Abstract
DNA damage (caused by direct cellular exposure and bystander signaling) and the complex pathways involved in its repair are critical events underpinning cellular and tissue response following radiation exposures. There are limited data addressing the dynamics of DNA damage induction and repair in the skin particularly in areas not directly exposed. Here we investigate the mechanisms regulating DNA damage, repair, intracellular signalling and their impact on premature differentiation and development of inflammatory-like response in the irradiated and surrounding areas of a 3D organotypic skin model. Following localized low-LET irradiation (225 kVp X-rays), low levels of 53BP1 foci were observed in the 3D model (3.8±0.28 foci/Gy/cell) with foci persisting and increasing in size up to 48 h post irradiation. In contrast, in cell monolayers 14.2±0.6 foci/Gy/cell and biphasic repair kinetics with repair completed before 24 h was observed. These differences are linked to differences in cellular status with variable level of p21 driving apoptotic signalling in 2D and accelerated differentiation in both the directly irradiated and bystander areas of the 3D model. The signalling pathways utilized by irradiated keratinocytes to induce DNA damage in non-exposed areas of the skin involved the NF-κB transcription factor and its downstream target COX-2.
Highlights
Ionizing radiation is a genotoxic agent producing wide range of DNA alterations which after processing through the cellular repair machinery determine the variety and severity of cellular and tissue effects
One of the important steps in evaluation the damage severity and cellular ability to advance through the cell cycle is activation of p21WAF1/Cip1. p21 inhibits the cell cycle dependent kinases (CDK) via supressing Cyclin E and Cyclin A-associated CDK2-activities, blocking the cell cycle progression [4]
DNA damage and repair in 2D N/TERT-1 keratinocytes Irradiation of N/TERT-1 keratinocytes in 2D culture induced linear dose dependent formation of 53BP1 foci with a peak at 30 min post irradiation
Summary
Ionizing radiation is a genotoxic agent producing wide range of DNA alterations (i.e. strand breaks, base damages and cross-links) which after processing through the cellular repair machinery determine the variety and severity of cellular and tissue effects. DSBs are repaired via two main repair pathways, the non-homologous end joining (NHEJ) and the homologous recombination (HR). These processes have been extensively studied and the main proteins involved identified and characterized. P21 inhibits the cell cycle dependent kinases (CDK) via supressing Cyclin E and Cyclin A-associated CDK2-activities, blocking the cell cycle progression [4]. It acts as cell cycle checkpoint and is able to block the cell cycle in both G1/S and G2/M phases. When sub-lethal DNA damages are induced, p21 acts as an inducer of cell cycle arrest and facilitates damage repair [4,5,6]
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