DNA Repair Interference with Sequence Specific DNA Binding Pyrrole Imidazole Polyamides; a Novel Radiation Sensitizer

Abstract

Pyrrole (Py)—Imidazole (Im) polyamides are a class of non-genotoxic DNA-binding small molecules with modular sequence recognition and high affinity. A polyamide targeted to the Androgen Receptor (AR) Response Element (ARE) half-site, suppresses AR positive prostate tumor xenografts. It is hypothesized that polyamides may displace DNA repair elements recruited after genotoxic insult and thereby sensitize cells to ionizing radiation. DNA double strand repair was assessed in a cell-free system using PC3 cell lysate, and a linearized pGEM3Z plasmid containing an insert with a BamH1 restriction site flanked by polyamide binding site motifs, and analyzed by qPCR. Rejoining of the linearized plasmid was reduced by 85 % and inhibited rejoining by DNA ligase compared to untreated controls. Neutral comet assay revealed no genomic fragmentation after 48 h of 10 µM polyamide treatment. Genomic fragmentation was statistically similar 1 h post radiation with 10 Gy, +/− polyamide. However, at 24 h post radiation, cells pretreated with 10 µM polyamide had persistently elevated tail lengths (181 vs. 129 p < 0.001) and tail moment (103 vs 74 p < 0.001). After radiation, greater than 95 % of cells +/− 10 µM polyamide pretreatment demonstrated increased γ-H2AX phosphorylation. 24 h post RT, 32% of polyamide treated cells continued to have elevated γ-H2AX phosphorylation, whereas only 19% of untreated cells continued to have elevated γ-H2AX phosphorylation. Polyamide treatment with 10 mM resulted in a sensitization enhancement ratio of 2.48 at 4 Gy by clonogenic assay. In conclusion Py-Im polyamides are non-genotoxic and sequence specific; potentially offering a strategy for sequence-directed interference with DNA repair and other DNA-depended processes.

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