title: Topoisomerase I and II inhibitors control caspase-2 pre-messenger RNA splicing in human cells.
abstract: 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.
authors.affiliation: INSERM U517, Faculty of Medicine, 7 Boulevard Jeanne d'Arc, 21000 Dijon, France.
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Solier
authors.forename: Amélie
authors.initials: A
authors.lastname: Lansiaux
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Logette
authors.forename: Jane
authors.initials: J
authors.lastname: Wu
authors.forename: Johann
authors.initials: J
authors.lastname: Soret
authors.forename: Jamal
authors.initials: J
authors.lastname: Tazi
authors.forename: Christian
authors.initials: C
authors.lastname: Bailly
authors.forename: Lydie
authors.initials: L
authors.lastname: Desoche
authors.forename: Eric
authors.initials: E
authors.lastname: Solary
authors.forename: Laurent
authors.initials: L
authors.lastname: Corcos
mesh_terms: D017398:Alternative Splicing / Q000187:drug effects*; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D017209:Apoptosis / Q000502:physiology; D015153:Blotting, Western; D053143:Caspase 2; D045744:Cell Line, Tumor / Q000378:metabolism; D003432:Cross-Linking Reagents; D003546:Cysteine Endopeptidases / Q000235:genetics*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005822:Genetic Vectors; D018922:HL-60 Cells; D006801:Humans; D019384:Nucleic Acid Synthesis Inhibitors; D010957:Plasmids; D012326:RNA Splicing / Q000187:drug effects*; D012333:RNA, Messenger / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D059004:Topoisomerase I Inhibitors; D059005:Topoisomerase II Inhibitors; D014162:Transfection; D020298:U937 Cells
chemical_list: D000903:Antibiotics, Antineoplastic; D003432:Cross-Linking Reagents; D004791:Enzyme Inhibitors; D019384:Nucleic Acid Synthesis Inhibitors; D012333:RNA, Messenger; D059004:Topoisomerase I Inhibitors; D059005:Topoisomerase II Inhibitors; C490360:CASP2 protein, human; D053143:Caspase 2; D003546:Cysteine Endopeptidases