PARP Inhibitors in Clinical Use Induce Genomic Instability in Normal Human Cells

<div><p>Poly(ADP-ribose) polymerases (PARPs) are the first proteins involved in cellular DNA repair pathways to be targeted by specific inhibitors for clinical benefit. Tumors harboring genetic defects in homologous recombination (HR), a DNA double-strand break (DSB) repair pathway, are hypersensitive to PARP inhibitors (PARPi). Early phase clinical trials with PARPi have been promising in patients with advanced BRCA1 or BRCA2-associated breast, ovary and prostate cancer and have led to limited approval for treatment of BRCA-deficient ovary cancer. Unlike HR-defective cells, HR-proficient cells manifest very low cytotoxicity when exposed to PARPi, although they mount a DNA damage response. However, the genotoxic effects on normal human cells when agents including PARPi disturb proficient cellular repair processes have not been substantially investigated. We quantified cytogenetic alterations of human cells, including primary lymphoid cells and non-tumorigenic and tumorigenic epithelial cell lines, exposed to PARPi at clinically relevant doses by both sister chromatid exchange (SCE) assays and chromosome spreading. As expected, both olaparib and veliparib effectively inhibited poly-ADP-ribosylation (PAR), and caused marked hypersensitivity in HR-deficient cells. Significant dose-dependent increases in SCEs were observed in normal and non-tumorigenic cells with minimal residual PAR activity. Clinically relevant doses of the FDA-approved olaparib led to a marked increase of SCEs (5-10-fold) and chromatid aberrations (2-6-fold). Furthermore, olaparib potentiated SCE induction by cisplatin in normal human cells. Our data have important implications for therapies with regard to sustained genotoxicity to normal cells. Genomic instability arising from PARPi warrants consideration, especially if these agents will be used in people with early stage cancers, in prevention strategies or for non-oncologic indications.</p></div

BRCA1- and BRCA2-deficient mES cells are hypersensitive to olaparib and veliparib but not BSI-201.

<p>The clonogenic survival assays of wild-type, <i>Brca1</i>^{-/- 236.44} and <i>Brca2</i>^{<i>lex1/lex2</i>} mES cells following treatment with olaparib, veliparib and BSI-201. BRCA1- and BRCA2-deficient cells were extremely sensitive to olaparib and veliparib as compared to wild-type cells. The concentration and arrows indicate the IC₅₀ for each cell line. Error bars depict mean with SD (n = 3).</p

Olaparib increases chromatid-type aberrations in repair-proficient human cells.

<p><b>A.</b> Representative chromatid-type aberrations in human cells include chromatid gap (a), chromatid break (b), radial chromosome (c and d) and telomere association (e). <b>B.</b> Olaparib-induced chromatid-type aberrations. Cells were exposed to vehicle or 1μM olaparib for 24 hrs. For each cell type, 100 metaphases were counted and the number of chromatid-type aberrations per metaphase was divided by chromosome number. Bars indicate the mean with SEM. The <i>P</i>-values were calculated using unpaired t-test.</p

SCE induction increases with dose and associates with inhibition of PARP activity.

<p>Induction of SCEs occurs with increasing dose of olaparib and veliparib but not BSI-201in MCF-10A (A) and EBV-BL (B) cells. Approximately 50 metaphases were counted per cell type. Fold increases of SCE per chromosome are shown compared to vehicle treated cells. Error bars depict mean with SD. One and two asterisks designate statistical significance compared with vehicle treated cells at <i>P</i> < 0.0001 and <i>P</i> < 0.001, respectively. The <i>P</i>-values were calculated using unpaired t-test. Dose response curves for cell survival are depicted on the left y-axis (solid circles) with corresponding SCE frequency on the right y-axis for MCF-10A (C) and EBV-BL (D) cells with olaparib, veliparib and BSI-201. Means with SD represent 3 survival assays per cell type. The IC₅₀ for olaparib, veliparib and BSI-201 were 4.7, 69.1 and 56.0 μM for MCF-10A, and 3.7, 42.5 and 48.9 μM for EBV-transformed B cells, respectively. <b>E.</b> Inhibition of cellular PARP activity in EBV-BL. Cells were incubated with or without increasing concentrations of olaparib, veliparib and BSI-201 24 hr before PARP activity was measured and values were normalized using protein concentration. Bars depict mean with SD of PARP activity for each concentration (μM) (n = 3). One asterisk designate statistical significance compared with untreated cells for paired <i>t</i>-test at <i>P</i> < 0.0001.</p

Olaparib increases the SCE frequency in repair-proficient human cells.

<p><b>A.</b> Cells were exposed with BrdU and 1μM olaparib during two cell cycle periods. Representatives of two metaphase spreads harvested from untreated (left) and olaparib treated (right) primary T cells 2. Arrows, e.g. site of SCE. <b>B.</b> Spontaneous and olaparib-induced SCEs by cell type. The y-axis is the number of SCE per chromosome for each metaphase counted. Approximately 50 metaphases were counted for each cell type. In order to compare each cell type, the number of SCEs per metaphase was divided by chromosome number. Fold increases of SCE per chromosome by olaparib for each cell type are shown. Error bars identify the mean with SD. Asterisks designate statistical significance at <i>P</i> < 0.0001 using unpaired t-test. <b>C.</b> Multiple SCEs per chromosome are observed following olaparib. Histograms show the percentage of chromosomes categorized by number of SCEs per chromosome by cell type and exposure. <b>D.</b> Temporal induction of SCEs by olaparib. (a) Spontaneous SCE, vehicle treated EBV-BL cells (90 hr—BrdU). (b) Acute olaparib-induced SCE (1μM olaparib and BrdU). (c) Olaparib-exposure (1μM) followed by removal and 2 cell cycle BrdU exposure. Fold increases of SCE per chromosome versus (a) of each exposure are shown. Error bars identify the mean with SD. Asterisks designate statistical significance at <i>P</i> < 0.0001 using unpaired t-test.</p

The high sensitivity of EC cell lines to cisplatin correlates with a reduced expression of ERCC1 but not XPF.

<p>A–B) Western blotting analysis of ERCC1 and XPF expression levels in five EC cell lines. U2OS, ERCC1-deficient 165TOR, XPF-deficient (XP2YO) and XP2YO-complemented (XP-F) cell lines were used as protein expression controls. β-tubulin was used as loading control. The U2OS lane comes from the same electrophoresis gel of the other cell lines. C–D) Densitometric analysis of ERCC1 and XPF expression. Results are presented as expression level respect to U2OS cell line, and normalized against the loading control (β-tubulin). Data are mean value ±s.d. of two independent experiments.</p

ECs are defective in HR repair.

<p>A) EC cells are defective in RAD51 foci assembly. The indicated cell lines were treated with a pulse of cisplatin (3.3 µM for 6 hs) and co-stained with anti-RAD51 (red) and anti-bromodeoxyuridine (BrdU, [green]) antibodies. Harrows points to representative BrdU-positive (S-phase) cells used for RAD51 quantification (see below). B) cisplatin induces a comparable damage in U2OS and EC cell lines. The indicated cell lines were treated as in A, co-stained with γH2AX (red) and BrdU (green) antibodies, and counterstained with DAPI (blue). Harrows points to representative BrdU-positive (S-phase) cells used for γH2AX quantification. C–D) quantification of the number of RAD51 (C) and γH2AX (D) foci, before and after cisplatin treatment. Data are mean value ±s.d. of two independent experiments. In C and D a minimum of 100 nuclei were counted for each cell line. Statistical analysis was performed using a paired two-tailed Student's <i>t</i>-test (P<0.05). E) 27x-1 and Tera-1 cell lines are defective in I-SceI-induced DSB,repair, by HR. Percentage of GFP+ cells measured by flow cytometry, 48 hs upon the transfection with both DR-GFP and I-SceI expression vectors (black bars, [DR-GFP+I-SceI]). Data were normalized against the transfection efficiency measured by transfecting a (constitutive) GFP-expressing vector (white bars [NZE CAG]). Data are mean value ± s.d. of three independent experiments. Statistical analysis was performed using a paired two-tail Student’s t-test (P<0.05). For more details see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051563#pone.0051563.s003" target="_blank">Fig. S3A</a>–B.</p

EC cell lines are sensitive to AZD2281 treatment.

<p>A) Colony assay of EC cells exposed over time to increasing doses of AZD2281. The somatic cell lines MCF10A was used as positive (resistant) control. The table shows the IC₅₀ value of AZD2281 for each cell line. Data are mean value ± s.d. of three (triplicates) independent experiments. B) AZD2281 treatment induces DNA damage. FACS analysis of γH2AX-positive cells upon AZD2281 treatment. The indicated cell lines were treated over time with the IC₅₀ dosage of AZD2281 and collected 6 hs up to 72 hs after initial treatment. Data are the mean value ± s.d. of two (triplicates) independent experiments. C–F) cell cycle profile of 27x-1 and NT2D1 cell lines following the treatment with AZD2281. The indicated cell lines were treated (or left untreated) as in B, and collected ad the indicated time points for cell cycle analysis. Data are the mean value ± s.d. of three independent experiments. G–H) cell cycle distribution of γH2AX-positive cells following AZD2281 treatment. Cells were treated as in B and collected, at the indicated time points, for the staining with the γH2AX antibody. Data are the mean value ± s.d. of three independent experiments.</p

Summary table indicating the IC₅₀ value (µM) for AZD2281 (left column) and cisplatin, in absence (mid-column) or in presence (right column) of AZD2281.

<p>Summary table indicating the IC₅₀ value (µM) for AZD2281 (left column) and cisplatin, in absence (mid-column) or in presence (right column) of AZD2281.</p

AZD2281 treatment enhances EC cell lines response to cisplatin.

<p>A) Colony assay. The indicated cell lines were treated with ½ of the IC₅₀ dose of AZD2281 for 14 days, in absence (open symbols) or in presence (filled symbols) of cisplatin. In the latter case cisplatin was given for 6 hs, in presence of AZD2281 (½ of the IC₅₀ dose). After initial treatment cisplatin was washed out and cells cultured for 14 days in presence of AZD2281. Data are mean value ±s.d. of three (triplicates) independent experiments. B) AZD2281 reduces the ability of EC cell lines to overcome cisplatin-induced damage. The indicated cell lines were either treated in continuous with the ½ of the IC₅₀ dose of AZD2281 (dashed lines) or with AZD2281 plus the IC₅₀ dose of cisplatin (non-dashed lines) as described in A) for up to 72 hs. At each indicated time point cells were harvested, and stained with the anti-γH2AX antibody for FACS analysis. Data are mean value ±s.d. of two independent experiments. C) AZD2281 enhances EC apoptotic response. The indicated cell lines were treated either with AZD2281 (½ of the IC₅₀ dose, white bar) or cispaltin (IC₅₀ dose, grey bar) or with combined therapy (black bar) as described in A, for up to 72 hs, and collected for FACS analysis of the sub-G1 fraction. Data are mean value ±s.d. of tree to four independent experiments.</p