Conserved genes and pathways in primary human fibroblast strains undergoing replicative and radiation induced senescence

BACKGROUND: Cellular senescence is induced either internally, for example by replication exhaustion and cell division, or externally, for example by irradiation. In both cases, cellular damages accumulate which, if not successfully repaired, can result in senescence induction. Recently, we determined the transcriptional changes combined with the transition into replicative senescence in primary human fibroblast strains. Here, by γ-irradiation we induced premature cellular senescence in the fibroblast cell strains (HFF and MRC-5) and determined the corresponding transcriptional changes by high-throughput RNA sequencing. RESULTS: Comparing the transcriptomes, we found a high degree of similarity in differential gene expression in replicative as well as in irradiation induced senescence for both cell strains suggesting, in each cell strain, a common cellular response to error accumulation. On the functional pathway level, "Cell cycle" was the only pathway commonly down-regulated in replicative and irradiation-induced senescence in both fibroblast strains, confirming the tight link between DNA repair and cell cycle regulation. However, "DNA repair" and "replication" pathways were down-regulated more strongly in fibroblasts undergoing replicative exhaustion. We also retrieved genes and pathways in each of the cell strains specific for irradiation induced senescence. CONCLUSION: We found the pathways associated with "DNA repair" and "replication" less stringently regulated in irradiation induced compared to replicative senescence. The strong regulation of these pathways in replicative senescence highlights the importance of replication errors for its induction.

Nucleotide excision repair pathway assessment in DNA exposed to low-intensity red and infrared lasers

Low-intensity lasers are used for prevention and management of oral mucositis induced by anticancer therapy, but the effectiveness of treatment depends on the genetic characteristics of affected cells. This study evaluated the survival and induction of filamentation of Escherichia coli cells deficient in the nucleotide excision repair pathway, and the action of T4endonuclease V on plasmid DNA exposed to low-intensity red and near-infrared laser light. Cultures of wild-type (strain AB1157) E. coli and strain AB1886 (deficient in uvrA protein) were exposed to red (660 nm) and infrared (808 nm) lasers at various fluences, powers and emission modes to study bacterial survival and filamentation. Also, plasmid DNA was exposed to laser light to study DNA lesions produced in vitro by T4endonuclease V. Low-intensity lasers:i) had no effect on survival of wild-type E. coli but decreased the survival of uvrA protein-deficient cells,ii) induced bacterial filamentation, iii) did not alter the electrophoretic profile of plasmids in agarose gels, andiv) did not alter the electrophoretic profile of plasmids incubated with T4 endonuclease V. These results increase our understanding of the effects of laser light on cells with various genetic characteristics, such as xeroderma pigmentosum cells deficient in nucleotide excision pathway activity in patients with mucositis treated by low-intensity lasers.

The effect of elemene on lung adenocarcinoma A549 cell radiosensitivity and elucidation of its mechanism

OBJECTIVE: To investigate the effect of elemene on the radiosensitivity of A549 cells and its possible molecular mechanism. METHODS: Apoptosis of A549 cells was detected by flow cytometry and fluorescence microscopy. The effect of double-strand break (DSB) damage repair in A549 cells was evaluated using the neutral comet assay. Protein expression levels were detected using western blotting, and the correlation between protein levels was analyzed. RESULTS: Elemene exhibited a radiosensitizing effect on A549 cells. The level of apoptosis induced by elemene combined with radiation was significantly greater (p<0.01) than that elicited by either radiation or elemene alone. Following radiation and subsequent repair for 24 h, the tail intensity of A549 cells treated with a combination of elemene and radiation was greater than that of cells treated with either elemene or radiation alone (p<0.01). This result indicates that elemene inhibits cellular DSB repair. Both elemene combined with radiation and radiation alone decreased the protein expression of DNA-PKcs and Bcl-2 compared to elemene alone (p<0.01), while p53 protein expression was increased (p<0.01). A negative correlation was observed between DNA-PKcs and p53 expression (r=−0.569, p=0.040), while a positive correlation was found between DNA-PKcs and Bcl-2 expression (r=0.755, p=0.012). CONCLUSIONS: Elemene exhibits a radiosensitizing effect on A549 cells, and its underlying molecular mechanism of action may be related to the downregulation of DNA-PKcs gene expression. .

MicroRNAs, cancer and ionizing radiation: Where are we? / MicroRNAs, câncer e radiação ionizante: em que ponto estamos?

Summary The aim of this study is to describe the biogenesis of microRNA, its relations with carcinogenesis, and the correlation between microRNA and ionizing radiation (IR), focusing on radioresponsiveness. It is known that microRNA biogenesis is well established and involves different enzymatic cleavages, resulting in the production of mature microRNA. MicroRNAs are involved in carcinogenesis. Their interaction is related to the genetic and epigenetic changes associated with activation of proto-oncogenes or inactivation of tumor suppressor genes. Several studies have shown that the levels of expression of some microRNAs vary significantly after irradiation. There are evidences that microRNAs can influence cellular response after IR. In addition, microRNAs are related to modulation of the expression of several post-transcriptional targets in DNA damage response pathways, and to the DNA damage repair regulation mechanism. Future studies can clarify a possible clinical use of microRNAs as a new class of radiosensitive agents.

Resumo O objetivo do presente estudo é descrever a biogênese do microRNA, suas relações na carcinogênese e a correlação do microRNA com a radiação ionizante (RI), com enfoque na radiorresponsividade. Observou-se que a biogênese do microRNA está bem estabelecida e envolve diversas clivagens enzimáticas que resultam na produção do microRNA maduro. Os microRNAs estão envolvidos na carcinogênese. Sua interação está relacionada às alterações genéticas e epigenéticas, associadas à ativação de proto- -oncogenes ou à inativação de genes supressores de tumor. Vários estudos demonstraram que os níveis de expressão de alguns microRNAs variam significativamente após a irradiação. Há evidências de que os microRNAs podem influenciar a resposta celular após a RI. Além disso, os microRNAs estão relacionados à modulação da expressão de vários alvos de pós-transcrição das vias de resposta aos danos no DNA e o do mecanismo de regulação de reparação de danos do DNA. Estudos futuros podem elucidar uma possível utilização clínica dos microRNAs como uma nova classe de agentes radiossensíveis.

Differential expression of thymic DNA repair genes in low-dose-rate irradiated AKR/J mice

We previously determined that AKR/J mice housed in a low-dose-rate (LDR) (137Cs, 0.7 mGy/h, 2.1 Gy) gamma-irradiation facility developed less spontaneous thymic lymphoma and survived longer than those receiving sham or high-dose-rate (HDR) (137Cs, 0.8 Gy/min, 4.5 Gy) radiation. Interestingly, histopathological analysis showed a mild lymphomagenesis in the thymus of LDR-irradiated mice. Therefore, in this study, we investigated whether LDR irradiation could trigger the expression of thymic genes involved in the DNA repair process of AKR/J mice. The enrichment analysis of Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes pathways showed immune response, nucleosome organization, and the peroxisome proliferator-activated receptors signaling pathway in LDR-irradiated mice. Our microarray analysis and quantitative polymerase chain reaction data demonstrated that mRNA levels of Lig4 and RRM2 were specifically elevated in AKR/J mice at 130 days after the start of LDR irradiation. Furthermore, transcriptional levels of H2AX and ATM, proteins known to recruit DNA repair factors, were also shown to be upregulated. These data suggest that LDR irradiation could trigger specific induction of DNA repair-associated genes in an attempt to repair damaged DNA during tumor progression, which in turn contributed to the decreased incidence of lymphoma and increased survival. Overall, we identified specific DNA repair genes in LDR-irradiated AKR/J mice.

Radiosensitivity and repair kinetics of gamma-irradiated leukocytes from sporadic prostate cancer patients and healthy individuals assessed by alkaline comet assay

Impaired DNA repair mechanism is one of the main causes of tumor genesis. Study of intrinsic radiosensitivity of cancer patients in a non-target tissue [e.g. peripheral blood] might show the extent of DNA repair deficiency of cells in affected individuals and might be used a predictor of cancer predisposition. Initial radiation-induced DNA damage [ratio of Tail DN A/Head DNA], dose-response curves and kinetics of DNA repair in leukocytes from healthy volunteers and prostate cancer patients were assessed using alkaline comet assay after exposure to [60]Co gamma rays. Results showed that higher levels of baseline and gamma rays induced DNA damage in leukocytes of prostate cancer cases than in controls. A similar dose response was obtained for both groups. After a repair time of 24 h following in vitro irradiation, samples from the healthy individuals showed no residual DNA damage in their leukocytes, whereas prostate cancer patients revealed more than 20%. Although similar initial radiosensitivity was observed for both groups, the repair kinetics of radiation induced DNA damage of leukocytes from prostate cancer cases and healthy subjects were statistically different. These results support the hypothesis that men affected by prostate cancer may have a constitutional genomic instability

Evaluation of radioinduced damage and repair capacity in blood lymphocytes of breast cancer patients

Genetic damage caused by ionizing radiation and repair capacity of blood lymphocytes from 3 breast cancer patients and 3 healthy donors were investigated using the comet assay. The comets were analyzed by two parameters: comet tail length and visual classification. Blood samples from the donors were irradiated in vitro with a 60Co source at a dose rate of 0.722 Gy/min, with a dose range of 0.2 to 4.0 Gy and analyzed immediately after the procedure and 3 and 24 h later. The basal level of damage and the radioinduced damage were higher in lymphocytes from breast cancer patients than in lymphocytes from healthy donors. The radioinduced damage showed that the two groups had a similar response when analyzed immediately after the irradiations. Therefore, while the healthy donors presented a considerable reduction of damage after 3 h, the patients had a higher residual damage even 24 h after exposure. The repair capacity of blood lymphocytes from the patients was slower than that of lymphocytes from healthy donors. The possible influence of age, disease stage and mutations in the BRCA1 and BRCA2 genes are discussed. Both parameters adopted proved to be sensitive and reproducible: the dose-response curves for DNA migration can be used not only for the analysis of cellular response but also for monitoring therapeutic interventions. Lymphocytes from the breast cancer patients presented an initial radiosensitivity similar to that of healthy subjects but a deficient repair mechanism made them more vulnerable to the genotoxic action of ionizing radiation. However, since lymphocytes from only 3 patients and 3 normal subjects were analyzed in the present paper, additional donors will be necessary for a more accurate evaluation

G2 repair and chromosomal damage in lymphocytes from workers occupationally exposed to low-level ionizing radiation

The effect of the G2 repair of chromosomal damage in lymphocytes from workers exposed to low levels of X- or g-rays was evaluated. Samples of peripheral blood were collected from 15 radiation workers, 20 subjects working in radiodiagnostics, and 30 healthy control donors. Chromosomal aberrations (CA) were evaluated by scoring the presence of chromatid and isochromatid breaks, dicentric and ring chromosomes in lymphocytes with/without 5mM caffeine plus 3mM-aminobenzamide (3-AB) treatment during G2. Our results showed that the mean value of basal aberrations in lymphocytes from exposed workers was higher than in control cells (p< 0.001). The chromosomal damage in G2, detected with caffeine plus 3-AB treatment was higher than the basal damage (untreated conditions), both in control and exposed populations (p< 0.05). In the exposed workers group, the mean value of chromosomal abnormalities in G2 was higher than in the control (p< 0.0001). No correlation was found between the frequency of chromosome type of aberrations (basal or in G2), and the absorbed dose. Nevertheless, significant correlation coefficients (p< 0.05) between absorbed dose and basal aberrations yield (r = 0.430) or in G2 (r = 0.448) were detected when chromatid breaks were included in the total aberrations yield. Under this latter condition no significant effect of age, years of employment or smoking habit on the chromosomal aberrations yield was detected. However, analysis of the relationship between basal aberrations yield and the efficiency of G2 repair mechanisms, defined as the percentage of chromosomal lesions repaired in G2, showed a significant correlation coefficient (r = -0.802; p< 0.001). These results suggest that in addition to the absorbed dose, the individual G2 repair efficiency may be another important factor affecting the chromosomal aberrations yield detected in workers exposed to low-level ionizing radiation

Mutational response of a diploid pso4-1 yeast strain defective in error-prone recombinational DNA repair

Foi examinada a resposta mutagênica de células diplóides de S. cerevisiae, homozigotas para a mutaçäo pso4-1 em comparaçäo com a cepa selvagem correspondente. Observou-se que o diplóide pso4-1 apresenta uma acentuada reduçäo nas freqüências de mutaçäo reversa induzidas após tratamento com radiaçäo UV, oy furocumarinas mono e bifuncionais mais UVA. Esta severa inibiçäo na mutagênese induzida ocorre principalmente em doses mais elevadas dos diferentes agentes genotóxicos utilizados. Em adiçäo ao seu efeito sobre o processo de mutagênese o mutante pso4-1 exibe um bloqueio generalizado na recombinaçäo miótica-recíproca (recombinaçäo) ou näo recíproca-espontânea e induzida. Estas observaçöes indicam que o gene PSO4 controla simultaneamente os processos de mutagênese de recombinaçäo, atuando sobre um mecanismo de reparo recombinacional sujeito a erro, comparável ao reparo SOS presente em E. coli

Aberraciones cromosómicas inducidas por la luz ultravioleta en células de pacientes con xeroderma pigmentosum / Chromosomical aberrations induced by ultraviolete light in cells from patients with pigmentosum xeroderma

Se examinó la respuesta citogética que presentan las células normales y las de pacientes con xeroderma pigmentosum ante la radiación ultravioleta. Los resultados mostraron un incremento significativo de brechas y rupturas cromatídicas, así como de la depresión de la división celular en las células afectadas en comparación con las normales. Estos datos confirman la fragilidad cromosómica de células de pacientes con xerocerma pigmentosum ante agentes ambientales, lo que se encuentra en el contexto de una secuencia que se inicia en deficiencias en la repación de DNA y continúa con la presencia de mutaciones y el desarrollo de cáncer

UV-induced growth inhibition in the trypanosomatid Crithidia fasciculata

The effects of short ultraviolet (253 mm) radiation on the growth of the trypanosomatid flagellate Crithidia fasciculada were studied in liquid medium and nutrient agar plates. Cell duplication was completely inhibited after exposure of the flagellates to doses equal to or higher than 50 J/m2. The UV-induced lag period was dose-dependent. Survival was reduced to 1% after exposure of the parasites to 200 J/m2. Ultrastructural changes after the lag period were studied by transmission electron microscopy. Changes of the kinetoplast network structure and sometimes of the mitochondrial matrix were observed. The existence of DNA repair mechanisms in this protozoan is discussed