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

M. Kotecha, J. Kluza, G. Wells, C. C. O'Hare, C. Forni, R. Mantovani, P. W. Howard, P. Morris, D. E. Thurston, J. A. Hartley, D. Hochhauser
2008 Molecular Cancer Therapeutics  
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)
more » ... , 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. Downloaded from Figure 4. Effects of GWL-78 on promoters using ChIP assay. NIH3T3 cells were treated for 24 h with 1 Amol/L GWL-78 and ChIP with control (Flag ) and anti-NF-Y-B antibodies. The results on CCAAT promoters for topo IIa, E2F1, mdr1, cdc25c, NF-Y-A, and nucleolin, are also shown. Analyses were done with semiquantitative PCR and the data were plotted after scanning of several amplifications in the linear range. Enrichments of the treated samples over the control Flag material were shown as percentage of enrichments of untreated controls: 14-fold (topo IIa), 7-fold (cdc2), 16-fold (E2F1), 4-fold (mdr1), and 5-fold (cdc25c). Enrichment of NF-Y-A was >10-fold. No enrichment was observed on the CCAAT-less nucleolin.
doi:10.1158/1535-7163.mct-07-0475 pmid:18483319 fatcat:oequvvltkbc3pd6p5lacdwdvfi