Inhibition of poly(ADP-ribose) polymerase down-regulates BRCA1 and RAD51 in a pathway mediated by E2F4 and p130

Abstract

Inhibitors of poly(ADP-ribose) polymerase (PARP) are in clinical trials for cancer therapy, on the basis of the role of PARP in recruitment of base excision repair (BER) factors to sites of DNA damage. Here we show that PARP inhibition to block BER is toxic to hypoxic cancer cells, in which homology-dependent repair (HDR) is known to be down-regulated. However, we also report the unexpected finding that disruption of PARP, itself, either via chemical PARP inhibitors or siRNAs targeted to PARP-1, can inhibit HDR by suppressing expression of BRCA1 and RAD51, key factors in HDR of DNA breaks. Mechanistically, PARP inhibition was found to cause increased occupancy of the BRCA1 and RAD51 promoters by repressive E2F4/p130 complexes, a pathway prevented by expression of HPV E7, which disrupts p130 activity, or by siRNAs to knock down p130 expression. Functionally, disruption of p130 by E7 expression or by siRNA knockdown also reverses the cytotoxicity and radiosensitivity associated with PARP inhibition, suggesting that the down-regulation of BRCA1 and RAD51 is central to these effects. Direct measurement of HDR using a GFP-based assay demonstrates reduced HDR in cells treated with PARP inhibitors. This work identifies a mechanism by which PARP regulates DNA repair and suggests new strategies for combination cancer therapies.

Fig. 1.

Cytotoxicity of PARP inhibition in hypoxic and normoxic cancer cells. (A) Survival by colony formation of RKO colon cancer cells and A549 lung cancer cells under either hypoxic or normoxic conditions to increasing doses of the PARP inhibitors PHEN [6(5H)-phenanthridinone] and ANI (4-amino-1,8-naphthalimide). (B) Survival of RKO, A549, and H460 cells grown under hypoxic or normoxic conditions and exposed to the PARP inhibitors KU00 (100 μM), ANI (200 μM), BZD (5 mM), PHEN (200 μM), 3AB (20 mM), or ABT (4 μM). (C) Effect of E7 expression on survival of RKO cells treated with PHEN. Cells expressing E7 or not were placed in hypoxia or normoxia and treated with PHEN for 48 h in normoxia, followed by growth to allow colony formation. Error bars represent SEs based on three replicates in all cases.

Fig. 2.

Inhibition or knockdown of PARP causes decreased levels of BRCA1 protein and BRCA1 mRNA in human cells. (A) A549 or H460 cells were exposed to the PARP inhibitors PHEN or ANI and harvested at 72 h for analysis of BRCA1 levels by immunoblot. (B) MCF7 and U2OS cells treated with ANI, PHEN, or ABT were analyzed for BRCA1 expression. (C) Analysis of BRCA1 mRNA levels by quantitative real-time RT-PCR in A549 cells after exposure to PARP inhibitors. (D) MCF7 cells were transfected with siRNAs targeting either PARP-1 or GAPDH or with a negative control siRNA pool. After 72 h cells were harvested for immunoblot analysis of the indicated proteins.

Fig. 3.

PARP inhibition or knockdown suppresses RAD51 expression. (A) A549, H460, or U2OS cells were treated with PARP inhibitors and harvested at 72 h for analysis of RAD51 levels by immunoblot. (B) Analysis of RAD51 mRNA levels by quantitative real-time RT-PCR in A549 cells after PARP inhibition. (C) A549 cells were transfected with siRNAs targeting either PARP-1 or GAPDH or with a negative control siRNA pool. After 72 h cells were harvested for immunoblot analyses of the indicated proteins. (D) A549 cells were exposed to either hypoxia or PHEN treatment, or both, as indicated, and analyzed for BRCA1 and RAD51.

Fig. 4.

PARP inhibitor–mediated suppression of BRCA1 and RAD51 is reversed by E7 expression or p130 knockdown, occurs via E2F4 and p130 occupancy of the respective promoters, and contributes to the cytotoxicity of PARP inhibition. (A and B) RKO cells expressing either E7 or a vector control (Neo) were treated or not with 200 μM PHEN and assayed by immunoblot for (A) BRCA1 or (B) RAD51 expression. ChIP assays of (C) BRCA1 or (D) RAD51 promoter occupancy were performed using antibodies to the indicated factors with lysates from A549 cells treated or not with 200 μM PHEN. Representative agarose gels containing BRCA1 or RAD51 promoter region PCR amplification products are shown. (E) Quantification by real-time PCR of BRCA1 or (F) RAD51 promoter occupancy by the indicated factors is shown, based on three independent ChIP assays, with error bars based on SEs. Promoter occupancy is expressed as the fold change relative to that observed in untreated cells. (G) BRCA1 and RAD51 levels were determined by Western blot in A549 cells that were treated or not with PHEN after transfection with either siRNAs targeting p130, GAPDH, nontargeting negative control pool, or no siRNAs (Mock). Note that the order of the lanes in the third panel is different from in the first and second panels. (H) Effect of p130 knockdown by siRNAs on survival of A549 cells treated with PHEN. Cells transfected or not with siRNAs targeting p130, as indicated, were placed in hypoxia or normoxia and then treated with increasing doses of PHEN for 72 h under normoxic conditions, followed by growth to allow colony formation. Error bars represent SEs based on three replicates. (I) Effect of forced BRCA1 or RAD51 expression on survival of MCF7 cells treated with PHEN. Cells transfected with cDNA expression vectors for BRCA1 or RAD51, or an empty vector control, were treated with increasing doses of PHEN for 72 h under normoxic conditions, followed by growth to allow colony formation. Error bars as above.

Fig. 5.

PARP inhibition stimulates interaction between p130 and E2F4, whereas PARP-1 binds to the BRCA1 promoter and physically interacts with E2F1. (A) Analysis of the association between p130 and E2F4 by coimmunoprecipitation using an E2F4 antibody in extracts from A549 cells treated or not with PHEN, followed by immunoblot with antibodies to either p130 or E2F4. (B) Western blot analysis of phosphorylation status of p130, as judged by gel mobility, after treatment or not with 200 μM PHEN for 72 h. (C) ChIP assay of BRCA1 promoter occupancy by PARP-1 in A549 cells treated or not with 200 μM PHEN. (D) Analysis of the association between PARP-1 and E2F1 by coimmunoprecipitation using an antibody specific to E2F1 in extracts from MCF7 cells treated or not with PHEN, followed by immunoblot with an antibody to PARP-1. (E) Immunoblot analysis of expression levels in A549 cells of p130, E2F1, and E2F4 after treatment or not with PHEN.

Fig. 6.

PARP inhibitor treatment sensitizes cells to ionizing radiation in a pathway dependent on p130 and suppresses HDR of DSBs. (A) Survival of A549 cells exposed to ionizing radiation. Cells were pretreated or not with PHEN at 200 μM for 48 h. (B) Radiosensitization by PARP inhibition is partially reversed by E7 expression. Survival of RKO cells with or without E7 expression to 7.5 Gy of ionizing radiation. Cells were pretreated or not with PHEN (200 μM for 48 h). (C) Radiosensitization by PARP inhibition is partially reversed by siRNA knockdown of p130. Cells transfected or not (Mock) with siRNAs targeting p130 were treated or not with PHEN for 48 h and exposed to ionizing radiation, followed by growth to allow colony formation. Error bars represent SEs based on three replicates. (D) Survival of A549 cells pretreated or not with PHEN (200 μM for 48 h) or pretreated with exposure to hypoxia (0.1% for 48 h), followed by treatment with PHEN (100 μM for 24 h), as indicated. Error bars as above. (E) Impaired HDR in MCF7 cells after treatment with PHEN, as detected using the DR-GFP recombination substrate. MCF7 cells containing the DR-GFP substrate were exposed or not to PHEN for 72 h, followed by transfection with either an I-SceI expression vector (+SceI) to induce a site-specific DSB or with an empty vector (-SceI) as a control. Cells were assayed 72 h later by FACS for HDR of the I-SceI-induced DSB, as indicated by production of GFP+ cells. Percentages of GFP+ cells are shown in each panel. (F) PARP inhibition produces radiosensitization even in cells already defective in BER. Mouse embryonic fibroblasts expressing wild-type or a dominant negative variant (E295K) of DNA polymerase β were treated or not with PHEN for 48 h and then exposed to 5 Gy of ionizing radiation. Survival was determined by colony formation, with SEs calculated on the basis of three replicates.