Pyrrole (P)-Imidazole (I) containing polyamides are small synthetic molecules which can target predetermined DNA sequences with high affinity and modulate gene expression by interfering with the binding of transcription factors to DNA. As a model system we have previously used the inverted CCAAT box 2 (ICB2) of the topoisomerase IIα promoter and shown that targeted polyamides inhibit the binding of the transcription factor NF-Y and induce expression of topo IIα in confluent cancer cells. Critical to this approach is the need for low molecular weight polyamides that readily enter in the nucleus. In this study, the fluorophore p-anisylbenzimidazolecarboxamido (Hx) moiety was rationally designed to mimic the recognition of A/T base pairs by two consecutive pyrrole units. The hybrid polyamide HxIP is the first example of a small fluorescent molecule that fluoresces significantly more intensely upon binding to its target sequence, 5′-TACGAT-3′ of the 5′-flank of ICB2. Sequence specificity was confirmed by DNAse I footprinting, with HxIP binding with increased affinity compared to the corresponding triamides/tetraamides (f-PIP/PPIP). Thermal denaturation, circular dichroism, and Surface Plasmon Resonance studies confirmed the sequence specificity of HxIP. A dose-dependent inhibition of protein binding to ICB2 by HxIP with complete abolition at concentrations >3 μM was seen in an Electrophoretic Mobility Shift Assay (EMSA). A supershift assay confirmed NF-Y binding to the target sequence. HxIP was also able to displace bound protein factors from DNA at the same concentrations. The incorporation of Hx in the hybrid molecule provides an intrinsic probe to directly monitor cellular uptake and migration into the nucleus. Confocal microscopy in both fixed and live NIH3T3 and A549 cells showed that HxIP is readily taken up by cells and quickly localises in the cell nucleus within 5 minutes. Exposure of confluent cells to 20-40 μM HxIP resulted in time-dependent upregulation of topo IIα expression, reaching levels comparable to those of proliferating cells within 24h as shown by immunoblotting analysis. Confluence-associated repression of topo IIα expression contributes to cellular resistance to agents such as etoposide. De-repression of topo IIα by pre-incubation with HxIP was shown to resensitise NIH3T3 cells to the cytotoxic effect of etoposide (MTT assay) and enhance its DNA damaging effects by increasing levels of etoposide-induced DNA strand breaks as assessed by the single cell gel electrophoresis (comet) assay and the phosphorylation of H2AX. Subcellular localisation of HxIP and potential synergies with etoposide treatment is currently being investigated in vivo against human tumour xenografts.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3521.