Visit our Expo - Redox and Inflammation signaling 2012

Session XIII : Cell signaling pathways leading to regulated chromatin modifications
Poster XIII, 9
Modulation of epigenetic processes in HRAS-transformed cells
Christine Sers1, Karen Weißhaupt1, Thomas Mikeska2, Diana Jammas2, Xiaohua
Chen1, Ralf-Jürgen Kuban3, Ute Ungethüm3, Ulf Krapfenbauer1, Hans-Peter Herzel4,
Reinhold Schäfer1,3, Jörn Walter2, Per Lund1.
1Laboratory of Molecular Tumor Pathology, Institute of Pathology, Charité, Berlin,
Schumannstr. 20/21, D-10117 Berlin, Germany; 2Department of Natural Sciences –
Technical Faculty III FR 8.3, Biological Sciences, Institute of Genetics/Epigenetics,
University of Saarland, Saarbrücken, Germany; 3Laboratory of Functional Genome
Research, Charité, Berlin, Schumannstr. 20/21, D-10117 Berlin, Germany; 4Institute for
Theoretical Biology, Humboldt University Berlin, Invalidenstrasse 43, D-10115 Berlin,
Germany;
Silencing of tumour suppressor genes is often caused by promoter hypermethylation, but little
is known about the impact of oncogenic signalling pathways on methylation. The aim of this
study was to investigate the role of signalling pathways downstream of RAS involved in
methylation-dependent down-regulation of genes. Immortalised 208F rat fibroblasts and
HRAS(V12)-transformed rat fibroblasts (FE-8) were treated with the de-methylating agent 5aza-deoxycytide
(5-aza-CdR) and with the HDAC-inhibitor Trichostatin A (TSA). The effect
of 5-aza-CdR on the expression of genes down-regulated in the HRAS-transformed cells was
investigated by conventional northern blot analysis (74 genes) and by microarray
hybridisation (7186 sequences). Fifty-three genes analysed by Northern blot analysis and 10
genes analysed by microarray hybridsation were re-expressed in FE-8 cells after treatment
with 5-aza-CdR. Among these genes, Clusterin, Mama, MMP2, TIMP2 and
Thrombospondin-1 were also re-expressed after inhibition of the MEK/ERK pathway.
Hypermethylation of putative regulatory elements in two independent HRAS-transforemd cell
lines and in cells harbouring an inducible HRAS, was detected within a CpG-island 14.5 kb
upstream of clusterin, within the clusterin promoter and within a CpG-island of the Mmp2promoter
by bisulphite sequencing. No significant hypermethylation was detected within the
promoter regions of Timp2 and syndecan 4. Our study shows that RAS oncogene-dependent
suppression and methylation-dependent silencing of genes are connected and impinge in part
on the same target genes.
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