RESUMEN
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.
Asunto(s)
Roturas del ADN de Doble Cadena , Histona Acetiltransferasas/fisiología , Histona Desacetilasas/fisiología , Recombinación Homóloga , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , Acetilación , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Histona Acetiltransferasas/genética , Histona Desacetilasas/genética , Humanos , Microscopía Fluorescente , Técnicas del Sistema de Dos HíbridosRESUMEN
High-linear energy transfer (LET) heavy ions have been increasingly employed as a useful alternative to conventional photon radiotherapy. As recent studies suggested that high LET radiation mainly affects the nonhomologous end-joining (NHEJ) pathway of DNA double strand break (DSB) repair, we further investigated this concept by evaluating the combined effect of an NHEJ inhibitor (NU7441) at a non-toxic concentration and carbon ions. NU7441-treated non-small cell lung cancer (NSCLC) A549 and H1299 cells were irradiated with X-rays and carbon ions (290 MeV/n, 50 keV/µm). Cell survival was measured by clonogenic assay. DNA DSB repair, cell cycle distribution, DNA fragmentation and cellular senescence induction were studied using a flow cytometer. Senescence-associated protein p21 was detected by western blotting. In the present study, 0.3 µM of NU7441, nontoxic to both normal and tumor cells, caused a significant radio-sensitization in tumor cells exposed to X-rays and carbon ions. This concentration did not seem to cause inhibition of DNA DSB repair but induced a significant G2/M arrest, which was particularly emphasized in p53-null H1299 cells treated with NU7441 and carbon ions. In addition, the combined treatment induced more DNA fragmentation and a higher degree of senescence in H1299 cells than in A549 cells, indicating that DNA-PK inhibitor contributes to various modes of cell death in a p53-dependent manner. In summary, NSCLC cells irradiated with carbon ions were radio-sensitized by a low concentration of DNA-PK inhibitor NU7441 through a strong G2/M cell cycle arrest. Our findings may contribute to further effective radiotherapy using heavy ions.
Asunto(s)
Cromonas/farmacología , Roturas del ADN de Doble Cadena , Reparación del ADN/efectos de los fármacos , Proteína Quinasa Activada por ADN/antagonistas & inhibidores , Morfolinas/farmacología , Fármacos Sensibilizantes a Radiaciones/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Reparación del ADN/efectos de la radiación , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de la radiación , Humanos , Transferencia Lineal de Energía , Neoplasias PulmonaresRESUMEN
The poly(ADP-ribose) polymerase (PARP)-1 regulates DNA damage responses and promotes base excision repair. PARP inhibitors have been shown to enhance the cytotoxicity of ionizing radiation in various cancer cells and animal models. We have demonstrated that the PARP inhibitor (PARPi) AZD2281 is also an effective radiosensitizer for carbon-ion radiation; thus, we speculated that the PARPi could be applied to a wide therapeutic range of linear energy transfer (LET) radiation as a radiosensitizer. Institutes for biological experiments using proton beam are limited worldwide. This study was performed as a cooperative research at heavy ion medical accelerator in Chiba (HIMAC) in National Institute of Radiological Sciences. HIMAC can generate various ion beams; this enabled us to compare the radiosensitization effect of the PARPi on cells subjected to proton and carbon-ion beams from the same beam line. After physical optimization of proton beam irradiation, the radiosensitization effect of the PARPi was assessed in the human lung cancer cell line, A549, and the pancreatic cancer cell line, MIA PaCa-2. The effect of the PARPi, AZD2281, on radiosensitization to Bragg peak was more significant than that to entrance region. The PARPi increased the number of phosphorylated H2AX (γ-H2AX) foci and enhanced G2/M arrest after proton beam irradiation. This result supports our hypothesis that a PARPi could be applied to a wide therapeutic range of LET radiation by blocking the DNA repair response.
Asunto(s)
Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/radioterapia , Inhibidores de Poli(ADP-Ribosa) Polimerasas/administración & dosificación , Terapia de Protones/métodos , Tolerancia a Radiación/efectos de los fármacos , Fármacos Sensibilizantes a Radiaciones/administración & dosificación , Células A549 , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta en la Radiación , Humanos , Neoplasias Experimentales/patología , Ftalazinas/administración & dosificación , Piperazinas/administración & dosificación , Dosificación Radioterapéutica , Resultado del TratamientoRESUMEN
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.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Proteínas de Ciclo Celular/metabolismo , Doxorrubicina/farmacología , Proteínas F-Box/metabolismo , Tolerancia a Radiación , Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Antígenos CD , Apoptosis/efectos de los fármacos , Cadherinas/genética , Cadherinas/metabolismo , Carcinoma de Células Escamosas/metabolismo , Proteínas Cdc20 , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Daño del ADN , Resistencia a Antineoplásicos , Proteínas F-Box/genética , Expresión Génica , Técnicas de Silenciamiento del Gen , Neoplasias de Cabeza y Cuello/metabolismo , Humanos , Poliploidía , ARN Interferente Pequeño/genéticaRESUMEN
The roles of various core components, including α/ß/γ-type small acid-soluble spore proteins (SASP), dipicolinic acid (DPA), core water content, and DNA repair by apurinic/apyrimidinic (AP) endonucleases or nonhomologous end joining (NHEJ), in Bacillus subtilis spore resistance to different types of ionizing radiation including X rays, protons, and high-energy charged iron ions have been studied. Spores deficient in DNA repair by NHEJ or AP endonucleases, the oxidative stress response, or protection by major α/ß-type SASP, DPA, and decreased core water content were significantly more sensitive to ionizing radiation than wild-type spores, with highest sensitivity to high-energy-charged iron ions. DNA repair via NHEJ and AP endonucleases appears to be the most important mechanism for spore resistance to ionizing radiation, whereas oxygen radical detoxification via the MrgA-mediated oxidative stress response or KatX catalase activity plays only a very minor role. Synergistic radioprotective effects of α/ß-type but not γ-type SASP were also identified, indicating that α/ß-type SASP's binding to spore DNA is important in preventing DNA damage due to reactive oxygen species generated by ionizing radiation.
Asunto(s)
Bacillus subtilis/efectos de la radiación , Reparación del ADN , ADN Bacteriano/efectos de la radiación , Radiación Ionizante , Esporas Bacterianas/efectos de la radiación , Proteínas Bacterianas/metabolismo , Ácidos Picolínicos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Agua/metabolismoRESUMEN
Poly(ADP-ribose) glycohydrolase (Parg) is the main enzyme involved in poly(ADP-ribose) degradation. Here, the effects of Parg deficiency on sensitivity to low and high linear-energy-transfer (LET) radiation were investigated in mouse embryonic stem (ES) cells. Mouse Parg(-/-) and poly(ADP-ribose) polymerase-1 deficient (Parp-1(-/-)) ES cells were used and responses to low and high LET radiation were assessed by clonogenic survival and biochemical and biological analysis methods. Parg(-/-) cells were more sensitive to γ-irradiation than Parp-1(-/-) cells. Transient accumulation of poly(ADP-ribose) was enhanced in Parg(-/-) cells. Augmented levels of phosphorylated H2AX (γ-H2AX) from early phase were observed in Parg(-/-) ES cells. The induction level of p53 phophorylation at ser18 was similar in wild-type and Parp-1(-/-) cells and apoptotic cell death process was mainly observed in the both genotypes. These results suggested that the enhanced sensitivity of Parg(-/-) ES cells to γ-irradiation involved defective repair of DNA double strand breaks. The effects of Parg and Parp-1 deficiency on the ES cell response to carbon-ion irradiation (LET13 and 70 keV/µm) and Fe-ion irradiation (200 keV/µm) were also examined. Parg(-/-) cells were more sensitive to LET 70 keV/µm carbon-ion irradiation than Parp-1(-/-) cells. Enhanced apoptotic cell death also accompanied augmented levels of γ-H2AX in a biphasic manner peaked at 1 and 24h. The induction level of p53 phophorylation at ser18 was not different between wild-type and Parg(-/-) cells. The augmented level of poly(ADP-ribose) accumulation was noted after carbon-ion irradiation compared to γ-irradiation even in the wild-type cells. An enhanced poly(ADP-ribose) accumulation was further observed in Parg(-/-) cells. Both Parg(-/-) cells and Parp-1(-/-) cells did not show sensitization to Fe-ion irradiation. Parg deficiency sensitizes mouse ES cells to a wide therapeutic range of LET radiation through the effects on DNA double strand break repair responses and enhanced cell death.
Asunto(s)
Apoptosis/efectos de la radiación , Células Madre Embrionarias/efectos de la radiación , Glicósido Hidrolasas/deficiencia , Poli(ADP-Ribosa) Polimerasas/deficiencia , Radiación Ionizante , Animales , Apoptosis/genética , Carbono , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Células Cultivadas , Roturas del ADN de Doble Cadena/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Citometría de Flujo , Rayos gamma , Glicósido Hidrolasas/genética , Iones Pesados , Histonas/metabolismo , Immunoblotting , Ratones , Ratones Noqueados , Fosforilación/efectos de la radiación , Poli Adenosina Difosfato Ribosa/metabolismo , Poli(ADP-Ribosa) Polimerasas/genética , Factores de Tiempo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
We have studied the induction of chromosome aberrations in human fibroblasts exposed in G0/G1 to X-rays or heavy ions to study the influence of G1 cell cycle arrest. Confluent normal fibroblasts were exposed to X-rays or accelerated particles with different LET values and chromosome aberrations were investigated in the first G0/G1 and G2//M phase. The particles used here were 490MeV/nucleon Si, 500MeV/nucleon Fe, and 200MeV/nucleon Fe ions. Cells were subcultured 24h after exposure and premature chromosome condensation (PCC) was performed by fusion-induced method for analysis of G0/G1 cells, and chemically-induced method for analysis of G2 and metaphase cells. Chromosome damage was assessed in chromosomes 1 and 3 using whole chromosome fluorescence in situ hybridization (FISH). Cell cycle was analyzed by flow cytometry at different incubation times following subculture. After irradiation with 2Gy of high-LET particles, the yields of chromosome aberrations and fragments were significantly higher in G0/G1 phase than in G2/M phase, whereas similar yields of damage were measured in both phases after exposure to X-rays. In contrast, the yield of misrepair, assessed by the number of color junctions, was similar in the G0/G1 and G2/M phases after exposure to either X-rays or high-LET particles. The yields of chromosome aberrations, fragments, and color junctions in both the G0/G1 and the G2/M phases, increased with LET up to 200keV/µm, then decreased for 440keV/µm Fe particles. A good correlation was found between chromosome aberrations in both G0/G1 and G2/M cells and survival fractions after 2Gy of different LET radiations, although the slopes were steeper for the G0/G1 cells. Flow cytometry analysis indicated that high-LET particles induce more non cycling G0/G1 cells within 48h of subculture than X-rays, suggesting that chromosome aberrations scored at the G2/M phase may not accurately describe the true radiation effect.
Asunto(s)
Ciclo Celular/genética , Ciclo Celular/efectos de la radiación , Aberraciones Cromosómicas/efectos de la radiación , Fibroblastos/efectos de la radiación , Piel/efectos de la radiación , Células Cultivadas , Fibroblastos/citología , Fibroblastos/metabolismo , Citometría de Flujo , Humanos , Procesamiento de Imagen Asistido por Computador , Hibridación Fluorescente in Situ , Transferencia Lineal de Energía , Piel/citología , Piel/metabolismo , Rayos XRESUMEN
The aim of this study was to measure the RBE (relative biological effectiveness) and OER (oxygen enhancement ratio) for survival of cells within implanted solid tumors following exposure to 290MeV/nucleon carbon-ion beams or X-rays. Squamous cell carcinoma cells (SCCVII) were transplanted into the right hind legs of syngeneic C3H male mice. Irradiation with either carbon-ion beams with a 6-cm spread-out Bragg peak (SOBP, at 46 and 80keV/µm) or X-rays was delivered to 5-mm or less diameter tumors. We defined three different oxygen statuses of the irradiated cells. Hypoxic and normoxic conditions in tumors were produced by clamping or not clamping the leg to avoid blood flow. Furthermore, single-cell suspensions were prepared from non-irradiated tumors and directly used to determine the radiation response of aerobic cells. Single-cell suspensions (aerobic condition) were fully air-saturated. Single-cell suspensions were prepared from excised and trypsinized tumors, and were used for in vivo-in vitro colony formation assays to obtain cell survival curves. The RBE values increased with increasing LET in SOBP beams. The maximum RBE values in three different oxygen conditions; hypoxic tumor, normoxic tumor and aerobic cells, were 2.16, 1.76 and 1.66 at an LET of 80keV/µm, respectively. After X-ray irradiation the OERh/n values (hypoxic tumor/normoxic tumor) were lower than the OERh/a (hypoxic tumor/aerobic cells), and were 1.87±0.13 and 2.52±0.11, respectively. The OER values of carbon-ion irradiated samples were small in comparison to those of X-ray irradiated samples. However, no significant changes of the OER at proximal and distal positions within the SOBP carbon-ion beams were observed. To conclude, we found that the RBE values for cell survival increased with increasing LET and that the OER values changed little with increasing LET within the SOBP carbon-ion beams.
Asunto(s)
Radioisótopos de Carbono/efectos adversos , Carcinoma de Células Escamosas/patología , Hipoxia/patología , Neoplasias/patología , Animales , Carcinoma de Células Escamosas/radioterapia , Supervivencia Celular , Ensayo de Unidades Formadoras de Colonias , Transferencia Lineal de Energía , Masculino , Ratones , Ratones Endogámicos CBA , Neoplasias/radioterapia , Efectividad Biológica Relativa , Células Tumorales Cultivadas , Rayos XRESUMEN
Increasing use of heavy ions for cancer therapy and concerns from exposure to heavy charged particles in space necessitate the study of the basic biological mechanisms associated with exposure to heavy ions. As the most critical damage induced by ionizing radiation is DNA double strand break (DSB), this review focuses on DSBs induced by heavy ions and their repair processes. Compared with X- or gamma-rays, high-linear energy transfer (LET) heavy ion radiation induces more complex DNA damage, categorized into DSBs and non-DSB oxidative clustered DNA lesions (OCDL). This complexity makes the DNA repair process more difficult, partially due to retarded enzymatic activities, leading to increased chromosome aberrations and cell death. In general, the repair process following heavy ion exposure is LET-dependent, but with nonhomologous end joining defective cells, this trend is less emphasized. The variation in cell survival levels throughout the cell cycle is less prominent in cells exposed to high-LET heavy ions when compared with low LET, but this mechanism has not been well understood until recently. Involvement of several DSB repair proteins is suggested to underlie this interesting phenomenon. Recent improvements in radiation-induced foci studies combined with high-LET heavy ion exposure could provide a useful opportunity for more in depth study of DSB repair processes. Accelerated heavy ions have become valuable tools to investigate the molecular mechanisms underlying repair of DNA DSBs, the most crucial form of DNA damage induced by radiation and various chemotherapeutic agents.
Asunto(s)
Daño del ADN , Reparación del ADN , Iones Pesados/efectos adversos , Ciclo Celular/efectos de la radiación , Supervivencia Celular/efectos de la radiación , Roturas del ADN de Doble Cadena , Humanos , Transferencia Lineal de Energía , Radiación IonizanteRESUMEN
Poly(ADP-ribose) polymerase (PARP)-1 promotes base excision repair and DNA strand break repair. Inhibitors of PARP enhance the cytotoxic effects of γ-irradiation and X-irradiation. We investigated the impact of PARP inhibition on the responses to γ-irradiation (low liner energy transfer [LET] radiation) and carbon-ion irradiation (high LET radiation) in the human pancreatic cancer cell line MIA PaCa-2. Cell survival was assessed by colony formation assay after combination treatment with the PARP inhibitor AZD2281 and single fraction γ-irradiation and carbon-ion irradiation (13 and 70 keV/µm [LET 13 and LET 70]). The DNA damage response (DDR) was assessed by pulse field gel electrophoresis, western blotting and flow cytometry. Treatment with a PARP inhibitor enhanced the cytotoxic effect of γ-irradiation and LET 13 and LET 70 carbon-ion irradiation. Moreover, the radiosensitization effect was greater for LET 70 than for LET 13 irradiation. Prolonged and increased levels of γ-H2AX were observed both after γ-irradiation and carbon-ion irradiation in the presence of the PARP inhibitor. Enhanced level of phosphorylated-p53 (Ser-15) was observed after γ-irradiation but not after carbon-ion irradiation. PARP inhibitor treatment induced S phase arrest and enhanced subsequent G2/M arrest both after γ-irradiation and carbon-ion irradiation. These results suggest that the induction of S phase arrest through an enhanced DDR and a local delay in DNA double strand break processing by PARP inhibition caused sensitization to γ-irradiation and carbon-ion irradiation. Taken together, PARP inhibitors might be applicable to a wide therapeutic range of LET radiation through their effects on the DDR.
Asunto(s)
Reparación del ADN/efectos de la radiación , Rayos gamma , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/radioterapia , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Fármacos Sensibilizantes a Radiaciones , Puntos de Control de la Fase S del Ciclo Celular/efectos de la radiación , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Daño del ADN/efectos de la radiación , Inhibidores Enzimáticos/farmacología , Histonas/biosíntesis , Humanos , Transferencia Lineal de Energía , Neoplasias Pancreáticas/metabolismo , Fosforilación/efectos de la radiación , Ftalazinas/farmacología , Piperazinas/farmacología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteína p53 Supresora de Tumor/biosíntesis , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The roles of DNA repair by apurinic/apyrimidinic (AP) endonucleases alone, and together with DNA protection by α/ß-type small acid-soluble spore proteins (SASP), in Bacillus subtilis spore resistance to different types of radiation have been studied. Spores lacking both AP endonucleases (Nfo and ExoA) and major SASP were significantly more sensitive to 254-nm UV-C, environmental UV (>280 nm), X-ray exposure, and high-energy charged (HZE)-particle bombardment and had elevated mutation frequencies compared to those of wild-type spores and spores lacking only one or both AP endonucleases or major SASP. These findings further implicate AP endonucleases and α/ß-type SASP in repair and protection, respectively, of spore DNA against effects of UV and ionizing radiation.
Asunto(s)
Bacillus subtilis/enzimología , Bacillus subtilis/efectos de la radiación , Reparación del ADN , Endonucleasas/metabolismo , Esporas Bacterianas/enzimología , Esporas Bacterianas/efectos de la radiación , Bacillus subtilis/fisiología , Proteínas Bacterianas/metabolismo , Viabilidad Microbiana/efectos de la radiación , Mutagénesis , Radiación , Radiación Ionizante , Esporas Bacterianas/fisiología , Rayos Ultravioleta , Rayos XRESUMEN
ZSTK474 is a novel orally applicable phosphoinositide 3-kinase-specific inhibitor that strongly inhibits cancer cell proliferation. To further explore the antitumor effect of ZSTK474 for future clinical usage, we studied its combined effects with radiation. The proliferation of HeLa cells was inhibited by treatment with X-rays alone or ZSTK474 alone. Combination treatment using X-rays then ZSTK474 given orally for 8 days, starting 24 h post-irradiation, significantly enhanced cell growth inhibition. The combined effect was also observed for clonogenic survival with continuous ZSTK474 treatment. Western blot analysis showed enhanced phosphorylation of Akt and GSK-3ß by X-irradiation, whereas phosphorylation was inhibited by ZSTK474 treatment alone. Treatment with ZSTK474 after X-irradiation also inhibited phosphorylation, and remarkably inhibited xenograft tumor growth. Combined treatment with X-rays and ZSTK474 has greater therapeutic potential than radiation or drug therapy alone, both in vitro and in vivo.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Neoplasias Experimentales/terapia , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-akt/metabolismo , Triazinas/farmacología , Animales , Western Blotting , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Terapia Combinada , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Células HeLa , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/radioterapia , Fosforilación/efectos de los fármacos , Fosforilación/efectos de la radiación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We examined lethal damages of X rays induced by direct and indirect actions, in terms of double-strand break (DSB) repair susceptibility using two kinds of repair-deficient Chinese hamster ovary (CHO) cell lines. These CHO mutants (51D1 and xrs6) are genetically deficient in one of the two important DNA repair pathways after genotoxic injury [homologous recombination (HR) and non-homologous end binding (NHEJ) pathways, respectively]. The contribution of indirect action on cell killing can be estimated by applying the maximum level of dimethylsulfoxide (DMSO) to get rid of OH radicals. To control the proportion of direct and indirect actions in lethal damage, we irradiated CHO mutant cells under aerobic and anoxic conditions. The contributions of indirect action on HR-defective 51D1 cells were 76% and 57% under aerobic and anoxic conditions, respectively. Interestingly, these percentages were similar to those of the wild-type cells even if the radiosensitivity was different. However, the contributions of indirect action to cell killing on NHEJ-defective xrs6 cells were 52% and 33% under aerobic and anoxic conditions, respectively. Cell killing by indirect action was significantly affected by the oxygen concentration and the DSB repair pathways but was not correlated with radiosensitivity. These results suggest that the lethal damage induced by direct action is mostly repaired by NHEJ repair pathway since killing of NHEJ-defective cells has significantly higher contribution by the direct action. In other words, the HR repair pathway may not effectively repair the DSB by direct action in place of the NHEJ repair pathway. We conclude that the type of DSB produced by direct action is different from that of DSB induced by indirect action.
Asunto(s)
Daño del ADN , Oxígeno/metabolismo , Aerobiosis/genética , Aerobiosis/efectos de la radiación , Animales , Células CHO , Muerte Celular/genética , Muerte Celular/efectos de la radiación , Cricetulus , Reparación del ADN por Unión de Extremidades/efectos de la radiación , Recombinación Homóloga/efectos de la radiación , Rayos X/efectos adversosRESUMEN
In previous studies we have shown that the linear energy transfer (LET)-relative biological effectiveness (RBE) curves were affected by LET and ion species [1,2]. In this paper we have examined the difference in the repair kinetics of G1-prematurely condensed chromosome breaks in normal human fibroblasts following irradiation with different heavy-ion beams of similar LET values. Normal human fibroblasts were irradiated with about 110 keV/microm of carbon (135 MeV/n), neon (400 MeV/n) and silicon ions (490 MeV/n), and the doses of carbon (3.25 Gy), neon (2.94+/-0.01 Gy) and silicon (2.31 Gy) were chosen to produce approximately the same number of initially measured G1-premature chromosome condensation (PCC) breaks (about 37 excess fragments per cell). The number of G1-PCC breaks was counted as excess fragments of prematurely condensed chromosomes using the PCC technique in the G1/G0 phase. The fractions of residual G1-PCC breaks after 24 h post-irradiation and half time, which is the time point where 50% of initially measured G1-PCC breaks are rejoined (t1/2), of the slow components of rejoining in carbon- and neon-ion irradiated cells were different from those of silicon-ion irradiated cells. However, no difference was observed in the half time of the fast components of rejoining in each ion beam. The results suggest that the difference in the fractions of residual G1-PCC breaks after 24 h post-irradiation reflect the result of the slow repair process for induced G1-PCC breaks, and that the repair process is dependent on the ion species, not the LET values.
Asunto(s)
Aberraciones Cromosómicas , Iones Pesados/efectos adversos , Transferencia Lineal de Energía , Reparación del ADN , Fibroblastos/efectos de la radiación , Fase G1 , HumanosRESUMEN
We studied the effects and mechanisms of ascorbic acid as a radiation protector. Cell survival, repair of DNA double strand breaks (DSBs), and sister chromatid exchanges (SCEs) were examined in normal human fibroblasts irradiated with X-rays and heavy ions. Post-irradiation treatment with 5mM ascorbic acid for 24 h in plateau phase (non-cycling) cells enhanced cell survival and DNA double strand break repair, and reduced SCEs after X-rays irradiation. On the other hand, only reduced SCEs were observed after heavy ion exposure such as to carbon ions. Judging from our data, it is possible that the radioprotective action of ascorbic acid would be effective in non-complex type DNA damage such as induced by X-rays. These findings provide new insight into the mechanism of DNA damage and repair produced by heavy ion irradiation.
Asunto(s)
Ácido Ascórbico/farmacología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Iones Pesados/efectos adversos , Protectores contra Radiación/farmacología , Rayos X/efectos adversos , Línea Celular , Roturas del ADN de Doble Cadena/efectos de los fármacos , Fibroblastos/efectos de la radiación , Humanos , Intercambio de Cromátides HermanasRESUMEN
We have observed that some of the DNA damage or damage product caused by irradiation of interphase cells persisted throughout the cell cycle, and resulted in the expression of gamma-H2AX foci on the mitotic chromosomes. These mitotic expressions of damage after gamma-irradiation of G1 or G2 phase cells were compared in wild-type CHO and their DNA repair deficient XR-1 and UV-1 cells. gamma-H2AX foci were located on one of the chromatids or on both chromatids as isolocus paired foci. DNA double strand break (DSB) repair deficient XR-1 cells exhibited greater persistence of gamma-H2AX foci than wild-type cells when irradiated at G1 phase. Delayed subculture after irradiation significantly reduced the persistence of damage in mitotic cells and the radiosensitivity in wild-type cells, but this was not the case for XR-1 cells. Interestingly, UV and crosslinking agents sensitive UV-1 cells which show similar sensitivity to gamma-irradiation as wild-type cells by gamma-irradiation, exhibited significantly higher gamma-H2AX persistence at mitosis when they were irradiated in G1-phase but not in G2-phase. One interpretation of this is that it is due to DNA damage accumulating at stalled replication forks. As in wild type cells, in delayed subculture after gamma-ray exposure of UV-1 cells, a reduced number of foci was also seen. Our results suggest that the persistence of gamma-H2AX foci does not always correspond with the radiosensitivities of cells, but rather depends on cells' ability to repair the different kinds of DNA damages. J. Cell. Physiol. 219: 760-765, 2009. (c) 2009 Wiley-Liss, Inc.
Asunto(s)
Roturas del ADN de Doble Cadena/efectos de la radiación , Fase G1/genética , Fase G1/efectos de la radiación , Fase G2/genética , Fase G2/efectos de la radiación , Mitosis/genética , Mitosis/efectos de la radiación , Animales , Células CHO , Cricetinae , Cricetulus , Reparación del ADN , Rayos gamma/efectos adversos , Histonas/metabolismo , Histonas/efectos de la radiación , FosforilaciónRESUMEN
Sulforaphane (SFN), an isothiocyanate derived from broccoli and other cruciferous vegetables, is a positive regulator of phase II detoxification enzymes and is highly effective in protection against chemically induced cancers by inducing apoptosis and cell cycle arrest. Here, we report that SFN also enhances radiosensitivity in human tumor cells. Cell survival in HeLa human cervix carcinoma cells pretreated with SFN was significantly lower than in cells treated with radiation only. Constant-field gel electrophoresis and a gamma-H2AX foci assay showed marked inhibition of DSB repair in irradiated cells treated with SFN, while little inhibition was observed in cells with DMSO (control). In addition, immunofluorescence experiments revealed a significant delay in Rad51 (a key protein for homologous recombination repair) foci formation and disappearance in irradiated cells treated with SFN when compared to the cells with X-irradiation alone. The dephosphorylation of DNA-PKcs (a critical nonhomologous end joining protein) was also markedly delayed by SFN pretreatment in irradiated cells. These DSB repair inhibition data partially support the high apoptotic frequency of irradiated cells pretreated with SFN. Furthermore, the combined treatment of X-rays and SFN (i.p. 300 micromol/kg) in the xenograft model with HeLa cells showed efficient inhibition of in vivo tumor growth. To the best of our knowledge, our study is the first report showing SFN-enhanced radiosensitivity of tumor cells in vitro and in vivo, which opens the door for a multitude of clinical applications for chemoradiotherapy using SFN.
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Anticarcinógenos/farmacología , Roturas del ADN de Doble Cadena/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Tolerancia a Radiación/efectos de los fármacos , Tiocianatos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Western Blotting , Ciclo Celular/efectos de los fármacos , Ciclo Celular/efectos de la radiación , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Ensayo de Unidades Formadoras de Colonias , Roturas del ADN de Doble Cadena/efectos de la radiación , Reparación del ADN/efectos de la radiación , Proteína Quinasa Activada por ADN/metabolismo , Técnica del Anticuerpo Fluorescente , Células HeLa/efectos de los fármacos , Células HeLa/efectos de la radiación , Humanos , Isotiocianatos , Proteínas Nucleares/metabolismo , Fosforilación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Recombinasa Rad51/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sulfóxidos , Células Tumorales Cultivadas , Rayos XRESUMEN
The biological effects of radiation originate principally in damages to DNA. DNA damages by X rays as well as heavy ions are induced by a combination of direct and indirect actions. The contribution of indirect action in cell killing can be estimated from the maximum degree of protection by dimethylsulfoxide (DMSO), which suppresses indirect action without affecting direct action. Exponentially growing Chinese hamster V79 cells were exposed to high-LET radiations of 20 to 2106 keV/mum in the presence or absence of DMSO and their survival was determined using a colony formation assay. The contribution of indirect action to cell killing decreased with increasing LET. However, the contribution did not reach zero even at very high LETs and was estimated to be 32% at an LET of 2106 keV/mum. Therefore, even though the radiochemically estimated G value of OH radicals was nearly zero at an LET of 1000 keV/mum, indirect action by OH radicals contributed to a substantial fraction of the biological effects of high-LET radiations. The RBE determined at a survival level of 10% increased with LET, reaching a maximum value of 2.88 at 200 keV/mum, and decreased thereafter. When the RBE was estimated separately for direct action (RBE(D)) and indirect action (RBE(I)); both exhibited an LET dependence similar to that of the RBE, peaking at 200 keV/mum. However, the peak value was much higher for RBE(D) (5.99) than RBE(I) (1.89). Thus direct action contributes more to the high RBE of high-LET radiations than indirect action does.
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Radiación Ionizante , Animales , Línea Celular , Cricetinae , CricetulusRESUMEN
High linear energy transfer (LET) radiation shows different biological effects from low-LET radiation. The complex nature of high LET radiation-induced damage, especially the clustered DNA damage, brings about slow repair of DNA double strand breaks (DSBs), which finally lead to higher lethality and chromosome aberration. Ionizing radiation (IR) induced DNA DSBs are repaired by both non-homologous end-joining (NHEJ) and homologous recombination repair (HRR) pathways in mammalian cells. The novel function of ataxia telangiectasia-mutated (ATM) protein is its involvement in the DSB repair of slow kinetics for "dirty" breaks rejoining by NHEJ, this suggests that ATM may play a more important role in high LET radiation-induced DNA damage. We show here that KU55933, an ATM inhibitor could distinctly lower the clonogenic survival in normal human skin fibroblast cells exposed to carbon ion radiation and dramatically impair the normal process for DSB repair. We also implicated the involvement of ATM in the two pathways of DNA DSB repair, with DNA-PKcs and Rad51 as the representative proteins. The phosphorylation of DNA-PKcs at Thr-2609 with both immunoblotting and immunofluorescent staining indicated an ATM-dependent change, while for Rad51, KU55933 pretreatment could postpone the formation of nuclear Rad51 foci. Interestingly, we also found that pretreatment with chloroquine, an ATM stimulator could protect cells from carbon ion radiation only at lower doses. For doses over 1Gy, protection was no longer observed. There was a dose-dependent increase for ATM kinase activity, with saturation at about 1Gy. Chloroquine pretreatment prior to 1Gy of carbon ion radiation did not enhance the autophosphorylation of ATM at serine 1981. The function of ATM in G2/M checkpoint arrest facilitated DSB repair in high-LET irradiation. Our results provide a possible mechanism for the direct involvement of ATM in DSB repair by high-LET irradiation.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Transferencia Lineal de Energía , Proteínas Serina-Treonina Quinasas/metabolismo , Tolerancia a Radiación/genética , Radiación Ionizante , Proteínas Supresoras de Tumor/metabolismo , Ataxia Telangiectasia/genética , Proteínas de la Ataxia Telangiectasia Mutada , Ciclo Celular , Línea Celular , Humanos , Modelos Genéticos , Recombinación GenéticaRESUMEN
Premature chromosome condensation (PCC) is a sensitive and unique way to detect interphase chromosome damage and its recovery in mammalian cells irradiated with ionizing radiation. In this chapter, we describe G1 PCC assay with which one can measure immediate chromosome breaks in G1 type chromosomes and their repair/rejoining. In order to induce G1 PCC, one needs to fuse mitotic cells with G1 cells to be tested. There are two methods to fuse cells; one is to use Sendai virus or its equivalent, and another method needs polyethylene glycol (PEG) as a fusing agent. The date obtained with PCC assay can bridge the gap between radiation-induced DNA damage (mainly double strand breaks) and chromosome aberrations observable at metaphase stage.