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Genomic profiling of enzastaurin‐treated B cell lymphoma RL cells

Follicular lymphoma (FL) is an indolent lymphoma associated with follicular centre B cells and typically contains the Bcl-2 chromosomal translocation t(14;18). FLs are sensitive to chemotherapy; however, the majority of patients eventually die from the disease. Thus, there is a need for new, less toxic and more active treatments. Enzastaurin (LY317615.HCl), an acyclic bisindolylmaleimide, was initially developed as an ATP-competitive selective inhibitor of PKC. Enzastaurin was shown to target the phosphoinositide 3-kinase (PI3K)/AKT pathway and to inactivate BAD, a pro-apoptotic member of the Bcl-2 family proteins. We recently investigated the effect of enzastaurin on proliferation and survival of myeloma and lymphoma cell lines[1,2]. We found that enzastaurin inhibits cell proliferation and induces apoptosis. These results are consistent with decreased phosphorylation of AKT pathway and its downstream targets as the glycogen synthase kinase-3 beta. To provide new insights into the molecular mechanisms of the antitumour action of enzastaurin in Non-Hodgkin lymphoma, we investigated its effects on the gene expression profiles (GEP) of the B cell lymphoma RL cell line, carrying t(14;18), by microarray analysis. Enzastaurin was a gift from Eli Lilly & Co. (Indianapolis, IN, USA). Total cell lysates were prepared and analysed by Western Blot analysis. The antibodies used for immunoblotting included anti-caspase 9, anti-caspase 8, anti-PARP, antiAKT, anti–Cyclin I, anti–Stat, anti–Myc and p44/MAPK. The RL cell line was treated with enzastaurin at the IC50 concentration (2.5mM) for 48 h. Total RNA was isolated from three independent replicas of RL cells, either treated or untreated, using the TRIzol reagent (Life Technologies Inc., Rockville, MD, USA) and then purified using the RNeasy total RNA Isolation Kit (Qiagen, Valencia, CA). TheWhole Transcript (WT) Assay (Affymetrix Inc., Santa Clara, CA) was used on 100 ng of purified total RNA to generate a single-stranded DNA sense target. Hybridization of the fragmented and labeled DNA targets on the Gene 1.0 ST Arrays (Affymetrix) and scanning of the chips (GeneChip Scanner 3000 7G, Affymetrix) were performed accordingly to the manufacturer’s protocols. Data were acquired using the GeneChip Operating Software (GCOS v1.4, Affymetrix Inc., Santa Clara, CA, USA), quality evaluation and RNA normalization were performed with the Expression Console Software (EC v1.1, Affymetrix Inc., SantaClara, CA,