Radiosensitization by a Pyrrole-Imidazole Polyamide Through Interference with DNA Repair

Abstract

Non-genotoxic small molecules that potentiate the effects of ionizing radiation in malignant cells could be used to augment radiotherapy for solid tumors. Pyrrole-imidazole (Py–Im) polyamides are synthetic non-genotoxic DNA minor groove-binding small molecules with programmable sequence specificity; binding to DNA induces allosteric changes that can interfere with protein-DNA interactions. We hypothesized that Py–Im polyamides can modulate the cellular response to ionizing radiation. The LNCaP and VCaP cell lines were used as model systems. Double (phosphorylated γ-H2AX) and single (XRCC1) DNA strand break foci were quantified by immunofluorescence. Cell viability was measured by xCELLIGence and PrestoBlue assays. Genomic fragmentation was measured by alkaline single cell gel electrophoresis. Gene expression changes were determined by RNA-sequencing. DNA ligase activity was assayed in vitro by gel-shift assay. A cell line expressing siRNA against DNA ligase 3 was used to determine if deficiency in this protein in vivo could potentiate cytotoxicity by a polyamide. Pre-treatment of cells with a Py-Im polyamide prior to exposure to ionizing radiation resulted in a delay in resolution of phosphorylated γ-H2AX foci, increase in XRCC1 foci, and reduced cellular replication potential. Polyamide treatment induced genes related to the ultraviolet radiation response. We observed that the polyamide is almost 10-fold more toxic to a cell line deficient in DNA ligase 3 as compared to the parental cell line. Alkaline single cell gel electrophoresis reveals that the polyamide induces genomic fragmentation in the ligase 3 deficient cell line but not the corresponding parental line. The polyamide interferes sequence-dependently with DNA ligation in vitro . These data support a model where a polyamide may interfere with repair of DNA strand breaks after ionizing radiation. We conclude that Py-Im polyamides may be further explored as sensitizers to genotoxic therapies.