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Analysis of DNA-damage response to ionizing radiation in serum-shock synchronized human fibroblasts.

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 ?- 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.

SUBMITTER: Corra S

PROVIDER: S-EPMC5493713 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Analysis of DNA-damage response to ionizing radiation in serum-shock synchronized human fibroblasts.

Corrà Samantha S Salvadori Riccardo R Bee Leonardo L Barbieri Vito V Mognato Maddalena M

Cell biology and toxicology 20170503 4

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 P ...[more]

PMID: 28466226

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Project description:Cell cycle arrest and transcriptional responses to ionizing radiation (IR)-induced DNA damage were quantified in telomerase-expressing human diploid fibroblasts. Assays of clonal expansion established 1.5 Gy IR as the D0 dose in three fibroblast lines and this dose was used in all subsequent analyses. Fibroblasts exhibited >90% arrest of progression from G2 to M at 2 h and from G1 to S at 6 and 12 h post-IR. Normal rates of DNA synthesis and mitosis 6 and 12 h after irradiation caused the S and M compartments to empty by over 70% at 24 h. Microarray monitored global gene expression in IR-treated cells and a new microarray analysis algorithm, EPIG, identified nine IR-responsive patterns of gene expression including a dominant p53-dependent G1 checkpoint response. Many p53 target genes, like CDKN1A, GADD45, BTG2 and PLK3, were significantly up-regulated at 2 h post-IR while many genes whose expression is regulated by E2F family transcription factors, including CDK2, CCNE1, CDC6, CDC2, MCM2, were significantly down-regulated at 24 h post-IR. Numerous genes that participate in DNA metabolism were also markedly repressed in arrested fibroblasts as a result of cell synchronization behind the G1 checkpoint. However, cluster and principal component analyses of gene expression revealed a profile of gene expression 24 h after IR with similarity to that of G0 growth quiescence. These results demonstrate a highly stereotypic pattern of response to IR that reflects primarily synchronization behind the G1 checkpoint but with prominent induction of additional markers of G0 quiescence such as GAS1. Experiment Overall Design: Logarithmically growing cells were treated with 1.5 Gy IR or sham, and harvested at 2, 6 or 24 h after the treatment. Total RNA was isolated using Qiagen RNeasy kit. The quality of all RNA samples was confirmed using an Agilent 2100 Bioanalyzer. Microarray analysis was then performed: briefly, 1 Âµg of sample RNA and global reference RNA (Stratagene) were converted to cDNA with reverse transcriptase then amplified using T7 RNA polymerase while labeling with either Cy3-dUTP or Cy5-dUTP (Agilentâs Low RNA Input Linear Amplification Kit). The quality of each labeled cRNA was evaluated using an Agilent 2100 Bioanalyzer prior to hybridization. 750ng of Cy3 and Cy5-labeled cRNA were used in the hybridization. The labeled cRNA from sham- or IR-treated samples was hybridized with the labeled global reference cRNA on an Agilent 22k human 1A array in a hybridization oven (Robbins Scientific Model 400, 1040-60-1AG) at 60oC for 17 h. Hybridization of sample RNA against reference RNA was done twice with dye swap. Following hybridization, the arrays were scanned using the Agilent DNA Microarray Scanner with SureScanÂ® Technology and microarray images were analyzed using Agilent Feature Extraction Software (v7.1). The gene expression level was presented as ratio of sample intensity against reference intensity.

Dataset Information

Analysis of DNA-damage response to ionizing radiation in serum-shock synchronized human fibroblasts.

Publications

Analysis of DNA-damage response to ionizing radiation in serum-shock synchronized human fibroblasts.

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