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Analysis of DNA-damage response to ionizing radiation in serum-shock synchronized human fibroblasts
Samantha Corrà1, Riccardo Salvadori1, Leonardo Bee1,2
1Department of Biology, School of Sciences, University of Padova, via U. Bassi 58 B, 35131, Padova, Italy.
Abstract:
Many aspects of cellular physiology, including cellular response to genotoxic stress, are related to the circadian rhythmicity induced by the molecular clock. The current study investigated if the cellular response to DNA damage is in relation to endogenous expression levels of the PER2 protein, a key component of the molecular regulatory system that confers rhythmicity in mammalian cells. Human normal fibroblasts (CCD-34Lu) were subjected to serum shock to induce circadian oscillations of the PER2 protein and then irradiated with γ- rays at times corresponding to the trough and peak expression of the PER2 protein. To better examine cellular response to DNA damage, the experiments performed in this study were carried out in non-proliferating CCD-34Lu fibroblasts in order to maintain the cell and circadian cycles separated while they were being exposed to genotoxic stress. Study results demonstrated that clonogenic cell survival, double-strand break repair kinetics, and TP53 protein levels were affected in the cells irradiated at the trough than in those irradiated at peak expression of the PER2 protein.
Insights
Cellular response to DNA damage varies with PER2 protein levels, a key circadian rhythm component. Cells irradiated at PER2 protein trough showed altered survival and DNA repair compared to peak expression.
Area of Science:
- Cellular and Molecular Biology
- Chronobiology
- Genetics and Genomics
Background:
- Circadian rhythms, regulated by molecular clocks, influence cellular physiology, including responses to genotoxic stress.
- The PER2 protein is a critical component of the molecular clock, conferring rhythmicity in mammalian cells.
- Understanding the interplay between circadian rhythms and DNA damage response is crucial for cellular health.
Purpose of the Study:
- To investigate the relationship between endogenous PER2 protein expression levels and the cellular response to DNA damage.
- To determine if DNA damage response, including cell survival and DNA repair, is modulated by the circadian phase.
Main Methods:
- Human normal fibroblasts (CCD-34Lu) were synchronized using serum shock to induce PER2 protein oscillations.
- Cells were irradiated with gamma rays at times corresponding to PER2 protein trough and peak expression.
- Experiments were conducted in non-proliferating fibroblasts to decouple cell cycle and circadian cycle during genotoxic stress exposure.
Main Results:
- Clonogenic cell survival was significantly affected by the timing of irradiation relative to PER2 expression levels.
- Double-strand break repair kinetics differed between cells irradiated at PER2 trough versus peak expression.
- TP53 protein levels exhibited distinct changes depending on whether cells were irradiated at PER2 trough or peak expression.
Conclusions:
- The cellular response to genotoxic stress, including DNA repair efficiency and cell survival, is influenced by the circadian rhythm, specifically by PER2 protein expression levels.
- Irradiation at the trough of PER2 expression leads to a more sensitive cellular response to DNA damage compared to irradiation at peak expression.
- These findings highlight the importance of circadian timing in DNA damage response pathways and suggest potential therapeutic implications.
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