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. 2017 Mar 16;12(3):e0173740.
doi: 10.1371/journal.pone.0173740. eCollection 2017.

Influence of a Pre-Stimulation With Chronic Low-Dose UVB on Stress Response Mechanisms in Human Skin Fibroblasts

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Free PMC article

Influence of a Pre-Stimulation With Chronic Low-Dose UVB on Stress Response Mechanisms in Human Skin Fibroblasts

Marie-Catherine Drigeard Desgarnier et al. PLoS One. .
Free PMC article

Abstract

Exposure to solar ultraviolet type B (UVB), through the induction of cyclobutane pyrimidine dimer (CPD), is the major risk factor for cutaneous cancer. Cells respond to UV-induced CPD by triggering the DNA damage response (DDR) responsible for signaling DNA repair, programmed cell death and cell cycle arrest. Underlying mechanisms implicated in the DDR have been extensively studied using single acute UVB irradiation. However, little is known concerning the consequences of chronic low-dose of UVB (CLUV) on the DDR. Thus, we have investigated the effect of a CLUV pre-stimulation on the different stress response pathways. We found that CLUV pre-stimulation enhances CPD repair capacity and leads to a cell cycle delay but leave residual unrepaired CPD. We further analyzed the consequence of the CLUV regimen on general gene and protein expression. We found that CLUV treatment influences biological processes related to the response to stress at the transcriptomic and proteomic levels. This overview study represents the first demonstration that human cells respond to chronic UV irradiation by modulating their genotoxic stress response mechanisms.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Schematic representation of the irradiation protocol.
Confluent NHDF were irradiated with different UVB irradiation protocols. Three conditions were used: (1) single UVB dose, (2) CLUV treatment and (3) CLUV followed by a single UVB dose. (1) Acute treatment is a single UVB irradiation of 400 J/m2; (2) CLUV treatment consists UVB irradiations of 75 J/m2 every 12 h for 7.5 days (15 irradiations). (3) Cells are irradiated with the CLUV treatment described in (2) followed by the single UVB irradiation described in (1) 12 h after the last CLUV irradiation. Cells subjected to the different irradiation protocols were then used for further analysis.
Fig 2
Fig 2. CPD repair rate is enhanced by the CLUV pre-stimulation in NHDF.
(A) Immuno-slot-blot showing the level of CPD and DNA. NHDF were irradiated with a single UVB dose (Acute), a CLUV irradiation or a CLUV followed by a single acute UVB dose (CLUV+Acute), as described in Fig 1. Zero and 24 h post-irradiation, DNA was harvested and applied on a membrane. Revelation of CPD and DNA was performed using specific monoclonal antibodies. NoUV is used as a negative control and DNA as a loading control. (B) Representation of the quantity of CPD repair after different UVB treatments. Quantitative analysis of the immuno-slot-blot detecting CPD is performed by measuring the signal intensity at each time points post-UV and compare it to the 0 h for each UVB treatment condition (Acute, CLUV, CLUV+Acute). The signal at 0 h corresponds to 100% of the CPD signal for each UVB treatment independently. The results then describe the CPD removal relative to the initial CPD amount for each UVB treatment (Acute, CLUV and CLUV+Acute). Normalization is performed using the corresponding DNA signal as previously [31]. Results are presented as means ± SEM. P-value was evaluated using the student’s t-test (*p < 0.05; **p < 0.01). Experiment was performed using 3 strain cells (N = 3) at least in duplicate (n = 2).
Fig 3
Fig 3. Cell division analysis after single UVB and CLUV irradiation.
Cells were either irradiated with either a CLUV treatment (CLUV) or a single UVB dose of 200 J/m2 (Acute). Un-irradiated cells (NoUV) were used as control. Synchronization in G0 was achieved by keeping the cells at full confluency for 12 days and then re-seeded at low density (8.3x103 cells/cm2). S-phase recovery derived as S/G2 ratio was assayed during 36 h using PI staining FACS analysis. Data are presented as means ± SEM using 4 independent cell strains (N = 4).
Fig 4
Fig 4. Effect of a CLUV pre-stimulation on UV-induced cell death sensitivity.
NHDF CLUV pre-stimulated (CLUV) or not (Acute) were irradiated with UVB doses ranging from 0 to 40,000 J/m2. Sensitivity to UV-induced apoptosis and necrosis was assessed by FACS analysis, 16 h post-irradiation. Dashed bars represent the percentage of necrotic cells and solid bars are apoptotic cells. Data are expressed as means ± SEM of four independent NHDF cell strains (N = 4). Significance was evaluated using the student’s t-test.
Fig 5
Fig 5. CLUV treatment induces transcriptomic changes.
After NHDF were subjected or not to the CLUV treatment, total RNA was extracted to analyze gene profiling. For this experiment, the CLUV treatment is performed using 100 J/m2 of UVB instead of 75 J/m2. The experiment was performed in triplicate using 3 different NHDF strains. (A) Heatmap depicting the significantly deregulated genes in CLUV treated NHDF and un-irradiated controls. This experiment was performed in 3 different NHDF strains, and the heatmap clearly shows the reproducibility of CLUV-induced changes between strains. The color scale is based on the log2 expression level values. Hierarchical clustering was performed on rows based on the Euclidian distance. Genes indicated in dark blue correspond to those whose expression is very low, whereas highly expressed genes are shown in red. (B) Scatter plot of log2 signal intensity for 60 000 targets covering the entire human transcriptome. The signal for CLUV cells at 0 h (y-axis) is plotted against un-irradiated cells (Control, No UVB) (x-axis). All the >2-fold deregulated genes between the 2 conditions are represented by black dots. The 3 blue points are 3 controls (B2M, TUBB, GOLGA1). The transcription level of those genes is known to be stable, independent of cell type and condition [39].
Fig 6
Fig 6. CLUV treatment induces proteomic changes.
Three cell strains of NHDF subjected or not to a CLUV treatment and proteins were extracted. Proteins from the triplicate were pooled and proteome change was analyzed. (A) 2D-DIGE depicting protein expression differences between CLUV and untreated NHDF. The control (NoUV) was labeled with cy3 (left panel) and CLUV-treated cells (CLUV) with cy5 (middle panel). After labeling, proteins were separated on a 2D-DIGE according to their molecular weight and pH. Gels were merged (cy3/cy5) (right panel) to see proteomics changes. (B) Merged 2D-DIGE gel (cy3/cy5) depicting proteomic changes. Proteins with equal abundance between the CLUV-treated and the untreated NHDF are shown in yellow spot, while up-regulated proteins by the CLUV treatment appears in red and down-regulated in green. Full circles display up-regulated protein and dashed circles exhibit down-regulated proteins. A total of 30 proteins were further analyzed by mass spectrometry.

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Grant support

This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada (NSERC) to PJR. Grant #:RGPIN-2016-05864. URL: http://nserc.ca/.
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