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In patients with non-resectable hepatic malignancies selective internal radiotherapy (SIRT) with yttrium-90 is an effective therapy. However, previous data indicate that SIRT leads to impaired immune function. The aim of the current study was to determine the extent of DNA lesions in peripheral blood mononuclear cells of SIRT patients and to correlate these lesions with cellular immune responses. In ten patients γH2AX and 53BP1 foci were determined. These foci are markers of DNA double-strand breaks (DSBs) and occur consecutively. In parallel, lymphocyte proliferation was assessed after stimulation with the T cell mitogen phytohemagglutinin. Analyses of vital cells were performed prior to and 1 h and 1 week after SIRT. 1 h and 1 week after SIRT numbers of γH2AX and of 53BP1 foci were more than threefold larger than before (p < 0.01). Already at baseline, foci were more abundant than published in healthy controls. Lymphocyte proliferation at baseline was below the normal range and further decreased after SIRT. Prior to therapy, there was an inverse correlation between lymphocyte proliferation and the quotient 53BP1/γH2AX; which could be considered as a measure of the course of DNA DSB repair (r = - 0.94, p < 0.0001). Proliferative responses were inversely correlated with 53BP1 foci prior to therapy and γH2AX and 53BP1 foci 1 h after therapy (r < - 0.65, p < 0.05). In conclusion, DNA foci in SIRT patients were correlated with impaired in vitro immune function. Unrepaired DNA DSBs or cell cycle arrest due to repair may cause this impairment.
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Braquiterapia/métodos , Roturas del ADN de Doble Cadena/efectos de la radiación , Reparación del ADN , Linfocitos/efectos de la radiación , Anciano , Anciano de 80 o más Años , Braquiterapia/efectos adversos , Puntos de Control del Ciclo Celular/genética , Puntos de Control del Ciclo Celular/efectos de la radiación , Proliferación Celular/genética , Proliferación Celular/efectos de la radiación , Femenino , Histonas/metabolismo , Humanos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/efectos de la radiación , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/radioterapia , Linfocitos/inmunología , Linfocitos/metabolismo , Masculino , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Radioisótopos de ItrioRESUMEN
INTRODUCTION: Radioprotective agents are of interest for application in radiotherapy for cancer and in public health medicine in the context of accidental radiation exposure. Methylproamine is the lead compound of a class of radioprotectors which act as DNA binding anti-oxidants, enabling the repair of transient radiation-induced oxidative DNA lesions. This study tested methylproamine for the radioprotection of both directly targeted and bystander cells. METHODS: T98G glioma cells were treated with 15 µM methylproamine and exposed to (137)Cs γ-ray/X-ray irradiation and He(2+) microbeam irradiation. Radioprotection of directly targeted cells and bystander cells was measured by clonogenic survival or γH2AX assay. RESULTS: Radioprotection of directly targeted T98G cells by methylproamine was observed for (137)Cs γ-rays and X-rays but not for He(2+) charged particle irradiation. The effect of methylproamine on the bystander cell population was tested for both X-ray irradiation and He(2+) ion microbeam irradiation. The X-ray bystander experiments were carried out by medium transfer from irradiated to non-irradiated cultures and three experimental designs were tested. Radioprotection was only observed when recipient cells were pretreated with the drug prior to exposure to the conditioned medium. In microbeam bystander experiments targeted and nontargeted cells were co-cultured with continuous methylproamine treatment during irradiation and postradiation incubation; radioprotection of bystander cells was observed. DISCUSSION AND CONCLUSION: Methylproamine protected targeted cells from DNA damage caused by γ-ray or X-ray radiation but not He(2+) ion radiation. Protection of bystander cells was independent of the type of radiation which the donor population received.
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Bencimidazoles/farmacología , Efecto Espectador , Supervivencia Celular/efectos de la radiación , Protectores contra Radiación/farmacología , Línea Celular Tumoral , HumanosRESUMEN
The migration of an electron-loss center (hole) in calf thymus DNA to bisbenzimidazole ligands bound in the minor groove is followed by pulse radiolysis combined with time-resolved spectrophotometry. The initially observed absorption spectrum upon oxidation of DNA by the selenite radical is consistent with spin on cytosine (C), as the GC⢠pair neutral radical, followed by the spectra of oxidized ligands. The rate of oxidation of bound ligands increased with an increase in the ratio (r) ligands per base pair from 0.005 to 0.04. Both the rate of ligand oxidation and the estimated range of hole transfer (up to 30 DNA base pairs) decrease with the decrease in one-electron reduction potential between the GC⢠pair neutral radical of ca. 1.54 V and that of the ligand radicals (E0', 0.90-0.99 V). Linear plots of log of the rate of hole transfer versus r give a common intercept at r = 0 and a free energy change of 12.2 ± 0.3 kcal mol-1, ascribed to the GC⢠pair neutral radical undergoing a structural change, which is in competition to the observed hole transfer along DNA. The rate of hole transfer to the ligands at distance, R, from the GC⢠pair radical, k2, is described by the relationship k2 = k0 exp(constant/R), where k0 includes the rate constant for surmounting a small barrier.
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Emparejamiento Base , ADN , ADN/química , Radicales Libres/química , Oxidación-Reducción , Bencimidazoles/química , Animales , Bovinos , Ligandos , Bisbenzimidazol/química , Reparación del ADN , Daño del ADN , Citosina/químicaRESUMEN
We describe a new approach for interactive analysis of time-lapse microscopy, and apply this approach to elucidating whether polarity regulation is conserved between epithelial cells and lymphocytes. A key advantage of our analysis platform, 'TACTICS', is the capacity to visualize individual data points in the context of large data sets, similar to standard approaches in flow cytometry. Scatter plots representing microscopic parameters or their derivations such as polarity ratios are linked to the original data such that clicking on each dot enables a link to images and movies of the corresponding cell. Similar to flow cytometric analysis, subsets of the data can be gated and reanalyzed to explore the relationships between different parameters. TACTICS was used to dissect the regulation of polarization of the cell fate determinant, Numb, in migrating lymphocytes. We show here that residues of Numb that are phosphorylated by atypical protein kinase C (aPKC) to mediate apicobasal polarity in epithelial cells are not required for polarization of Numb in T cells, indicating that the role of aPKC is not conserved between lymphocytes and epithelia.
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Procesamiento de Imagen Asistido por Computador/métodos , Linfocitos/citología , Linfocitos/inmunología , Transducción de Señal/inmunología , Animales , Polaridad Celular/inmunología , Células Epiteliales/citología , Células Epiteliales/inmunología , Procesamiento de Imagen Asistido por Computador/instrumentación , Proteínas de la Membrana/inmunología , Ratones , Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/métodos , Proteínas del Tejido Nervioso/inmunología , Especificidad de Órganos/inmunología , Fosforilación/inmunología , Proteína Quinasa C/inmunologíaRESUMEN
Purpose: The interest in exploiting Auger emitters in cancer therapy stems from their high linear energy transfer (LET)-type radiation damage to DNA. However, the design of Auger-emitter labeled vehicles that target the Auger cascade specifically to the DNA of tumour cells is challenging. Here we suggest a possible approach to evaluate tumour-targeting Auger-labeled conjugates by assessing the impact of a radioprotector known to be effective in protecting from low LET radiation, but not high LET radiation. Given some similarity between the energy spectrum of Auger electrons and that of secondary electrons from soft X-rays, we report the results of radioprotection experiments with 25 kVp X-rays. Materials and methods: Clonogenic survival curves for cultured human keratinocytes were established for three different irradiation conditions: 137Cs γ-rays, 25 kVp X-rays and 320 kVp X-rays, and the effect of including a new radioprotector, denoted "2PH", was investigated.Results: The extent of radioprotection by 2PH was comparable for all radiation conditions, although RBE was higher (about 1.7) for soft X-rays. Conclusions: Radioprotectors like 2PH will help to evaluate Auger endoradiotherapy strategies, by determining the relative contributions of the high-LET effects (not protected), compared to other components, such as Auger electrons not effectively targeted to DNA.
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Neoplasias , Humanos , Neoplasias/radioterapia , Electrones , Rayos X , Rayos gammaRESUMEN
The γH2AX focus assay represents a fast and sensitive approach for the detection of one of the critical types of DNA damage - double-strand breaks (DSB) induced by various cytotoxic agents including ionising radiation. Apart from research applications, the assay has a potential in clinical medicine/pathology, such as assessment of individual radiosensitivity, response to cancer therapies, as well as in biodosimetry. Given that generally there is a direct relationship between numbers of microscopically visualised γH2AX foci and DNA DSB in a cell, the number of foci per nucleus represents the most efficient and informative parameter of the assay. Although computational approaches have been developed for automatic focus counting, the tedious and time consuming manual focus counting still remains the most reliable way due to limitations of computational approaches. We suggest a computational approach and associated software for automatic focus counting that minimises these limitations. Our approach, while using standard image processing algorithms, maximises the automation of identification of nuclei/cells in complex images, offers an efficient way to optimise parameters used in the image analysis and counting procedures, optionally invokes additional procedures to deal with variations in intensity of the signal and background in individual images, and provides automatic batch processing of a series of images. We report results of validation studies that demonstrated correlation of manual focus counting with results obtained using our computational algorithm for mouse jejunum touch prints, mouse tongue sections and human blood lymphocytes as well as radiation dose response of γH2AX focus induction for these biological specimens.
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Biología Computacional/métodos , Roturas del ADN de Doble Cadena , Daño del ADN , Histonas/análisis , Algoritmos , Animales , Relación Dosis-Respuesta en la Radiación , Humanos , Procesamiento de Imagen Asistido por Computador , Yeyuno/efectos de la radiación , Linfocitos/efectos de la radiación , Ratones , Lengua/efectos de la radiación , Estudios de Validación como AsuntoRESUMEN
Synchrotron radiation, especially microbeam radiotherapy (MRT), has a great potential to improve cancer radiotherapy, but non-targeted effects of synchrotron radiation have not yet been sufficiently explored. We have previously demonstrated that scattered synchrotron radiation induces measurable γ-H2AX foci, a biomarker of DNA double-strand breaks, at biologically relevant distances from the irradiated field that could contribute to the apparent accumulation of bystander DNA damage detected in cells and tissues outside of the irradiated area. Here, we quantified an impact of scattered radiation to DNA damage response in "naïve" cells sharing the medium with the cells that were exposed to synchrotron radiation. To understand the effect of genetic alterations in naïve cells, we utilised p53-null and p53-wild-type human colon cancer cells HCT116. The cells were grown in two-well chamber slides, with only one of nine zones (of equal area) of one well irradiated with broad beam or MRT. γ-H2AX foci per cell values induced by scattered radiation in selected zones of the unirradiated well were compared to the commensurate values from selected zones in the irradiated well, with matching distances from the irradiated zone. Scattered radiation highly impacted the DNA damage response in both wells and a pronounced distance-independent bystander DNA damage was generated by broad-beam irradiations, while MRT-generated bystander response was negligible. For p53-null cells, a trend for a reduced response to scattered irradiation was observed, but not to bystander signalling. These results will be taken into account for the assessment of genotoxic effects in surrounding non-targeted tissues in preclinical experiments designed to optimise conditions for clinical MRT and for cancer treatment in patients.
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Abscopal effects are an important aspect of targeted radiation therapy due to their implication in normal tissue toxicity from chronic inflammatory responses and mutagenesis. Gene expression can be used to determine abscopal effects at the molecular level. Synchrotron microbeam radiation therapy utilizing high-intensity X rays collimated into planar microbeams is a promising cancer treatment due to its reported ability to ablate tumors with less damage to normal tissues compared to conventional broadbeam radiation therapy techniques. The low scatter of synchrotron radiation enables microbeams to be delivered to tissue effectively, and is also advantageous for out-of-field studies because there is minimal interference from scatter. Mouse legs were irradiated at a dose rate of 49 Gy/s and skin samples in the out-of-field areas were collected. The out-of-field skin showed an increase in Tnf expression and a decrease in Mdm2 expression, genes associated with inflammation and DNA damage. These expression effects from microbeam exposure were similar to those found with broadbeam exposure. In immune-deficient Ccl2 knockout mice, we identified a different gene expression profile which showed an early increase in Mdm2, Tgfb1, Tnf and Ccl22 expression in out-of-field skin that was not observed in the immune-proficient mice. Our results suggest that the innate immune system is involved in out-of-field tissue responses and alterations in the immune response may not eliminate abscopal effects, but could change them.
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Daño del ADN/genética , Expresión Génica/efectos de la radiación , Inmunidad Innata/efectos de la radiación , Sincrotrones , Animales , Quimiocina CCL2/genética , Perfilación de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Thoracic radiotherapy (RT) is required for the curative management of inoperable lung cancer, however, treatment delivery is limited by normal tissue toxicity. Prior studies suggest that using radiation-induced DNA damage response (DDR) in peripheral blood mononuclear cells (PBMC) has potential to predict RT-associated toxicities. We collected PBMC from 38 patients enrolled on a prospective clinical trial who received definitive fractionated RT for non-small cell lung cancer. DDR was measured by automated counting of nuclear γ-H2AX foci in immunofluorescence images. Analysis of samples collected before, during and after RT demonstrated the induction of DNA damage in PBMC collected shortly after RT commenced, however, this damage repaired later. Radiation dose to the tumour and lung contributed to the in vivo induction of γ-H2AX foci. Aliquots of PBMC collected before treatment were also irradiated ex vivo, and γ-H2AX kinetics were analyzed. A trend for increasing of fraction of irreparable DNA damage in patients with higher toxicity grades was revealed. Slow DNA repair in three patients was associated with a combined dysphagia/cough toxicity and was confirmed by elevated in vivo RT-generated irreparable DNA damage. These results warrant inclusion of an assessment of DDR in PBMC in a panel of predictive biomarkers that would identify patients at a higher risk of toxicity.
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In microorganisms, evolutionarily conserved mechanisms facilitate adaptation to harsh conditions through stress-induced mutagenesis (SIM). Analogous processes may underpin progression and therapeutic failure in human cancer. We describe SIM in multiple in vitro and in vivo models of human cancers under nongenotoxic drug selection, paradoxically enhancing adaptation at a competing intrinsic fitness cost. A genome-wide approach identified the mechanistic target of rapamycin (MTOR) as a stress-sensing rheostat mediating SIM across multiple cancer types and conditions. These observations are consistent with a two-phase model for drug resistance, in which an initially rapid expansion of genetic diversity is counterbalanced by an intrinsic fitness penalty, subsequently normalizing to complete adaptation under the new conditions. This model suggests synthetic lethal strategies to minimize resistance to anticancer therapy.
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Adaptación Fisiológica/genética , Antineoplásicos/farmacología , Resistencia a Antineoplásicos/genética , Mutagénesis , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Serina-Treonina Quinasas TOR/metabolismo , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Reparación del ADN/genética , Aptitud Genética , Estudio de Asociación del Genoma Completo , Humanos , Selección Genética , Transducción de Señal , Serina-Treonina Quinasas TOR/genéticaRESUMEN
PURPOSE: There is growing interest in developing individually tailored cancer radiation therapy (RT), wherein patients with high intrinsic radiosensitivity are identified before commencing treatment, to minimize severe adverse reactions. In a previous retrospective study of severely radiosensitive RT patients, we established a functional assay with a high predictive capability. The assay involves ex vivo irradiation of peripheral blood mononuclear cells and analysis of DNA repair using the γ-H2AX assay. It is unknown whether RS is a fixed phenomenon or is modulated under different conditions. We now report the impact of RT on the apparent radiosensitivity, as reflected by the assay. METHODS AND MATERIALS: Peripheral blood mononuclear cells of 11 patients with non-small cell lung cancer were collected before, during, and after RT. Quantitative parameters derived from the nonlinear regression analysis of γ-H2AX foci were applied to examine the cellular radiation response. RESULTS: Although the repair rate and foci yield remained constant during and after RT, the "unrepairable" component of γ-H2AX foci decreased over the course of treatment in 7 patients, signifying a generally enhanced DNA repair capacity. Interestingly, enhanced repair capacity tended to be associated with a poorer response to RT. CONCLUSIONS: Although generalization of these results into normal and tumor tissues warrants further investigation, the findings of this study have important implications in future strategies for identifying radiosensitive individuals before exposure to RT. We can anticipate that the threshold values that will discriminate radiosensitive patients in a future prospective trial will differ from those established in the retrospective study.
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Carcinoma de Pulmón de Células no Pequeñas/sangre , Carcinoma de Pulmón de Células no Pequeñas/radioterapia , Reparación del ADN/efectos de la radiación , Leucocitos Mononucleares/citología , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/radioterapia , Anciano , Apoptosis , Daño del ADN , Femenino , Histonas/metabolismo , Humanos , Cinética , Masculino , Persona de Mediana Edad , Tomografía de Emisión de Positrones , Estudios Prospectivos , Tolerancia a Radiación , Análisis de Regresión , Resultado del TratamientoRESUMEN
PURPOSE: Roberts syndrome (RBS) is a rare, recessively transmitted developmental disorder characterized by growth retardation, craniofacial abnormalities, and truncation of limbs. All affected individuals to date have mutations in the ESCO2 (establishment of cohesion 2) gene, a key regulator of the cohesin complex, which is involved in sister chromatid cohesion and DNA double-strand break (DSB) repair. Here we characterize DNA damage responses (DDRs) for the first time in an RBS-affected family. METHODS AND MATERIALS: Lymphoblastoid cell lines were established from an RBS family, including the proband and parents carrying ESCO2 mutations. Various DDR assays were performed on these cells, including cell survival, chromosome break, and apoptosis assays; checkpoint activation indicators; and measures of DNA breakage and repair. RESULTS: Cells derived from the RBS-affected individual showed sensitivity to ionizing radiation (IR) and mitomycin C-induced DNA damage. In this ESCO2 compound heterozygote, other DDRs were also defective, including enhanced IR-induced clastogenicity and apoptosis; increased DNA DSB induction; and a reduced capacity for repairing IR-induced DNA DSBs, as measured by γ-H2AX foci and the comet assay. CONCLUSIONS: In addition to its developmental features, RBS can be, like ataxia telangiectasia, considered a DDR-defective syndrome, which contributes to its cellular, molecular, and clinical phenotype.
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Acetiltransferasas/genética , Cromátides/genética , Proteínas Cromosómicas no Histona/genética , Anomalías Craneofaciales/genética , Roturas del ADN de Doble Cadena , Trastornos por Deficiencias en la Reparación del ADN/genética , Ectromelia/genética , Hipertelorismo/genética , Tolerancia a Radiación/genética , Línea Celular , Supervivencia Celular , Cromátides/efectos de la radiación , Ensayo Cometa , Anomalías Craneofaciales/patología , ADN/efectos de la radiación , Ectromelia/patología , Femenino , Histonas/análisis , Humanos , Hipertelorismo/patología , Inmunoprecipitación/métodos , Recién Nacido , Mitomicina/farmacología , Mutación/genética , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , FenotipoRESUMEN
PURPOSE: Nontargeted effects of ionizing radiation, by which unirradiated cells and tissues are also damaged, are a relatively new paradigm in radiobiology. We recently reported radiation-induced abscopal effects (RIAEs) in normal tissues; namely, DNA damage, apoptosis, and activation of the local and systemic immune responses in C57BL6/J mice after irradiation of a small region of the body. High-dose-rate, synchrotron-generated broad beam or multiplanar x-ray microbeam radiation therapy was used with various field sizes and doses. This study explores components of the immune system involved in the generation of these abscopal effects. METHODS AND MATERIALS: The following mice with various immune deficiencies were irradiated with the microbeam radiation therapy beam: (1) SCID/IL2γR-/- (NOD SCID gamma, NSG) mice, (2) wild-type C57BL6/J mice treated with an antibody-blocking macrophage colony-stimulating factor 1 receptor, which depletes and alters the function of macrophages, and (3) chemokine ligand 2/monocyte chemotactic protein 1 null mice. Complex DNA damage (ie, DNA double-strand breaks), oxidatively induced clustered DNA lesions, and apoptotic cells in tissues distant from the irradiation site were measured as RIAE endpoints and compared with those in wild-type C57BL6/J mice. RESULTS: Wild-type mice accumulated double-strand breaks, oxidatively induced clustered DNA lesions, and apoptosis, enforcing our RIAE model. However, these effects were completely or partially abrogated in mice with immune disruption, highlighting the pivotal role of the immune system in propagation of systemic genotoxic effects after localized irradiation. CONCLUSIONS: These results underline the importance of not only delineating the best strategies for tumor control but also mitigating systemic radiation toxicity.
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Apoptosis , Roturas del ADN de Doble Cadena , Sistema Inmunológico/fisiología , Traumatismos Experimentales por Radiación/inmunología , Animales , Efecto Espectador , Quimiocina CCL2/sangre , Quimiocina CCL2/genética , ADN/aislamiento & purificación , Femenino , Ligandos , Macrófagos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Estrés Oxidativo , Dosis de Radiación , Traumatismos Experimentales por Radiación/etiología , Receptor de Factor Estimulante de Colonias de Macrófagos/antagonistas & inhibidores , Sincrotrones , Factor de Crecimiento Transformador beta1/sangreRESUMEN
PURPOSE: The aim of the study is to establish the relationship between the efficiency of DNA double-stranded breakage by (125)I-labelled DNA ligands and the distance from the decaying atom to the helical axis. MATERIALS AND METHODS: Two new iodinated minor groove binding ligands were synthesized which, on the basis of molecular modelling studies, place the iodine atom at different distances from the DNA helical axis (namely 7.4 and 11.2 A degrees ). Plasmid DNA breakage experiments, in both buffer-only and buffer + 2M dimethylsulfoxide (DMSO), were used to determine the efficiency of induction of internal double-stranded breaks (DSB) of the two new ligands, as well as that for (125)I-Hoechst 33258, which is characterized by a helical axis-iodine atom distance of 9.1 A degrees . RESULTS: The results showed a progressive decrease in the efficiency of DNA DSB induction with the axis-iodine atom distance, for both incubation conditions. The distance-damage relationship was somewhat steeper than previously predicted from the theoretical studies by Humm and Charlton, based on radical-mediated damage. Another distinctive trend was revealed by comparison of breakage efficiency with and without DMSO. The extent of DMSO protection increased significantly with DNA-iodine distance. CONCLUSIONS: The steeper than predicted decrease in DSB induction with DNA-iodine distance is consistent with a substantial contribution to DNA breakage of the charge neutralization effect (arising from the transient positive charge left on the daughter Te atom), and the expectation that this contribution would be very dependent on the distance of the site of hole injection from the base-pair pi-stack. An important caveat to the results and conclusions is the need to confirm the estimated helical axis-iodine distances with X-ray crystallography studies, and for further exemplification with a more extensive collection of DNA ligands.
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Bencimidazoles/química , Bisbenzimidazol/análogos & derivados , Roturas del ADN de Doble Cadena/efectos de la radiación , Radioisótopos de Yodo/química , Bisbenzimidazol/química , Ligandos , Modelos Moleculares , Conformación de Ácido Nucleico , Plásmidos/química , Plásmidos/efectos de la radiación , RadiactividadRESUMEN
The role of gene mutations in tumourigenesis is well understood, however, the mechanism(s) by which they arise are less clear. Indeed, the common assumption that tumourigenic mutations are the result of DNA replication errors is apparently contradicted by the very low division frequency of the cells from which tumours are thought to arise (i.e. deep stem cells). As a potential solution to this paradox, we tested a model whereby clustered DNA lesion sites (CLS) (where several lesions occur within a few base pairs of each other on opposing strands) could give rise to mutations in quiescent cells. We used statistical analyses to search for sets of dinucleotide sequences (designated target sequences) that are present at and in close proximity to mutation sites in four genes associated with human colorectal tumourigenesis (adenomatosis polyposis coli (APC), v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS), phosphoinositide-3-kinase, catalytic, alpha polypeptide (PIK3CA), and tumour protein p53 (TP53)). The dinucleotides CG, AC-GT, TG, and GC were identified as target sequences in at least three of the genes analysed. Consistent with their designation as target sequences, these dinucleotides have all been identified as high probability sites of oxidative damage formation in in vitro studies. Our results strongly suggest a statistical association between the presence of multiple, clustered target sequences and mutational events. We propose that CLS are a major source of mutations during human tumourigenesis.
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Neoplasias Colorrectales/etiología , Neoplasias Colorrectales/genética , ADN/genética , Mutación , Secuencia de Bases , Fosfatidilinositol 3-Quinasa Clase I , Análisis por Conglomerados , Análisis Mutacional de ADN , Interpretación Estadística de Datos , Genes p53 , Genes ras , Humanos , Modelos Genéticos , Datos de Secuencia Molecular , Fosfatidilinositol 3-Quinasas , Proteína p53 Supresora de Tumor/genéticaRESUMEN
We show the efficacy of a therapeutic strategy that combines the potency of a DNA-binding photosensitizer, UV(A)Sens, with the tumor-targeting potential of receptor-mediated endocytosis. The photosensitizer is an iodinated bibenzimidazole, which, when bound in the minor groove of DNA and excited by UV(A) irradiation, induces cytotoxic lesions attributed to a radical species resulting from photodehalogenation. Although reminiscent of photochemotherapy using psoralens and UV(A) irradiation, an established treatment modality in dermatology particularly for the treatment of psoriasis and cutaneous T-cell lymphoma, a critical difference is the extreme photopotency of the iodinated bibenzimidazole, approximately 1,000-fold that of psoralens. This feature prompted consideration of combination with the specificity of receptor-mediated targeting. Using two in vitro model systems, we show the UV(A) cytotoxicity of iodo ligand/protein conjugates, implying binding of the conjugate to cell receptors, internalization, and degradation of the conjugate-receptor complex, with release and translocation of the ligand to nuclear DNA. For ligand-transferrin conjugates, phototoxicity was inhibited by coincubation with excess native transferrin. Receptor-mediated UV(A)-induced cytotoxicity was also shown with the iodo ligand conjugate of an anti-human epidermal growth factor receptor monoclonal antibody, exemplifying the potential application of the strategy to other cancer-specific targets to thus improve the specificity of phototherapy of superficial lesions and for extracorporeal treatments.
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ADN/metabolismo , Endocitosis , Receptores ErbB/metabolismo , Fotoquimioterapia/métodos , Fármacos Fotosensibilizantes/uso terapéutico , Receptores de Transferrina/metabolismo , Rayos Ultravioleta , Animales , Células CHO , Cricetinae , Humanos , Inmunoterapia , Células K562RESUMEN
PURPOSE: A priori identification of the small proportion of radiation therapy patients who prove to be severely radiosensitive is a long-held goal in radiation oncology. A number of published studies indicate that analysis of the DNA damage response after ex vivo irradiation of peripheral blood lymphocytes, using the γ-H2AX assay to detect DNA damage, provides a basis for a functional assay for identification of the small proportion of severely radiosensitive cancer patients undergoing radiotherapy. METHODS AND MATERIALS: We introduce a new, more rigorous, integrated approach to analysis of radiation-induced γ-H2AX response, using Bayesian statistics. RESULTS: This approach shows excellent discrimination between radiosensitive and non-radiosensitive patient groups described in a previously reported data set. CONCLUSIONS: Bayesian statistical analysis provides a more appropriate and reliable methodology for future prospective studies.
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Teorema de Bayes , Neoplasias/sangre , Tolerancia a Radiación/genética , Biomarcadores , Daño del ADN , Bases de Datos Factuales , Relación Dosis-Respuesta en la Radiación , Femenino , Histonas/química , Humanos , Linfocitos/efectos de la radiación , Masculino , Modelos Teóricos , Método de Montecarlo , Neoplasias/radioterapia , Estudios Prospectivos , Radioterapia , Reproducibilidad de los Resultados , Estudios Retrospectivos , Factores de TiempoRESUMEN
The importance of nontargeted (systemic) effects of ionizing radiation is attracting increasing attention. Exploiting synchrotron radiation generated by the Imaging and Medical Beamline at the Australian Synchrotron, we studied radiation-induced nontargeted effects in C57BL/6 mice. Mice were locally irradiated with a synchrotron X-ray broad beam and a multiplanar microbeam radiotherapy beam. To assess the influence of the beam configurations and variations in peak dose and irradiated area in the response of normal tissues outside the irradiated field at 1 and 4 days after irradiation, we monitored oxidatively induced clustered DNA lesions (OCDL), DNA double-strand breaks (DSB), apoptosis, and the local and systemic immune responses. All radiation settings induced pronounced persistent systemic effects in mice, which resulted from even short exposures of a small irradiated area. OCDLs were elevated in a wide variety of unirradiated normal tissues. In out-of-field duodenum, there was a trend for elevated apoptotic cell death under most irradiation conditions; however, DSBs were elevated only after exposure to lower doses. These genotoxic events were accompanied by changes in plasma concentrations of macrophage-derived cytokine, eotaxin, IL10, TIMP1, VEGF, TGFß1, and TGFß2, along with changes in tissues in frequencies of macrophages, neutrophils, and T lymphocytes. Overall, our findings have implications for the planning of therapeutic and diagnostic radiation treatments to reduce the risk of radiation-related adverse systemic effects. Cancer Res; 77(22); 6389-99. ©2017 AACR.
Asunto(s)
Roturas del ADN de Doble Cadena/efectos de la radiación , Piel/efectos de la radiación , Sincrotrones , Rayos X , Animales , Apoptosis/genética , Apoptosis/efectos de la radiación , Citocinas/sangre , Citocinas/metabolismo , Relación Dosis-Respuesta en la Radiación , Macrófagos/metabolismo , Macrófagos/efectos de la radiación , Ratones Endogámicos C57BL , Traumatismos Experimentales por Radiación/genética , Traumatismos Experimentales por Radiación/metabolismo , Traumatismos Experimentales por Radiación/prevención & control , Piel/inmunología , Piel/metabolismo , Factores de TiempoRESUMEN
Previous studies have described UVA-induced DNA strand breakage at the binding sites of iodinated DNA minor groove binding bisbenzimidazoles. The DNA breakage, presumably mediated by the carbon-centred ligand radical produced by photodehalogenation, was also shown to be cytotoxic. The earlier studies included a comparison of three ligand isomers, designated ortho-, meta- and para-iodoHoechst, and the efficiency of photo-induction of strand breaks in plasmid DNA proved to be much higher for the ortho-isomer. We have now extended the comparison of the three isomers with respect to photo-induced cytotoxicity in K562 cells. Although the relationship between the extent of nuclear uptake and the concentration of the ligand in the medium was similar for the three isomers, assay of in situ dehalogenation in drug-treated cells indicated that the apparent cross-section for dehalogenation of the ortho-isomer was greater than 5-fold higher than that for the meta- and para-isomers. Also, analysis of clonogenic survival data showed that the dehalogenation event associated with ortho-iodoHoechst was a more efficient mediator of UVA-induced cytotoxicity in K562 cells than that for meta- or para-iodoHoechst. The number of dehalogenation events associated with 50% cell-kill for ortho-iodoHoechst (1.23+/-0.04 x 10(4)) was less than that for the para- (3.92+/-0.29 x 10(4)) and meta- (11.6+/-0.90 x 10(4)) isomers. Thus it is concluded that the photopotency of ortho-iodoHoechst, which is an important feature in the context of its potential use in clinical phototherapy, is due not only to more efficient UVA-mediated dehalogenation of the ligand, but also to greater cytotoxic potency per dehalogenation event.
Asunto(s)
Bisbenzimidazol/farmacología , Daño del ADN , ADN/efectos de la radiación , Yodo/metabolismo , Rayos Ultravioleta , Sitios de Unión , Bisbenzimidazol/química , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Supervivencia Celular/efectos de la radiación , ADN/metabolismo , Colorantes Fluorescentes , Humanos , Yodo/química , Isomerismo , Células K562/efectos de los fármacos , Células K562/patología , Células K562/efectos de la radiación , Ligandos , Efectividad Biológica Relativa , Espectrometría de Fluorescencia , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
New analogues of the minor groove binding ligand Hoechst 33342 have been investigated in an attempt to improve radioprotective activity. The synthesis, DNA binding, and in vitro radioprotective properties of methylproamine, the most potent derivative, are reported. Experiments with V79 cells have shown that methylproamine is approximately 100-fold more potent than the classical aminothiol radioprotector WR1065. The crystal structures of methylproamine and proamine complexes with the dodecamer d(CGCGAATTCGCG)(2) confirm that the new analogues also are minor groove binders. It is proposed that the DNA-bound methylproamine ligand acts as a reducing agent by an electron transfer mechanism, repairing transient radiation-induced oxidizing species on DNA.