DNA hypermethylation silences TRAIL in transformed cells.

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DNA hypermethylation silences TRAIL in transformed cells. DNA hypermethylation silences TRAIL in transformed cells. A, left, RT-qPCR for induction of TRAIL mRNA by decitabine in immortalized (gray bars) and transformed (black bars) HEK, HMEC, BJ, and SALE cells, n = 3. TRAIL mRNA levels are normalized relative to the corresponding untreated transformed cell line (set to 1). Right, ELISA measurement for TRAIL protein after treatment with decitabine in immortalized (gray bars) and transformed (black bars) HEK cells, n = 2. Bottom left, structure of decitabine. B, regions of the TRAIL promoter analyzed by bisulfite sequencing. Exon1 (white/gray box), the TSS (arrow), a CpG-island 2 kb upstream (dark gray box), and the localization of amplified sequences with individual CpGs (circles) numbered in 5′ to 3′ direction are shown. Bisulfite sequencing of the exon1/intron1 TRAIL–promoter region and 2 regions covering a CpG-island 2 kb upstream of the TSS with the methylation rate for each individual CpG in HA1E (gray bars) and HA1ER (black bars); n = numbers of clones analyzed. C, top, RT-qPCR for TRAIL mRNA in HA1E after retroviral infection with a HRASG12V- and a HRASG12V/S35-expressing pBabe-plasmid (black bars) and an empty control vector (gray bar), n = 2. Bottom, Western blot for TRAIL, phospho-p42/p44 MAPK, and p42/p44 MAPK. D, left: RT-qPCR for TRAIL mRNA after treatment of HA1E and HA1ER with the MAPKK inhibitor U0126 or decitabine or combination of both drugs, n = 3. Right, Western blot for TRAIL in HA1ER. Data are means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Per Lund et al. Mol Cancer Ther 2011;10:1611-1623 ©2011 by American Association for Cancer Research