C/EBPß expression in ALK-positive anaplastic large cell lymphomas is required for cell proliferation and is induced by the STAT3 signaling pathway.

BACKGROUND: Anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma is characterized by the t(2;5) chromosomal translocation, resulting in the expression of a fusion protein formed of nucleophosmin (NPM) and ALK. Recently, we reported the abnormal expression of the transcription factor CCAAT/enhancer binding protein-beta (C/EBPbeta) in ALK-positive anaplastic large cell lymphomas, and demonstrated its dependence on NPM-ALK activity. DESIGN AND METHODS: In this study, the role of C/EBPbeta in proliferation and survival of ALK-positive anaplastic large cell lymphomas was investigated, as well as the mechanism of its expression and activity. Highly effective short hairpin RNA sequences and/or pharmacological inhibitors were used to abrogate the expression or activity of C/EBPbeta, signal transducer and activator of transcription 3 (STAT3), AKT, extracellular signal-related kinase 1/2 (ERK1/2) and mammalian target of rapamycin (mTOR). RESULTS: Interference with C/EBPbeta expression resulted in a dramatic decrease in cell proliferation in ALK-positive anaplastic large cell lymphomas, with a mild induction of apoptosis after 6 days. Down-regulation of STAT3 resulted in a marked decrease in C/EBPbeta mRNA and protein levels with impairment in cell proliferation and viability, underscoring the important role of these two proteins in ALK-mediated oncogenesis. Additionally, we demonstrated that reduction of ERK1/2 activity led to C/EBPbeta Thr(235) dephosphorylation and moderate growth retardation. The AKT/mTOR signaling pathway did not have any influence on C/EBPbeta expression or C/EBPbeta phosphorylation. CONCLUSIONS: These findings reveal the convergence of STAT3 and ERK1/2 signaling pathways activated by NPM-ALK in mediating the regulation of C/EBPbeta expression, a transcription factor central to NPM-ALK transformation.

Efficient shRNA delivery into B and T lymphoma cells using lentiviral vector-mediated transfer.

RNA interference is a powerful tool for the functional analysis of proteins by specific gene knockdown. In this study, we devised a rapid and efficient way to screen suitable siRNA sequences and subsequently employ them for specific gene knockdown in usually hard-to-transfect lymphoid cell lines, using a self-inactivating lentiviral vector. Two proteins with different half-lives were chosen, cyclin D1 and STAT3. A specific lacZ reporter fusion assay was used to identify highly effective siRNA sequences. Only siRNA molecules with more than 85% of knockdown efficiency were selected for the generation of lentiviral transfer vectors. Transduction rates of 75-99% were achieved in the lymphoma cell lines Granta 519 (mantle cell lymphoma), Karpas 299, and SUDHL-1 (anaplastic large T cell lymphoma), as demonstrated by green fluorescent protein expression in fluorescence-activated cell sorting analysis. The high level of transduction efficiency allows RNA interference studies to be performed on transduced cells without further manipulation, such as cell sorting or cloning. The LacZ reporter system together with the lentivirus technology is a very important tool in the hematology field, which enables experiments in lymphoid cells that were not possible before.