Apoptosis Is Associated with Modifications of bcl-2 mRNA AU-Binding Proteins Martino Donnini,* ,1 Andrea Lapucci,* ,1 Laura Papucci,* Ewa Witort,* Alessio Tempestini,* Gary Brewer,† Anna Bevilacqua,† Angelo Nicolin,‡ Sergio Capaccioli,* ,2 and Nicola Schiavone* ,2 *Department of Experimental Pathology and Oncology, School of Medicine, University of Florence, Italy; ‡Department of Pharmacology, School of Medicine, University of Milan, Italy; and †Department of Molecular Genetics and Microbiology, UMDNJ, Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, New Jersey 08854 Received September 1, 2001 The expression of genes requiring finely tuned con- trol is regulated by a posttranscriptional mechanism involving mRNA A  U-rich elements (AREs) cooper- ating with ARE-binding proteins (AUBPs) in modula- tion of mRNA stability. We reported previously that an ARE in the bcl-2 mRNA 3-untranslated region (3- UTR) had destabilizing activity and was involved in bcl-2 downregulation during apoptosis in vitro. Here we demonstrate that the bcl-2 ARE complexes with a number of specific AUBPs, whose pattern undergoes changes following application of apoptotic stimuli. The caspase inhibitor Z-VAD-fmk strongly attenuates both bcl-2 mRNA decay and bcl-2 AUBP pattern changes elicited by apoptotic stimuli, indicating the involvement of bcl-2 AUBPs in bcl-2 mRNA stability control. © 2001 Academic Press Key Words: apoptosis; bcl-2; gene expression; post- transcriptional control; AU-rich elements; AU-binding proteins. Cell growth, differentiation, and death are finely reg- ulated by complex genetic programs in which bcl-2 gene expression often plays a central role (1). The bcl-2 expression has to be downregulated to activate the apoptotic process (2, 3). A number of proto-oncogenes (bcl-X L , c-Myb, ras), oncosuppressors (p53), cytokines (IL-2, IL-3, IL-4) and growth factors (bFGF, NGF) are known to either up- or down-regulate bcl-2 expression (4). The bcl-2 expression can be modulated at different levels, depending either on bcl-2 transcriptional rate (5, 6), or mRNA stability (7) or protein activity (8, 9). The mRNA stability is determined by cis-elements located either in the coding or noncoding regions of mRNA of many transiently expressed genes (10). Some of these elements fall in to the A + U-rich elements (ARE) category, in that they contain several AUUUA motifs located in the 3'-untranslated regions (10 –14). Deregulation of mRNA stability has been observed in neoplastic transformation. In particular, mutations re- sponsible for stabilization of some mRNAs such as c-fos and c-myc are frequently associated with human tu- mors (15, 16). A new mechanism of posttranscriptional control of bcl-2 expression based on mRNA stability has been recently identified in our laboratory following the ob- servation that the 3'-untranslated region (3'-UTR) of bcl-2 mRNA harbors an evolutionary conserved A + U-rich element (17). The bcl-2 ARE possesses a mod- erate destabilizing activity that can be dramatically enhanced by apoptotic stimuli (7). The modulating ac- tivity of many AREs involves some trans-acting pro- teins or protein complexes that bind to these elements and therefore are named ARE-binding proteins (AUBPs). A number of AUBPs have been described and are known to possess multifunctional activity (18). Here we demonstrate that the bcl-2 ARE binds pro- teins or protein complexes, ranging from 35 to 100 kDa, with high affinity. Apoptotic stimuli (UVC-irradiation or cycloheximide treatment) modify the pattern of bcl- 2-AUBPs by a mechanism involving activation of caspases. In summary, these results suggest the in- volvement of specific AUBPs in modulation of bcl-2 mRNA stability following apoptotic stimuli. MATERIALS AND METHODS Cell lines and treatments. The Jurkat T-cell leukemia line (E61, ECACC) was cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, 50 IU/ml penicillin and 50 g/ml streptomycin, in a humidified atmosphere of 5% CO 2 at 37°C. Cells (2  10 6 /100-mm plate) were induced to apoptosis by irradiation with UVC (15 J/m 2 , 254 nm), following a 2-h pretreat- ment with 100 M Z-Val-Ala-DL-Asp(OMe)-fluoromethylketone (Z- VAD-fmk) (Bachem AG, Switzerland), when required and collected at various time points. Cycloheximide (50 M) was used as an 1 Both authors have equally contributed to this work. 2 To whom correspondence and reprint requests may be addressed. Fax: +39 55 4282333. E-mail: sergio@unifi.it, nicola@unifi.it. Biochemical and Biophysical Research Communications 287, 1063–1069 (2001) doi:10.1006/bbrc.2001.5700, available online at http://www.idealibrary.com on 1063 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.