Selective inhibition of transcription of the Ets2 gene in prostate cancer cells by a triplex-forming oligonucleotide

Abstract

The transcription factor Ets2 has a role in cancer development and represents an attractive therapeutic target. In this study, we designed a triplex-forming oligonucleotide (TFO) directed to a homopurine:homopyrimidine sequence in the Ets2 promoter. Transcription factors of the Sp family bound to this sequence and mutation of the Sp1 site reduced Ets2 promoter activity. The Ets2-TFO had high binding affinity for the target sequence and inhibited binding of Sp1/Sp3 to the overlapping site. This effect occurred with a high degree of sequence specificity. Mismatched oligonucleotides did not inhibit Sp1/Sp3 binding and mutations in the target sequence that abolished triplex formation prevented inhibition of Sp1/Sp3 binding by the TFO. The Ets2-TFO inhibited Ets2 promoter activity and expression of the endogenous gene in prostate cancer cells at nanomolar concentrations. The TFO did not affect reporter constructs with mutations in the TFO binding site and promoters of non-targeted genes. Expression of non-targeted genes was also not affected in TFO-treated cells. Collectively, these data demonstrated that the anti-transcriptional activity of the Ets2-TFO was sequence- and target-specific, and ruled out alternative, non-triplex mediated mechanisms of action. This anti-transcriptional approach may be useful to examine the effects of selective downregulation of Ets2 expression and may have therapeutic applications.

DNA triple helices: Biological consequences and therapeutic potential

“…Among these are chemical modifications that result in neutral or cationic backbones to reduce the electrostatic repulsion between the negatively charged phosphodiester backbone of the TFO with that of the target DNA duplex. Examples of such modifications include thioate linkages [56,57], N 3' − P 5' phosphoramidates [58,59], and morpholino phosphoramidate linkages [60]. The cationic phosphoramidate linkages, N,Ndiethylethylenediamine and N,N-dimethylaminopropylamine, confer increases in TFO binding affinity and intracellular activity [61].…”

Small RNA-directed transcriptional control: New insights into mechanisms and therapeutic applications

“…52 The PAR was proposed to bind through the formation of triple helix DNA with a purine-rich sequence element in the DHFR promoter 52 reminiscent of the TFO-induced transcriptional repression. 53,66 Another example of transcriptional regulation by ncRNA was reported in relation to the CCND1 gene, which encodes the cell cycle regulatory protein cyclin D1. 38 Cyclin D1 expression was reduced in cells exposed to ionizing radiation.…”

A model for triple helix formation on human telomerase reverse transcriptase (hTERT) promoter and stabilization by specific interactions with the water soluble perylene derivative, DAPER

“…Homopurine-homopyrimidine tracts are frequently found in the upstream regulatory regions of genes and several TFOs have been shown to down-regulate expression of targeted genes by blocking transcription initiation [13][14][15][16][17].…”