Fanconi anemia cells with unrepaired DNA damage activate components of the checkpoint recovery process.

BACKGROUND: The FA/BRCA pathway repairs DNA interstrand crosslinks. Mutations in this pathway cause Fanconi anemia (FA), a chromosome instability syndrome with bone marrow failure and cancer predisposition. Upon DNA damage, normal and FA cells inhibit the cell cycle progression, until the G2/M checkpoint is turned off by the checkpoint recovery, which becomes activated when the DNA damage has been repaired. Interestingly, highly damaged FA cells seem to override the G2/M checkpoint. In this study we explored with a Boolean network model and key experiments whether checkpoint recovery activation occurs in FA cells with extensive unrepaired DNA damage. METHODS: We performed synchronous/asynchronous simulations of the FA/BRCA pathway Boolean network model. FA-A and normal lymphoblastoid cell lines were used to study checkpoint and checkpoint recovery activation after DNA damage induction. The experimental approach included flow cytometry cell cycle analysis, cell division tracking, chromosome aberration analysis and gene expression analysis through qRT-PCR and western blot. RESULTS: Computational simulations suggested that in FA mutants checkpoint recovery activity inhibits the checkpoint components despite unrepaired DNA damage, a behavior that we did not observed in wild-type simulations. This result implies that FA cells would eventually reenter the cell cycle after a DNA damage induced G2/M checkpoint arrest, but before the damage has been fixed. We observed that FA-A cells activate the G2/M checkpoint and arrest in G2 phase, but eventually reach mitosis and divide with unrepaired DNA damage, thus resolving the initial checkpoint arrest. Based on our model result we look for ectopic activity of checkpoint recovery components. We found that checkpoint recovery components, such as PLK1, are expressed to a similar extent as normal undamaged cells do, even though FA-A cells harbor highly damaged DNA. CONCLUSIONS: Our results show that FA cells, despite extensive DNA damage, do not loss the capacity to express the transcriptional and protein components of checkpoint recovery that might eventually allow their division with unrepaired DNA damage. This might allow cell survival but increases the genomic instability inherent to FA individuals and promotes cancer.

Trimethoprim-sulfamethoxazole increase micronuclei formation in peripheral blood from weanling well-nourished and malnourished rats.

The combination of trimethoprim and sulfamethoxazole (TMP-SMX) is a widely used drug. In spite of this, there are few reports on its genotoxicity, and the results are controversial. Severe malnutrition is a complex condition that increases the susceptibility to infections. Consequently, drugs are extensively used in malnutrition cases. Experimental animal models have been widely used to study the effects of malnutrition. Neonatal rats were experimentally malnourished (UN) during lactation. The UN rats weighed 51.1% less than the well-nourished (WN) controls and had lower concentrations of serum protein and blood lipids. The micronucleus (MN) assay is useful for detecting chromosome damage induced by nutritional deficiencies. In vivo rodent MN assays have been widely used to screen genotoxic agents. In this study, we have evaluated the frequency of spontaneous and TMP-SMX-induced micronuclei in the peripheral blood of weanling (21 days of age) rats using a flow cytometric analysis technique. The spontaneous frequency of micronucleated reticulocytes (MN-RETs) was 2.7 times greater in the UN rats than in the WN rats. In rats that were not treated with TMP-SMX, the percentage of reticulocytes was significantly lower (41.1%) in the UN rats than the WN controls. A therapeutic dose of TMP-SMX (80 mg/kg (TMP), 400 mg/kg (SMX) for 48 hr) increased MN-RETs in the WN and in the UN rats. The data demonstrate the genotoxic effect of this drug. The results indicate that severe protein-calorie restriction and drug treatment enhance DNA damage in rat peripheral blood reticulocytes, potentially increasing the risk of negative effects on health.

Propiedades psicométricas del cuestionario de auto reporte de la calidad de vida KIDSCREEN-27 en adolescentes chilenos / Psychometric properties of the quality of life questionnaire KIDSCREEEN-27 adapted to Chilean adolescents

Se evalúa, las propiedades psicométricas del cuestionario de Calidad de Vida KIDSCREEN-27 adaptado a niños/as y adolescentes chilenos. Participaron 1678 sujetos escolarizados de entre 8 y 18 años. Se evaluó tanto la consistencia interna como indicadores de validez de constructo, discriminante y convergente. El alfa de Cronbach de la escala total como de las distintas dimensiones es superior a 0.70. Los análisis factoriales exploratorio y confirmatorio arrojan evidencia de una estructura similar a la teórica de cinco dimensiones. El instrumento es capaz de discriminar entre hombres y mujeres, así como entre rangos de edad, en diferentes dimensiones. Los resultados permiten decir que el KIDSCREEN-27 presentó coeficientes de fiabilidad y validez aceptables, similares a los de la versión original, permitiendo disponer de un nuevo instrumento que evalúe calidad de vida en Chile y a la vez comparar los resultados obtenidos con otros países que utilicen este instrumento.

The aim of this research was to evaluate the psychometric properties of the quality of life questionnaire KIDSCREEEN-27 adapted to Chilean children and adolescents. The sample was 1678 participants from schools between 8 and 18 years old. The internal consistency and indicators for construct, discriminant and convergent validity was evaluated. Cronbach alphas for total scale and the domains were higher to .70. The exploratory and confirmatory factorial analysis confirmed the five domains proposed in the theoretical structure. The questionnaire was capable of discriminate by gender, as well as among ages, in different domains. In conclusion, results show that the KIDSCREEN-27 presents acceptable coefficients of reliability and validity, similar to those of the original version. This allows Chilean researchers to have a new instrument to evaluate quality of life, and simultaneously to compare their results with other countries that use this instrument.

Spontaneous and mitomycin C-induced micronuclei in peripheral blood reticulocytes from severely malnourished rats.

Severe malnutrition caused by deficiencies in protein, calorie, and micronutrient intake is widely distributed throughout the world and is a particular problem in developing countries. Animal models have been useful for studying the effects of malnutrition under different experimental conditions. In this study, we have evaluated the effect of malnutrition on the frequency of spontaneous and mitomycin C (MMC)-induced micronuclei in the peripheral blood of rats measured using a flow cytometric analysis technique. Neonatal rats were experimentally malnourished during lactation and assayed at weaning (21 days of age). The malnourished rats weighed 49.2% less than well-nourished controls and had lower concentrations of serum protein, triglycerides, and cholesterol. In rats not treated with MMC, the frequency of micronucleated reticulocytes (MN-RETs) was 1.6 times greater in malnourished rats than in well-nourished rats (0.48% +/- 0.16% vs. 0.31% +/- 0.09%). The mean MN-RET frequency measured 32 hr after treatment with single i.p. doses of 0.5, 0.75, or 1.0 mg/kg of MMC was 0.60 +/- 0.10 vs. 0.84 +/- 0.14, 1.21 +/- 0.52 vs. 2.36 +/- 0.47, and 2.50 +/- 0.06 vs. 4.64 +/- 1.14 for well-nourished vs. malnourished rats, respectively. Statistical comparisons indicate significant differences between the two groups of rats at all doses tested. Malnourishment and MMC treatment had no significant effects on the frequencies of RETs or micronucleated normochromatic erythrocytes. The data indicate that protein-calorie malnutrition during lactation is associated with increased frequencies of MN-RETs, which are indicative of chromosome damage. These findings suggest that malnutrition could result in greater susceptibility to environmental damage.