The PARP-1 Inhibitor Olaparib Causes Retention of γ-H2AX Foci in <i>BRCA</i>1 Heterozygote Cells Following Exposure to Gamma Radiation

Abstract

A novel treatment for cancer patients with homozygous deletions of BRCA 1 and BRCA 2 is to use drugs that inhibit the enzyme poly(ADP-ribose) polymerase (PARP). Specific inhibition of PARP-1 can induce synthetic lethality in irradiated cancer cells while theoretically leaving normal tissue unaffected. We recently demonstrated in a cell survival assay that lymphoblastoid cells with mono-allelic mutations of BRCA 1 were hypersensitive to gamma radiation in the presence of the PARP-1 inhibitor Olaparib compared to normal cells and mono-allelic BRCA 2 cells. To determine if the enhanced radiation sensitivity was due to a persistence of DNA strand breaks, we performed γ -H2AX foci analysis in cells derived from two normal individuals, three heterozygous BRCA 1 and three heterozygous BRCA 2 cell lines. Cells were exposed to 2 Gy gamma radiation in the presence or absence of 5 μM Olaparib. Using immunofluorescence and imaging flow cytometry, foci were measured in untreated cells and at 0.5, 3, 5 and 24 hours post-irradiation. In all lymphoblastoid cells treated with 2 Gy gamma radiation, there was a predictable induction of DNA strand breaks, with a modest but significant retention of foci over 24 hours in irradiated cells treated with Olaparib (ANOVA P < 0.05). However , in mono-allelic BRCA 1 cells , there was a failure to fully repair DNA double-strand breaks (DSB) in the presence of Olaparib, evidenced by a significant retention of foci at 24 hours ’ post irradiation ( t -Test P < 0.05). These data show that the cellular hypersensitivity of mono-allelic BRCA 1 lymphoblastoid cells to gamma radiation in the presence of the Olaparib is due to the retention of DNA DSB. These data may indicate that patients with inherited mutations in the BRCA 1 gene treated with radiotherapy and PARP-1 inhibitors may experience elevated radiation-associated normal tissue toxicity.