Modulation of topoisomerase IIα expression and chemosensitivity through targeted inhibition of NF-Y:DNA binding by a diamino p-anisyl-benzimidazole (Hx) polyamide

Abstract

Background: Sequence specific polyamide HxIP 1, targeted to the inverted CCAAT Box 2 (ICB2) on the topoisomerase IIα (topo IIα) promoter can inhibit NF-Y binding, re-induce gene expression and increase sensitivity to etoposide. To enhance biological activity, diamino-containing derivatives (HxI*P 2 and HxIP* 3) were synthesised incorporating an alkyl amino group at the N1-heterocyclic position of the imidazole/pyrrole.

Methods: DNase I footprinting was used to evaluate DNA binding of the diamino Hx-polyamides, and their ability to disrupt the NF-Y:ICB2 interaction assessed using EMSAs. Topo IIα mRNA (RT-PCR) and protein (Immunoblotting) levels were measured following 18h polyamide treatment of confluent A549 cells. γH2AX was used as a marker for etoposide-induced DNA damage after pre-treatment with HxIP* 3 and cell viability was measured using Cell-Titer Glo®.

Results: Introduction of the N1-alkyl amino group reduced selectivity for the target sequence 5'-TACGAT-3' on the topo IIα promoter, but increased DNA binding affinity. Confocal microscopy revealed both fluorescent diamino polyamides localised in the nucleus, yet HxI*P 2 was unable to disrupt the NF-Y:ICB2 interaction and showed no effect against the downregulation of topo IIα. In contrast, inhibition of NF-Y binding by HxIP* 3 stimulated dose-dependent (0.1-2μM) re-induction of topo IIα and potentiated cytotoxicity of topo II poisons by enhancing DNA damage.

Conclusions: Polyamide functionalisation at the N1-position offers a design strategy to improve drug-like properties. Dicationic HxIP* 3 increased topo IIα expression and chemosensitivity to topo II-targeting agents.

General significance: Pharmacological modulation of topo IIα expression has the potential to enhance cellular sensitivity to clinically-used anticancer therapeutics. This article is part of a Special Issue entitled: Nuclear Factor Y in Development and Disease, edited by Prof. Roberto Mantovani.

Keywords: Chemosensitisation; DNA-binding polyamides; Gene modulation; NF-Y; Sequence selectivity; Topoisomerase IIα (topo IIα); Transcription factor-DNA interactions.

Figure 1

Schematic representation of the confluence-induced downregulation of topo IIα, mediated by the repressive binding of NF-Y to the ICB2. The ICB sequence ATTGG is highlighted in blue and the 5′-flanking sequence of the ICB2 is outlined with a dashed box.

Figure 2

Design of the diamino Hx-polyamides 2 and 3. (A) Structures of the monoamino Hx-polyamide, HxIP 1 and the orthogonally positioned diamino-containing derivatives, HxI*P 2 and HxIP* 3. The asterisk (*) denotes position of the N-alkyl amino group. (B) Schematic models of polyamides 2 and 3 binding to the 5′-TACGAT-3′ sequence on the 5′-flank of the ICB2 in an antiparallel 2:1 fashion as an overlapped stacked dimer.

Figure 3

Biophysical comparison of the diamino Hx-polyamide DNA binding characteristics. (A) CD spectra for HxIP* 3 (top panel) and HxI*P 2 (bottom) binding with the target sequence 5′-ATCGAT-3′ and non-consensus sequences 5′-AAATTT′-3 and 5′-ACGCGT-3′. (B) SPR sensorgram of HxIP* 3 with the target 5′-ATCGAT-3′ sequence at 100, 200 and 300 nM concentrations of compound. Thick grey lines represent true association and dissociation and thin black lines represent global kinetics fits. (C) Steady-state analyses of HxIP* 3 with 5′-ATGCAT-3′ (•, solid line), 5′-ATCGAT-3′ (▲, dashed line), and 5′-ACGCGT-3′ (◆, dotted line) sequences. SPR experiments were run in 10 mM CCL, 1 mM EDTA, 200 mM NaCl, 0.05% P20 (pH 6.5) at 25 °C.

Figure 4

Binding of diamino Hx-polyamides 2 and 3 to the topo IIα promoter. Autoradiograms of DNase I footprinting gels, HxI*P 2 (left) and HxIP* 3 (right). The concentrations (μM) used are shown at the top of the gel. G+A represents a formic acid-piperidine marker specific for purines. The positions of the ICB1, ICB2, ICB3 and the target sequence are indicated.

Figure 5

Inhibition of NF-Y binding to ICB2 by the diamino Hx-polyamides. Electrophoretic mobility shift assays (EMSAs) using a radiolabelled oligonucleotide containing the ICB2 and target sequence 5′-TACGAT-3′ were pre-incubated with increasing concentrations of polyamide 2 or 3 for 1 h at room temperature prior to addition of the NIH3T3 nuclear extract. 0, control reaction containing the oligonucleotide and nuclear extract without polyamide; C, reaction in the presence of an excess of unlabelled competitor oligonucleotide of the same sequence as the control reaction; M, reaction in the presence of an excess of unlabelled oligonucleotide with a mutation to the ICB2 motif. (A) Comparative analysis of the inhibitory effects of HxI*P 2 and HxIP* 3 on the binding of NF-Y containing protein complexes to the ICB2. (B) EMSA showing HxIP* 3 is able to displace NF-Y already bound to the ICB2 when the radiolabelled oligonucleotide is pre-incubated with the nuclear extract prior to the addition of the polyamide. (C) Supershift analysis using an anti-NF-YA antibody confirmed the presence of NF-Y in the protein complex bound to the radiolabelled oligonucleotide.

Figure 6

Visualisation of HxI*P 2 and HxIP* 3 nuclear localisation. A549 (top panels) and NIH3T3 (bottom panels) cells were treated with the indicated concentrations of HxI*P (left) or HxIP* (right) for 24 h, washed with PBS, and fixed with 2% paraformaldehyde (PFA). They were subsequently permeabilised and the nuclei were stained with propidium iodide (PI) before confocal microscopy imaging. The composite image presents the superimposed overlay of diamino Hx-polyamide fluorescence and the PI fluorescence. No polyamide fluorescent signal was detected in the control, untreated cells under the same observation settings.

Figure 7

Effect of the diamino HxIP derivatives on topo IIα expression in confluent A549 cells. (A) Quantification of topo IIα mRNA levels via RT-PCR analysis. Cells were maintained at confluency before 18 h treatment with increasing concentrations of HxI*P 2 or HxIP* 3. mRNA levels are compared relative to untreated confluent A549 cells (CON). Error bars represent the SEM from three biological replicates. Statistical significance was calculated using one-way ANOVA analysis (* p < 0.05). (B) Immunoblot analysis of confluent A549 nuclear extracts probed with topo IIα antibody following 18 h treatment with HxI*P 2 or HxIP* 3. Lamin is shown as a loading control.

Figure 8

Potentiation of topo II poison induced-cytotoxicity by HxIP*. A549 cells were pre-treated with the indicated concentrations of HxIP* 3 for 24 h and exposed to etoposide (50 μM) for 2 h. After 24 h in drug free medium, they were analysed for γH2AX levels by confocal microscopy and immunoblotting. (A) Representative images of A549 cells. (B) Number of H2AX foci per nuclei for the indicated treatment combinations, as quantified by CellProfiler Software. (C) Immunoblot analysis of the nuclear levels of γH2AX and topo IIα, 24 h after pre-treatment with HxIP* for 24 h, treatment with etoposide for 2 h and their combination. (D) Confluent A549 cells were treated with HxIP* (2 and 5 μM) or etoposide (400 μM) and HxIP*-etoposide combination, where 6 h etoposide exposure followed 24 h pre-treatment with HxIP*. Cell viability (%) was measured using the CellTiter-Glo® assay. Luminescence values are normalised to those of the untreated control. (E) Viability was also assessed after 6 h treatment with doxorubicin (75 μM) and HxIP*-doxorubicin combinations, where 6 h exposure followed 24 h HxIP* pre-treatment. All data are represented as mean ± SEM (n=3). Statistical significance was calculated using one-way ANOVA analysis (* p < 0.05, ** p < 0.01, *** p < 0.001).