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1.
Mol Pharmacol ; 68(5): 1496-503, 2005 Nov.
Article in English | MEDLINE | ID: mdl-15961672

ABSTRACT

Yondelis is a potent DNA-binding anticancer drug isolated from the tunicate Ecteinascidia turbinata currently undergoing phase III clinical trials. We and others have shown selective inhibition to the transcriptional induction of several genes. We tested the hypothesis that Yondelis specifically targets cell-cycle genes. Our analysis on endogenous and transfected reporter systems revealed complex patterns of transcriptional inhibition and, surprisingly, activation. Other inducible systems-the metallothionein and the CYP3A4 promoters-were little affected. We assayed whether interference of DNA binding of the common nuclear factor Y (NF-Y) activator was responsible for the observed inhibition: in vivo chromatin immunoprecipitation analysis in NIH3T3 and HCT116 cells indicates that NF-Y binding is little affected by Yondelis addition. Finally, histone acetylation was modestly affected only on Cdc2 and cyclin B2 but not on other repressed promoters. These data prove that Yondelis is not a general inhibitor of inducible genes, and its selective effects are exerted downstream from transcription factors binding and histone acetyl transferases recruitment.


Subject(s)
Antineoplastic Agents/pharmacology , Cell Cycle/genetics , Dioxoles/pharmacology , Isoquinolines/pharmacology , Promoter Regions, Genetic/drug effects , Animals , CCAAT-Binding Factor/metabolism , DNA/metabolism , Mice , NIH 3T3 Cells , Tetrahydroisoquinolines , Trabectedin , Transcription, Genetic/drug effects
2.
J Biol Chem ; 277(8): 5841-8, 2002 Feb 22.
Article in English | MEDLINE | ID: mdl-11689552

ABSTRACT

The nuclear factor y (NF-Y) trimer and TFIID contain histone fold subunits, and their binding to the CCAAT and Initiator elements of the major histocompatibility complex class II Ea promoter is required for transcriptional activation. Using agarose-electrophoretic mobility shift assay we found that NF-Y increases the affinity of holo-TFIID for Ea in a CCAAT- and Inr-dependent manner. We began to dissect the interplay between NF-Y- and TBP-associated factors PO1II (TAF(II)s)-containing histone fold domains in protein-protein interactions and transfections. hTAF(II)20, hTAF(II)28, and hTAF(II)18-hTAF(II)28 bind to the NF-Y B-NF-YC histone fold dimer; hTAF(II)80 and hTAF(II)31-hTAF(II)80 interact with the trimer but not with the NF-YB-NF-YC dimer. The histone fold alpha2 helix of hTAF(II)80 is not required for NF-Y association, as determined by interactions with the naturally occurring splice variant hTAF(II)80 delta. Expression of hTAF(II)28 and hTAF(II)18 in mouse cells significantly and specifically reduced NF-Y activation in GAL4-based experiments, whereas hTAF(II)20 and hTAF(II)135 increased it. These results indicate that NF-Y (i) recruits purified holo-TFIID in vitro and (ii) can associate multiple TAF(II)s, potentially accommodating different core promoter architectures.


Subject(s)
CCAAT-Binding Factor/metabolism , Histones/chemistry , Transcription Factors, TFII/metabolism , Animals , Dimerization , Mice , Promoter Regions, Genetic , Protein Binding , Protein Folding , Protein Subunits , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Transcription Factor TFIID , Transcription Factors, TFII/chemistry , Transcription Factors, TFII/isolation & purification , Transfection
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