RESUMO
Intense electromagnetic fields (EMFs) induce DNA double stranded breaks (DSBs) in exposed lymphocytes.We study developing pre-B lymphocytes following V(D)J recombination at their Immunoglobulin light chain loci (IgL). Recombination physiologically induces DNA DSBs, and we tested if low doses of EMF irradiation affect this developmental stage. Recombining pre-B cells, were exposed for 48 h to low intensity EMFs (maximal radiative power density flux S of 9.5 µW/cm2 and electric field intensity 3 V/m) from waves of frequencies ranging from 720 to 1224 MHz. Irradiated pre-B cells show decreased levels of recombination, reduction which is dependent upon the power dose and most remarkably upon the frequency of the applied EMF. Although 50% recombination reduction cannot be obtained even for an S of 9.5 µW/cm2 in cells irradiated at 720 MHz, such an effect is reached in cells exposed to only 0.45 µW/cm2 power with 950 and 1000 MHz waves. A maximal four-fold recombination reduction was measured in cells exposed to 1000 MHz waves with S from 0.2 to 4.5 µW/cm2 displaying normal levels of γH2AX phosphorylated histone. Our findings show that developing B cells exposure to low intensity EMFs can affect the levels of production and diversity of their antibodies repertoire.
Assuntos
Campos Eletromagnéticos , Células Precursoras de Linfócitos B/efeitos da radiação , Ondas de Rádio , Animais , Anticorpos/efeitos da radiação , Linhagem Celular , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Camundongos , Terapia por Radiofrequência/tendênciasRESUMO
DNA damage can be generated in multiple ways from genotoxic and physiologic sources. Genotoxic damage is known to disrupt cellular functions and is lethal if not repaired properly. We compare the transcriptional programs activated in response to genotoxic DNA damage induced by ionizing radiation (IR) in abl pre-B cells from mice deficient in DNA damage response (DDR) genes Atm, Mre11, Mdc1, H2ax, 53bp1, and DNA-PKcs. We identified a core IR-specific transcriptional response that occurs in abl pre-B cells from WT mice and compared the response of the other genotypes to the WT response. We also identified genotype specific responses and compared those to each other. The WT response includes many processes involved in lymphocyte development and immune response, as well as responses associated with the molecular mechanisms of cancer, such as TP53 signaling. As expected, there is a range of similarity in transcriptional profiles in comparison to WT cells, with Atm-/- cells being the most different from the core WT DDR and Mre11 hypomorph (Mre11A/A) cells also very dissimilar to WT and other genotypes. For example, NF-kB-related signaling and CD40 signaling are deficient in both Atm-/- and Mre11A/A cells, but present in all other genotypes. In contrast, IR-induced TP53 signaling is seen in the Mre11A/A cells, while these responses are not seen in the Atm-/- cells. By examining the similarities and differences in the signaling pathways in response to IR when specific genes are absent, our results further illustrate the contribution of each gene to the DDR. The microarray gene expression data discussed in this paper have been deposited in NCBI's Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/) and are accessible under accession number GSE116388.
Assuntos
Dano ao DNA/genética , Células Precursoras de Linfócitos B/metabolismo , Animais , Ciclo Celular/genética , Ciclo Celular/efeitos da radiação , Pontos de Checagem do Ciclo Celular/genética , Regulação da Expressão Gênica/efeitos da radiação , Genótipo , Camundongos , Células Precursoras de Linfócitos B/imunologia , Células Precursoras de Linfócitos B/efeitos da radiação , Radiação Ionizante , Transdução de Sinais , Transcrição Gênica/efeitos da radiaçãoRESUMO
DNA double strand break (DSB) repair is critical for generation of B-cell receptors, which are pre-requisite for B-cell progenitor survival. However, the transcription factors that promote DSB repair in B cells are not known. Here we show that MEF2C enhances the expression of DNA repair and recombination factors in B-cell progenitors, promoting DSB repair, V(D)J recombination and cell survival. Although Mef2c-deficient mice maintain relatively intact peripheral B-lymphoid cellularity during homeostasis, they exhibit poor B-lymphoid recovery after sub-lethal irradiation and 5-fluorouracil injection. MEF2C binds active regulatory regions with high-chromatin accessibility in DNA repair and V(D)J genes in both mouse B-cell progenitors and human B lymphoblasts. Loss of Mef2c in pre-B cells reduces chromatin accessibility in multiple regulatory regions of the MEF2C-activated genes. MEF2C therefore protects B lymphopoiesis during stress by ensuring proper expression of genes that encode DNA repair and B-cell factors.
Assuntos
Quebras de DNA de Cadeia Dupla , Hematopoese/fisiologia , Células Precursoras de Linfócitos B/fisiologia , Recombinação V(D)J/fisiologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Sobrevivência Celular/efeitos da radiação , Cromatina/metabolismo , Feminino , Fluoruracila/farmacologia , Hematopoese/efeitos dos fármacos , Hematopoese/efeitos da radiação , Fatores de Transcrição MEF2/fisiologia , Masculino , Camundongos , Células Precursoras de Linfócitos B/efeitos dos fármacos , Células Precursoras de Linfócitos B/efeitos da radiação , Irradiação Corporal Total/efeitos adversosRESUMO
Ionizing radiation (IR) induces cellular stress responses, such as signal transduction, gene expression, protein modification, and metabolite change that affect cellular behavior. We analyzed X-irradiated human Epstein-Barr virus-transformed B lymphoblastoid cells and normal fibroblasts to search for metabolites that would be suitable IR-responsive markers by Liquid Chromotography-Mass spectrometry (LC-MS). Mass spectra, as analyzed with principal component analysis, showed that the proportion of peaks with IR-induced change was relatively small compared with the influence of culture time. Dozens of peaks that had either been upregulated or downregulated by IR were extracted as candidate IR markers. The IR-changed peaks were identified by comparing mock-treated groups to 100 mGy-irradiated groups that had recovered after 10 h, and the results indicated that the metabolites involved in nucleoside synthesis increased and that some acylcarnitine levels decreased in B lymphoblastoids. Some peaks changed by as much as 20 mGy, indicating the presence of an IR-sensitive signal transduction/metabolism control mechanism in these cells. On the other hand, we could not find common IR-changed peaks in fibroblasts of different origin. These data suggest that cell phenotype-specific pathways exist, even in low-dose responses, and could determine cell behavior.
Assuntos
Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Ácidos Nucleicos/metabolismo , Células Precursoras de Linfócitos B/metabolismo , Células Precursoras de Linfócitos B/efeitos da radiação , Transdução de Sinais/fisiologia , Relação Dose-Resposta à Radiação , Humanos , Doses de Radiação , Transdução de Sinais/efeitos da radiação , Raios XRESUMO
In the present study, the protein microarray was used to investigate the protein expression in human B-cell lymphoblastoid cells intermittently exposed to 1.8-GHz GSM radiofrequency radiation (RFR) at the specific absorption rate (SAR) of 2.0 W/kg for 24 h. The differential expression of 27 proteins was found, which were related to DNA damage repair, apoptosis, oncogenesis, cell cycle and proliferation (ratio >1.5-fold, P<0.05). The results validated with Western blot assay indicated that the expression of RPA32 was significantly down-regulated (P<0.05) while the expression of p73 was significantly up-regulated in RFR exposure group (P<0.05). Because of the crucial roles of those proteins in DNA repair and cell apoptosis, the results of present investigation may explain the biological effects of RFR on DNA damage/repair and cell apoptosis.
Assuntos
Células Precursoras de Linfócitos B/metabolismo , Células Precursoras de Linfócitos B/efeitos da radiação , Proteínas/metabolismo , Proteínas/efeitos da radiação , Ondas de Rádio/efeitos adversos , Apoptose/efeitos da radiação , Ciclo Celular/efeitos da radiação , Linhagem Celular , Proliferação de Células/efeitos da radiação , Transformação Celular Neoplásica/efeitos da radiação , Reparo do DNA/efeitos da radiação , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/efeitos da radiação , Regulação para Baixo/efeitos da radiação , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/efeitos da radiação , Proteínas Nucleares/metabolismo , Proteínas Nucleares/efeitos da radiação , Análise Serial de Proteínas , Proteína de Replicação A/metabolismo , Proteína de Replicação A/efeitos da radiação , Proteína Tumoral p73 , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/efeitos da radiação , Regulação para Cima/efeitos da radiaçãoRESUMO
Little is known about the effects of ionizing radiation on the transition and the related signal transduction of progenitor B cells in the bone marrow. Thus, using an NIH Swiss mouse model, we explored the impact of ionizing radiation on the early stage of B-cell development via an examination of the transition of CLP to pro-B to pre-B cells within bone marrow as a function of radiation doses and times. Our results showed that while the total number of bone marrow lymphoid cells at different stages were greatly reduced by subtotal body irradiation (sub-TBI), the surviving cells continued to transition from common lymphoid progenitors to pro-B and then to pre-B in a reproducible temporal pattern. The rearrangement of the immunoglobulin heavy chain increased significantly 1-2 weeks after irradiation, but no change occurred after 3-4 weeks. The rearrangement of the immunoglobulin light chain decreased significantly 1-2 weeks after sub-TBI but increased dramatically after 3-4 weeks. In addition, several key transcription factors and signaling pathways were involved in B-precursor transitions after sub-TBI. The data indicate that week 2 after irradiation is a critical time for the transition from pro-B cells to pre-B cells, reflecting that the functional processes for different B-cell stages are well preserved even after high-dose irradiation.
Assuntos
Medula Óssea/fisiologia , Medula Óssea/efeitos da radiação , Células Precursoras de Linfócitos B/metabolismo , Células Precursoras de Linfócitos B/efeitos da radiação , Regeneração/efeitos da radiação , Irradiação Corporal Total , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/efeitos da radiação , Contagem de Células , Rearranjo Gênico do Linfócito B , Masculino , Camundongos , Células Precursoras de Linfócitos B/citologia , Receptores de Interleucina-7/metabolismo , Transdução de Sinais/efeitos da radiação , Fatores de Transcrição/metabolismoAssuntos
Ciclosporina/toxicidade , Reparo do DNA/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/efeitos da radiação , Imunossupressores/toxicidade , Células Precursoras de Linfócitos B/efeitos dos fármacos , Células Precursoras de Linfócitos B/efeitos da radiação , Sirolimo/análogos & derivados , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Dano ao DNA/efeitos da radiação , Reparo do DNA/efeitos da radiação , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Ensaio de Imunoadsorção Enzimática , Everolimo , Fibroblastos/citologia , Humanos , Luciferases de Vaga-Lume/genética , Luciferases de Vaga-Lume/metabolismo , Células Precursoras de Linfócitos B/citologia , Proteínas Quinases/efeitos dos fármacos , Dímeros de Pirimidina/análise , Dímeros de Pirimidina/química , Dímeros de Pirimidina/imunologia , Sirolimo/uso terapêutico , Serina-Treonina Quinases TOR , Fatores de Tempo , Raios Ultravioleta/efeitos adversosRESUMO
Artemis is a recently identified factor involved in V(D)J recombination and nonhomologous end joining (NHEJ) of DNA double-strand break (DSB) repair. Here, we performed targeted disruption of the Artemis gene (ARTEMIS) in the human pre-B cell line Nalm-6. Unexpectedly, we found that cells lacking Artemis exhibit increased sensitivity to low doses, but not high doses, of ionizing radiation. We also show that ARTEMIS-deficient cells are hypersensitive to the topoisomerase II inhibitor etoposide, but to a much lesser extent than cells lacking DNA ligase IV, a critical component of NHEJ. Unlike DNA ligase IV-deficient cells, ARTEMIS-deficient cells were not hypersensitive to ICRF-193, a topoisomerase II inhibitor that does not stabilize topoisomerase II-DNA cleavable complexes. Collectively, our results suggest that Artemis only partially participates in the NHEJ pathway to repair DSBs in human somatic cells.
Assuntos
Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , Inibidores Enzimáticos/farmacologia , Proteínas Nucleares/fisiologia , Southern Blotting , Western Blotting , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/efeitos da radiação , Ensaio de Unidades Formadoras de Colônias , Primers do DNA/química , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA , Dicetopiperazinas , Relação Dose-Resposta à Radiação , Endonucleases , Etoposídeo/farmacologia , Citometria de Fluxo , Humanos , Piperazinas/farmacologia , Células Precursoras de Linfócitos B/efeitos dos fármacos , Células Precursoras de Linfócitos B/metabolismo , Células Precursoras de Linfócitos B/efeitos da radiação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Tolerância a Radiação , Radiação Ionizante , Recombinação Genética/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Inibidores da Topoisomerase IIRESUMO
Radiation exposure is an occupational hazard for military personnel, some health care professionals, airport security screeners, and medical patients, with some individuals at risk for acute, high-dose exposures. Therefore, the biological effects of radiation, especially the potential for chromosome damage, are major occupational and health concerns. However, the biophysical mechanisms of chromosome instability subsequent to radiation-induced DNA damage are poorly understood. It is clear that interphase chromosomes occupy discrete structural and functional subnuclear domains, termed chromosome territories (CT), which may be organized into 'neighborhoods' comprising groups of specific CTs. We directly evaluated the relationship between chromosome positioning, neighborhood composition, and translocation partner choice in primary lymphocytes, using a cell-based system in which we could induce multiple, concentrated DNA breaks via high-dose irradiation. We critically evaluated mis-rejoining profiles and tested whether breaks occurring nearby were more likely to fuse than breaks occurring at a distance. We show that CT neighborhoods comprise heterologous chromosomes, within which inter-CT distances directly relate to translocation partner choice. These findings demonstrate that interphase chromosome arrangement is a principal factor in genomic instability outcomes in primary lymphocytes, providing a structural context for understanding the biological effects of radiation exposure, and the molecular etiology of tumor-specific translocation patterns.