Camptothecin releases P-TEFb from the inactive 7SK snRNP complex.
An immediate effect of DNA Topoisomerase I inhibitors camptothecin (CPT) and its derivates is the inhibition of transcription. These fast-acting drugs are believed to inhibit transcription by blocking topoisomerase-mediated relief of DNA supercoiling that occurs during transcription elongation. The CPT effects are commonly considered to be due to a collision between the drug-trapped enzyme on the DNA template and the elongating RNAPII. Here we present evidences that CPT treatment induces an early affect on the positive elongation factor b (P-TEFb). The P-TEFb activity is tightly and dynamically regulated, and a reservoir of P-TEFb is kept in an inactive state in the multisubunit 7SK snRNP. We found that, shortly after treatment, CPT disrupts the large inactive P-TEFB complex, and such effect is reversible and independent from DNA replication. Thus, CPT modulates P-TEFb equilibrium in a manner similar to Flavopiridol (FP), a pan-Cdk inhibitor proposed as chemotherapeutic agents against cancers. We determined that while FP inhibits Cdk9 leading to hypo-phosphorylation of RNA polymerase II, CPT-mediated release of free P-TEFb correlates with a concomitant hyper-phosphorylation of RNAPII, which in turn alters the levels and distribution of the RNAPII along transcribed genes. The findings that CPT affects P-TEFb activity provide a direct evidence of the mechanism of this drug to inhibit transcription.
Amente
Stefano
S
Department of Structural and Functional Biology, University of Naples Federico II, via Cinthia, Naples, Italy.
Gargano
Barbara
B
Napolitano
Giuliana
G
Lania
Luigi
L
Majello
Barbara
B
D000818:Animals; D002166:Camptothecin / Q000494:pharmacology*; D042863:Cyclin-Dependent Kinase 9 / Q000037:antagonists & inhibitors; D005419:Flavonoids / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D010766:Phosphorylation / Q000187:drug effects; D010880:Piperidines / Q000494:pharmacology; D042925:Positive Transcriptional Elongation Factor B / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D012319:RNA Polymerase II / Q000378:metabolism; D051381:Rats; D017411:Ribonucleoproteins, Small Nuclear / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects
D005419:Flavonoids; D010880:Piperidines; D017411:Ribonucleoproteins, Small Nuclear; C077990:alvocidib; D042925:Positive Transcriptional Elongation Factor B; D042863:Cyclin-Dependent Kinase 9; D012319:RNA Polymerase II; D002166:Camptothecin