Modulation of topoisomerase IIα expression by a DNA sequence-specific polyamide

Abstract

Topoisomerase IIA (topo IIa) is an important target for several chemotherapeutic agents, including etoposide and doxorubicin. Confluent cells express low levels of topo IIa and are resistant to etoposide treatment. Repression of transcription in confluent cells is mediated by binding of the transcription factor NF-Y to inverted CCAAT motifs within the topo IIa promoter. To block the repressive binding of NF-Y, a polyamide (JH-37) was designed to bind to the flanking regions of selected CCAAT sites within the topo IIa promoter. Electrophoretic mobility shift assays and DNase I footprinting assays showed occupancy of the inverted CCAAT sites by JH-37. Chromatin immunoprecipitation assays confirmed in vivo inhibition of NF-Y binding to the topo IIa promoter. Following incubation of confluent NIH3T3 cells with JH-37, increased expression of topo IIa mRNA and protein was detectable. This correlated both with increased DNA double-strand breaks as shown by comet assay and decreased cell viability following exposure to etoposide. Polyamides can modulate gene expression and chemosensitivity of cancer cells.

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

“…We recently showed that a polyamide directed against the binding site of NF-Y in the topo IIa promoter was able to inhibit NF-Y binding and alter gene expression in confluent cells (2). Other studies have shown that polyamides can modulate gene expression in vivo (24).…”

“…CaCo2 cells were also obtained from Cancer Research UK London Research Institute and grown in RPMI (Bioclear) supplemented with 10% FCS and 1% glutamine and incubated at 37jC in 5% CO 2 .…”

“…There is considerable evidence of preferred DNA sequences to which several of these agents bind. For example, doxorubicin has been shown to bind preferentially to the CAAT quartet sequence and to the alternating polynucleotide d(TATATA) 2 (1). However, the ability of these agents to recognize specific DNA sequences has not been previously considered a significant factor in their anticancer activity.…”

“…Such agents could be used to modulate chemosensitivity by overcoming resistance mechanisms such as reduced expression of topoisomerase IIa (topo IIa; ref. 2).…”

Development of a Novel Gene Silencer Pyrrole-Imidazole Polyamide Targeting Human Connective Tissue Growth Factor

“…10,13) The hCTGF gene promoter contains several regulatory elements, such as Smad, AP1, TATA, NF-kB, GATA, CBP and Oct-1. [14][15][16][17] We designed a PI polyamide specific for bp Ϫ157 to Ϫ151 of the hCTGF promoter sequence.…”