RESUMEN
The study of rare human syndromes characterized by radiosensitivity has been instrumental in identifying novel proteins and pathways involved in DNA damage responses to ionizing radiation. In the present study, a mutation in mitochondrial poly-A-polymerase (MTPAP), not previously recognized for its role in the DNA damage response, was identified by exome sequencing and subsequently associated with cellular radiosensitivity. Cell lines derived from two patients with the homozygous MTPAP missense mutation were radiosensitive, and this radiosensitivity could be abrogated by transfection of wild-type mtPAP cDNA into mtPAP-deficient cell lines. Further analysis of the cellular phenotype revealed delayed DNA repair, increased levels of DNA double-strand breaks, increased reactive oxygen species (ROS), and increased cell death after irradiation (IR). Pre-IR treatment of cells with the potent anti-oxidants, α-lipoic acid and n-acetylcysteine, was sufficient to abrogate the DNA repair and clonogenic survival defects. Our results firmly establish that mutation of the MTPAP gene results in a cellular phenotype of increased DNA damage, reduced repair kinetics, increased cell death by apoptosis, and reduced clonogenic survival after exposure to ionizing radiation, suggesting a pathogenesis that involves the disruption of ROS homeostasis.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN/efectos de los fármacos , ARN Polimerasas Dirigidas por ADN/genética , Homocigoto , Linfocitos/efectos de la radiación , Proteínas Mitocondriales/genética , Mutación Missense , Amish/genética , Antioxidantes/farmacología , Apoptosis/efectos de la radiación , Línea Celular , Supervivencia Celular/efectos de la radiación , Citoprotección , Roturas del ADN de Doble Cadena/efectos de los fármacos , ARN Polimerasas Dirigidas por ADN/metabolismo , Relación Dosis-Respuesta en la Radiación , Genotipo , Humanos , Cinética , Linfocitos/efectos de los fármacos , Linfocitos/enzimología , Linfocitos/patología , Proteínas Mitocondriales/metabolismo , Fenotipo , Especies Reactivas de Oxígeno/metabolismo , TransfecciónRESUMEN
Surgical biopsies from ten head and neck squamous cell carcinomas were labeled in vitro with bromodeoxyuridine. In histological sections, bromodeoxyuridine-positive nuclei and beta-fibroblast growth factor (beta-FGF) were stained using immunohistochemistry. In clearly discernible clusters of tumor cells, the cytoplasm shows strong positive beta-FGF staining, whereas other tumor regions are completely beta-FGF negative. Within positively stained areas, the tumor cell bromodeoxyuridine labeling index is higher in comparison to beta-FGF-negative areas by a factor of 5 +/- 0.8. This is reflected in a positive correlation of the tumor cell labeling index and the relative extent of beta-FGF-positive tumor areas. Viable tumor areas bordering on necrosis, which are known to be hypoxic, are beta-FGF negative. The average tumor endothelial cell labeling index was 1.8 +/- 0.6%, as compared to 0.16% in adjacent normal mucosa. Since endothelial cell pulse labeling indices are too low for a further quantitative analysis, the relationship of beta-FGF expression and endothelial cell turnover was studied in more detail in two fairly well-differentiated murine squamous cell carcinoma lines (AT 84 and AT 478). Labeling indices were higher and endothelial cell doubling times were significantly shorter in beta-FGF-positive as compared to beta-FGF-negative tumor areas (AT 84, 9.3 h versus 25.4 h; AT 478/25, 6.8 h versus 16 h). Thus, the discrete expression of beta-FGF is associated with regional differences in endothelial cell kinetics. In two generations of the tumor line AT 478, characterized by different volume doubling times of 18 days (AT 478/25) and 36 days (AT 478/4), beta-FGF-positive areas represent 75.5 +/- 6% and 19.7 +/- 7% of the viable tumor tissue, respectively. This indicates a correlation between beta-FGF production of tumor cells and growth rate.