RESUMEN
BACKGROUND: Renal cell carcinoma (RCC) was historically considered to be less responsive to radiation therapy (RT) compared to other cancer indications. However, advancements in precision high-dose radiation delivery through single-fraction and multi-fraction stereotactic ablative radiotherapy (SABR) have led to better outcomes and reduced treatment-related toxicities, sparking renewed interest in using RT to treat RCC. Moreover, numerous studies have revealed that certain therapeutic agents including chemotherapies can increase the sensitivity of tumors to RT, leading to a growing interest in combining these treatments. Here, we developed a rational combination of two radiosensitizers in a tumor-targeted liposomal formulation for augmenting RT in RCC. The objective of this study is to assess the efficacy of a tumor-targeted liposomal formulation combining the mTOR inhibitor everolimus (E) with the survivin inhibitor YM155 (Y) in enhancing the sensitivity of RCC tumors to radiation. EXPERIMENTAL DESIGN: We slightly modified our previously published tumor-targeted liposomal formulation to develop a rational combination of E and Y in a single liposomal formulation (EY-L) and assessed its efficacy in RCC cell lines in vitro and in RCC tumors in vivo. We further investigated how well EY-L sensitizes RCC cell lines and tumors toward radiation and explored the underlying mechanism of radiosensitization. RESULTS: EY-L outperformed the corresponding single drug-loaded formulations E-L and Y-L in terms of containing primary tumor growth and improving survival in an immunocompetent syngeneic mouse model of RCC. EY-L also exhibited significantly higher sensitization of RCC cells towards radiation in vitro than E-L and Y-L. Additionally, EY-L sensitized RCC tumors towards radiation therapy in xenograft and murine RCC models. EY-L mediated induction of mitotic catastrophe via downregulation of multiple cell cycle checkpoints and DNA damage repair pathways could be responsible for the augmentation of radiation therapy. CONCLUSION: Taken together, our study demonstrated the efficacy of a strategic combination therapy in sensitizing RCC to radiation therapy via inhibition of DNA damage repair and a substantial increase in mitotic catastrophe. This combination therapy may find its use in the augmentation of radiation therapy during the treatment of RCC patients.
Asunto(s)
Carcinoma de Células Renales , Reparación del ADN , Neoplasias Renales , Survivin , Serina-Treonina Quinasas TOR , Ensayos Antitumor por Modelo de Xenoinjerto , Carcinoma de Células Renales/patología , Carcinoma de Células Renales/radioterapia , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/metabolismo , Animales , Survivin/metabolismo , Humanos , Ratones , Línea Celular Tumoral , Neoplasias Renales/patología , Neoplasias Renales/radioterapia , Neoplasias Renales/tratamiento farmacológico , Reparación del ADN/efectos de los fármacos , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo , Mitosis/efectos de los fármacos , Mitosis/efectos de la radiación , Imidazoles/farmacología , Daño del ADN , Everolimus/farmacología , Naftoquinonas/farmacología , Naftoquinonas/uso terapéutico , Fármacos Sensibilizantes a Radiaciones/farmacología , Fármacos Sensibilizantes a Radiaciones/uso terapéutico , Liposomas/farmacología , Inhibidores mTOR/farmacología , Inhibidores mTOR/uso terapéuticoRESUMEN
Purpose: Tumor treating fields (TTFields) is a novel antimitotic treatment that was first proven effective for glioblastoma multiforme, now with trials for several extracranial indications underway. Several studies focused on concurrent TTFields therapy with radiation in the same time period, but were not given simultaneously. This study evaluates the targeting accuracy of simultaneous radiation therapy while TTFields arrays are in place and powered on, ensuring that radiation does not interfere with TTFields and TTFields does not interfere with radiation. This is one of several options to enable TTFields to begin several weeks sooner, and opens potential for synergistic effects of combined therapy. Methods: TTFields arrays were attached to a warm saline water bath and salt was added until the TTFields generator reached the maximal 2000â mA peak-to-peak current. A ball cube phantom containing 2 orthogonal films surrounded by fiducials was placed in the water phantom, CT scanned, and a radiation treatment plan with 58 isocentric beams was created using a 3â cm circular collimator. Fiducial tracking was used to deliver radiation, the films were scanned, and end-to-end targeting error was measured with vendor-supplied software. In addition, radiation effects on electric fields generated by the TTFields system were assessed by examining logfiles generated from the field generator. Results: With TTFields arrays in place and powered on, the robotic radiosurgery system achieved a final targeting result of 0.47â mm, which was well within the submillimeter specification. No discernible effects on TTFields current output beyond 0.3% were observed in the logfiles when the radiation beam pulsed on and off. Conclusion: A robotic radiosurgery system was used to verify that radiation targeting was not adversely affected when the TTFields arrays were in place and the TTFields delivery device was powered on. In addition, this study verified that radiation delivered simultaneously with TTFields did not interfere with the generation of the electric fields.
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Neoplasias Encefálicas/radioterapia , Glioblastoma/radioterapia , Radioterapia/métodos , Terapia Combinada/métodos , Marcadores Fiduciales , Cabeza , Humanos , Mitosis/efectos de la radiación , Fantasmas de Imagen , Hipofraccionamiento de la Dosis de Radiación , Radiocirugia/instrumentación , Planificación de la Radioterapia Asistida por Computador , RobóticaRESUMEN
Mitotic cell fusion induced Premature Chromosome Condensation (G0-PCC) assay in human lymphocytes allows rapid detection of cytogenetic damage in interphase stage, within few hours after blood collection. Hence, it is the most suitable method for rapid and high dose biodosimetry. Mitotic cells, used for G0-PCC could be either freshly isolated or previously cryo-preserved. However, under emergency scenarios, only cryo-preserved cells can be relied upon, fresh isolation will only delay the process by 18-24 h. Impact of cryopreservation on mitotic cells and their efficacy to induce PCC are not reported. In the present study, we investigated effect of cryopreservation on mitotic cells and refined the parameters for G0-PCC. More than 95% of the cells were recoverable after 4 months of cryopreservation, within 20 min recovery at 37 °C, without significant change in the mitotic index or viability. Recovered mitotic cells have shown mitotic index of 89 ± 4% and viability of 90 ± 4%, similar to that of freshly isolated cells. Decrease in metaphases was observed within 40 min after recovery as the mitotic cells progressed through cell cycle and reduced to 21% at 1.5 h. Nevertheless, in presence of Colcemid, the cells progressed slowly and considerably high metaphase index (60%) persisted up to ~ 2 h. The recovered cells efficiently fused with lymphocytes and induced PCC. Average PCC index varied from 10 to 20%, which did not change with cryopreservation duration. Post fusion incubation duration of 2 h was found to be optimum for proper chromosome condensation. In conclusion, use of cryo-preserved mitotic cells is the most practical approach for rapid biodosimetry. The cells can be recovered quickly and efficiently without alteration in viability or mitotic index. Recovered cells are fully competent to induce G0-PCC.
Asunto(s)
Aberraciones Cromosómicas/efectos de la radiación , Cromosomas Humanos , Criopreservación , Rayos gamma/efectos adversos , Linfocitos/metabolismo , Mitosis/efectos de la radiación , Humanos , Linfocitos/patología , Dosis de Radiación , RadiometríaRESUMEN
Aberrant cell proliferation is a hallmark of cancer, including glioblastoma (GBM). Here we report that protein arginine methyltransferase (PRMT) 6 activity is required for the proliferation, stem-like properties, and tumorigenicity of glioblastoma stem cells (GSCs), a subpopulation in GBM critical for malignancy. We identified a casein kinase 2 (CK2)-PRMT6-regulator of chromatin condensation 1 (RCC1) signaling axis whose activity is an important contributor to the stem-like properties and tumor biology of GSCs. CK2 phosphorylates and stabilizes PRMT6 through deubiquitylation, which promotes PRMT6 methylation of RCC1, which in turn is required for RCC1 association with chromatin and activation of RAN. Disruption of this pathway results in defects in mitosis. EPZ020411, a specific small-molecule inhibitor for PRMT6, suppresses RCC1 arginine methylation and improves the cytotoxic activity of radiotherapy against GSC brain tumor xenografts. This study identifies a CK2α-PRMT6-RCC1 signaling axis that can be therapeutically targeted in the treatment of GBM.
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Neoplasias Encefálicas , Carcinogénesis , Proteínas de Ciclo Celular , Glioblastoma , Factores de Intercambio de Guanina Nucleótido , Mitosis/efectos de la radiación , Proteínas de Neoplasias , Proteínas Nucleares , Proteína-Arginina N-Metiltransferasas , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/radioterapia , Carcinogénesis/genética , Carcinogénesis/metabolismo , Carcinogénesis/efectos de la radiación , Quinasa de la Caseína II/genética , Quinasa de la Caseína II/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Femenino , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/radioterapia , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Células HEK293 , Humanos , Masculino , Ratones , Mitosis/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína-Arginina N-Metiltransferasas/genética , Proteína-Arginina N-Metiltransferasas/metabolismo , Transducción de Señal/genética , Transducción de Señal/efectos de la radiación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Enhanced UV-B radiation can lead to a variety of stress responses, including effects on cell cycle regulation and mitosis. Aurora kinases are part of the serine/threonine kinase family and play important roles in cell cycle regulation and mitosis. We hypothesize that there may be a connection between these two processes. In this study, the dynamics of chromosomal (H2B-YFP) and AUR1-GFP changes after enhanced UV-B radiation were observed using confocal microscopy, and gene and protein expression patterns under UV-B stress were quantified using RT-qPCR and Western blotting techniques. We analyzed the responses of the AUR1 overexpression to UV-B stress. We measured maximum quantum yield of photosystem â ¡ as a proxy for UV-B stress. The recovery capacity of AUR1 overexpression strains was analyzed. In our research, we observed that enhanced UV-B radiation affects the subcellular positioning of AUR1, resulting in abnormalities in the positioning and location of the spindle at the poles, which ultimately affects the separation of chromosomes, resulting in "partition-bundle division" and the incorrect direction of division. At the same time, our results also indicated that low-dose UV-B can induce the expression of AUR1, and this overexpression of AUR1 can alleviate the damage caused by UV-B radiation. In summary, the results of our study show that enhanced UV-B radiation can change the activity and expression of AUR1, which is one of the causes of abnormal chromosome segregation. AUR1 participates in the response to UV-B stress, and, to a certain extent, can improve the UV-B tolerance of plants.
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Proteínas de Arabidopsis , Arabidopsis , Regulación de la Expresión Génica de las Plantas , Mitosis , Proteínas Serina-Treonina Quinasas , Rayos Ultravioleta , Arabidopsis/genética , Arabidopsis/efectos de la radiación , Proteínas de Arabidopsis/genética , Segregación Cromosómica/genética , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Mitosis/genética , Mitosis/efectos de la radiación , Proteínas Serina-Treonina Quinasas/genética , Huso Acromático/genéticaRESUMEN
This study aims to investigate whether ionizing radiation combined with doxorubicin-conjugated iron oxide nanoparticles (NP-DOX) improves the internalization and cytotoxic effects of the nano-carrier-mediated drug delivery in MG-63 human osteosarcoma cells. NP-DOX was designed and synthesized using the co-precipitation method. Highly stable and crystalline nanoparticles conjugated with DOX were internalized in MG-63 cells through macropinocytosis and located in the perinuclear area. Higher nanoparticles internalization in MG-63 cells previously exposed to 1 Gy X-rays was correlated with an early accumulation of cells in G2/M, starting at 12 h after treatment. After 48 h, the application of the combined treatment led to higher cytotoxic effects compared to the individual treatment, with a reduction in the metabolic capacity and unrepaired DNA breaks, whilst a low percent of arrested cells, contributing to the commitment of mitotic catastrophe. NP-DOX showed hemocompatibility and no systemic cytotoxicity, nor histopathological alteration of the main organs.
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Doxorrubicina/farmacología , Sistemas de Liberación de Medicamentos , Nanopartículas/química , Osteosarcoma/tratamiento farmacológico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Terapia Combinada , Doxorrubicina/química , Endocitosis/efectos de los fármacos , Endocitosis/efectos de la radiación , Compuestos Férricos/química , Compuestos Férricos/farmacología , Humanos , Mitosis/efectos de los fármacos , Mitosis/efectos de la radiación , Osteosarcoma/patología , Osteosarcoma/radioterapia , Radiación IonizanteRESUMEN
Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase that serves as a key regulator of cellular physiology in the context of apoptosis, mitosis, and DNA damage responses. Canonically, PP2A functions as a tumor suppressor gene. However, recent evidence suggests that inhibiting PP2A activity in tumor cells may represent a viable approach to enhancing tumor sensitivity to chemoradiotherapy as such inhibition can cause cells to enter a disordered mitotic state that renders them more susceptible to cell death. Indeed, there is evidence that inhibiting PP2A can slow tumor growth following radiotherapy in a range of cancer types including ovarian cancer, liver cancer, malignant glioma, pancreatic cancer, and nasopharyngeal carcinoma. In the present review, we discuss current understanding of the role of PP2A in tumor radiotherapy and the potential mechanisms whereby it may influence this process.
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Neoplasias/genética , Neoplasias/metabolismo , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/metabolismo , Animales , Apoptosis/genética , Apoptosis/efectos de la radiación , Puntos de Control del Ciclo Celular/genética , Puntos de Control del Ciclo Celular/efectos de la radiación , Daño del ADN/genética , Daño del ADN/efectos de la radiación , Humanos , Mitosis/genética , Mitosis/efectos de la radiación , Neoplasias/patología , Neoplasias/radioterapia , Tolerancia a Radiación/genética , Radioterapia , Resultado del TratamientoRESUMEN
Understanding the mitotic DNA damage response (DDR) is critical to our comprehension of cancer, premature aging and developmental disorders which are marked by DNA repair deficiencies. In this study we use a micro-focused laser to induce DNA damage in selected mitotic chromosomes to study the subsequent repair response. Our findings demonstrate that (1) mitotic cells are capable of DNA repair as evidenced by DNA synthesis at damage sites, (2) Repair is attenuated when DNA-PKcs and ATM are simultaneously compromised, (3) Laser damage may permit the observation of previously undetected DDR proteins when damage is elicited by other methods in mitosis, and (4) Twenty five percent of mitotic DNA-damaged cells undergo a subsequent mitosis. Together these findings suggest that mitotic DDR is more complex than previously thought and may involve factors from multiple repair pathways that are better understood in interphase.
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Roturas del ADN/efectos de la radiación , Reparación del ADN , ADN/biosíntesis , Fase G1/genética , Mitosis/genética , Animales , Línea Celular , ADN/genética , ADN/efectos de la radiación , Fase G1/efectos de la radiación , Humanos , Rayos Infrarrojos/efectos adversos , Rayos Láser/efectos adversos , Mitosis/efectos de la radiación , PotoroidaeRESUMEN
Background Management of locoregionally recurrent head and neck squamous cell carcinomas (HNSCC) is challenging due to potential radioresistance. Pulsed low-dose rate (PLDR) irradiation exploits phenomena of increased radiosensitivity, low-dose hyperradiosensitivity (LDHRS), and inverse dose-rate effect. The purpose of this study was to evaluate LDHRS and the effect of PLDR irradiation in isogenic HNSCC cells with different radiosensitivity. Materials and methods Cell survival after different irradiation regimens in isogenic parental FaDu and radioresistant FaDu-RR cells was determined by clonogenic assay; post irradiation cell cycle distribution was studied by flow cytometry; the expression of DNA damage signalling genes was assesed by reverse transcription-quantitative PCR. Results Radioresistant Fadu-RR cells displayed LDHRS and were more sensitive to PLDR irradiation than parental FaDu cells. In both cell lines, cell cycle was arrested in G2/M phase 5 hours after irradiation. It was restored 24 hours after irradiation in parental, but not in the radioresistant cells, which were arrested in G1-phase. DNA damage signalling genes were under-expressed in radioresistant compared to parental cells. Irradiation increased DNA damage signalling gene expression in radioresistant cells, while in parental cells only few genes were under-expressed. Conclusions We demonstrated LDHRS in isogenic radioresistant cells, but not in the parental cells. Survival of LDHRS-positive radioresistant cells after PLDR was significantly reduced. This reduction in cell survival is associated with variations in DNA damage signalling gene expression observed in response to PLDR most likely through different regulation of cell cycle checkpoints.
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Neoplasias de Cabeza y Cuello/radioterapia , Recurrencia Local de Neoplasia/radioterapia , Tolerancia a Radiación , Carcinoma de Células Escamosas de Cabeza y Cuello/radioterapia , Ciclo Celular/efectos de la radiación , Línea Celular Tumoral , Supervivencia Celular/efectos de la radiación , Daño del ADN/genética , Fase G1/efectos de la radiación , Fase G2/efectos de la radiación , Expresión Génica , Humanos , Mitosis/efectos de la radiación , Dosificación Radioterapéutica , Factores de Tiempo , Ensayo de Tumor de Célula Madre/métodosRESUMEN
PURPOSE: Esophageal cancer is a deadly malignancy with a 5-year survival rate of only 5% to 20%, which has remained unchanged for decades. Esophageal cancer possesses a high frequency of TP53 mutations leading to dysfunctional G1 cell-cycle checkpoint, which likely makes esophageal cancer cells highly reliant upon G2-M checkpoint for adaptation to DNA replication stress and DNA damage after radiation. We aim to explore whether targeting Wee1 kinase to abolish G2-M checkpoint sensitizes esophageal cancer cells to radiotherapy. EXPERIMENTAL DESIGN: Cell viability was assessed by cytotoxicity and colony-forming assays, cell-cycle distribution was analyzed by flow cytometry, and mitotic catastrophe was assessed by immunofluorescence staining. Human esophageal cancer xenografts were generated to explore the radiosensitizing effect of AZD1775 in vivo. RESULTS: The IC50 concentrations of AZD1775 on esophageal cancer cell lines were between 300 and 600 nmol/L. AZD1775 (100 nmol/L) as monotherapy did not alter the viability of esophageal cancer cells, but significantly radiosensitized esophageal cancer cells. AZD1775 significantly abrogated radiation-induced G2-M phase arrest and attenuation of p-CDK1-Y15. Moreover, AZD1775 increased radiation-induced mitotic catastrophe, which was accompanied by increased γH2AX levels, and subsequently reduced survival after radiation. Importantly, AZD1775 in combination with radiotherapy resulted in marked tumor regression of esophageal cancer tumor xenografts. CONCLUSIONS: Abrogation of G2-M checkpoint by targeting Wee1 kinase with AZD1775 sensitizes esophageal cancer cells to radiotherapy in vitro and in mouse xenografts. Our findings suggest that inhibition of Wee1 by AZD1775 is an effective strategy for radiosensitization in esophageal cancer and warrants clinical testing.
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Proteínas de Ciclo Celular/antagonistas & inhibidores , Quimioradioterapia/métodos , Neoplasias Esofágicas/terapia , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirazoles/farmacología , Pirimidinonas/farmacología , Fármacos Sensibilizantes a Radiaciones/farmacología , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Neoplasias Esofágicas/patología , 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 , Concentración 50 Inhibidora , Masculino , Ratones , Mitosis/efectos de los fármacos , Mitosis/efectos de la radiación , Pirazoles/uso terapéutico , Pirimidinonas/uso terapéutico , Fármacos Sensibilizantes a Radiaciones/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In order to preserve genome integrity and their ploidy, cells must ensure that the duplicated genome has been faithfully replicated and evenly distributed before they complete their division by mitosis. To this end, cells have developed highly elaborated checkpoints that halt mitotic progression when problems in DNA integrity or chromosome segregation arise, providing them with time to fix these issues before advancing further into the cell cycle. Remarkably, exit from mitosis constitutes a key cell cycle transition that is targeted by the main mitotic checkpoints, despite these surveillance mechanisms being activated by specific intracellular signals and acting at different stages of cell division. Focusing primarily on research carried out using Saccharomyces cerevisiae as a model organism, the aim of this review is to provide a general overview of the molecular mechanisms by which the major cell cycle checkpoints control mitotic exit and to highlight the importance of the proper regulation of this process for the maintenance of genome stability during the distribution of the duplicated chromosomes between the dividing cells.
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Proteínas de Ciclo Celular/metabolismo , Daño del ADN/genética , Puntos de Control de la Fase M del Ciclo Celular/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Huso Acromático/metabolismo , Puntos de Control del Ciclo Celular/genética , Puntos de Control del Ciclo Celular/efectos de la radiación , Proteínas de Ciclo Celular/genética , Daño del ADN/efectos de la radiación , Mitosis/genética , Mitosis/efectos de la radiación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Saccharomyces cerevisiae/fisiología , Huso Acromático/genética , Quinasa Tipo Polo 1RESUMEN
Human oxidation resistance 1 (OXR1) was identified as a protein that decreases genomic mutations in Escherichia coli caused by oxidative DNA damage. However, the mechanism by which OXR1 defends against genome instability has not been elucidated. To clarify how OXR1 maintains genome stability, the effects of OXR1-depletion on genome stability were investigated in OXR1-depleted HeLa cells using gamma-rays (γ-rays). The OXR1-depleted cells had higher levels of superoxide and micronucleus (MN) formation than control cells after irradiation. OXR1-overexpression alleviated the increases in reactive oxygen species (ROS) level and MN formation after irradiation. The increased MN formation in irradiated OXR1-depleted cells was partially attenuated by the ROS inhibitor N-acetyl-L-cysteine, suggesting that OXR1-depeletion increases ROS-dependent genome instability. We also found that OXR1-depletion shortened the duration of γ-ray-induced G2/M arrest. In the presence of the cell cycle checkpoint inhibitor caffeine, the level of MN formed after irradiation was similar between control and OXR1-depleted cells, demonstrating that OXR1-depletion accelerates MN formation through abrogation of G2/M arrest. In OXR1-depleted cells, the level of cyclin D1 protein expression was increased. Here we report that OXR1 prevents genome instability by cell cycle regulation as well as oxidative stress defense.
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Puntos de Control de la Fase G2 del Ciclo Celular/efectos de la radiación , Rayos gamma , Inestabilidad Genómica/efectos de la radiación , Proteínas Mitocondriales/metabolismo , Mitosis/efectos de la radiación , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Inestabilidad Genómica/efectos de los fármacos , Células HeLa , Humanos , Peróxido de Hidrógeno/toxicidad , Micronúcleo Germinal/efectos de los fármacos , Micronúcleo Germinal/metabolismo , Micronúcleo Germinal/efectos de la radiación , Proteínas Mitocondriales/deficiencia , Mitosis/efectos de los fármacos , Modelos Biológicos , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/efectos de la radiación , Superóxidos/metabolismoRESUMEN
Mitochondrial dynamics are crucial for cellular survival in response to various stresses. Previously, we reported that Drp1 promoted mitochondrial fission after x-irradiation and its inhibition resulted in reduced cellular radiosensitivity and mitotic catastrophe. However, the mechanisms of radiation-induced mitotic catastrophe related to mitochondrial fission remain unclear. In this study, we investigated the involvement of cellular ATP production, ROS generation, and Ca2+ levels in mitotic catastrophe in EMT6 cells. Knockdown of Drp1 and Fis1, which are mitochondrial fission regulators, resulted in elongated mitochondria and significantly attenuated cellular radiosensitivity. Reduced mitochondrial fission mainly decreased mitotic catastrophe rather than necrosis and apoptosis after irradiation. Cellular ATP contents in Drp1 and Fis1 knockdown cells were similar to those in control cells. N-acetylcysteine and 2-glucopyranoside ascorbic acid have no effect on mitotic catastrophe after irradiation. The cellular [Ca2+]i level increased after irradiation, which was completely suppressed by Drp1 and Fis1 inhibition. Furthermore, BAPTA-AM significantly reduced radiation-induced mitotic catastrophe, indicating that cellular Ca2+ is a key mediator of mitotic catastrophe induction after irradiation. These results suggest that mitochondrial fission is associated with radiation-induced mitotic catastrophe via cytosolic Ca2+ regulation.
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Neoplasias de la Mama/metabolismo , Calcio/metabolismo , Dinámicas Mitocondriales , Adenosina Trifosfato/metabolismo , Animales , Neoplasias de la Mama/patología , Neoplasias de la Mama/radioterapia , Línea Celular Tumoral , Femenino , Ratones , Dinámicas Mitocondriales/efectos de la radiación , Mitosis/efectos de la radiación , Tolerancia a Radiación , Especies Reactivas de Oxígeno/metabolismo , Rayos XRESUMEN
Peripheral nerve damage, such as that found after surgery or trauma, is a substantial clinical challenge. Much research continues in attempts to improve outcomes after peripheral nerve damage and to promote nerve repair after injury. In recent years, low-intensity pulsed ultrasound (LIPUS) has been studied as a potential method of stimulating peripheral nerve regeneration. In this review, the physiology of peripheral nerve regeneration is reviewed, and the experiments employing LIPUS to improve peripheral nerve regeneration are discussed. Application of LIPUS following nerve surgery may promote nerve regeneration and improve functional outcomes through a variety of proposed mechanisms. These include an increase of neurotrophic factors, Schwann cell (SC) activation, cellular signaling activations, and induction of mitosis. We searched PubMed for articles related to these topics in both in vitro and in vivo animal research models. We found numerous studies, suggesting that LIPUS following nerve surgery promotes nerve regeneration and improves functional outcomes. Based on these findings, LIPUS could be a novel and valuable treatment for nerve injury-induced erectile dysfunction.
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Disfunción Eréctil/terapia , Regeneración Nerviosa , Pene/inervación , Traumatismos de los Nervios Periféricos/terapia , Nervio Pudendo/lesiones , Terapia por Ultrasonido/métodos , Animales , Proliferación Celular/efectos de la radiación , Supervivencia Celular/efectos de la radiación , Disfunción Eréctil/etiología , Humanos , Masculino , Mitosis/efectos de la radiación , Factores de Crecimiento Nervioso , Traumatismos de los Nervios Periféricos/complicaciones , Células de Schwann/efectos de la radiación , Transducción de Señal , Ondas UltrasónicasRESUMEN
OBJECTIVE: To study the frequency and spectrum of chromosome aberrations in human peripheral blood lymphocytes depending on the radiation dose, the stage of the mitotic cycle and the periods of fixation of the cell culture. MATERIALS AND METHODS: The test system of blood lymphocytes of donors, the metaphase analysis of chromosome aberrations (a uniform staining of the drugs). The gamma-irradiation was performed on the device with the source 60Co at the dose rate of 0.5 Gy/min, the dose range was 0.25-4.0 Gy. The lymphocyte culture was irradiated after 0, 24, 40 and 48 hours from the beginning of the incubation, which corresponds to G0-, G1-, S-, G2-stages of the mitot- ic cycle. The cells were fixed after 52 hours and 62 hours from the beginning of the incubation. RESULTS: The author's experimental data on the regularities of chromosome aberrations formation during irradiation at the different periods of the mitotic cycle of human lymphocyte culture are presented. The character of the dose dependences of structural damages of chromosomes during the mitotic cycle with an application of the linear, lin- ear-square and parabolic models is analyzed. The greatest yield of exchange-type aberrations is registered at the irradiation in G0- and G1-stages of the mitotic cycle, which submits to the linear-square dependence on an irradia- tion dose. When irradiating cells in the S- and G2-stages, the main contribution to the spectrum of radiation-induced chromosome aberrations is made by deletions, the level of which increases linearly with the dose. This is evidenced by the negative values of the quadratic term in the regression equations for these stages. The analysis of the curves using the parabolic model Y = k · Dn has shown that, at the irradiation of lymphocytes in G0- and G1-stages, the dose dependences approach the quadratic ones, which confirms the reasonableness of the interpretation of the obtained cytogenetic data from the point of view of the «classical¼ theory of the radiation-induced chromosome aberrations formation. However, the model of the spline regression is more accurate at the approximation of the dependences of the cytogenetic effects in the region of low doses of irradiation. CONCLUSIONS: The results obtained by the irradiation of human lymphocyte culture at different stages of the mitot- ic cycle indicate that the same radiation dose induces different qualitative and quantitative cytogenetic effects depending on the physiological state of the cell at the time of irradiation. The combination of such factors as a radi- ation dose rate, a mitotic cycle stage, the post-irradiation conditions, and an individual radiosensitivity are reflect- ed in quantitative and qualitative variations of the cellular radiosensitivity.
Asunto(s)
Aberraciones Cromosómicas/efectos de la radiación , Rayos gamma , Linfocitos/efectos de la radiación , Mitosis/efectos de la radiación , Técnicas de Cultivo de Célula , Células Cultivadas , Radioisótopos de Cobalto , Análisis Citogenético , Relación Dosis-Respuesta en la Radiación , Humanos , Dosis de RadiaciónRESUMEN
18F-FDG PET/CT imaging is used in the diagnosis of diseases, including cancers. The principal photons used for imaging are 511â¯keâ¯V gamma photons resulting from positron annihilation. The absorbed dose varies among body organs, depending on administered radioactivity and biological clearance. We have attempted to evaluate DNA double-strand breaks (DSB) and toxicity induced in V79 lung fibroblast cells in vitro by 18F-FDG, at doses which might result from PET procedures. Cells were irradiated by 18F-FDG at doses (14.51 and 26.86â¯mGy), comparable to absorbed doses received by critical organs during PET procedures. The biological endpoints measured were formation of γ-H2AX foci, mitochondrial stress, chromosomal aberrations, and cell cycle perturbation. Irradiation induced DSB (γH2AX assay), mitochondrial depolarization, and both chromosome and chromatid types of aberrations. At higher radiation doses, increased aneuploidy and reduced mitotic activity were also seen. Thus, significant biological effects were observed at the doses delivered by the 18F-FDG exposure and the effects increased with dose.
Asunto(s)
Aberraciones Cromosómicas , Daño del ADN , Fibroblastos/efectos de la radiación , Radioisótopos de Flúor/toxicidad , Fluorodesoxiglucosa F18/toxicidad , Rayos gamma/efectos adversos , Radiofármacos/toxicidad , Aneuploidia , Animales , Bencimidazoles , Carbocianinas , Ciclo Celular/efectos de la radiación , Línea Celular , Cromátides/efectos de la radiación , Cromátides/ultraestructura , Cromosomas/efectos de la radiación , Cromosomas/ultraestructura , Cricetulus , Roturas del ADN de Doble Cadena , Reparación del ADN , Relación Dosis-Respuesta en la Radiación , Fibroblastos/ultraestructura , Histonas/genética , Cariotipificación , Pulmón/citología , Masculino , Potencial de la Membrana Mitocondrial/efectos de la radiación , Mitosis/efectos de la radiaciónRESUMEN
Recent studies suggest that cancer cell death accompanied by organelle dysfunction might be a promising approach for cancer therapy. The Golgi apparatus has a key role in cell function and may initiate signaling pathways to mitigate stress and, if irreparable, start apoptosis. It has been shown that Golgi disassembly and fragmentation under oxidative stress act as indicators for stress-mediated cell death pathways through cell cycle arrest in the G2/M phase. The present study shows that UV-induced reactive oxygen species (ROS) generation by Ag@ZnO nanoparticles (NPs) transform the Golgi structures from compressed perinuclear ribbons into detached vesicle-like structures distributed in the entire cytoplasm of melanoma cells. This study also demonstrates that Ag@ZnO NP-induced Golgi fragmentation cooccurs with G2 block of cell cycle progression, preventing cells from entering the mitosis phase. Additionally, the increased intracellular ROS production triggered by Ag@ZnO NPs upon UV exposure promoted autophagy. Taken together, Ag@ZnO NPs induce stress-related Golgi fragmentation and autophagy, finally leading to melanoma cell apoptosis. Intracellular oxidative stress generated by Ag@ZnO NPs upon UV irradiation may thus represent a targeted approach to induce cancer cell death through organelle destruction in melanoma cells, while fibroblast cells remained largely unaffected.
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Proliferación Celular/efectos de los fármacos , Aparato de Golgi/efectos de los fármacos , Melanoma/tratamiento farmacológico , Estrés Oxidativo/efectos de los fármacos , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Autofagia/efectos de los fármacos , Autofagia/efectos de la radiación , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de la radiación , Línea Celular Tumoral , Proliferación Celular/efectos de la radiación , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Aparato de Golgi/genética , Humanos , Melanoma/genética , Melanoma/patología , Nanopartículas del Metal/química , Nanopartículas del Metal/uso terapéutico , Mitosis/efectos de los fármacos , Mitosis/efectos de la radiación , Especies Reactivas de Oxígeno/química , Transducción de Señal/efectos de los fármacos , Transducción de Señal/efectos de la radiación , Plata/química , Plata/farmacología , Rayos Ultravioleta , Óxido de Zinc/química , Óxido de Zinc/farmacologíaRESUMEN
Chromosome analysis is a fundamental technique for a wide range of cytogenetic studies. Chromosome aberrations are easily introduced by many kinds of clastogenic agents such as ionizing irradiation, UV, or alkylating agents, and damaged chromosomes may be prone to cancer. Chromosomes are conventionally prepared from mitotic cells arrested by the colcemid block method. However, obtaining of mitotic chromosomes is sometimes hampered under several circumstances, for example after high-dose (over several Gys of γ-rays) ionizing irradiation exposure accident. As a result, cytogenetic analysis will be often difficult or even impossible in such cases. Premature chromosome condensation (PCC) is an alternative technique that has proved to be a unique and useful way in chromosome analysis. Previously, PCC has been achieved following cell fusion mediated either by fusogenic viruses (for example Sendai virus) or by polyethylene glycol (PEG) (cell-fusion PCC), but the cell-fusion PCC has several drawbacks. The novel drug-induced PCC use of specific inhibitors for serine/threonine protein phosphatase was introduced about 20 years ago. This method is much simple and easy even than the conventional mitotic chromosome preparation using colcemid block protocol and the obtained PCC index (equivalent to mitotic index for metaphase chromosome) is much higher. Furthermore, this method allows the interphase chromatin to be condensed and visualized like mitotic chromosomes, and thus has been opening the way for chromosome analysis not only in metaphase chromosomes but also in interphase chromatin. The drug-induced PCC has therefore proven the usefulness in cytogenetics and other many cell biology fields. Since the first version of drug-induced PCC protocol has been published in 2009 (Gotoh, Methods in molecular biology. Humana Press, New York, 2009), many newer applications of drug-induced PCC in radiation biology and chromosome science fields in a wide range of species from animal to plant have been reported (Gotoh et al., Biomed Res 16:63-68, 1995; Lamadrid Boada et al., Mutat Res 757:45-51, 2013; Ravi et al., Biochimie 95:124-33, 2013; Ono et al., J Cell Biol 200:429-41, 2013; Vagnarelli, Exp Cell Res 318:1435-41, 2012; Roukos et al., Nat Protoc 9:2476-92, 2014; Miura and Blakely, Cytometry A 79:1016-22, 2013; Zabka et al., J Plant Physiol 174:62-70, 2015; Samaniego et al., Planta 215:195-204, 2002; Rybaczek et al., Folia Histochem Cytobiol 40:51-9, 2002; Gotoh and Durante J Cell Physiol 209:297-304, 2006). Therefore as a new edition, I will write in this chapter the drug-induced PCC technique with newer findings, in particular focused drug-induced PCC protocols in radiation biology with referring updated articles published recently.
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Cromatina/metabolismo , Análisis Citogenético/métodos , Fase G2/efectos de la radiación , Interfase/efectos de la radiación , Mitosis/efectos de la radiación , Preparaciones Farmacéuticas/metabolismo , Radiobiología/métodos , Animales , Adhesión Celular/efectos de la radiación , Células Cultivadas , Aberraciones Cromosómicas/efectos de la radiación , Reparación del ADN/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Humanos , Cinética , Radiación IonizanteRESUMEN
Despite the success for the treatment of melanoma such as targeted molecular therapy, the use of such treatments are expensive For this reason, this study was carried out to explore the anti-cancer properties of available drugs that are able to modify the melanoma prognosis. The study was conducted in two phases: Evaluation of pharmacological effects of pentoxifylline (PTX) administered above (60â¯mg/kg) which is the therapeutic dose that is aimed at reducing the side-effect of radiotherapy, and of α- galactosylceramide (GalCer) administered at 100⯵g/kg, as well as their combination using a murine model (BDF1 mice) of melanoma cell line (B16-F1, ATCC). For the radiotherapy phase, 9â¯Gy was applied in the tumor area, before (3 days), during (30â¯min) and after (3 days) the PTXâ¯+â¯GalCer treatment. In both study phases, the mitosis rate, leukocyte infiltration and necro-apoptosis were assessed using histological and immunohistochemical approach and tumor volume evaluation as biomarkers. All treatments showed good prognosis results estimated as reduction of mitosis rate (PTXâ¯+â¯GalCer after radiotherapy and GalCer), increased leukocyte infiltrate (PTXâ¯+â¯GalCer after radiotherapy and GalCer) and necro-apoptosis augmentation (PTXâ¯+â¯GalCer after radiotherapy and radiotherapy control). Nevertheless, a lower development of tumor volume was found in GalCer treatment. In this way, it is possible to suggest that the integrated treatment with immuno-stimulators such as GalCer, plus drug used for peripheral vascular disease (PTX) after radiotherapy is probably an alternative for controlling aggressive melanoma in murine model.
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Apoptosis , Quimioradioterapia , Galactosilceramidas/farmacología , Leucocitos , Melanoma Experimental , Mitosis , Pentoxifilina/farmacología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Línea Celular Tumoral , Leucocitos/metabolismo , Leucocitos/patología , Masculino , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Melanoma Experimental/terapia , Ratones , Mitosis/efectos de los fármacos , Mitosis/efectos de la radiaciónRESUMEN
The binding of DNA-dependent protein kinase catalytic subunit (DNA-PKcs, also known as PRKDC) to Ku proteins at DNA double-strand breaks (DSBs) has long been considered essential for non-homologous end joining (NHEJ) repair, providing a rationale for use of DNA-PKcs inhibitors as cancer therapeutics. Given lagging clinical translation, we reexamined mechanisms and observed instead that DSB repair can proceed independently of DNA-PKcs. While repair of radiation-induced DSBs was blocked in cells expressing shRNAs targeting Ku proteins or other NHEJ core factors, DSBs were repaired on schedule despite targeting DNA-PKcs. Although we failed to observe a DSB repair defect, the γH2AX foci that formed at sites of DNA damage persisted indefinitely after irradiation, leading to cytokinesis failure and accumulation of binucleated cells. Following this mitotic slippage, cells with decreased DNA-PKcs underwent accelerated cellular senescence. We identified downregulation of ataxia-telangiectasia mutated kinase (ATM) as the critical role of DNA-PKcs in recovery from DNA damage, insofar as targeting ATM restored γH2AX foci resolution and cytokinesis. Considering the lack of direct impact on DSB repair and emerging links between senescence and resistance to cancer therapy, these results suggest reassessing DNA-PKcs as a target for cancer treatment.