Analysis of ionizing radiation-induced DNA damage and repair in three-dimensional human skin model system.
Knowledge of cellular responses in tissue microenvironment is crucial for the accurate prediction of human health risks following chronic or acute exposure to ionizing radiation (IR). With this objective, we investigated the radio responses for the first time in three-dimensional (3D) artificial human skin tissue microenvironment after gamma-rays radiation. IR-induced DNA damage/repair response was assessed by immunological analysis of well-known DNA double strand break (DSB) repair proteins, i.e. 53BP1 and phosphorylated ataxia telangiectasia mutated(ser1981) (ATM(ser1981)). Efficient 53BP1 and phosphorylated ATM foci formation was observed in human EpiDerm tissue constructs after low and high doses of gamma-rays. Interestingly, EpiDerm tissue constructs displayed less 53BP1 and ATM foci number at all radiation doses (0.1, 1, 2.5 and 5 Gy) than that observed for 2D human fibroblasts. DSB repair efficiency judged by the disappearance of 53BP1 foci declined with increasing doses of gamma-rays and tissue constructs irradiated with 2.5 and 5 Gy of gamma-rays displayed 53BP1 foci persisting up to 72 h of analysis. Pretreatment of EpiDerm tissue constructs with LY294002, [an inhibitor of phosphatidylinositol-3 kinase and PI-3 kinase like kinases (PIKK)] completely abolished IR-induced 53BP1 foci formation and increased the apoptotic death. This observation indicates the importance of PIKK signalling pathway for efficient radiation responses in intact tissue constructs. In summary, we have successfully demonstrated the feasibility of monitoring the DNA damage response in human skin tissue microenvironment. In this system, 53BP1 can be used as a useful marker for monitoring the DSB repair efficiency.
Su
Yanrong
Y
Department of Radiation Oncology, College of Physicians and Surgeons, Columbia University Medical Center, New York, NY 10032, USA.
Meador
Jarah A
JA
Geard
Charles R
CR
Balajee
Adayabalam S
AS
D015415:Biomarkers / Q000378:metabolism; D002460:Cell Line; D002867:Chromones / Q000494:pharmacology; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects / Q000528:radiation effects; D004249:DNA Damage / Q000187:drug effects / Q000528:radiation effects*; D004260:DNA Repair / Q000187:drug effects / Q000528:radiation effects*; D004307:Dose-Response Relationship, Radiation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects; D005720:Gamma Rays / Q000009:adverse effects*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D008954:Models, Biological; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011839:Radiation, Ionizing; D015398:Signal Transduction / Q000187:drug effects / Q000528:radiation effects; D012867:Skin / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects*; D000071857:Tumor Suppressor p53-Binding Protein 1
D015415:Biomarkers; D002867:Chromones; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D009025:Morpholines; C498301:TP53BP1 protein, human; D000071857:Tumor Suppressor p53-Binding Protein 1; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D019869:Phosphatidylinositol 3-Kinases