RESUMEN
The aim of the present work was to investigate the mechanisms of radiation-induced bystander signalling leading to apoptosis in non-irradiated co-cultured cells. Cultured non-transformed cells were irradiated, and the effect on the apoptosis rate on co-cultured non-irradiated malignant cells was determined. For this, two different levels of the investigation are presented, i.e. release of signalling proteins and transcriptomic profiling of the irradiated and non-irradiated co-cultured cells. Concerning the signalling proteins, in this study, the attention was focussed on the release of the active and latent forms of the transforming growth factor-ß1 protein. Moreover, global gene expression profiles of non-transformed and transformed cells in untreated co-cultures were compared with those of 0.5-Gy-irradiated non-transformed cells co-cultured with the transformed cells. The results show an effect of radiation on the release of signalling proteins in the medium, although no significant differences in release rates were detectable when varying the doses in the range from 0.25 to 1 Gy. Moreover, gene expression results suggest an effect of radiation on both cell populations, pointing out specific signalling pathways that might be involved in the enhanced induction of apoptosis.
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Apoptosis/fisiología , Apoptosis/efectos de la radiación , Línea Celular Transformada/efectos de la radiación , Fibroblastos/patología , Fibroblastos/efectos de la radiación , Radiación Ionizante , Factor de Crecimiento Transformador beta/metabolismo , Animales , Línea Celular Transformada/metabolismo , Células Cultivadas , Técnicas de Cocultivo , Relación Dosis-Respuesta a Droga , Perfilación de la Expresión Génica , Humanos , Modelos Biológicos , RatasRESUMEN
The ultimate response of a cell or tissue to radiation is dependent in part on intercellular signalling. This becomes increasingly important at low doses, or at low dose rates, associated with typical human exposures. In order to help characterise the underlying mechanism of intercellular signalling, and how they are perturbed following exposure to ionising radiation, a previously well-defined model system of intercellular induction of apoptosis (IIA) (Portess et al. 2007, Cancer Res. 67, 1246-1253) was adopted. The aim of the present work is to evaluate the signalling mechanisms underpinning this process through exploring the variables that can affect the IIA, i.e. dose, time and space.
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Apoptosis/fisiología , Apoptosis/efectos de la radiación , Línea Celular Transformada/efectos de la radiación , Fibroblastos/patología , Fibroblastos/efectos de la radiación , Radiación Ionizante , Animales , Células Cultivadas , Técnicas de Cocultivo , Relación Dosis-Respuesta a Droga , Humanos , Modelos Biológicos , RatasRESUMEN
TGFß is a key modulator of the Epithelial-Mesenchymal Transition (EMT), a process important in cancer progression and metastasis, which leads to the suppression of epithelial genes and expression of mesenchymal proteins. Ionizing radiation was found to specifically induce expression of the TGF-ß1 isoform, which can modulate late post-radiation changes and increase the risk of tumor development and metastasis. Interactions between TGFß induced EMT and DNA damage responses have not been fully elucidated, particularly at low doses and following different radiation quality exposures. Further characterization of the relationship between radiation quality, EMT and cancer development is warranted. We investigated whether space radiation induced TGFß dependent EMT, using hTERT immortalized human esophageal epithelial cells (EPC2-hTERT) and non-transformed mink lung epithelial cells (Mv1Lu). We have observed morphologic and molecular alterations in EPC2 and Mv1Lu cells consistent with EMT after pre-treatment with TGFß1. This effect could be efficiently inhibited in both cell lines by the use of a TGFßRI inhibitor. High-energy silicon or iron nuclei were each able to cause a mild induction of EMT, with the inclusion of TGFß1 inducing a greatly enhanced EMT phenotype even when cells were irradiated with doses as low as 0.1 Gy. A further enhancement of EMT was achieved at a higher dose of 2 Gy. TGFßRI inhibitor was able to reverse the EMT induced by the combination of TGFß1 and radiation. These studies indicate that heavy ions, even at a low dose, may trigger the process of TGFß1-induced EMT, and suggest further studies are needed to determine whether the chronic exposures received in space may potentiate this process in astronauts, leading to an increased risk of cancer.
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Células Epiteliales/efectos de la radiación , Transición Epitelial-Mesenquimal/efectos de la radiación , Iones Pesados/efectos adversos , Hierro , Silicio , Factor de Crecimiento Transformador beta1/farmacología , Animales , Línea Celular Transformada/efectos de los fármacos , Línea Celular Transformada/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Células Epiteliales/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Esófago/citología , Proteínas de la Matriz Extracelular/metabolismo , Humanos , Pulmón/citología , Visón , Fosforilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Pteridinas/farmacología , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Proteínas Recombinantes/farmacología , Proteína Smad2/metabolismo , Proteína smad7/metabolismo , beta Catenina/metabolismoRESUMEN
BACKGROUND: Inter-individual variation in DNA repair capacity is thought to modulate breast cancer risk. The phenotypic mutagen sensitivity assay (MSA) measures DNA strand breaks in lymphocytes; women with familial and sporadic breast cancers have a higher mean number of breaks per cell (MBPC) than women without breast cancer. Here, we explore the relationships between the MSA and the Rad51 gene, which encodes a DNA repair enzyme that interacts with BRCA1 and BRCA2, in BRCA1 mutation carriers and women with sporadic breast cancer. METHODS: Peripheral blood lymphoblasts from women with known BRCA1 mutations underwent the MSA (n = 138 among 20 families). BRCA1 and Rad51 genotyping and sequencing were performed to identify SNPs and haplotypes associated with the MSA. Positive associations from the study in high-risk families were subsequently examined in a population-based case-control study of breast cancer (n = 1170 cases and 2115 controls). RESULTS: Breast cancer diagnosis was significantly associated with the MSA among women from BRCA1 families (OR = 3.2 95%CI: 1.5-6.7; p = 0.004). The Rad51 5'UTR 135 C>G genotype (OR = 3.64; 95% CI: 1.38, 9.54; p = 0.02), one BRCA1 haplotype (p = 0.03) and in a polygenic model, the E1038G and Q356R BRCA1 SNPs were significantly associated with MBPC (p = 0.009 and 0.002, respectively). The Rad51 5'UTR 135C genotype was not associated with breast cancer risk in the population-based study. CONCLUSIONS: Mutagen sensitivity might be a useful biomarker of penetrance among women with BRCA1 mutations because the MSA phenotype is partially explained by genetic variants in BRCA1 and Rad51.
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Proteína BRCA1/fisiología , Neoplasias de la Mama/genética , Roturas del ADN , Reparación del ADN/fisiología , ADN de Neoplasias/efectos de la radiación , Genes BRCA1 , Mutación , Polimorfismo de Nucleótido Simple , Recombinasa Rad51/fisiología , Adulto , Anciano , Estudios de Casos y Controles , Línea Celular Transformada/efectos de la radiación , Línea Celular Transformada/ultraestructura , Células Cultivadas/efectos de la radiación , Células Cultivadas/ultraestructura , Reparación del ADN/genética , ADN de Neoplasias/genética , Salud de la Familia , Femenino , Predisposición Genética a la Enfermedad , Haplotipos/genética , Heterocigoto , Humanos , Linfocitos/efectos de la radiación , Linfocitos/ultraestructura , Persona de Mediana Edad , Mutágenos/farmacología , New York/epidemiología , Recombinasa Rad51/genética , Tolerancia a Radiación/genética , Sistema de Registros , RiesgoRESUMEN
NAD(P)H:quinone acceptor oxidoreductase (NQO) 1 polymorphism is associated with various hematological malignancies, especially infant leukemia or therapy-related leukemias, which involve the rearrangement of mixed lineage leukemia (MLL) gene. Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (EBV-LCLs) with either of 2 well known polymorphic variations of C609T and C465T were selected from our archives of EBV-LCL clones and studied the induction of p53 expression after DNA damage. Irradiation of cells with C609T/C609T polymorphism (NQO1 *2*2) did not affect the induction of p53 expression. However, irradiation of cells with C465T/WT polymorphism (NQO1 *1*3) resulted in attenuation of p53 and p21 induction. Our results suggest that increased risk of infant leukemia development in patients with NQO1 *1*3 polymorphism is partially dependent on the inhibition of p53 pathway, though further studies are needed to fully understand the pathological role of C465T variant in the development of childhood leukemia.
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Regulación Neoplásica de la Expresión Génica , Leucemia/genética , Proteína de la Leucemia Mieloide-Linfoide/genética , NAD(P)H Deshidrogenasa (Quinona)/genética , Proteína p53 Supresora de Tumor/genética , Sustitución de Aminoácidos , Western Blotting , Línea Celular Transformada/efectos de la radiación , Daño del ADN , Predisposición Genética a la Enfermedad , Humanos , Mutación Missense , Proteína Oncogénica p21(ras)/genética , Polimorfismo de Nucleótido Simple , Proteína p53 Supresora de Tumor/biosíntesisRESUMEN
As first task of a comprehensive investigation on DNA repair genotype-phenotype correlations, the suitability of Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs) as surrogate of cryopreserved peripheral blood mononuclear cells (PBMCs) in DNA repair phenotypic assays was evaluated. To this aim the amount of DNA damage induced by gamma-rays and DNA repair capacity were evaluated in unstimulated (G(0)) and mitogen-simulated (G(2)) PBMC from 20 healthy subjects and in EBV-transformed LCL obtained from the same individuals. Phosphorylation of histone H2AX, micronuclei and chromosomal aberrations were the end-points investigated. The results obtained show higher basal frequencies of binucleated cells bearing micronuclei and nucleoplasmic bridge (NPB) in LCL with respect to PBMC, suggesting that EBV transformation may be associated with chromosomal instability. After irradiation, higher levels of micronuclei were induced in G(0)-treated PBMC compared to cycling LCL; conversely, NPB were more frequent in LCL than in PBMC. Moreover, higher levels of chromosomal aberrations were observed in G(2)-treated PBMC compared to LCL. Concerning gamma-H2AX measurements, phosphorylation levels 1h after treatment and dephosphorylation kinetics were basically similar in LCL and in PBMC. However, while Spearman's test showed a strong correlation between the results obtained in replicated experiments with PBMC, high inter-experimental variability and poor reproducibility was observed in the experiments performed with LCL, possibly due to the intrinsic instability of LCL. In summary, both the analysis of gamma-H2AX and the evaluation of chromosome damage highlighted a larger inter-experimental variability in the results obtained with LCL compared to PBMC. Noteworthy, the two set of results proved to lack any significant correlation at the individual level. These results indicate that LCL may be unsuitable for investigating genotype-phenotype correlations with phenotypic DNA repair assays, especially when low impact functional genetic variants are involved.
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Línea Celular , Roturas del ADN de Doble Cadena , Reparación del ADN , Leucocitos Mononucleares/efectos de la radiación , Linfocitos , Línea Celular Transformada/efectos de la radiación , Transformación Celular Viral , Aberraciones Cromosómicas , Citometría de Flujo/métodos , Fase G2 , Estudios de Asociación Genética , Histonas/metabolismo , Linfocitos/efectos de la radiación , FosforilaciónRESUMEN
To establish immortal human cells, we introduced the cDNA of the human telomere reverse transcriptase (hTERT) gene into skin fibroblast cells obtained from normal and ataxia telangiectasia (AT) individuals of Japanese origin. hTERT-immortalized cells retained their original characteristics and radiosensitivity except for immortalization, suggesting that these cells might be useful for analyzing the effects of radiation on human cells.hTERT-immortalized cells from a normal individual showed a greater resistance after low-dose-rate irradiation than after high-dose-rate irradiation. In contrast, cells from AT patients irradiated at a low-dose rate showed virtually the same survival as those irradiated at a high-dose rate. In hTERT-immortalized normal cells, the genetic effects of low-dose-rate radiation were quantitatively and qualitatively less severe than those of high-dose-rate radiation. In hTERT-immortalized AT cells, some fraction of DNA damage such as DNA double-strand breaks might not be repaired, and AT cells were severely affected by low-dose-rate radiation. The activation of ataxia telangiectasia mutated (ATM) protein was confirmed during low-dose-rate radiation, and may play an important role in repair of DNA damage induced by low-dose-rate radiation. This paper reviews briefly the current research at our laboratory. The hTERT-immortalized cells may be useful in determining the effects of low-dose and low-dose-rate radiation on human cells.
Asunto(s)
Línea Celular Transformada , Dosis de Radiación , Telomerasa/genética , Ataxia Telangiectasia/genética , Línea Celular Transformada/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Técnicas de Transferencia de Gen , HumanosRESUMEN
Alternative and aberrant splicing of hdm2 occurs in tumor and normal tissues. However, the factors that induce these splice variants and whether they are translated to protein products in vivo is unknown, making it difficult to decipher which of these hdm2 transcripts have a normal physiologic function or contribute to carcinogenesis. We investigated the conditions that induce this post-transcriptional modification of hdm2 in tumor and nontumorigenic cell lines. We showed that UV and gamma radiation as well as cisplatin treatment induced alternative splicing of hdm2, which resulted in a single splice variant, hdm2(alt1), irrespective of the cell type. Interestingly, the mechanism of UV-induced splicing is independent of p53 status. Immunoanalysis revealed that, after UV radiation, HDM2(ALT1) protein was expressed and interacted with HDM2 that correlated to increased p53 protein levels and its accumulation in the nucleus, whereas HDM2 localized more to the cytoplasm with a decrease in its RNA and protein level. We propose that stress-induced HDM2(ALT1) regulates HDM2 at two levels, RNA and protein, further modulating the p53-HDM2 interaction or interactions of HDM2 with other cell cycle regulatory proteins. This kind of regulation may possibly restrict oncogenic functions of HDM2 and contribute to the many protective responses triggered by certain stress signals. Our data imply that HDM2(ALT1) possesses a normal physiologic function in damaged cells, perhaps facilitating cellular defense.
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Empalme Alternativo , Carcinoma/metabolismo , Daño del ADN , Células Epiteliales/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/fisiología , Proteína p53 Supresora de Tumor/metabolismo , Empalme Alternativo/efectos de los fármacos , Empalme Alternativo/efectos de la radiación , Carcinoma/genética , Línea Celular Transformada/efectos de los fármacos , Línea Celular Transformada/metabolismo , Línea Celular Transformada/efectos de la radiación , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/metabolismo , Línea Celular Tumoral/efectos de la radiación , Cisplatino/farmacología , ADN/efectos de los fármacos , ADN/efectos de la radiación , Células Epiteliales/efectos de los fármacos , Células Epiteliales/efectos de la radiación , Femenino , Rayos gamma , Homeostasis , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiología , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Proteínas Proto-Oncogénicas c-mdm2/genética , ARN Mensajero/metabolismo , ARN Neoplásico/metabolismo , Fracciones Subcelulares/metabolismo , Transcripción Genética/efectos de los fármacos , Transcripción Genética/efectos de la radiación , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genética , Rayos UltravioletaRESUMEN
We collected peripheral blood (PB) from 556 patients with various types of cancer who had undergone radiotherapy and from 81 healthy volunteers. We exposed whole PB and Epstein-Barr virus-transformed lymphoblastoid cell lines (EBLs) derived from the PB mononucleocytes to X-irradiation (5 Gy). Using the alkaline comet assay, we measured the immediate DNA damage and, at 15 min, the % residual damage. In PB, the immediate damage was similar in patients and healthy volunteers while the % residual damage (mean+/-S.D.) was significantly higher in patients with breast (54.3+/-A23.9), cervical (54.7+/-A23.9), head/neck (56.8+/-A24.4), lung (60.1+/-23.5), or esophageal cancers (59.5+/-A33.7) than in healthy donors (42.9+/-19.6) (P<0.05). We did not observe such differences in the EBV-transformed cell lines. Thus, radiation sensitivity of fresh PB cells measured by the alkaline comet assay was related to cancer status.
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Ensayo Cometa/métodos , Daño del ADN/efectos de la radiación , Reparación del ADN , Leucocitos Mononucleares/efectos de la radiación , Neoplasias/genética , Tolerancia a Radiación/genética , Donantes de Sangre , Línea Celular Transformada/efectos de la radiación , Femenino , Herpesvirus Humano 4 , Humanos , Neoplasias/sangre , Neoplasias/radioterapiaAsunto(s)
Hipoxia de la Célula/fisiología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Tolerancia a Radiación/fisiología , Factores de Transcripción/metabolismo , Animales , Línea Celular Transformada/metabolismo , Línea Celular Transformada/efectos de la radiación , Supervivencia Celular , Fibroblastos/metabolismo , Fibroblastos/efectos de la radiación , RatonesRESUMEN
PURPOSE: We sought to determine whether hypoxia-induced radioresistance is mediated by the transcription factor hypoxia-inducible factor-1alpha (HIF-1alpha). METHODS AND MATERIALS: We used 2 mouse embryonic fibroblast cell lines transformed with H-ras and TAg, 1 HIF-1alpha+/+ and the other HIF-1alpha-/-. Cell were exposed to either 95% air and 5% CO2 (normoxic conditions) or 0.2% O2, 94.8% N2, and 5% CO2 (hypoxic conditions) for 4 hours. Cells were then irradiated and subjected to clonogenic survival assays. RESULTS: Whereas neither +/+ ras/TAg nor -/- ras/TAg cells expressed HIF-1alpha under normoxic conditions, hypoxia induced expression of HIF-1alpha only in +/+ ras/TAg cells, confirming the absence of HIF-1alpha in -/- ras/TAg cells. Clonogenic survival curves for +/+ ras/TAg and -/- ras/TAg cells under normoxia and hypoxia demonstrated that hypoxia increased radioresistance in both cell lines to the same degree. At 1-log cell kill, the +/+ ras/TAg and -/- ras/TAg cells had an identical oxygen enhancement ratio of 1.28 +/- 0.09 and nearly identical oxygen enhancement ratios at 2-log cell kill. CONCLUSION: In our system of transformed mouse embryonic fibroblasts, hypoxia-mediated radiation resistance is independent of HIF-1alpha.
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Hipoxia de la Célula/fisiología , Tolerancia a Radiación/fisiología , Factores de Transcripción/metabolismo , Animales , Línea Celular Transformada/metabolismo , Línea Celular Transformada/efectos de la radiación , Fibroblastos/metabolismo , Fibroblastos/efectos de la radiación , Subunidad alfa del Factor 1 Inducible por Hipoxia , RatonesRESUMEN
P21(Waf1) cyclin-dependent kinase inhibitor blocks cell cycle transition from G1 phase into DNA replication after DNA damage. The main targets of p21(Waf1) are Cyc 1E--Cdk2 and Cyc 1A--Cdk2 complexes, PCNA (proliferating cell nuclear antigen), a subunit of DNA polymerase delta, and E2F-1 transcription factor. The universal mechanism of cell cycle arrest in normal cells is determined as p21(Waf1) interaction with positive regulators of G1 phase. As a rule, DNA integrity control mechanisms are destroyed in the process of oncogenic transformation, which results in proliferation of genetically defective cells. The purpose of our study was to investigate molecular mechanisms of cell cycle regulation in transformants that are able (E1A + E1B-19kDa) or unable (E1A(+) + cHa-ras) to be arrested at G1/S checkpoint. We have shown that p21(Waf1) is able to form complexes with cyclins and Cdks, PCNA and E2F-1 transcryption factor, although it interacts with E1A oncoproducts in both transformants. The presence of E1A bound p21(Waf1) in cyclin-kinase complexes seems to be the cause of activating phosphorilation of Cdk2 at Thr-160 in cyclin A/E--Cdk2 complexes in both control and X-ray irradiated cells. Thus, the absence of G1/S arrest following irradiation in E1A + cHa-ras transformants and its presence in E1A(+) + E1B-19kDa transformants is not connected with differences in interaction of p21(waf1) with the main regulators of G1-to-S transition, but is realized through other not yet identified ways.
Asunto(s)
Inhibidor p21 de las Quinasas Dependientes de la Ciclina/fisiología , Oncogenes , Proteínas Proto-Oncogénicas/metabolismo , Proteínas E1A de Adenovirus , Animales , Ciclo Celular , Línea Celular Transformada/citología , Línea Celular Transformada/efectos de la radiación , Ciclina A/metabolismo , Ciclina E/metabolismo , Quinasa 2 Dependiente de la Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Ciclinas/metabolismo , Factor de Transcripción E2F1/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de la radiación , Fase G1/efectos de la radiación , Fosforilación , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas Proto-Oncogénicas/genética , Ratas , Fase S/efectos de la radiación , Treonina , Rayos XRESUMEN
Ectopic expression of telomerase results in an immortal phenotype in various types of normal cells, including primary human fibroblasts. In addition to its role in telomere lengthening, telomerase has now been found to have various functions, including the control of DNA repair, chromatin modification, and the control of expression of genes involved in cell cycle regulation. The investigations on the long-term effects of telomerase expression in normal human fibroblast highlighted that these cells show low frequencies of chromosomal aberrations. In this paper, we describe the karyotypic stability of human fibroblasts immortalized by expression of hTERT. The ectopic overexpression of telomerase is associated with unusual spontaneous as well as radiation-induced chromosome stability. In addition, we found that irradiation did not enhance plasmid integration in cells expressing hTERT, as has been reported for other cell types. Long-term studies illustrated that human fibroblasts immortalized by telomerase show an unusual stability for chromosomes and for plasmid integration sites, both with and without exposure to ionizing radiation. These results confirm a role for telomerase in genome stabilisation by a telomere-independent mechanism and point to the possibility for utilizing hTERT-immortalized normal human cells for the study of gene targeting.
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Cromosomas Humanos/efectos de la radiación , Fibroblastos/efectos de la radiación , Telomerasa/fisiología , Línea Celular Transformada/enzimología , Línea Celular Transformada/efectos de la radiación , Línea Celular Transformada/ultraestructura , Aberraciones Cromosómicas , Cromosomas Humanos/metabolismo , Células Clonales/enzimología , Células Clonales/efectos de la radiación , Células Clonales/ultraestructura , Proteínas de Unión al ADN , Fibroblastos/enzimología , Fibroblastos/ultraestructura , Marcación de Gen , Humanos , Cariotipificación , Plásmidos/genética , Tolerancia a Radiación , Proteínas Recombinantes de Fusión/fisiología , Telomerasa/genética , Telómero/ultraestructura , Transfección , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
The effects of ionizing radiation (IR) on the temporal transcriptional response of lymphoblastoid cells were investigated in this study. We used oligonucleotide microarrays to assess mRNA levels of genes in lymphoblastoid cells at various time points within 24 h following gamma-irradiation. We identified 319 and 816 IR-responsive genes following 3 Gy and 10 Gy of IR exposure, respectively, with 126 genes in common between the two doses. A high percentage of IR-responsive genes are involved in the control of cell cycle, cell death, DNA repair, DNA metabolism, and RNA processing. We determined the temporal expression profiles of the IR-responsive genes and assessed effects of IR dose on this temporal pattern of expression. By combining dose-response data with temporal profiles of expression, we have identified sets of coordinately responding genes. Through a genomic approach, we characterized a set of genes that are implicated in cellular adaptation to IR stress. These findings will allow a better understanding of complex processes such as radiation-induced carcinogenesis and the development of biomarkers for radiation exposure.
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Línea Celular Transformada/efectos de la radiación , Rayos gamma , Regulación de la Expresión Génica/efectos de la radiación , Linfocitos/efectos de la radiación , Transcripción Genética/efectos de la radiación , Línea Celular Transformada/metabolismo , Relación Dosis-Respuesta en la Radiación , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/genética , Humanos , Linfocitos/metabolismo , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/metabolismo , ARN Mensajero/efectos de la radiación , Dosis de Radiación , Radiación IonizanteRESUMEN
Exposure to ionizing radiation is believed to cause cell injury via the production of free radicals that are thought to induce oxidative damage. It has been proposed that exposure to agents that enhance oxidative stress-induced injury by disrupting thiol metabolism may sensitize cells to the cytotoxic effects of ionizing radiation. Recently, it has been shown that glucose deprivation selectively induces cell injury in transformed human cells via metabolic oxidative stress (J. Biol. Chem., 273: 5294-5299; Ann. N.Y. Acad. Sci., 899: 349-362), resulting in profound disruptions in thiol metabolism. Because 2-deoxy-D-glucose (2DG) is a potent inhibitor of glucose metabolism thought to mimic glucose deprivation in vivo, the hypothesis that exposure to 2DG might be capable of inducing radiosensitization in transformed cells via perturbations in thiol metabolism was tested. When HeLa cells were exposed to 2DG (4-10 mM) for 4-72 h, cell survival decreased (20-90%) in a dose- and time-dependent fashion. When HeLa cells were treated with 6 mM 2DG for 16 h before ionizing radiation exposure, radiosensitization was observed with a sensitizer enhancement ratio of 1.4 at 10% isosurvival. Treatment with 2DG was also found to cause decreases in intracellular total glutathione content (50%). Simultaneous treatment with the thiol antioxidant N-acetylcysteine (NAC; 30 mM) protected HeLa cells against the cytotoxicity and radiosensitizing effects of 2DG, without altering radiosensitivity in the absence of 2DG. Furthermore, treatment with NAC partially reversed the 2DG-induced decreases in total glutathione content, as well as augmented intracellular cysteine content. Finally, the cytotoxicity and radiosensitizing effects of 2DG were more pronounced in v-Fos-transformed versus nontransformed immortalized rat cells, and this radiosensitization was also inhibited by treatment with NAC. These results support the hypothesis that exposure to 2DG causes cytotoxicity and radiosensitization via a mechanism involving perturbations in thiol metabolism and allows for the speculation that these effects may be more pronounced in transformed versus normal cells.
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Desoxiglucosa/toxicidad , Fármacos Sensibilizantes a Radiaciones/toxicidad , Compuestos de Sulfhidrilo/metabolismo , Acetilcisteína/farmacología , Animales , Antioxidantes/farmacología , Línea Celular Transformada/efectos de los fármacos , Línea Celular Transformada/efectos de la radiación , Transformación Celular Viral , Cisteína/metabolismo , Desoxiglucosa/farmacología , Relación Dosis-Respuesta a Droga , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/efectos de la radiación , Radicales Libres , Genes fos , Glucosa/antagonistas & inhibidores , Glucosa/metabolismo , Glutatión/metabolismo , Células HeLa/efectos de los fármacos , Células HeLa/metabolismo , Células HeLa/efectos de la radiación , Humanos , Proteínas Oncogénicas v-fos/fisiología , Oxidación-Reducción , Estrés Oxidativo , Protectores contra Radiación/farmacología , Fármacos Sensibilizantes a Radiaciones/farmacología , Ratas , Ensayo de Tumor de Célula MadreRESUMEN
PURPOSE: To determine if clinically relevant doses of ionizing radiation are capable of inducing extrachromosomal DNA loss in transformed human cell lines. MATERIALS AND METHODS: The multidrug-resistant (MDR) human epidermoid KB-C1 cell line and the human neuroendocrine colon carcinoma line COLO320, which contain extrachromosomally amplified MDR1 drug resistance genes and MYCC oncogenes, were irradiated with 2 Gy fractions up to a total dose of 28 Gy. To track the fate of extrachromosomally amplified genes, cells surviving radiation therapy and unirradiated control cells were analyzed by fluorescent in situ hybridization of chromosomes using MDR1 and MYCC-specific cosmid DNA probes. In addition, total DNA and protein isolated from irradiated and control cells was subjected to Southern and Western blotting procedures, respectively, to determine amplified gene copy number and protein expression levels. Dose-response assays to follow loss of function of the MDR1 gene from KB-C1 cells were also performed. RESULTS: A significant reduction in extrachromosomal DNA, amplified gene copy number, and expression was detected in surviving cells after relatively low doses of radiation. Entrapment of extrachromosomal DNA into micronuclei was a consistent feature of irradiated cells. CONCLUSIONS: Clinically relevant doses of radiation can deplete extrachromosomal DNA in viable human malignant cells and alter their phenotype. Depletion of extrachromosomally amplified genes from tumor cells occurs via entrapment in radiation-induced micronuclei.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/análisis , Resistencia a Antineoplásicos/efectos de la radiación , Amplificación de Genes , Eliminación de Gen , Genes MDR/efectos de la radiación , Genes myc/efectos de la radiación , Proteínas Proto-Oncogénicas c-myc/análisis , Línea Celular Transformada/efectos de los fármacos , Línea Celular Transformada/efectos de la radiación , Fraccionamiento de la Dosis de Radiación , Relación Dosis-Respuesta en la Radiación , Resistencia a Antineoplásicos/genética , Citometría de Flujo , Genes MDR/efectos de los fármacos , Genes myc/efectos de los fármacos , Humanos , Pruebas de Micronúcleos , Tolerancia a Radiación/efectos de los fármacos , Tolerancia a Radiación/genética , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/efectos de la radiación , Ensayo de Tumor de Célula MadreRESUMEN
Malignant gliomas are extremely aggressive cancers currently lacking effective treatment modalities. Gene therapy represents a promising approach for this disease. A requisite component for improving gene-based therapies of brain cancer includes tumor suppressor genes that exhibit cancer constrained inhibitory activity. Subtraction hybridization identified melanoma differentiation associated gene-7 (mda-7) as a gene associated with melanoma cell growth, differentiation and progression. Ectopic expression of mda-7 by means of a replication-incompetent adenovirus (Ad), Ad.mda-7, induces growth suppression and apoptosis selectively in diverse human cancers, without producing any apparent harmful effect in normal cells. We presently demonstrate that Ad.mda-7 induces growth inhibition and apoptosis in malignant human gliomas expressing both mutant and wild-type p53, and these effects correlate with an elevation in expression of members of the growth arrest and DNA damage (GADD) gene family. In contrast, infection with a recombinant Ad expressing wild-type p53, Ad.wtp53, specifically affects mutant p53 expressing gliomas. When tested in early passage normal and immortal human fetal astrocytes, growth inhibition resulting from infection with Ad.mda-7 or Ad.wtp53 is significantly less than in malignant gliomas and no toxicity is evident in these normal cells. Moreover, infection of gliomas with Ad.mda-7 or treatment with purified GST-MDA-7 protein sensitizes both wild-type and mutant p53 expressing tumor cells to the growth inhibitory and antisurvival effects of ionizing radiation, and this response correlates with increased expression of specific members of the GADD gene family. Since heterogeneity in p53 expression is common in evolving gliomas, the present findings suggest that Ad.mda-7 may, in many instances, prove more beneficial for the gene-based therapy of malignant gliomas than administration of wild-type p53.
Asunto(s)
Apoptosis , Astrocitos/citología , Neoplasias Encefálicas/patología , Glioma/patología , Interleucinas/fisiología , Apoptosis/genética , Apoptosis/efectos de la radiación , Astrocitos/efectos de la radiación , Neoplasias Encefálicas/terapia , División Celular/efectos de la radiación , Línea Celular Transformada/citología , Línea Celular Transformada/efectos de la radiación , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus , Daño del ADN , Genes Supresores de Tumor , Genes p53 , Terapia Genética , Vectores Genéticos/farmacología , Glioma/terapia , Humanos , Interleucinas/genética , Tolerancia a Radiación/genética , Receptores Virales/análisis , Proteínas Recombinantes de Fusión/fisiología , Transducción Genética , Células Tumorales Cultivadas/citología , Células Tumorales Cultivadas/efectos de la radiación , Proteína p53 Supresora de Tumor/fisiologíaRESUMEN
Expression of human adenovirus type 5 E1A oncogene in normal rodent cells leads to disruption of the G1/S cell cycle arrest realization in response to DNA damage. It has been shown here that rat embryo fibroblasts transformed by E1Aad5 oncogene in complementation with E1B-19 kDa gene realize the irradiation-induced transient G1/S arrest, which depends on selective suppression of CyclinE-Cdk2 activity despite functional inactivation of p21Waf1 inhibitor. Inhibitor p21Waf1 is not revealed in complexes with cyclins E and A in E1A + E1B-19 kDa transformants, however, it is not due to p21Waf1 interaction with E1A oncoproteins, because the E1A-p21Waf1 complex formation in E1A + cHa-ras transformants does not prevent the high level of CycIE, A-p21Waf1 association. In the case of p21Waf1 inactivation, the main way of cyclin-kinase activity regulation in E1A + E1B-19 kDa cells may be Cdk2 phosphorylation. However, irradiation of E1A + E1B-19 kDa transformed cells induces no changes in CAK (Cdk7-associated) kinase activity and in the protein level of Cdc25A phosphatase, which are responsible for activating Thr160 phosphoralation and Tyr15 dephosphorylation on Cdk2. Using phospho-Tyr15-Cdk2 specific antibodies, no increase of phosphorylation at Tyr15 position on immunoprecipitated Cdk2 was detected after irradiation. It seems likely that in the case of inactivated inhibitor p21Waf1 the transient G1/S block after irradiation in E1A + E1B-19 kDa transformants depends on suppression of Cycl-E-Cdk2 activity caused by inhibition of Thr160 Cdk2 phosphorylation, but his occurs with the involvement of other kinases rather than CAK.
Asunto(s)
Línea Celular Transformada/efectos de la radiación , Transformación Celular Neoplásica/efectos de la radiación , Daño del ADN , Fase G1 , Fase S , Proteínas E1A de Adenovirus/biosíntesis , Proteínas E1A de Adenovirus/genética , Proteínas E1B de Adenovirus/biosíntesis , Proteínas E1B de Adenovirus/genética , Animales , Quinasas CDC2-CDC28/análisis , Línea Celular Transformada/citología , Ciclina A/análisis , Ciclina B/análisis , Quinasa 2 Dependiente de la Ciclina , Embrión de Mamíferos , Fibroblastos/citología , Fibroblastos/efectos de la radiación , Fosforilación , RatasRESUMEN
Y-family DNA polymerases can replicate past a variety of damaged bases in vitro but, with the exception of DNA polymerase eta (poleta), which is defective in xeroderma pigmentosum variants, there is little information on the functions of these polymerases in vivo. Here, we show that DNA polymerase iota (poliota), like poleta, associates with the replication machinery and accumulates at stalled replication forks following DNA-damaging treatment. We show that poleta and poliota foci form with identical kinetics and spatial distributions, suggesting that localization of these two polymerases is tightly co-ordinated within the nucleus. Furthermore, localization of poliota in replication foci is largely dependent on the presence of poleta. Using several different approaches, we demonstrate that poleta and poliota interact with each other physically and that the C-terminal 224 amino acids of poliota are sufficient for both the interaction with poleta and accumulation in replication foci. Our results provide strong evidence that poleta targets poliota to the replication machinery, where it may play a general role in maintaining genome integrity as well as participating in translesion DNA synthesis.
Asunto(s)
Replicación del ADN/fisiología , ADN Polimerasa Dirigida por ADN/fisiología , Transporte Activo de Núcleo Celular , Animales , Cafeína/toxicidad , Línea Celular , Línea Celular Transformada/efectos de los fármacos , Línea Celular Transformada/efectos de la radiación , Núcleo Celular/enzimología , ADN/efectos de los fármacos , ADN/efectos de la radiación , Daño del ADN , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/deficiencia , ADN Polimerasa Dirigida por ADN/genética , Genes Reporteros , Prueba de Complementación Genética , Humanos , Microscopía Fluorescente , Mapeo de Interacción de Proteínas , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de Secuencia , Spodoptera/citología , Transfección , Técnicas del Sistema de Dos Híbridos , Rayos Ultravioleta , Xerodermia Pigmentosa/enzimología , Xerodermia Pigmentosa/genética , Xerodermia Pigmentosa/patología , ADN Polimerasa iotaRESUMEN
OBJECTIVE: Fanconi anemia (FA) is a genetically heterogeneous disorder associated with defects in at least eight genes. The biochemical function(s) of the FA proteins are unknown, but together they define the FA pathway, which is involved in cellular responses to DNA damage and in other cellular processes. It is currently unknown whether all FA proteins are involved in controlling a single function or whether some of the FA proteins have additional roles. The aim of this study was 1) to determine whether the FA group A and group C genes have identical or partially distinct functions, and 2) to have a better model for human FA. MATERIALS AND METHODS: We generated mice with a targeted mutation in fanca and crossed them with fancc disrupted animals. Several phenotypes including sensitivity to DNA cross linkers and ionizing radiation, hematopoietic colony growth, and germ cell loss were analyzed in fanca-/-, fancc-/-, fanca/fancc double -/-, and controls. RESULTS: Fibroblast cells and hematopoietic precursors from fanca/fancc double-mutant mice were not more sensitive to MMC than those of either single mutant. fanca/fancc double mutants had no evidence for an additive phenotype at the cellular or organismal level. CONCLUSIONS: These results support a model where both FANCA and FANCC are part of a multi-protein nuclear FA complex with identical function in cellular responses to DNA damage and germ cell survival.