Poly(ADP-Ribose) Polymerase Inhibitor Induces Accelerated Senescence in Irradiated Breast Cancer Cells and Tumors

A 53BP1 IRIF binding domain GFP reporter reveals IR dose–dependent foci persistence in living cells

γH2AX foci and 53BP1 localization to IRIF can serve as proxies for unrepaired DSBs and the DNA damage response (8). The functional elements of the 53BP1 IRIF binding domain are a dimerizing domain, paired Tudor domains that recognize the stable histone marks H4-diMeK20 and/or H3-diMeK79, and a nuclear localization signal (10, 11). Cells lacking PARP activity display a delay in H2AX phosphorylation and persistence of γH2AX foci (12). 53BP1 binding at IRIF is partly dependent on H2AX phosphorylation and chromatin remodeling, also influenced by PARP activity. Thus, to examine PARP inhibitor effects on IRIF kinetics in living cells, we placed GFP fused to the 53BP1 IRIF binding domain (10) under tetracycline-inducible control (GFP-IBD; Supplementary Fig. S1) in a lentiviral vector. We transduced MCF7 Tet-On Advanced (MCF7^Tet-On, Clontech), which is a cell line derived from MCF7, a p53-positive, caspase-3–negative, and apoptosis-resistant human breast cancer–derived cell line that stably expresses the Tet-On Advanced transactivator.

Following induction with doxycycline, unirradiated MCF7^Tet-On cells expressing inducible GFP-IBD (MCF7^Tet-On GFP-IBD) displayed pan-nuclear fluorescence, with only rare nuclear foci (mean, 0.4 ± 0.7 per cell). Consistent with previous reports, the GFP-IBD reporter relocalized within minutes after IR to form nuclear foci that colocalized with γH2AX, endogenous 53BP1, and MDC1 proteins (Supplementary Fig. S2). The GFP-IBD foci then slowly resolved over the next 24 hours. The ATM kinase inhibitors KU-55933 and CGK733 decreased GFP-IBD foci formation (data not shown). In turn, shRNA knockdown of proteins required for 53BP1 relocalization to IRIF, including ATM, MDC1, and RNF8, blocked the formation of GFP-IBD foci after IR (Fig. 1A). Significantly, knockdown of endogenous 53BP1 increased the number of GFP-IBD foci in unirradiated cells and slowed their resolution after IR, indicating that 53BP1 remains active in MCF7^Tet-On GFP-IBD cells.

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Figure 1.

GFP-IBD reporter reveals IRIF formation and kinetics. A, shRNA knockdown of the upstream DNA damage response protein ATM, MDC1, or RNF8 abrogates GFP-IBD reporter relocalization to IRIF. shRNA knockdown of endogenous 53BP1 increases foci numbers at 3 and 24 h. Bar, 10 μm. B, the formation and resolution of GFP-IBD foci in response to IR depend on time and dose. Bar, 10 μm. C, points, mean number of GFP-IBD foci per cell (n > 50) at increasing IR doses evaluated at 3 h (solid circles) and 24 h (solid squares); bars, SD. D, clonogenic survival of MCF7^Tet-On GFP-IBD cells treated with increasing doses of IR. Points, mean (n = 3); bars, SD. Clonogenicity is modeled as distinct regimens of lower lethality from 0 to 6 Gy [percent survival = 330 × e^{−0.89 × (dose in Gy)}, R² = 0.958] and higher lethality from 6 to 12 Gy [percent survival = 20.0 × e^{−1.99 × (dose in Gy)}, R² = 0.999]. Clonogenic efficiency of untreated MCF7^Tet-On GFP-IBD cells represents 100% control.

We examined IRIF formation and resolution in relation to IR dose and time in MCF7^Tet-On GFP-IBD cells in vitro (Fig. 1B and C). Most GFP-IBD foci resolved by 24 hours at doses up to 8 Gy. For 2 Gy, the mean of 47 ± 13 IRIF at 3 hours decreased to 3.2 ± 1.7 at 24 hours, whereas for 8 Gy, the mean of 55 ± 15 IRIF at 3 hours decreased to 12 ± 6 at 24 hours. After 12 Gy, the mean of 53 ± 14 IRIF at 3 hours decreased only to 37 ± 18 at 24 hours. The increased IRIF persistence with higher IR dose suggests saturation of repair capacity or other damage responses. Indeed, doses above 6 Gy had greater effects on clonogenicity, a likely consequence of persistent DNA damage (Fig. 1D).

PARP1 inhibitor ABT-888 markedly enhances IRIF persistence, suppressing cell proliferation

Treating MCF7^Tet-On GFP-IBD cells with IR in the presence of the PARP1 inhibitor ABT-888 (veliparib; 2-[(R)-2-methylpyrrolidin-2-yl]-1H-benzimidazole-4-carboxamide; ref. 13) prevented PARP activation (Supplementary Fig. S3) and markedly increased residual IRIF at 3 and 24 hours (Fig. 2A and B). Neither the GFP-IBD reporter nor ABT-888 seemed to alter γH2AX localization or the recruitment of MDC1 and endogenous 53BP1 to IRIF (Fig. 2B). Time-lapse live-cell imaging of GFP-IBD revealed that in cells treated with 6 Gy, IRIF appeared within 15 minutes and began to decrease noticeably by 60 minutes (Fig. 2C; Supplementary Fig. S4). However, after 6 Gy + ABT-888, IRIF continued to appear up to 60 minutes, perhaps via conversion of single-strand breaks to DSBs (7), but remained largely unchanged thereafter. Previous data suggest that the growth of IRIF might maintain DNA damage signaling from unrepaired DSBs (14). Whereas the mean size of IRIF formed in cells treated with ABT-888 was clearly smaller (Supplementary Fig. S5), the total volume of IRIF per cell at 24 hours with IR + ABT-888 (187 ± 11/μm³) was significantly greater than that with IR alone (87 ± 10/μm³; P = 0.005, t test).

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Figure 2.

PARP1 inhibitor ABT-888 (veliparib) alters IRIF dynamics and suppresses cell proliferation. A, ABT-888 increased the number of residual IRIF 24 h after IR. Cells were pretreated with DMSO (control) or 10 μmol/L ABT-888 (inset) for 30 min before IR. Live-cell images shown at 3 and 24 h. Bar, 5 μm. B, immunofluorescence reveals colocalization of GFP-IBD with γH2AX, MDC1, and endogenous full-length 53BP1 at IRIF in cells treated with 6 Gy ± ABT-888. Nuclei indicated by 4′,6-diamidino-2-phenylindole (DAPI) staining (blue). Bar, 10 μm. C, time-lapse live-cell imaging of GFP-IBD localization in MCF7^Tet-On GFP-IBD cells treated with 6 Gy ± ABT-888. Bar, 10 μm. D, ABT-888 suppresses the growth of irradiated MCF7^Tet-On GFP-IBD cells. Cells were treated as shown, fixed at 10 d, and stained with crystal violet.

ABT-888 alone slightly decreased colony formation at 10 μmol/L (100 ± 1% for control versus 88 ± 0.6% for ABT-888), but significantly reduced colony formation following 2 Gy (29.7 ± 1.5% for IR alone versus 11.3 ± 0.6% for IR + ABT-888; P < 0.001, t test), with similar fold reductions at each IR dose up to 6 Gy. We next examined the potential mechanisms of growth suppression after IR + ABT-888. PARP inhibition did not dramatically affect MCF7^Tet-On GFP-IBD cell death after IR. Even 7 days later, few cells exhibited propidium iodide permeability, suggesting that PARP inhibition might induce MCF7^Tet-On GFP-IBD cell cycle arrest rather than apoptosis or necrosis. This is consistent with the previous observation that inhibition of ADP-ribosylation could block apoptosis, and a transient burst of PARP activity was required for apoptosis (15). Indeed, whereas ABT-888 alone did not appreciably decrease proliferating cells at 24 hours (58 ± 1% BrdUrd⁺ for control versus 56 ± 1% for ABT-888; not shown), the antiproliferative effects of 3 Gy (41 ± 1%) were enhanced by ABT-888 (27 ± 1%). In turn, MCF7^Tet-On GFP-IBD cells treated with 3 or 6 Gy alone showed a higher recovery of proliferative capacity compared with IR + ABT-888 (Fig. 2D).

ABT-888 accelerates senescence in irradiated MCF7^Tet-On GFP-IBD cells in vitro

Unrepaired DNA damage can promote accelerated or premature senescence, even in cells with otherwise unlimited proliferative capacity (16–18). Accelerated senescence following IR has been observed in MCF7^Tet-On GFP-IBD cells both in vitro and in vivo (19, 20). At 4 days after IR + ABT-888, cells displaying persistent GFP-IBD foci began to exhibit a morphology characteristic of senescence. At 7 days, surviving cells remained adherent, became enlarged with a flat morphology, and displayed multiple nuclear GFP-IBD foci (Fig. 3A). We investigated other hallmarks of accelerated senescence (16, 17), including SA-βGal staining (Fig. 3B) and increased expression of the cyclin-dependent kinase inhibitor p21^Cip1/WAF1 (Fig. 3C). After 6 Gy + ABT-888, 76 ± 4% of surviving cells showed SA-βGal staining compared with 1.2 ± 1.0% for ABT-888 and 2.5 ± 2.0% for 6 Gy (P < 0.001), and p21^Cip1/WAF1 gene expression was significantly upregulated following IR + ABT-888 compared with IR alone (P < 0.02, t test). Immunocytochemistry suggested that the accumulation of p21^Cip1/WAF1 was greatest in cells with persistent IRIF (Fig. 3D). Accelerated senescence following IR and ABT-888 treatment was not limited to cells with wild-type p53, as we observed the same phenotype in MCF7^Tet-On GFP-IBD cells treated with p53 inhibitor pifithrin (data not shown), as well as in breast and other cancer cell lines with mutations in p53 (Supplementary Fig. S6).

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Figure 3.

ABT-888 induces accelerated senescence in irradiated MCF7^Tet-On GFP-IBD cells. A, MCF7^Tet-On GFP-IBD cells treated with IR + ABT-888 display persistent IRIF and develop senescent morphology. Bar, 20 μm. B, IR + ABT-888 increases SA-βGal activity. Cells were treated with 6 Gy ± ABT-888, fixed at 7 d, and stained. Bar, 20 μm. C, columns, mean relative p21^Cip1/WAF1 transcript levels after IR ± ABT-888 determined by quantitative PCR; bars, SD. White columns, IR; gray columns, IR + ABT-888. D, immunofluorescence staining for p21^Cip1/WAF1 at 4 d after 3 Gy ± ABT-888. Bar, 20 μm.

ABT-888 accelerates the senescence and suppresses the growth of irradiated MCF7^Tet-On GFP-IBD tumors

To visualize IRIF in vivo, MCF7^Tet-On GFP-IBD cells were injected into nude mice to form xenograft tumors. Imaging of GFP-IBD by two-photon microscopy revealed that the kinetics of IRIF formation and resolution in tumors were comparable to that observed in MCF7^Tet-On GFP-IBD cells in vitro (Fig. 4A). When mice were treated with ABT-888 twice daily for 2 days before IR and then twice daily thereafter, we observed no increase in IRIF number at early time points but the number of cells with residual IRIF increased at 24 hours. Twenty-two percent of tumor cells treated with 3 Gy exhibited ≥4 IRIF/cell whereas 42% of tumor cells with 3 Gy + ABT-888 had ≥4 IRIF/cell (P < 0.001, t test; Fig. 4B). To evaluate DNA damage–induced senescence in vivo, we examined SA-βGal staining in frozen tumor sections at 7 days (Fig. 4C). ABT-888 alone slightly enhanced SA-βGal staining above background but markedly increased staining when combined with IR. To compare in vivo growth delay with that observed in vitro, we performed a tumor regrowth experiment. Mice bearing MCF7^Tet-On GFP-IBD tumors were treated with ABT-888 for 2 days before a single 6-Gy dose and then only for 2 days after IR. This short course of PARP inhibition significantly slowed MCF7^Tet-On GFP-IBD tumor regrowth compared with 6 Gy alone [day 9, P = 0.021; day 12, P = 0.013; day 14, P = 0.001 (t test); Fig. 4D].

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Figure 4.

IR + ABT-888 induces persistent IRIF and senescence in vivo and suppresses MCF7^Tet-On GFP-IBD tumor regrowth. A, dose response of IRIF formation in xenograft tumor cells 24 h after IR. Bar, 10 μm. B, IR + ABT-888 increases residual IRIF compared with IR alone. Intravital imaging of GFP-IBD foci in tumors at 3 and 24 h after 3 Gy ± ABT-888. Bar, 10 μm. C, SA-βGal activity in tumors treated with IR ± ABT-888. Frozen sections of excised tumors 7 d after IR were fixed and stained. Bar, 20 μm. D, ABT-888 + IR suppresses tumor regrowth. Tumor growth was significantly delayed after 6 Gy + ABT-888 (solid squares) compared with 6 Gy (solid circles). Points, mean fractional volume (V/V_o); bars, SEM (n = 4 per group).

Our data confirm previously reported enhancement of IR effects by PARP inhibition (6, 11) and implicate IRIF persistence as a potential mechanism of accelerated tumor cell senescence. Persistent cell cycle arrest and accelerated senescence are ascribed to accumulation of unrepaired DNA damage and chromatin perturbation, among other inducers (17, 18). We speculate that the efficacy of PARP inhibitors toward homologous recombination–deficient BRCA1-, BRCA2-, or PTEN- negative cancer cells (21) may similarly reflect a cellular response to accumulation of unrepaired endogenous DNA damage. Indeed, preliminary analysis of the PTEN mutant cell line PC-3 suggests that ABT-888 accelerates senescence, particularly in combination with radiation. Whereas it remains a dogma that IR and genotoxic agents mediate their lethal effects via enhanced apoptosis, necrosis, or mitotic catastrophe, senescence is an alternative terminal phenotype that may be highly relevant as a determinant of outcomes for cancer treatment (16, 22, 23). Alone or in combination with other epigenetic drugs such as histone deacetylase inhibitors that promote IRIF persistence and accelerated senescence (24), PARP inhibitors may have a significant effect by inducing senescence as a novel mechanism for sensitization to radiation and chemotherapy.