Specific inhibition of serine- and arginine-rich splicing factors phosphorylation, spliceosome assembly, and splicing by the antitumor drug NB-506

Citation
B. Pilch et al., Specific inhibition of serine- and arginine-rich splicing factors phosphorylation, spliceosome assembly, and splicing by the antitumor drug NB-506, CANCER RES, 61(18), 2001, pp. 6876-6884
Citations number
39
Categorie Soggetti
Oncology,"Onconogenesis & Cancer Research
Journal title
CANCER RESEARCH
ISSN journal
00085472 → ACNP
Volume
61
Issue
18
Year of publication
2001
Pages
6876 - 6884
Database
ISI
SICI code
0008-5472(20010915)61:18<6876:SIOSAA>2.0.ZU;2-T
Abstract
Specific phosphorylation of serine- and arginine-rich pre-m RNA splicing fa ctors (SR proteins) is one of the key determinants regulating splicing even ts. Several kinases involved in SR protein phosphorylation have been identi fied and characterized, among which human DNA topoisomerase I is known to h ave DNA-relaxing activity. In this study, we have investigated the mechanis m of splicing inhibition by a glycosylated indolocarbazole derivative (NB-5 06), a potent inhibitor of both kinase and relaxing activities of topoisome rase 1. NB-506 completely inhibits the capacity of topoisomerase I to phosp horylate, in vitro, the human splicing factor 2/alternative splicing factor (SF2/ASF). This inhibition is specific, because NB-506 does not demonstrat e activity against other kinases known to phosphorylate SF2/ASF such as SR protein kinase 1 and cdc2 kinase. Importantly, HeLa nuclear extracts compet ent in splicing but not splicing-deficient cytoplasmic S100 extracts treate d with the drug fail to phosphorylate SF2/ASF and to support splicing of pr e-mRNA substrates containing SF2/ASF-target sequences. Native gel analysis of splicing complexes revealed that the drug affects the formation of the s pliceosome. a dynamic ribonucleoprotein structure where splicing takes plac e. In the presence of the drug, neither pre-spliceosome nor spliceosome is formed, demonstrating that splicing inhibition occurs at early steps of spl iceosome assembly. Splicing inhibition can be relieved by adding phosphoryl ated SF2/ASF, showing that extracts treated with NB-506 lack a phosphorylat ing activity required for splicing. Moreover, NB-506 has a cytotoxic effect on murine P388 leukemia cells but not on P388CPT5 camptothecin-resistant c ells that carry two point mutations in conserved regions of topoisomerase I gene (Gly361Val and Asp709Tyr). After drug treatment, P388 cells accumulat ed hypophosphorylated forms of SR proteins and polyadenylated RNA in the nu cleus. In contrast, neither SR protein phosphorylation nor polyadenylated m RNA distribution was affected in P388 CPT5-treated cells. Consistently, NB5 06 treatment altered the mRNA levels and/or splicing pattern of several tes ted genes (Bel-X, CD 44, SC35, and Sty) in P388 cells but not in P388 CPT5 cells. The study shows for the first time that indolocarbazole drugs target ing topoisomerase I can affect gene expression by modulating pre-mRNA splic ing through inhibition of SR proteins phosphorylation.