Event Abstract Back to Event Impact of acidosis on ζ-crystallin-mediated bcl-2 expression in leukemic cell lines Rosa Loffredo1*, Matteo Lulli1, Ewa Witort1, IIrene Granucci1, Federico Di Gesualdo1, Andrea Lapucci1, Amedeo Amedei2 and Sergio Capaccioli1 1 University of Florence, Italy 2 Università di Firenze, Department of Internal Medicine, Section of Immunology, Italy Background and aim. Acidosis, a feature of the tumour microenvironment, has been demonstrated in numerous solid tumours and in haematological malignancies. Moreover, extracellular acidity exudes cytoprotective effect on mouse lymphoma and human T-cell Acute Lymphoblastic Leukemia (T-cell ALL) against apoptotosis by increasing Bcl-2 expression through acid-sensing GPR65 (G protein-coupled receptor) via MEK/ERK pathway (1). In most malignancies, Bcl-2 is overexpressed in the absence of chromosomal rearrangements, by mechanisms remained unclear until we demonstrated in 2003 that its expression is controlled at post-transcriptional level (2) by interactions of a destabilizing, adenine and uracil-rich element (AU-rich element, ARE) located in the 3’UTR of bcl-2 mRNA and specific ARE-Binding Proteins (AUBPs). We have also identified ζ-crystallin as a important player in bcl-2 post-transcriptional control and an AUBP endowed with bcl-2 mRNA stabilizing activity (3). ζ-Crystallin prevents cellular acidosis by binding to an AU-rich pH-responsive element located in several pH-responsive transcripts (4). In acidosis, its expression increases and enhances the stability of target mRNAs (5). Considering that Bcl-2 overexpression is a feature of a variety of human cancers, the impact of deregulation of bcl-2 post-transcriptional control on clinical outcome is evident. Therefore, our aim was to confirm that acidosis-affected ζ-crystallin expression could positively regulate bcl-2 expression in leukemia cells. Methods. The Jurkat, MOLT-4 and SUP-T1 cell lines were used as model of T-cell ALL. Jurkat cells were grown in acidic medium (pH 6.3) for 6h, exposed to apoptotic stimuli (UV radiations, H2O2 and cycloheximide) and their viability was analysed by FACS. The ζ-crystallin and Bcl-2 expression were evaluated by WB, while bcl-2 mRNA stability measured by ratio of bcl-2 mRNA/hnRNA levels in all three cell lines and T-cells from ALL patients was determined by RT-PCR. In order to extend our studies, quantification and correlation plot of ζ-Crystallin and Bcl-2 expression levels in B-cells from healthy donors and Chronic Lymphocytic Leukemia (CLL) patients were done by quantitative WB analysis. Results. The extracellular acidity increased viability of Jurkat cells exposed to all three apoptotic stimuli. Moreover, acidosis induced over-expression both of ζ-crystallin and Bcl-2 in Jurkat cells. In addition, the bcl-2 mRNA stability in ALL T-cells from leukemic patients compared to control lymphocytes demonstrated much higher stability of bcl-2 mRNA in ALL T-cells with respect to controls. Our preliminary analysis carried out in B cells from CLL patients also indicated a positive correlation between Bcl-2 and ζ-crystallin. Conclusions. Based on our data, ζ-crystallin enhances Bcl-2 expression in acidic microenviroment and emerges as an important pH-responsive element that confers a cytoprotective effect on lymphoma/leukemic cells against apoptotic stimuli. These acidosis-induced changes render cells exquisitely sensitive to Bcl-2 targeted chemotherapy and represent an interesting attribute for development of innovative therapeutic tools. Acknowledgements Associazione Italiana per la Ricerca sul Cancro (AIRC), Ente Cassa di Risparmio di Firenze