RESUMO
Fanconi Anemia (FA) is a rare autosomal recessive disorder characterized by hypersensitivity to inter-strand crosslinks (ICLs). FANCD2, a central factor of the FA pathway, is essential for the repair of double strand breaks (DSBs) generated during fork collapse at ICLs. While lesions different from ICLs can also trigger fork collapse, the contribution of FANCD2 to the resolution of replication-coupled DSBs generated independently from ICLs is unknown. Intriguingly, FANCD2 is readily activated after UV irradiation, a DNA-damaging agent that generates predominantly intra-strand crosslinks but not ICLs. Hence, UV irradiation is an ideal tool to explore the contribution of FANCD2 to the DNA damage response triggered by DNA lesions other than ICL repair. Here we show that, in contrast to ICL-causing agents, UV radiation compromises cell survival independently from FANCD2. In agreement, FANCD2 depletion does not increase the amount of DSBs generated during the replication of UV-damaged DNA and is dispensable for UV-induced checkpoint activation. Remarkably however, FANCD2 protects UV-dependent, replication-coupled DSBs from aberrant processing by non-homologous end joining, preventing the accumulation of micronuclei and chromatid aberrations including non-homologous chromatid exchanges. Hence, while dispensable for cell survival, FANCD2 selectively safeguards chromosomal stability after UV-triggered replication stress.
Assuntos
Reparo do DNA/genética , Replicação do DNA/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Anemia de Fanconi/genética , Cromátides/genética , Cromátides/efeitos da radiação , Instabilidade Cromossômica/efeitos da radiação , Cromossomos/genética , Cromossomos/efeitos da radiação , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Reparo do DNA por Junção de Extremidades/genética , Reparo do DNA por Junção de Extremidades/efeitos da radiação , Reparo do DNA/efeitos da radiação , Replicação do DNA/efeitos da radiação , Anemia de Fanconi/patologia , Instabilidade Genômica/genética , Instabilidade Genômica/efeitos da radiação , Humanos , RNA Interferente Pequeno , Raios UltravioletaRESUMO
In a recent paper we reported the results of an experiment carried out by analysing chromosomal damage in Chinese hamster (CHO) cells exposed to low doses of X-rays. The present investigation was undertaken in order to validate those results using a different approach, the single cell gel electrophoresis assay (comet assay) immediately after irradiation. Cells were cultured during 14 cycles, irradiation treatment was performed once per cycle when the cells were at 90-95% of confluence. Doses of 2.5, 5.0 and 10.0 mSv were used. Sequential irradiation of CHO cells induced a decrease of cells without migration and an increase of cells showing DNA damage with the three doses employed. Significant increases of low-level damaged cells (p < 0.001) were found for the 14 exposures when compared to controls except for the first irradiations with 2.5 and 10 mSv, respectively. No significant increase of the frequency of cells with severe damage was observed in any case. These findings could be explained by assuming a complex interactive process of cell recovery, DNA damage and repair together with the induction of genomic instability, the incidence of bystander effects as well as some kind of radioadaptative response of the cells. If these phenomena are limited to the cell line employed deserves further investigation.
Assuntos
Células CHO/efeitos da radiação , Dano ao DNA , Adaptação Fisiológica , Animais , Células CHO/ultraestrutura , Cromátides/efeitos da radiação , Cromátides/ultraestrutura , Aberrações Cromossômicas , Quebra Cromossômica , Cromossomos/efeitos da radiação , Cromossomos/ultraestrutura , Ensaio Cometa , Cricetinae , Cricetulus , DNA/efeitos da radiação , Reparo do DNA , Relação Dose-Resposta à Radiação , Processamento de Imagem Assistida por Computador , Transferência Linear de Energia , Microscopia de Fluorescência , Tolerância a RadiaçãoRESUMO
SORB (selected observed residual breakpoints) induced by ionizing radiation or endonucleases are often non-randomly distributed in mammalian chromosomes. However, the role played by chromatin structure in the localization of chromosome SORB is not well understood. Anti-topoisomerase drugs such as etoposide are potent clastogens and unlike endonucleases or ionizing radiation, induce DNA double-strand breaks (DSB) by an indirect mechanism. Topoisomerase II (Topo II) is a main component of the nuclear matrix and the chromosome scaffold. Since etoposide leads to DSB by influencing the activity of Topo II, this compound may be a useful tool to study the influence of the chromatin organization on the distribution of induced SORB in mammalian chromosomes. In the present work, we compared the distribution of SORB induced during S-phase by etoposide or X-rays in the short euchromatic and long heterochromatic arms of the CHO9 X chromosome. The S-phase stage (early, mid or late) at which CHO9 cells were exposed to etoposide or X-rays was marked by incorporation of BrdU during treatments and later determined by immunolabeling of metaphase chromosomes with an anti-BrdU FITC-coupled antibody. The majority of treated cells were in late S-phase during treatment either with etoposide or X-rays. SORB induced by etoposide mapped preferentially to Xq but random localization was observed for SORB produced by X-rays. Possible explanations for the uneven distribution of etoposide-induced breakpoints along Xq are discussed.
Assuntos
Células CHO/efeitos dos fármacos , Células CHO/efeitos da radiação , Quebra Cromossômica , Inibidores Enzimáticos/toxicidade , Etoposídeo/toxicidade , Inibidores da Topoisomerase II , Cromossomo X/efeitos dos fármacos , Cromossomo X/efeitos da radiação , Animais , Células CHO/ultraestrutura , Cromátides/efeitos dos fármacos , Cromátides/efeitos da radiação , Cromátides/ultraestrutura , Aberrações Cromossômicas , Mapeamento Cromossômico , Cricetinae , Cricetulus , DNA/efeitos dos fármacos , DNA/efeitos da radiação , Dano ao DNA , Feminino , Fase S/efeitos dos fármacos , Fase S/efeitos da radiação , Cromossomo X/genética , Cromossomo X/ultraestruturaRESUMO
PURPOSE: It is important to develop simple experimental models to assess the induction of DNA damage and study the different factors involved under controlled conditions. This paper describes the cytogenetic analysis carried out in Chinese hamster cells (CHO) sequentially exposed to very low doses of X-rays. MATERIALS AND METHODS: CHO cells were cultured for 14 passages. Irradiation treatment was performed once per passage, and three irradiation doses were employed: 2.5, 5.0 and 10.0 mSv. RESULTS: Sequential irradiation of CHO cells did not increase the yield of chomatid- or chromosome-type aberrations. However, a significant increase of achromatic lesions (gaps) was found after the first or second X-ray dose, with all three irradiation doses employed. CONCLUSIONS: The variation in the frequency of gaps as well as that in the mitotic index during the 14 cycles of radiation could be an indication of the induction of genomic instability. According to this, continuous rises and falls in the frequency of gaps as well as in the mitotic index reflects the simultaneous induction of endogenous DNA damage, cell death and cell survival.
Assuntos
Apoptose/efeitos da radiação , Cromátides/efeitos da radiação , Aberrações Cromossômicas , Dano ao DNA , Relação Dose-Resposta à Radiação , Metáfase/efeitos da radiação , Raios X , Animais , Células CHO/citologia , Sobrevivência Celular/efeitos da radiação , Cricetinae , Análise Citogenética , Doses de RadiaçãoRESUMO
The effect of turmeric and curcumin, two natural antioxidants, on the frequencies of chromosome aberrations induced in Chinese hamster ovary (CHO) cells by gamma-radiation was investigated. Cells were treated with three concentrations of each drug, turmeric (100, 250, and 500 microg/ml) and curcumin (2.5, 5, and 10 microg/ml), and then irradiated (2.5 Gy) during different phases of the cell cycle. Turmeric was not clastogenic by itself, whereas curcumin at 10 microg/ml enhanced the chromosomal damage frequency. Neither of the two antioxidants showed protective effect against the clastogenicity of gamma-radiation. Instead, an obvious increase in the frequencies of chromosome aberrations was observed when turmeric at 500 microg/ml was associated with gamma-radiation during G2/S phase, and curcumin at 10 microg/ml plus gamma-radiation during S and G2/S phases of the cell cycle. The results clearly indicate the exacerbated effect of turmeric and curcumin on radiation-induced clastogenicity, suggesting that these antioxidants are also potentiating agents depending on the experimental conditions.