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.

Camptothecin releases P-TEFb from the inactive 7SK snRNP complex.

AMENTE, STEFANO;NAPOLITANO, GIULIANA;LANIA, LUIGI;MAJELLO, BARBARA
2009

Abstract

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.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11588/350343
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