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Cell Cycle, Cell Death, and Senescence

Topoisomerase I and II Inhibitors Control Caspase-2 Pre-Messenger RNA Splicing in Human Cells¹

¹ Inserm U517, Faculty of Medicine, Dijon, France;
² Service d' Hématologie Clinique, CHRU le Bocage, Dijon, France;
³ Inserm U524, Institut de Recherches sur le Cancer de Lille, Lille Cedex, France;
⁴ Department of Pediatrics, Washington University School of Medicine, McDonnell Pediatric Research Building Rm 3107, St. Louis, MO; and
⁵ Metazoan Messenger RNAs Metabolism, IGM, UMR 5535 CNRS, Montpellier, France

Requests for reprints: Laurent Corcos, Inserm U517, Faculty of Medicine, 7 Boulevard Jeanne d'Arc, 21000 Dijon, France. Phone: 33-3-80-39-34-40; Fax: 33-3-80-39-34-34. E-mail: Laurent.corcos{at}u-bourgogne.fr

We have recently shown that the topoisomerase II inhibitor, etoposide (VP16), could trigger caspase-2 pre-mRNA splicing in human leukemic cell lines. This leads to increased inclusion of exon 9, which is specifically inserted into the short caspase-2S isoform mRNA and absent from the long caspase-2L isoform mRNA. One of the consequences of this alternative splicing is a decrease in the total amount of the mature form of caspase-2L mRNA and protein. In this study, we analyzed the effects of several representative molecules of various classes of cytotoxic agents on caspase-2 pre-mRNA splicing in both U937 leukemic cells and in HeLa cervix carcinoma cells. Very strikingly, both topoisomerase I (camptothecin and homocamptothecin derivatives) and II (VP16, amsacrine, doxorubicin, mitoxantrone) inhibitors induced exon 9 inclusion. DNA intercalating glycosyl indolocarbazole derivatives as well as DNA alkylating agents, such as cisplatin and melphalan, antimetabolites like 5-fluorouracil, and mitotic spindle poisons like vinblastine had no effect. Therefore, both classes of DNA topoisomerases can control pre-mRNA splicing of the caspase-2 transcript. In addition, the splicing reaction brought about by camptothecin was hampered in human CEM/C2 and in murine P388-45R leukemic deficient in topoisomerase I activity. Conversely, VP16 did not trigger caspase-2 alternative splicing in human HL60/MX2 leukemic cells harboring a mutant topoisomerase II. Minigene transfection analysis revealed that topoisomerase inhibitors did not change the splicing profile when cis-acting elements in intron-9, reported to control exon 9 inclusion independently of drug treatment, were removed. Rather, our experiments suggest that exon 9 inclusion induced by topoisomerase inhibitors reflects the activity exerted by topoisomerase I or II on proteins that control splicing reactions, or their direct involvement in pre-mRNA splicing.

Key Words: topoisomerases inhibitors • caspase-2 • mRNA splicing

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