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 BRCA1 and BRCA2 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 BRCA1 were hypersensitive to gamma radiation in the presence of the PARP-1 inhibitor Olaparib compared to normal cells and mono-allelic BRCA2 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 BRCA1 and three heterozygous BRCA2 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 BRCA1 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 BRCA1 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 BRCA1 gene treated with radiotherapy and PARP-1 inhibitors may experience elevated radiation-associated normal tissue toxicity.

Effects of Combined Treatment with Ionizing Radiation and the PARP Inhibitor Olaparib in BRCA Mutant and Wild Type Patient-Derived Pancreatic Cancer Xenografts

“…Previous studies have suggested that treatment with PARP inhibitors in combination with DNA damaging agents such as cisplatin or IR can result in the accumulation of DSB and γH2AX foci [19, 20]. In the BRCA wild type model OCIP23, treatment with both olaparib and IR as single agents resulted in the accumulation of γH2AX foci (AZD-2281 p = 0.029, IR p = 0.029) when compared to untreated control tumors.…”

Embracing synthetic lethality of novel anticancer therapies

“…The reduction in the BRCA2 levels by its negative regulators was dependent on the mitochondrial stress-induced elevated cytosolic Ca+ levels. Bourton and co-workers have showed that gamma radiation increased the g-H2AX foci (a histone protein) by imaging flow cytometry in normal and BRCA1-mutated lymphoblastoid cells [39]. Combination therapy with gamma radiation and olaparib was conducted in both normal as well as BRCA1-mutated cancer cells and g-H2AX foci were retained in both.…”

Radiosensitivity of Human Breast Cancer Cell Lines Expressing the Breast Tumor Kinase (Brk)

“…This is exemplified in cells derived from individuals with inherited mutations in genes controlling the recognition and repair of DNA double strand break repair. For example in A-T cells there is a deficiency in the repair of ionising radiation induced DSB shown by a persistence of γ−Η2ΑΞ foci in the nuclei of cells when compared to normal cells [21]. Based upon these observations, we might expect that the breast cancer cells lines in this study which are sensitive to radiation such as the MDA-MB-157 Brk-KM and vector cell lines (when compared to the Brk-WT derivative), may exhibit a defect in the repair of DSB in the foci assay.…”

“…Image compensation has been described in detail previously elsewhere [20,21]. In brief, cells that had been fixed 30 minutes postirradiation where γ−Η2ΑΞ staining or ATM protein staining intensity was likely to be highest were used to capture images of cells with the 488 nm laser only and without brightfield illumination.…”

“…Imaging flow cytometry was carried out using the ImagestreamX system (Amnis Inc., Seattle, Washington, USA) as described previously [20,21]. Images of 10,000 cells were captured on channel 1 for brightfield (BF); on channel 2 for Alexa Fluor ® 488 (AF), depicting staining of γ-H2AX foci or ATM protein localisation; on channel 5 for DRAQ5™ (D5), depicting staining of the nucleus.…”