Inhibition of DNA binding of the NF-Y transcription factor by the pyrrolobenzodiazepine-polyamide conjugate GWL-78

Abstract

Many genes involved in cell cycle control have promoters that bind the heterotrimeric transcription factor NF-Y. Several minor-groove binding drugs have been shown to block interactions of transcription factors with cognate DNA-binding sequences. We showed previously that noncovalent minor-groove binding agents block interactions of NF-Y with the promoter of topoisomerase IIA (topo IIA). In this study, we investigated the ability of GWL-78, a pyrrolobenzodiazepine-poly(N-methylpyrrole) conjugate, to inhibit the binding of NF-Y to DNA. Electrophoretic mobility shift assays showed that GWL-78 could displace NF-Y bound to several CCAAT motifs within promoters of genes involved in cell cycle progression. DNase I footprinting of the topo IIA promoter confirmed binding of GWL-78 to AT-rich sequences corresponding to the preferred binding site of NF-Y. Incubation with GWL-78 resulted in displacement of NF-Y binding to DNA. Chromatin immunoprecipitation assays on the topo IIA promoter showed that GWL-78 was able to enter the nucleus and interact with specific DNA sequences. Treatment of NIH3T3 cells with GWL-78 resulted in a block of cell cycle progression, which did not involve activation of p53. Thus, agents such as GWL-78 may be useful in modulating transcription and blocking cellular proliferation.

Nuclear Localization and Gene Expression Modulation by a Fluorescent Sequence-Selective p-Anisyl-benzimidazolecarboxamido Imidazole-Pyrrole Polyamide

“…Previous studies of the in vitro interaction of the polyamides with the topo IIa promoter utilized numerous biophysical assays. Cellular permeation and promoter interaction were indirectly assumed by increased levels of topo IIa in confluent cells as assessed by immunoblotting, chromatin immunoprecipitation, and cellular sensitization to etoposide (Hochhauser et al, 2007;Le et al, 2006;Kotecha et al, 2008). In this study, the nuclear accumulation and DNA binding of the polyamide is directly verified by detecting the increased fluorescence output.…”

“…Our research groups have previously employed the topo IIa promoter as a model system for proof of principle of the ability of polyamides to modulate gene expression in cells (Tolner et al, 2001;Le et al, 2006;Wells et al, 2006;Hochhauser et al, 2007;Kotecha et al, 2008;Franks et al, 2010).…”

Genetic Variants in CCNB1 Associated With Differential Gene Transcription and Risk of Coronary In-Stent Restenosis

“…factor) is a ubiquitous heterotrimeric transcription factor formed from NF-YA, B, and C subunits that is required for cell proliferation. [26][27][28][29][30][31] We recently found that NF-Y is induced during atherosclerosis and restenosis in rodent models and humans and promotes platelet-derived growth factor-BB-dependent cyclin B1 expression, vascular smooth muscle cell proliferation, and neointimal lesion development in a mouse femoral artery denudation model. 14 The results in the current study demonstrate that the restenosis-risk T allele of −957[T/C], but not the C allele, generates a CCAAT box at position −959/−955 relative to the CCNB1 transcription initiation site that supports specific binding of NF-Y and drives NF-YA-dependent transcription of luciferase reporter plasmids.…”

Entwicklung Pyrrolobenzodiazepin(PBD)‐haltiger Antikörper‐Wirkstoff‐Konjugate (ADCs) ausgehend von Anthramycin

“…B. Endonuclease BamH1, [21] RNA-Polymerase [3a, 22] und Ligase 1 [23] ) und spezifischen Transkriptionsfaktoren (z. B. Sp1, [24] NF-Y [25] und NF-kB) [25a, 26] als auch die Regulierung verschiedener Signalwege (z. B. p53-abhän-gige und -unabhängige apoptogene, [27] JNK/AP-1-, [8] VEGF- [28] und SDF1a-Signalwege).…”