RNF8 promotes high linear energy transfer carbon-ion-induced DNA double-stranded break repair in serum-starved human cells.

The cell-killing effect of radiotherapy largely depends on unrepaired DNA double-stranded breaks (DSBs) or lethal chromosome aberrations induced by DSBs. Thus, the capability of DSB repair is critically important for the cancer-cell-killing effect of ionizing radiation. Here, we investigated the involvement of the DNA damage signaling factors ataxia telangiectasia mutated (ATM), ring finger protein 8 (RNF8), and RNF168 in quiescent G0/G1 cells, which are expressed in the majority of cell populations in tumors, after high linear energy transfer (LET) carbon-ion irradiation. Interestingly, ATM inhibition caused a substantial DSB repair defect after high-LET carbon-ion irradiation. Similarly, RNF8 or RNF168 depletion caused a substantial DSB repair defect. ATM inhibition did not exert an additive effect in RNF8-depleted cells, suggesting that ATM and RNF8 function in the same pathway. Importantly, we found that the RNF8 RING mutant showed a similar DSB repair defect, suggesting the requirement of ubiquitin ligase activity in this repair pathway. The RNF8 FHA domain was also required for DSB repair in this axis. Furthermore, the p53-binding protein 1 (53BP1), which is an important downstream factor in RNF8-dependent DSB repair, was also required for this repair. Importantly, either ATM inhibition or RNF8 depletion increased the frequency of chromosomal breaks, but reduced dicentric chromosome formation, demonstrating that ATM/RNF8 is required for the rejoining of DSB ends for the formation of dicentric chromosomes. Finally, we showed that RNF8 depletion augmented radiosensitivity after high-LET carbon-ion irradiation. This study suggests that the inhibition of RNF8 activity or its downstream pathway may augment the efficacy of high-LET carbon-ion therapy.

Significance of PD-L1 expression in carbon-ion radiotherapy for uterine cervical adeno/adenosquamous carcinoma.

OBJECTIVE: Programmed cell death-ligand 1 (PD-L1) is expressed in tumor cells and has been shown to predict clinical outcomes of several types of malignancies. The aim of this study was to investigate the effects of carbon-ion (C-ion) beam irradiation on PD-L1 expression in human uterine cervical adeno/adenosquamous carcinoma (UCAA) cells and clinical samples and to identify the prognostic factors for outcomes after C-ion radiotherapy (CIRT). METHODS: The effects of C-ion irradiation on PD-L1 expression in human UCAA and cervical squamous cell carcinoma cells were examined by flow cytometry. We examined PD-L1 expression in UCAA biopsy specimens from 33 patients before CIRT started (pre-CIRT) and after 12 Gy (relative biological effectiveness [RBE]) irradiation (post-12Gy-C) in 4 fractions of CIRT to investigate the correlation between PD-L1 status and clinical outcomes. RESULTS: The PD-L1 expression was upregulated by C-ion beam in a dose-dependent manner in HeLa and SiHa cells through phosphorylated Chk1. The overall frequencies of pre-CIRT and post-12Gy-C PD-L1 positivity were 45% (15/33) and 67% (22/33), respectively. The post-12Gy-C PD-L1 expression was significantly elevated compared to the pre-CIRT PD-L1 expression. There was no significant relationship between the pre-CIRT PD-L1 status and clinical outcomes, such as local control (LC), progression-free survival (PFS), and overall survival (OS). However, the post-12Gy-C PD-L1 expression had better correlation with PFS, but not with LC and OS. CONCLUSION: CIRT can induce PD-L1 expression in UCAA and we propose that PD-L1 expression after starting CIRT may become as a predictive prognostic marker in CIRT for UCAA.

Targeted cancer cell ablation in mice by an α-particle-emitting astatine-211-labeled antibody against major histocompatibility complex class I chain-related protein A and B.

Major histocompatibility complex class I chain-related protein A and B (MICA/B) are ligands of the immune receptor, natural-killer group 2 member D. MICA/B expression is often found in several types of cancer but is restricted in normal tissues. Here, we show that an α-particle emitting astatine-211 (211At)-labeled antibody targeting MICA/B (211At-anti MICA/B Ab) efficiently ablates cancer cells in vitro and in vivo. We generated 211At-anti MICA/B Ab, an anti-MICA/B antibody conjugated with a highly cytotoxic α-particle emitting radionuclide 211At. 211At-anti MICA/B Ab binds to human osteosarcoma SaOS2 and U2OS cells that exhibit high levels of MICA/B expression and efficiently kills those cells in vitro. Biodistribution analysis using xenograft mouse models of HCT116 p53-/- positive for MICA/B expression, showed increased 211At in the xenografts for up to 22 h after injection as time proceeded. A single dose of 211At-anti MICA/B Ab (1 MBq) showed significant reduction in the tumor growth rate of HCT116 p53-/- xenografts compared to 211At-labeled mouse IgG (1 MBq) at 21 days after injection. No body weight loss and erythrocytopenia was evident in mice that received 211At-anti MICA/B. Leukocytopenia and thrombocytopenia were observed within a week after 211At-anti MICA/B injection, but counts of red blood cells and platelets were recovered to control levels at about 3-4 weeks after injection. Taken together, these data strongly demonstrate that targeted α-particle therapy using 211At-anti-MICA/B Ab emitting highly cytotoxic α-particles is a potential new therapeutic option for several types of cancer.

Novel function of HATs and HDACs in homologous recombination through acetylation of human RAD52 at double-strand break sites.

The p300 and CBP histone acetyltransferases are recruited to DNA double-strand break (DSB) sites where they induce histone acetylation, thereby influencing the chromatin structure and DNA repair process. Whether p300/CBP at DSB sites also acetylate non-histone proteins, and how their acetylation affects DSB repair, remain unknown. Here we show that p300/CBP acetylate RAD52, a human homologous recombination (HR) DNA repair protein, at DSB sites. Using in vitro acetylated RAD52, we identified 13 potential acetylation sites in RAD52 by a mass spectrometry analysis. An immunofluorescence microscopy analysis revealed that RAD52 acetylation at DSBs sites is counteracted by SIRT2- and SIRT3-mediated deacetylation, and that non-acetylated RAD52 initially accumulates at DSB sites, but dissociates prematurely from them. In the absence of RAD52 acetylation, RAD51, which plays a central role in HR, also dissociates prematurely from DSB sites, and hence HR is impaired. Furthermore, inhibition of ataxia telangiectasia mutated (ATM) protein by siRNA or inhibitor treatment demonstrated that the acetylation of RAD52 at DSB sites is dependent on the ATM protein kinase activity, through the formation of RAD52, p300/CBP, SIRT2, and SIRT3 foci at DSB sites. Our findings clarify the importance of RAD52 acetylation in HR and its underlying mechanism.

Inhibition of the HDAC/Suv39/G9a pathway restores the expression of DNA damage-dependent major histocompatibility complex class I-related chain A and B in cancer cells.

Immunotherapy is expected to be promising as a next generation cancer therapy. Immunoreceptors are often activated constitutively in cancer cells, however, such levels of ligand expression are not effectively recognized by the native immune system due to tumor microenvironmental adaptation. Studies have demonstrated that natural-killer group 2, member D (NKG2D), a major activating immunoreceptor, responds to DNA damage. The upregulation of major histocompatibility complex class I-related chain A and B (MICA/B) (members of NKG2D ligands) expression after DNA damage is associated with NK cell-mediated killing of cancer cells. However, the regulation of DNA damage-induced MICA/B expression has not been fully elucidated in the context of the types of cancer cell lines. In the present study, we found that MICA/B expression varied between cancer cell lines after DNA damage. Screening in terms of chromatin remodeling identified that inhibitors related to chromatin relaxation via post-translational modification on histone H3K9, i.e. HDAC, Suv39 or G9a inhibition, restored DNA damage-dependent MICA/B expression in insensitive cells. In addition, we revealed that the restored MICA/B expression was dependent on ATR as well as E2F1, a transcription factor. We further revealed that low­dose treatment of an HDAC inhibitor was sufficient to restore MICA/B expression in insensitive cells. Finally, we demonstrated that HDAC inhibition restored DNA damage­dependent cytotoxic NK activity against insensitive cells. Thus, the present study revealed that DNA damage­dependent MICA/B expression in insensitive cancer cells can be restored by chromatin relaxation via the HDAC/Suv39/G9a pathway. Collectively, manipulation of chromatin status by therapeutic cancer drugs may potentiate the antitumor effect by enhancing immune activation following radiotherapy and DNA damage-associated chemotherapy.

Low- and High-LET Ionizing Radiation Induces Delayed Homologous Recombination that Persists for Two Weeks before Resolving.

Genome instability is a hallmark of cancer cells and dysregulation or defects in DNA repair pathways cause genome instability and are linked to inherited cancer predisposition syndromes. Ionizing radiation can cause immediate effects such as mutation or cell death, observed within hours or a few days after irradiation. Ionizing radiation also induces delayed effects many cell generations after irradiation. Delayed effects include hypermutation, hyper-homologous recombination, chromosome instability and reduced clonogenic survival (delayed death). Delayed hyperrecombination (DHR) is mechanistically distinct from delayed chromosomal instability and delayed death. Using a green fluorescent protein (GFP) direct repeat homologous recombination system, time-lapse microscopy and colony-based assays, we demonstrate that DHR increases several-fold in response to low-LET X rays and high-LET carbon-ion radiation. Time-lapse analyses of DHR revealed two classes of recombinants not detected in colony-based assays, including cells that recombined and then senesced or died. With both low- and high-LET radiation, DHR was evident during the first two weeks postirradiation, but resolved to background levels during the third week. The results indicate that the risk of radiation-induced genome destabilization via DHR is time limited, and suggest that there is little or no additional risk of radiation-induced genome instability mediated by DHR with high-LET radiation compared to low-LET radiation.

VE-821, an ATR inhibitor, causes radiosensitization in human tumor cells irradiated with high LET radiation.

BACKGROUND: High linear energy transfer (LET) radiation such as carbon ion particles is successfully used for treatment of solid tumors. The reason why high LET radiation accomplishes greater tumor-killing than X-rays is still not completely understood. One factor would be the clustered or complex-type DNA damages. We previously reported that complex DNA double-strand breaks produced by high LET radiation enhanced DNA end resection, and this could lead to higher kinase activity of ATR protein recruited to RPA-coated single-stranded DNA. Although the effect of ATR inhibition on cells exposed to low LET gamma-rays has recently been reported, little is known regarding the effect of ATR inhibitor on cells treated with high LET radiation. The purpose of this study is to investigate the effects of the ATR inhibitor VE-821 in human tumor and normal cells irradiated with high LET carbon ions. FINDINGS: HeLa, U2OS, and 1BR-hTERT (normal) cells were pre-treated with 1 µM VE-821 for 1 hour and irradiated with either high LET carbon ions or X-rays. Cell survival, cell cycle distribution, cell growth, and micronuclei formation were evaluated. VE-821 caused abrogation of G2/M checkpoint and forced irradiated cells to divide into daughter cells. We also found that carbon ions caused a higher number of multiple micronuclei than X-rays, leading to decreased cell survival in tumor cells when treated with VE-821, while the survival of irradiated normal cells were not significantly affected by this inhibitor. CONCLUSIONS: ATR inhibitor would be an effective tumor radiosensitizer with carbon ion irradiation.

Pre-exposure to ionizing radiation stimulates DNA double strand break end resection, promoting the use of homologous recombination repair.

The choice of DNA double strand break (DSB) repair pathway is determined at the stage of DSB end resection. Resection was proposed to control the balance between the two major DSB repair pathways, homologous recombination (HR) and non-homologous end joining (NHEJ). Here, we examined the regulation of DSB repair pathway choice at two-ended DSBs following ionizing radiation (IR) in G2 phase of the cell cycle. We found that cells pre-exposed to low-dose IR preferred to undergo HR following challenge IR in G2, whereas NHEJ repair kinetics in G1 were not affected by pre-IR treatment. Consistent with the increase in HR usage, the challenge IR induced Replication protein A (RPA) foci formation and RPA phosphorylation, a marker of resection, were enhanced by pre-IR. However, neither major DNA damage signals nor the status of core NHEJ proteins, which influence the choice of repair pathway, was significantly altered in pre-IR treated cells. Moreover, the increase in usage of HR due to pre-IR exposure was prevented by treatment with ATM inhibitor during the incubation period between pre-IR and challenge IR. Taken together, the results of our study suggest that the ATM-dependent damage response after pre-IR changes the cellular environment, possibly by regulating gene expression or post-transcriptional modifications in a manner that promotes resection.

The complexity of DNA double strand breaks is a critical factor enhancing end-resection.

DNA double strand breaks (DSBs) induced by ionizing radiation (IR) are deleterious damages. Two major pathways repair DSBs in human cells, DNA non-homologous end-joining (NHEJ) and homologous recombination (HR). It has been suggested that the balance between the two repair pathways varies depending on the chromatin structure surrounding the damage site and/or the complexity of damage at the DNA break ends. Heavy ion radiation is known to induce complex-type DSBs, and the efficiency of NHEJ in repairing these DSBs was shown to be diminished. Taking advantage of the ability of high linear energy transfer (LET) radiation to produce complex DSBs effectively, we investigated how the complexity of DSB end structure influences DNA damage responses. An early step in HR is the generation of 3'-single strand DNA (SSD) via a process of DNA end resection that requires CtIP. To assess this process, we analyzed the level of phosphorylated CtIP, as well as RPA phosphorylation and focus formation, which occur on the exposed SSD. We show that complex DSBs efficiently activate DNA end resection. After heavy ion beam irradiation, resection signals appear both in the vicinity of heterochromatic areas, which is also observed after X-irradiation, and additionally in euchromatic areas. Consequently, ~85% of complex DSBs are subjected to resection in heavy ion particle tracks. Furthermore, around 20-40% of G1 cells exhibit resection signals. Taken together, our observations reveal that the complexity of DSB ends is a critical factor regulating the choice of DSB repair pathway and drastically alters the balance toward resection-mediated rejoining. As demonstrated here, studies on DNA damage responses induced by heavy ion radiation provide an important tool to shed light on mechanisms regulating DNA end resection.

Visualisation of γH2AX foci caused by heavy ion particle traversal; distinction between core track versus non-track damage.

Heavy particle irradiation produces complex DNA double strand breaks (DSBs) which can arise from primary ionisation events within the particle trajectory. Additionally, secondary electrons, termed delta-electrons, which have a range of distributions can create low linear energy transfer (LET) damage within but also distant from the track. DNA damage by delta-electrons distant from the track has not previously been carefully characterised. Using imaging with deconvolution, we show that at 8 hours after exposure to Fe (∼200 keV/µm) ions, γH2AX foci forming at DSBs within the particle track are large and encompass multiple smaller and closely localised foci, which we designate as clustered γH2AX foci. These foci are repaired with slow kinetics by DNA non-homologous end-joining (NHEJ) in G1 phase with the magnitude of complexity diminishing with time. These clustered foci (containing 10 or more individual foci) represent a signature of DSBs caused by high LET heavy particle radiation. We also identified simple γH2AX foci distant from the track, which resemble those arising after X-ray exposure, which we attribute to low LET delta-electron induced DSBs. They are rapidly repaired by NHEJ. Clustered γH2AX foci induced by heavy particle radiation cause prolonged checkpoint arrest compared to simple γH2AX foci following X-irradiation. However, mitotic entry was observed when ∼10 clustered foci remain. Thus, cells can progress into mitosis with multiple clusters of DSBs following the traversal of a heavy particle.

Selective enhancing effect of early mitotic inhibitor 1 (Emi1) depletion on the sensitivity of doxorubicin or X-ray treatment in human cancer cells.

Chemotherapy and radiation in addition to surgery has proven useful in a number of different cancer types, but the effectiveness in normal tissue cannot be avoided in these therapies. To improve the effectiveness of these therapies selectively in cancer tissue is important for avoiding side effects. Early mitotic inhibitor 1 (Emi1) is known to have the function to inhibit anaphase-promoting complex/cyclosome ubiquitin ligase complex, which ubiquitylates the cell cycle-related proteins. It recently has been shown that Emi1 knockdown prevents transition from S to G2 phase by down-regulating geminin via anaphase-promoting complex/cyclosome activation. At present, anticancer drugs for targeting DNA synthesis to interfere with rapidly dividing cells commonly are used. As Emi1 depletion interferes with completion of DNA synthesis in cancer cells, we thought that Emi1 knockdown might enhance the sensitivity for anticancer agents. Here, we confirmed that Emi1 siRNA induced polyploidy for preventing transition from S to G2 phase in several cancer cell lines. Then, we treated Emi1 depleted cells with doxorubicin. Interestingly, increased apoptotic cells were observed after doxorubicin treatment in Emi1 siRNA-treated cancer cells. In addition, Emi1 depletion enhanced the sensitivity of x-ray irradiation in cancer cells. Importantly, synergistic effect of Emi1 knockdown in these combination therapies was not observed in normal cells. These results suggest that Emi1 siRNA can be a useful tool for enhancing of sensitivity of cancer cells to anticancer reagents and radiation.