Sensitization to radiation and alkylating agents by inhibitors of poly(ADP-ribose) polymerase is enhanced in cells deficient in DNA double-strand break repair

Abstract

As single agents, chemical inhibitors of poly(ADP-ribose) polymerase (PARP) are nontoxic and have clinical efficacy against BRCA1- and BRCA2-deficient tumors. PARP inhibitors also enhance the cytotoxicity of ionizing radiation and alkylating agents but will only improve clinical outcomes if tumor sensitization exceeds effects on normal tissues. It is unclear how tumor DNA repair proficiency affects the degree of sensitization. We have previously shown that the radiosensitizing effect of PARP inhibition requires DNA replication and will therefore affect rapidly proliferating tumors more than normal tissues. Because many tumors exhibit defective DNA repair, we investigated the impact of double-strand break (DSB) repair integrity on the sensitizing effects of the PARP inhibitor olaparib. Sensitization to ionizing radiation and the alkylating agent methylmethane sulfonate was enhanced in DSB repair-deficient cells. In Artemis(-/-) and ATM(-/-) mouse embryo fibroblasts, sensitization was replication dependent and associated with defective repair of replication-associated damage. Radiosensitization of Ligase IV(-/-) mouse embryo fibroblasts was independent of DNA replication and is explained by inhibition of "alternative" end joining. After methylmethane sulfonate treatment, PARP inhibition promoted replication-independent accumulation of DSB, repair of which required Ligase IV. Our findings predict that the sensitizing effects of PARP inhibitors will be more pronounced in rapidly dividing and/or DNA repair defective tumors than normal tissues and show their potential to enhance the therapeutic ratio achieved by conventional DNA-damaging agents.

Figure 1

A. Mean percentage of cells in G1, S phase and G2 phases of the cell cycle in exponential phase populations of MEFs. Cells fixed and stained with propidium iodide for flow cytometric analysis of DNA content. B. WT, ATM^−/−, Artemis^−/− and Ligase IV^−/−p53^−/− MEFs were pre-treated with 0.5 μM olaparib for 2 hours before treatment with 10 mM H₂O₂ (with or without 0.5 μM olaparib) for 10 min in the dark. Cells were stained for poly(ADP-ribose) (pAR) and counterstained with DAPI. Fluorescence intensity of pAR signal in each nucleus was quantified using NIH Image-J. Mean values of 100-200 cells +/− SD are presented. C. Clonogenic survival of exponential phase populations of WT, ATM^−/−, Artemis^−/− and Ligase IV^−/−p53^−/− MEFs exposed to olaparib at the indicated doses for 24 hours after plating. D. Clonogenic survival of WT, ATM^−/−, Artemis^−/− and Ligase IV^−/−p53^−/− MEFs continuously exposed to olaparib for the duration of the assay. To maintain levels of olaparib, medium was replaced every 48 hours. *p<0.01 (ATM^−/− compared with WT, Artemis^−/− and Ligase IV^−/−p53^−/− at each olaparib dose).

Figure 2

Clonogenic survival of exponential phase populations of WT, ATM^−/−, Artemis^−/− and Ligase IV^−/−p53^−/− MEFs treated with A. MMS for 1 hour at 37°C +/− 0.5 μM olaparib for 2 hours pre-, during and 21 hours post-treatment B. 250 kV X-rays +/− 0.5 μM olaparib for 2 hours pre- and 22 hours post-irradiation. C. Clonogenic survival curves derived from exponential HeLa cells exposed to 0.5 μM olaparib and/or 10 μM ATM inhibitor, KU55933 for 2 hours pre- and 22 hours post-irradiation.

Figure 3

A-D. Clonogenic survival curves derived from MEFs exposed to 0.5 μM olaparib and/or 5 μM aphidicolin (APH) for 1 hour pre- and 2 hours post-irradiation with 250 kV X-rays at the doses shown. A. WT MEFs, B. Artemis^−/− MEFs, C. ATM^−/− MEFs, D. Ligase IV^−/−p53^−/− MEFs.

Figure 4

A. Representative images of γH2AX immunofluorescence in S-phase WT or Artemis^−/− MEFs. Cells were exposed to 0.5 μM olaparib for one hour prior to treatment with MMS (0.1 mM, 1 hour) and until fixation at the timepoints shown. B. Quantitative analysis of γH2AX signal in S-phase WT or Artemis^−/− MEFs. Mean fluorescence intensity per nucleus +/− SEM from 3 experiments shown, at least 25 nuclei per timepoint. *p<0.05. C. Representative images of γH2AX and phospho-histone H3 immunofluorescence in G2-phase WT and Artemis^−/− MEFs 24 hours after MMS +/− olaparib treatment as in A. G2-phase cells were identified by speckled p-histone H3 staining. D. Quantitative analysis of γH2AX signal in G2-phase cells 24 hours after MMS treatment +/− olaparib. *p<0.05.

Figure 5

A. Quantitative analysis of γH2AX foci in G1 phase 48BR hTERT (wild type), CJ176 hTERT (Artemis deficient) and AT5-BIVA hTERT (ATM deficient) fibroblast cell lines exposed to 10 μM PJ-34 (PARP inhibitor) for one hour pre-irradiation (3 Gy, gamma rays), treated with 3 μM aphidicolin then fixed and co-stained for γH2AX and CENP-F. B. Quantitative analysis of γ-H2AX foci in G1 phase MEFs exposed to 10 μM PJ-34 for one hour pre-irradiation (3 Gy, gamma rays), fixed and stained for γ-H2AX and phospho-histone H3. G2 cells distinguished by phospho-histone H3 staining and S phase cells identified by background γ-H2AX staining were excluded. *p<0.05, **p<0.01 for comparison between cells treated or not with PARP inhibitor.

Figure 6

A. Quantitative analysis of γH2AX foci in G1 phase 48BR primary and 1BR hTERT fibroblasts exposed to 10 μM PJ-34 for one hour prior to treatment with MMS at the indicated doses. B-E. Quantitative analysis of γH2AX foci in 48BR (B), Ligase IV^−/−p53^−/− MEFs (C), CJ176 (Artemis deficient) (D) and HSC62 (BRCA2 deficient) (E) primary fibroblast cell lines and exposed to 10 μM PJ-34 for one hour prior to and during damage induction with 1 mM MMS and for the indicated repair periods. Cells were fixed and co-stained for γH2AX and CENP-F or phospho-histone H3. *p<0.05, **p<0.01, ***p<0.001 for comparison between cells treated or not with PARP inhibitor. Representative images are shown in Supplementary Figure S5.