RESUMEN
Molecular responses to genotoxic stress, such as ionizing radiation, are intricately complex and involve hundreds of genes. Whether targeted overexpression of an endogenous gene can enhance resistance to ionizing radiation remains to be explored. In the present study we take an advantage of the CRISPR/dCas9 technology to moderately overexpress the RPA1 gene that encodes a key functional subunit of the replication protein A (RPA). RPA is a highly conserved heterotrimeric single-stranded DNA-binding protein complex involved in DNA replication, recombination, and repair. Dysfunction of RPA1 is detrimental for cells and organisms and can lead to diminished resistance to many stress factors. We demonstrate that HEK293T cells overexpressing RPA1 exhibit enhanced resistance to cell killing by gamma-radiation. Using the alkali comet assay, we show a remarkable acceleration of DNA breaks rejoining after gamma-irradiation in RPA1 overexpressing cells. However, the spontaneous rate of DNA damage was also higher in the presence of RPA1 overexpression, suggesting alterations in the processing of replication errors due to elevated activity of the RPA protein. Additionally, the analysis of the distributions of cells with different levels of DNA damage showed a link between the RPA1 overexpression and the kinetics of DNA repair within differentially damaged cell subpopulations. Our results provide knew knowledge on DNA damage stress responses and indicate that the concept of enhancing radioresistance by targeted alteration of the expression of a single gene is feasible, however undesired consequences should be considered and evaluated.
RESUMEN
PURPOSE: Exposure to high dose ionizing radiation leads to premature cell senescence and suppression of cell proliferation. In contrast, low dose and low dose-rate gamma-irradiation can lead to stimulation of cell proliferation. We aimed to examine whether the low dose radiation-induced proliferation of normal human fibroblasts can lead to a progressive depletion of proliferation potential and to an early onset of senescence. MATERIALS AND METHODS: Normal human embryonic lung fibroblasts (HELF-104) at passage 22-24 were gamma-irradiated with doses of 0 (sham-irradiation), 10, 30, 50, 90, 120, 150, 200, and 500 mGy as well as 1 and 2 Gy. After irradiation, the fraction of cells positively stained for senescence-associated ß-galactosidase activity was measured weekly until the cell culture completely ceased to proliferate. RESULTS: We show that single irradiation of HELF-104 cells with 30 and 50 mGy resulted in deceleration of senescence. The suppression of senescence was observed during almost the entire length of the study up to a complete arrest of cell growth. CONCLUSIONS: Our data, together with the previously published observation of delayed stimulation of proliferation in HELF-104 cells exposed to 30 mGy, suggest that low dose gamma-irradiation can increase the overall proliferative potential of normal human fibroblasts.
