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Distinct inhibitory effects on mTOR signaling by ethanol and INK128 in diffuse large B-cell lymphoma

ABSTRACT: The mechanistic target of rapamycin, (mTOR) kinase plays a pivotal role in controlling critical cellular growth and survival pathways, and its aberrant induction is implicated in cancer pathogenesis. Therefore, suppression of active mTOR signaling has been of great interest to researchers; several mTOR inhibitors have been discovered to date. Ethanol (EtOH), similar to pharmacologic mTOR inhibitors, has been shown to suppress the mTOR signaling pathway, though in a non-catalytic manner. Despite population studies showing that the consumption of EtOH has a protective effect against hematological malignancies, the mechanisms behind EtOH’s modulation of mTOR activity in cells and its downstream consequences are largely unknown. Here we evaluated the effects of EtOH on the mTOR pathway, in comparison to the active-site mTOR inhibitor INK128, and compared translatome analysis of their downstream effects in diffuse large B-cell lymphoma (DLBCL). SUDHL-2 and SUDHL-4 diffuse large B-cell lymphoma (DLBCL) cell lines (ATCC) were cultured in RPMI Medium 1640 (Gibco BRL) supplemented with 10% fetal bovine serum (FBS) at 37°C in 5% CO2. EtOH-treated cells were cultured in sealed flasks to maintain EtOH concentrations, 20mM in the culture medium, water was added to EtOH control wells. INK128-treated cells were treated with INK128 at 40nM (Selleckchem) dissolved in dimethyl sulfoxide (DMSO) or 0.01% DMSO was added for the control wells. To create polysomal fractions, cells were lysed in cytoplasmic lysis buffer (5mM Tris, 2.5mM MgCl2, 1.5mM KCl, 1% Triton X-100, 0.5% Sodium Deoxycholate and 2 mM DTT), loaded on 10 - 50% linear sucrose gradients and fractionated. The RNA in each fraction was monitored by optical density measurement (A254) and eleven fractions were collected with a fraction collector (Brandel). The RNA from each fraction was isolated by Trizol (Invitrogen) and used for RT-qPCR analysis. RNA from high molecular weight polysomal fractions, with actively translated mRNAs (fractions 9 - 11) were pooled and used for microarray analysis. RNA was isolated using Qiagen RNeasy protocols and quality and quantity were checked with an Agilent 2100 Bio-Analyzer using RNA 6000 Nano chips. Labeling and amplification were done with the standard Illumina protocol and the biotin-labeled cRNA was hybridized to Illumina's HumanHT-12 V4.0 Expression BeadChip. The arrays were washed, blocked and the biotin-labeled cRNA was detected by staining with streptavidin-Cy3.

ORGANISM(S): Homo sapiens  

SUBMITTER: Kevin G Becker   Bojie Dai  Raymond J Peroutka  Simone Houng  Elin Lehrmann  James J Steinhardt  Krystyna Mazan-Mamczarz  Yongqing Zhang  Parameswary A Muniandy  Ronald B Gartenhaus  Efroni Sol  Ari L Landon  Moriah Gidoni 

PROVIDER: E-GEOD-62790 | ArrayExpress | 2015-03-03



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