. Conclusion We confirmed an inverse correlation between the expression of wild-type CEBPA and of CD7 in AML cells. Our results contradict the hypothesis that C/EBPα acts as repressor for CD7, and instead show that epigenetic mechanisms are responsible for CD7 regulation, in AML cells as well as in T-cells, the typical CD7 expressing cell type.</p

Cone mosaic façades in Mesopotamia (4th millenium BCE): archaeometric approach to an architectural innovation

We present an archaeometric study of clay mosaic cones from the archaeological site of Uruk, excavated in the earlier 20th century and currently kept in the Museum of the Ancient Near East in Berlin (Vorderasiatisches Museum – VAM). Clay cones have been found in major and distant centres of the 4th millennium BCE Uruk period and are characteristic for monumental architecture. They were used to create colourful geometric patterns on the façades of buildings. Research focused on reconstructing the colouring of the cone heads by investigating colourants and the technical processes of their application. From microscopic observations and spectroscopic analyses, the main colouring materials were identified as dolomite for the white-coloured clay cones, hematite/ochre for the red ones, and bitumen for the black ones. These colouring materials are assumed to have been applied cold, after the firing of the ceramic, and in association with an organic binder for the white- and the red-coloured cones