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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.
Citation: Drigeard Desgarnier M-C, Fournier F, Droit A, Rochette PJ (2017) Influence of a pre-stimulation with chronic low-dose UVB on stress response mechanisms in human skin fibroblasts. PLoS ONE 12(3): e0173740. https://doi.org/10.1371/journal.pone.0173740
Editor: Andrzej T. Slominski, University of Alabama at Birmingham, UNITED STATES
Received: January 4, 2017; Accepted: February 24, 2017; Published: March 16, 2017
Copyright: © 2017 Drigeard Desgarnier et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: 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/.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Skin cancers represent the most frequent type of cancer [1]. Exposure to solar ultraviolet (UV), through the induction of pre-mutagenic DNA lesions, is the major risk factor for cutaneous cancer development [2]. More precisely, UVB (280–315 nm) are the most carcinogenic wavelengths reaching the Earth surface [3]. The two UVB-induced mutagenic DNA damage are the cyclobutane pyrimidine dimer (CPD) and the pyrimidine (6–4) pyrimidine photoproducts (6-4PP) [4]. If UV-induced DNA damage remain unrepaired, they can lead to UVB signature mutations found in skin cancer [5]. However, the main and most mutagenic UV-induced DNA damage is the CPD [4, 6, 7], which is responsible for C→T and CC→TT transition mutations found in skin cancer [8–12].
Even if UVB are the major contributor of skin cancer, they have also positive effects and applications. First, they are used in dermatology for phototherapy in order to treat different skin conditions [13]. They are also critical for vitamin D3 fixation [14, 15]. Also, in response to UVB, the skin neuroendocrine system responds differently with, among others, the stimulation of corticotropin-releasing factor (CRF) expression [16].
In human cells, UVB-induced DNA damage stimulate various molecular mechanisms to prevent the conversion of pre-mutagenic lesions such as the CPD into cancer driver mutations. These mechanisms signal the DNA damage to the cell, and then mediate DNA lesions removal or their tolerance [17]. When the decision is made to remove the lesion, the DNA damage response (DDR) is activated to either restore DNA by the nucleotide excision repair (NER) or to safely discard the damaged cell by programmed cell death [17, 18]. An early mechanism involved in CPD repair is the activation of DNA damage checkpoint that activates cell cycle delay to allow efficient repair. The regulation of those mechanisms is important to avoid mutagenicity. NER pathway is particularly important to prevent mutagenesis and is a critical mechanism for UVB cancer prevention. Indeed, patient deficient in the NER pathway (Xeroderma Pigmentosum; XP) have 1 000-fold increase of UV-induced skin cancer [19]. The protection mechanisms against UV-induced genotoxic stress have been extensively studied after subjecting cells or animal models with single acute UVB irradiation reviewed in [17, 20]. However, little is known about how those mechanisms are influenced by chronic exposure to UVB light. More precisely, the potential adaptive response of cells after chronic UVB irradiation is poorly understood [21, 22]. Knowing that we are physiologically exposed to repeated chronic low dose of UV (CLUV), it becomes crucial and relevant to understand how molecular mechanisms respond to recurrent irradiations.
In many studies, it has been found that pre-treating cells with chronic low amount of mutagenic agents lead to an adaptive cellular response render the cells more resistant to a subsequent stress against mutagenic agents [21–24]. However, most studies focused on adaptive response with non-physiological agents [21, 23, 25]. For example, one of the first studies demonstrating an adaptive response in human cells has shown that pre-stimulating dermal fibroblasts with chronic low doses of quinacrine mustard enhances CPD repair [23]. Notwithstanding, the effect of a physiological exposure to a CLUV dose has been studied [26, 27]. However, the effect of a CLUV treatment on DNA repair efficiency is somehow controversial. Indeed, it has been reported in mouse skin, that a CLUV dose decreases DNA repair capacity [26] with CPD accumulation and persistence [28, 29]. On the other hand, it has been revealed that CLUV treatment can lead to a faster DNA repair in skin type IV [30].
Although those studies have reported an effect of the CLUV treatment on DNA repair of UV-induced CPD, the consequences of this CLUV treatment and how cells respond after subsequent UVB dose, particularly on the DDR pathway has not been studied. Here, we have investigated whether an exposure of human cells to a CLUV treatment induces some changes at different stress response level. More precisely, we have determined the effect of a CLUV treatment of normal human dermal fibroblasts (NHDF) on the cellular response mechanisms to genotoxic stress, including CPD repair rate, cell cycle arrest and sensitivity to UV-induced apoptosis. In this study, NHDF were used as a model of human non-transformed cell strains and UVB were used as the most mutagenic UV wavelengths reaching the Earth surface. This study does not aim to evaluate each pathway in detail but rather offers an overview of the genotoxic stress response modulated by a chronic UV irradiation. Our data revealed that a CLUV treatment induces persistent residual CPD. Furthermore, our results show that CLUV treatment enhances the repair of newly formed CPD, delay cell cycle progression, but does not sensitize cells to apoptosis. Taken together, our results demonstrate that NHDF cells are able to modulate their DDR pathways following a chronic UVB pre-stimulation.
Results
1. Effect of a CLUV pre-stimulation on DNA repair
We first aimed to determine the influence of CLUV pre-stimulation on UV-induced CPD repair. CPD were induced by an acute UVB treatment (400 J/m2) following or not a CLUV pre-stimulation (schematically represented in Fig 1). We first showed that the CLUV treatment induces CPD that still remain unrepaired 24 h post-irradiation (Fig 2A, left panel and Fig 2B). On the other hand, we can observe that a CLUV pre-stimulation enhance CPD repair (Fig 2B). More precisely, 72% of CPD are repaired 24 h post-irradiation in cells when they are pre-stimulated with a CLUV treatment where it reaches only 50.5% in non-pre-stimulated cells (p < 0.05). Moreover, since the CLUV treatment induces persistent CPD that remain in the genome 24 h post-irradiation, the repair rate derived in CLUV pre-treated cells take into account the newly formed CPD by the acute irradiation and the persistent CPD, thus the rate of newly formed CPD repair is underestimated (Fig 2A and 2B).
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.
(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).
Cells subjected to the single UVB dose were removed from the incubator at the same frequency and length than the CLUV treated cells and the culture media was replaced at the frequency as well. This was done to ensure that the CLUV effect was not the result of the stress induced by the experimental procedure.
2. Consequence of a CLUV treatment on cell cycle
Previous studies have shown that under UV stress, cell cycle progression is halted to allow an effective DNA repair or to induce efficient apoptosis, thus preventing replication over mutagenic DNA damage [17, 32]. Indeed, previous analysis on human dermal fibroblasts demonstrated that a halt in cell cycle is required for effective UV-induced CPD repair [33]. Thereby, to determine the influence of a CLUV treatment on cell cycle progression, we have analyzed cell cycle using flow cytometry in CLUV treated cells and compared with acute UVB treated and un-irradiated cells. For this experiment, we used 200 J/m2 of UVB as acute dose to induced a similar amount of CPD as the residual CPD induced by the CLUV treatment. CPD are known to block cell cycle progression [32] and therefore, it was important to compare conditions (CLUV vs single acute UVB) with the same amount of CPD. As shown in Fig 2A, there are 2 times more CPD induced by the single acute UVB irradiation of 400 J/m2 than the residual CPD induced by the CLUV treatment. For the CLUV treatment, we used the protocol depicted in Fig 1.
Cells were synchronized by keeping them at full confluency for 12 days and then re-seeded at low density to measure the S-phase recovery time. As shown in Fig 3, un-irradiated cells enter in S-phase 16h after their release, where it takes between 16 and 24 h for the acute irradiated cells. We can observe a S-phase initiation after 36 h in CLUV treated cells, but the recovery is still not completed at the longest time point analyzed (36 h). It is important to note that we have confirmed that the CLUV treated cells are not senescent and can replicate post-treatment (data not shown).
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).
3. Consequence of a CLUV pre-stimulation on UVB-induced cell death
It is well established that DNA damage trigger apoptosis [34, 35]. Indeed, various mechanisms take place after DNA damage induction to protect cells against the conversion of those mutagenic DNA damage into mutations. Programmed cell death is one of the most important protection mechanism against genotoxic stress [36]. Thus, to evaluate whether a CLUV pre-stimulation affects cell death, we examined cell sensitivity to UVB in CLUV pre-stimulated and un-stimulated cells (i.e. not receiving the CLUV pre-stimulation). We found no significant difference in UVB-induced cell death sensitivity in CLUV pre-stimulated cells when compared to un-stimulated cells (Fig 4). Indeed, at UVB doses ranging from 0 to 20,000 J/m2, the level of apoptotic and necrotic cells is virtually identical between CLUV pre-treated and un-stimulated cells. At the highest UVB dose use (40,000 J/m2), an increase in UV-induced necrosis and apoptosis sensitivity can be observed in CLUV pre-stimulated cells. However, this difference is not statistically significant (p = 0.08).
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.
4. Microarray analysis of CLUV-induced transcriptomic changes
It has been reported that stress induces gene expression changes [37] and particularly that UV induces the expression of genes involved in DDR response [38]. Thus, as the CLUV pre-stimulation influences DNA repair capacity, cell death sensitivity and cell cycle, we investigated the changes at the transcriptome level induced by this CLUV treatment. Therefore, a gene profiling analysis was performed to determine the entire human gene expression in CLUV pre-stimulated cells and in un-irradiated controls (Fig 5). The heat-map depicting all 2-fold deregulated genes for each replicate displays that the gene expression differences found between the CLUV pre-treated cells and the un-irradiated controls are reproducible (Fig 5A).
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].
Scatter plot analysis comparing gene expression of CLUV pre-stimulated cells and un-irradiated cells highlight on up-regulated and down-regulated genes expression caused by the CLUV pre-treatment (Fig 5B). Microarray analysis underlines a total of 948 deregulated genes caused by the CLUV treatment. To evaluate correlation between gene expression patterns after CLUV treatment and their biological consequences, A BiNGO analysis was performed. A list of annotation was generated and classified according to biological processes that indicate different modulated cellular pathways significantly deregulated after a CLUV treatment (Table 1). Particularly, this induces important changes including some that are directly related to the stress response pathway (Table 1). More precisely, this comprises 151 deregulated genes implicated in the “response to stress” goID process (S1 Table). This represents 15.9% of all deregulated genes.
5. CLUV-induced proteomic changes
It is well documented that UV exposure leads to protein expression changes in skin cells [40]. More precisely, proteomic analysis reveals that a single acute UVB irradiation induces protein expression changes in skin fibroblasts [41, 42]. Since we discovered variations in CLUV-induced gene expression, and in accordance with previous studies observing the effect of UVB on proteome [41, 42], we sought to investigate proteomics changes in response to a CLUV treatment. A 2D-DIGE protein expression profiling analysis shows that the CLUV treatment induces proteomics changes (Fig 6). The 2D-DIGE/MS protein identification assay allows the identification of only a small fraction of the entire human proteome and most of the low-expressed proteins are not detected using this technique. Nonetheless, we have identified 2,500 proteins from which 30 were found deregulated by the CLUV irradiation. Indeed, we can observe some up-regulated and down-regulated protein induced by the CLUV pre-stimulation (Fig 6A, right panel). Spots with the highest expression were further analyzed (surrounded spots in Fig 6B) using Maldi TOF Mass spectrometry (S2 Table). Some redundancy in protein identification can be observed (#spot 3 and 5; #spot 6 and 9; #spot 23 and 25; and #spot 24 and 28), but these spots are close to each other and they are most likely artifacts from the spot identification.
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.
Using the Reactome pathway database, we have identified biological process associated with the identified deregulated proteins (Table 2). While the gene expression profiling demonstrates a deregulation associated with stress response (Fig 5, Table 1), the proteome expression profiling emphasize on this process (Fig 6, Table 2). Indeed, we found 2 deregulated proteins in the programmed cell death process. However, this is in contradiction with our result depicted in Fig 4 showing that CLUV pre-stimulation does not influence UV-induced cell death sensitivity. On the other hand, the cell cycle progression delay induced by the CLUV treatment (Fig 3) is in accordance with the fact that we found protein deregulation in cell cycle process (Fig 6). Interestingly, we discovered an up-regulation of the DOT1 like histone lysine methyltransferase (DOT1L) (S2 Table), which is a protein involved in chromatin organization during DNA damage repair and the silencing of this protein exacerbate UV sensitivity [43]. We also found 5 of the 30 proteins modified by the CLUV treatment were involved in immune system biological process (Table 2 and S2 Table).
Discussion
UVB, a complete carcinogen, is the major factor involved in human skin cancer [44]. Cells react to UVB irradiation by triggering stress response at different molecular levels to protect themselves against this genotoxic stress [45, 46]. More precisely, cell cycle delay, DNA repair and apoptosis are amongst the most important protection mechanisms against UVB-induced skin cancer driver mutations [34, 47]. Nonetheless, even if these molecular mechanisms are well documented, most of previous studies were focused on the effect of single UVB irradiation [20, 48]. Even though repeated low dose of UVB are more representative of what humans are exposed to, only few studies have used this regimen [26, 49, 50]. Moreover, those previous studies have been conducted in different models (yeast, mice) and were mainly focused on the effect of CLUV alone, but not on the consequence of a CLUV pre-stimulation on a subsequent acute irradiation. In the present study, we used a CLUV irradiation protocol to mimic chronic irradiation and to understand how cells can cope with subsequent irradiation. We were aiming to determine whether the CLUV irradiation treatment would influence stress response mechanisms. To our knowledge, this is the first report studying the effect of a CLUV pre-stimulation on primary human diploid fibroblasts.
1. CLUV pre-stimulation enhance CPD repair
Using a slot-blot immunoassay, we measured the repair of UVB-induced CPD (Fig 2) after or not a CLUV pre-stimulation (Fig 1). Our results revealed that CPD repair is improved when cells are pre-stimulated with a CLUV regimen. This suggests an enhancement of the nucleotide excision repair (NER), the repair mechanism responsible for the CPD removal in human. Previous studies have demonstrated an improvement of DNA repair after a pre-stimulation with a chronic low dose of carcinogenesis, but none of them used CLUV irradiation [23, 24]. Although some studies have evaluated CLUV-induced CPD repair [26, 49], it has never been shown that a CLUV pre-stimulation enhance CPD repair. Indeed, none of the previous studies have investigated the effect of a CLUV treatment on DNA repair of newly formed CPD i.e. when cells are subsequently irradiated with an acute UVB dose.
We have also found that the CLUV treatment generates persistent CPD that remains on the DNA, at least 24 h post irradiation (Fig 2). It has been previously shown that some DNA regions are refractory to DNA repair [51, 52] and we suspect the CLUV-induced remaining CPD to accumulate in those regions. However, more investigation needs to be performed to determine the localization and implication of those residual CPD. Localization of those residual CPD might be important to determine their mutagenicity. Indeed, CPD can be induced in 4 types of dipyrimidine sites, i.e. TT, CC, CT and TC [53]. Since C→T transitions are the skin cancer causing mutations, T-containing CPD can be considered non-mutagenic [54]. If the residual CLUV-induced CPD are preferentially localized on T-containing dipyrimidine sites, the consequences on carcinogenesis is minimal. More work should be performed to determine the localization and implication of residual CPD.
2. Cell replication is delayed after a CLUV pre-stimulation
We have examined the effect of UVB irradiation (single UVB or CLUV) on cell replication and, as expected, we found that a single acute UVB dose is causing a delay in S-phase recovery when compared to un-irradiated cells (Fig 3). This delay is longer when cells are subjected to a CLUV treatment rather than a single dose (Fig 3). The delay in cell cycle for the single UVB irradiated cells could be explained by the DDR that halt cell cycle to allow efficient CPD repair [32, 55]. In CLUV treated cells, the longer delay might be the consequence of the residual CPD induced by the CLUV irradiation, which is consistent with previous study [56]. Indeed, it is well documented that DNA lesions are delaying DNA replication progression by blocking DNA polymerase [57]. When it happens, translesional DNA polymerase are needed to bypass the CPD [58, 59]. The translesion DNA synthesis (TLS) has been shown to be crucial for DNA damage tolerance [60] and also after a CLUV exposure [27]. TLS is much slower than the replication polymerases, mainly due to the lower processivity of TLS polymerase [60, 61]. We think this would explain, at least in part, the longer recovery time needed when cells are subjected to a CLUV irradiation.
3. CLUV pre-stimulation does not sensitize cells to UV-induced cell death
We investigated whether the CLUV pre-stimulation would influence cell sensitivity to UV-induced cell death. Surprisingly, our results revealed that the CLUV pre-treatment does not influence UV-induced cell death sensitivity (Fig 4). It has been previously shown that CPD are the principal UV-induced apoptosis inductor [17, 62, 63] and that unrepaired DNA damage could trigger cell death [64]. Since we found persistent residual CPD after the CLUV treatment (Fig 2), we were expecting to have a higher sensitivity to UV-induced apoptosis in CLUV pre-stimulated cells. However, the amount of CLUV-induced residual CPD is relatively minimal (Fig 2) compared to the amount generated by the UVB doses used to induce cell death (up to 40,000 J/m2) (Fig 4). This might explain why the CLUV pre-stimulation does not lead to higher UV-induced apoptosis sensitivity.
4. CLUV treatment induces transcriptomic and proteomic changes
Since we found that CLUV treatment had an influence on major stress response mechanisms, including cell cycle and DNA repair, we pursued the investigation by analyzing transcriptome and proteome changes induced by the CLUV. We first notice that CLUV pre-stimulation induces important changes in gene expression (Fig 5). It has been previously reported that CPD induce different transcriptional response associated to replication and DNA damage repair, suggesting that CPD are by themselves a cause of gene expression changes [65]. Since CLUV pre-stimulation leads to the accumulation of residual CPD (Fig 2), the observed gene expression changes induced by our CLUV treatment might be attributed to those residual CPD.
A further analysis of deregulated genes using categorization by gene ontology showed that 151 are directly related to cellular stress response, which represents 15.9% of all deregulated genes (Fig 5, Table 1). Despite the influence of cellular stress in general to gene expression [37], the UV rays are also known to induce transcriptomic changes [38]. Amongst the genes deregulated by a single UV irradiation, a large amount is associated with inflammation [37], senescence, cell cycle, DNA damage response and p53 signaling [66]. Noteworthy, all the studies done previously have been conducted using a single UV irradiation and we are the first one reporting the effect of chronic irradiation on human transcriptome. In our study, we have found that a CLUV treatment induces response to stress as described in previous study [66]. Indeed, our transcriptomic analysis found a deregulation in the ‘‘cell death”; “inflammatory response”; “response to wounding” and “response to stimulus” in the ‘‘response to stress” biological process. By example, we found that XRCC2 gene 2.480 up deregulated. This protein is known to be crucial for DNA double strand break (DSB) repair by homologous recombination [67]. This result is in accordance with previous study where Garinis, G.A et al., demonstrate the role of unrepaired CPD in UV-induced DNA breaks [65]. Furthermore, IL-33 and CRH gene, both important in the inflammatory response are deregulated (S1 Table). IL-33 plays a role in the activation of innate immune system [68] and CRH is a major coordinator of the stress response. Previous studies pointed out the key role of CRH in skin response to stress [16].
We did not find any difference in NER-related gene expression, which was expected since the regulation of NER efficiency is mainly driven at the post-translational level rather than the transcriptional level.
Since our results showed that CLUV irradiation affects gene expression, we further analyzed whether the CLUV treatment affects protein levels (Fig 6). The large-scale study of protein revealed 2 500 potential proteins on the 2D-gel, from which 30 proteins were at least 2 times deregulated (Fig 6B). Those 30 proteins were identified by mass spectrometry, 21 are up-regulated and 9 down-regulated by the CLUV pre-stimulation (S2 Table). We found the antioxidant enzyme Superoxide dismutase mitochondrial (SODM) to be down-regulated (S2 Table). In addition to protect against oxidative stress, SODM is known to confer resistance to apoptosis [69]. Furthermore, previous work has demonstrated that UVB induces proteomics changes, especially an up-regulation of vimentin [42]. This has been suggested to contribute to cells resistance to UVB-induced damage. Vimentin is also found to be up-regulated in our present work (Table 2 and S2 Table), and is known as a contributor of apoptosis [70], which is in contradiction with our data showing that CLUV pre-stimulation does not influence UV-induced cell death sensitivity (Fig 4). In fact, we could see a higher sensitivity to UV-induced cell death at 40,000 J/m2, but it was not significant. More work would need to be done to determine the exact influence of vimentin upregulation and SODM down-regulation in UV-induced cell stress response post-CLUV irradiation.
Furthermore, the up-regulation of DOT1L was of interest since its role in chromatin structure to regulate DNA damage response is well established [71]. In addition to be crucial in chromatin organization, recent study demonstrates its critical role on cell cycle regulation [72] but also on UV sensitivity [43]. Furthermore, DOT1L plays a role in DSB repair [73], which is in accordance with our transcriptomic results (S1 Table). Additional analysis should be performed to clarify the role of those proteins in cellular response to a CLUV pre-stimulation.
Finally, 5 of the 30 proteins found in our analysis are implicated in the immune system process (Table 2). This is in accordance with previous report showing the involvement of UV exposure in immune system changes [74]. However, further investigation is needed to correlate the effect of CLUV treatment on the immune system.
To our knowledge, this study represents the first demonstration that a chronic irradiation can influence genotoxic stress response. Indeed, using 4 different strains of NHDF, we have shown that cells can respond to chronic UV irradiation by adapting their genotoxic stress response mechanisms, i.e. cell cycle and DNA repair. This cellular adaptability might reflect the potential human skin adaptation to chronic exposure to sunlight. However, the transcriptome and proteome analysis have shown that those stress response mechanisms are not the only ones affected by the CLUV treatment and further analysis need to be done to shed light on those mechanisms and their consequence for cells.
Materials and methods
Ethic statement
All experiments performed in this study were conducted in accordance with our institution's guidelines and the Declaration of Helsinki. The research protocols received approval by the Centre de Recherche du CHU de Québec (CRCHUQ) institutional committee for the protection of human subjects.
Cell culture
NHDF are from human skin biopsies (mastectomy) of 4 healthy women from 18 to 38 years old. Cells were used between passage 11 and 13 [75]. They were cultured in Dulbecco’s modified Eagle’s Medium (DMEM) (Corning cellgro, VA, USA) complemented with 10% FBS and 1% penicillin/streptomycin (Wisent, QC, CA) at 37°C, 5% CO2.
UVB irradiation and CLUV treatment
NHDF were irradiated using RPR-3000 UVB lamps (Southern New England Ultraviolet Co.) with an emission peak of 300 nm [76]. A cellulose acetate sheet (Kodacel TA-407, clear 0.015 in.; Eastman-Kodak Co.) was used to filter out wavelength below 295 nm [76]. The acute UVB irradiation of 400 J/m2 has been chosen according previous studies [31, 77] and corresponds to 5–10 min of solar exposure at zenith sun [78]. The UVB dose chosen for the chronic irradiation (CLUV) has been selected based on previous experiments and cell sensitivity [79]. Each irradiation in of the chronic protocol corresponds to around 1 min of direct solar exposure.
CLUV.
Confluent NHDF were first exposed to a CLUV treatment consisting of 75 J/m2 of UVB (100 J/m2 for the gene profiling experiment, Fig 5) every 12 h for 7.5 days. Each day, cells then received a total of 150 J/m2 of UVB (200 J/m2 for the gene profiling experiment, Fig 5). After 7.5 days, cells have received a total of 15 irradiations (Fig 1.2 and 1.3). After the CLUV treatment, cells were incubated for 12 h and were either harvested (corresponding to the 0 h time point) or 24 h later (Fig 1.2). Irradiation was performed in PBS to avoid cell dehydration and oxidative stress (Corning cellgro). The DMEM medium was filtered and reuse between irradiations.
Acute.
Confluent NHDF were exposed to a single acute UVB irradiation of 400J/m2 (200 J/m2 for the cell cycle experiment, Fig 3). Prior to the single acute UVB irradiation, medium was filtered and replace with PBS at the same frequency (every 12 h, for 7.5 days) as the CLUV treated cells in order to mimic experimental stress.
DNA damage and repair
DNA extraction.
Total DNA was extracted using a DNeasy Blood and Tissue Kit (QIAGEN) following the manufacturer’s protocol with an additional RNase treatment. DNA concentration was determined using a spectrophometer (NanoDrop 2000; Thermoscientific).
DNA slot blots.
The immuno-slot-blot technique was performed as previously described [52]. Briefly, after alkaline DNA denaturation (10 min 56°C, followed by 3N of NaOH), DNA was blotted on positively charged nitrocellulose membranes (Bio-Dot® SF Microfiltration Apparatus), and DNA was heat fixed to the membrane (80°C, 3 h). Membranes were then blocked with 5% nonfat dry milk and hybridized with a mouse anti-CPD monoclonal antibody (Cosmo Bio Co., clone TDM-2) diluted 1:5 000 in 1% milk + 0.05% tween. The secondary HRP-conjugated antibody (Rabbit anti-mouse) (Jackson ImmunoResearch) was diluted 1:5 000 in 1% milk + 0.05% tween. A mouse anti-ssDNA monoclonal antibody (EMD Millipore, clone 16–19) diluted 1:1 000 was used to detect DNA. Membranes analysis and quantification were performed with C-DiGit® Blot Scanner (LI-COR Biosciences).
NHDF from 3 different cell strains were used for this experiment, and the slot blot was performed at least twice for each NHDF strain cells. P-values were derived from the two-tailed heteroscedastic Student’s t-test.
Cell cycle analysis by flow cytometry
NHDF cells were subjected to either an acute dose (acute), a CLUV treatment (CLUV) or un-irradiated (NoUV). Cells were then re-seeded at a density of 8.3x103 cells/cm2 and incubated for different time points (0 to 36 h). They were then fixed with ethanol 70% and stained using propidium iodide (PI). Cell cycle distribution was analyzed by flow cytometry. Four NHDF cell strains were used for this experiment.
UVB-induced cell death assay
Confluent NHDF pre-stimulated or not by the CLUV treatment were subjected to acute UVB dose, ranging from 0 to 40,000 J/m2. Sixteen h after acute irradiation, cells were harvested and stained with Annexin V/PI apoptosis kit (Molecular probes, Eugene, OR) as previously described [31, 75]. Briefly, 16 h after acute UVB dose, cells were harvested and resuspended in Annexin V binding buffer. The staining annexin V and PI are added and cells are incubated 15 min at room temperature. Analysis of apoptotic (Annexin V positive cells) and necrotic (PI positive cells) was performed by flow cytometry. Four different NHDF cell strains were used and p-values were derived from the two-tailed heteroscedastic Student’s t-test.
Gene expression profiling analysis
RNA isolation.
Total RNA was isolated from CLUV pre-stimulated and un-irradiated controls (NoUV) using TRIzol® Reagent (Life Technologies) according to manufacturer’s instructions. RNA quantity and quality were assessed using a 2100 Bioanalyzer Instruments (Agilent Technologies) according to the manufacturer’s protocol.
Sample preparation and procedure.
Sample preparation was performed following to One-color Microarray-Based Gene Expression Analysis protocol and as described in [80].
Microarray hybridization and analysis.
150 ng of amplified cRNA was incubated on a G4851A SurePrint G3 Human Ge 8 x 60 K array slide (Agilent Technologies). Slides have over 60 000 targets of the global human genome. After 18 h of slide’s hybridization, they were washed and scanned on an Agilent SureScan Scanner.
Data analysis.
A data report was produced by Arraystar v 4.1 software (DNASTAR), which includes the scatter plot and heat map of deregulated genes. Further statistical analyses were performed in collaboration with the Bioinformatics Platform of the CHU de Québec. GO enrichment analyses were done on the list of variant genes (fold-change ≥ 2) using the "biological process" ontology. Enrichment analyses were performed using BiNGO (v 3.0.3) on the Cytoscape software platform (v 3.2.1) [81, 82]. Enrichment was tested by hypergeometric test (without replacement) with Bonferroni correction for multiple testing, and annotations are said to be significantly enriched at corrected p-value ≤ 0.01.
Proteome expression analysis
Sample preparation and procedure.
Three cell strains of CLUV treated NHDF were pooled together. The same 3 cell strains un-irradiated (NoUV) were also pooled and were used as baseline. Proteins were extracted using RIPA buffer (Thermo scientific) and protein concentration was determined by the BCA assay using Bio-Rad protein kit.
Two-Dimensional Difference Gel Electrophoresis (2D-DIGE).
The two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry analysis were performed by Applied Biomics (Hayward, CA). Before proceeding with the 2D-DIGE, a 5 kDa MWCO spin column was used to replace RIPA buffer with the 2D lysis buffer (7M urea, 2M thiourea, 4% CHAPS, 30 mM Tris-HCl). Procedure of 2D-DIGE has been performed as previously described [83]. Briefly, samples were first labeled with CyDye DIGE fluors (NoUV: Cy3; CLUV: Cy5). Then, for the first dimension, the isoelectric focusing (IEF) has been used to separate both samples, and then a SDS polyacrylamide gel electrophoresis (SDS-PAGE) has been used for the second dimension. Image acquisition is performed with Typhoon image scanner and analysis of scan was performed using ImageQuant software. Using DeCyder analysis software, the protein levels were determined. The DeCyder software found the 70 most deregulated proteins. Only proteins deregulated at least 1.5 times were sent for mass spectrometry analysis.
Data analysis.
MS analysis is based on peptide fingerprint mass mapping. MASCOT software was used to identify proteins according to their peptide fingerprint. Proteins were accepted on the basis of peptide count and total ion confidence interval (C.I. %) (S2 Table). Protein identifications were considered accurate if there was at least 3 peptides match and if total ion C.I. % was greater than 95,0% when calculated from MS data.
Proteins functional analysis was performed using the Reactome pathway knowledge base (Reactome v54), an open-source, open-data and peer-reviewed database of human pathways and reactions [84, 85].
Supporting information
S1 Table. Compilation of deregulated genes related to stress response.
https://doi.org/10.1371/journal.pone.0173740.s001
(TIF)
S2 Table. List of the 30 proteins deregulated and their mass spectrometry characteristics.
https://doi.org/10.1371/journal.pone.0173740.s002
(TIF)
Author Contributions
- Conceptualization: MCDD PJR.
- Data curation: MCDD FF.
- Formal analysis: MCDD FF PJR.
- Funding acquisition: PJR.
- Investigation: MCDD PJR.
- Methodology: MCDD.
- Project administration: PJR.
- Resources: PJR AD.
- Software: PJR AD MCDD FF.
- Supervision: PJR.
- Validation: MCDD PJR.
- Visualization: MCDD PJR.
- Writing – original draft: MCDD.
- Writing – review & editing: MCDD PJR.
References
- 1. Bibbins-Domingo K, Grossman DC, Curry SJ, Davidson KW, Ebell M, Epling JW Jr., et al. Screening for Skin Cancer: US Preventive Services Task Force Recommendation Statement. Jama. 2016;316(4):429–35. Epub 2016/07/28. pmid:27458948
- 2. Black HS, deGruijl FR, Forbes PD, Cleaver JE, Ananthaswamy HN, deFabo EC, et al. Photocarcinogenesis: an overview. Journal of photochemistry and photobiology B, Biology. 1997;40(1):29–47. Epub 1997/08/01. pmid:9301042
- 3. de Gruijl FR. Photocarcinogenesis: UVA vs UVB. Methods in enzymology. 2000;319:359–66. Epub 2000/07/25. pmid:10907526
- 4. Pfeifer GP. Formation and processing of UV photoproducts: effects of DNA sequence and chromatin environment. Photochemistry and photobiology. 1997;65(2):270–83. Epub 1997/02/01. pmid:9066304
- 5. Schuch AP, Menck CF. The genotoxic effects of DNA lesions induced by artificial UV-radiation and sunlight. Journal of photochemistry and photobiology B, Biology. 2010;99(3):111–6. Epub 2010/04/08. pmid:20371188
- 6. Rochette PJ, Bastien N, Todo T, Drouin R. Pyrimidine (6–4) pyrimidone photoproduct mapping after sublethal UVC doses: nucleotide resolution using terminal transferase-dependent PCR. Photochemistry and photobiology. 2006;82(5):1370–6. Epub 2006/06/17. pmid:16776547
- 7. Tornaletti S, Pfeifer GP. UV damage and repair mechanisms in mammalian cells. Bioessays. 1996;18(3):221–8. Epub 1996/03/01. pmid:8867736
- 8. Brash DE. UV signature mutations. Photochemistry and photobiology. 2015;91(1):15–26. Epub 2014/10/30. pmid:25354245
- 9. Hutchinson F. Induction of tandem-base change mutations. Mutat Res. 1994;309(1):11–5. Epub 1994/08/01. pmid:7519728
- 10. Lebkowski JS, Clancy S, Miller JH, Calos MP. The lacI shuttle: rapid analysis of the mutagenic specificity of ultraviolet light in human cells. Proceedings of the National Academy of Sciences of the United States of America. 1985;82(24):8606–10. Epub 1985/12/01. pmid:3001711
- 11. McGregor WG, Chen RH, Lukash L, Maher VM, McCormick JJ. Cell cycle-dependent strand bias for UV-induced mutations in the transcribed strand of excision repair-proficient human fibroblasts but not in repair-deficient cells. Mol Cell Biol. 1991;11(4):1927–34. Epub 1991/04/01. pmid:2005888
- 12. Miller JH. Mutational specificity in bacteria. Annu Rev Genet. 1983;17:215–38. Epub 1983/01/01. pmid:6364961
- 13. Singh RK, Lee KM, Jose MV, Nakamura M, Ucmak D, Farahnik B, et al. The Patient's Guide to Psoriasis Treatment. Part 1: UVB Phototherapy. Dermatology and therapy. 2016;6(3):307–13. Epub 2016/07/31. pmid:27474029
- 14. Michaels CJ, Antwis RE, Preziosi RF. Impacts of UVB provision and dietary calcium content on serum vitamin D3, growth rates, skeletal structure and coloration in captive oriental fire-bellied toads (Bombina orientalis). Journal of animal physiology and animal nutrition. 2015;99(2):391–403. Epub 2014/05/09. pmid:24810567
- 15. Slominski AT, Zmijewski MA, Skobowiat C, Zbytek B, Slominski RM, Steketee JD. Sensing the environment: regulation of local and global homeostasis by the skin's neuroendocrine system. Advances in anatomy, embryology, and cell biology. 2012;212:v, vii, 1–115. Epub 2012/08/17. pmid:22894052
- 16. Slominski AT, Zmijewski MA, Zbytek B, Tobin DJ, Theoharides TC, Rivier J. Key role of CRF in the skin stress response system. Endocrine reviews. 2013;34(6):827–84. Epub 2013/08/14. pmid:23939821
- 17. Sancar A, Lindsey-Boltz LA, Unsal-Kacmaz K, Linn S. Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annu Rev Biochem. 2004;73:39–85. Epub 2004/06/11. pmid:15189136
- 18. Harper JW, Elledge SJ. The DNA damage response: ten years after. Mol Cell. 2007;28(5):739–45. Epub 2007/12/18. pmid:18082599
- 19. Boyle J, Ueda T, Oh KS, Imoto K, Tamura D, Jagdeo J, et al. Persistence of repair proteins at unrepaired DNA damage distinguishes diseases with ERCC2 (XPD) mutations: cancer-prone xeroderma pigmentosum vs. non-cancer-prone trichothiodystrophy. Hum Mutat. 2008;29(10):1194–208. Epub 2008/05/13. pmid:18470933
- 20. Jackson SP, Bartek J. The DNA-damage response in human biology and disease. Nature. 2009;461(7267):1071–8. Epub 2009/10/23. pmid:19847258
- 21. Kadhim MA, Moore SR, Goodwin EH. Interrelationships amongst radiation-induced genomic instability, bystander effects, and the adaptive response. Mutat Res. 2004;568(1):21–32. Epub 2004/11/09. pmid:15530536
- 22. Takahashi A, Ohnishi T. Molecular mechanisms involved in adaptive responses to radiation, UV light, and heat. J Radiat Res. 2009;50(5):385–93. Epub 2009/06/16. pmid:19525615
- 23. Ye N, Bianchi MS, Bianchi NO, Holmquist GP. Adaptive enhancement and kinetics of nucleotide excision repair in humans. Mutat Res. 1999;435(1):43–61. Epub 1999/10/20. pmid:10526216
- 24. Cramers P, Filon AR, Pines A, Kleinjans JC, Mullenders LH, van Zeeland AA. Enhanced nucleotide excision repair in human fibroblasts pre-exposed to ionizing radiation. Photochemistry and photobiology. 2012;88(1):147–53. Epub 2011/10/25. pmid:22017241
- 25. Vijayalaxmi , Cao Y, Scarfi MR. Adaptive response in mammalian cells exposed to non-ionizing radiofrequency fields: A review and gaps in knowledge. Mutat Res Rev Mutat Res. 2014. Epub 2014/02/20.
- 26. Mitchell DL, Greinert R, de Gruijl FR, Guikers KL, Breitbart EW, Byrom M, et al. Effects of chronic low-dose ultraviolet B radiation on DNA damage and repair in mouse skin. Cancer Res. 1999;59(12):2875–84. Epub 1999/06/26. pmid:10383149
- 27. Hishida T, Kubota Y, Carr AM, Iwasaki H. RAD6-RAD18-RAD5-pathway-dependent tolerance to chronic low-dose ultraviolet light. Nature. 2009;457(7229):612–5. Epub 2008/12/17. pmid:19079240
- 28. Mitchell DL, Byrom M, Chiarello S, Lowery MG. Effects of chronic exposure to ultraviolet B radiation on DNA repair in the dermis and epidermis of the hairless mouse. The Journal of investigative dermatology. 2001;116(2):209–15. Epub 2001/02/17. pmid:11179995
- 29. Mitchell DL, Volkmer B, Breitbart EW, Byrom M, Lowery MG, Greinert R. Identification of a non-dividing subpopulation of mouse and human epidermal cells exhibiting high levels of persistent ultraviolet photodamage. The Journal of investigative dermatology. 2001;117(3):590–5. Epub 2001/09/21. pmid:11564164
- 30. Sheehan JM, Cragg N, Chadwick CA, Potten CS, Young AR. Repeated ultraviolet exposure affords the same protection against DNA photodamage and erythema in human skin types II and IV but is associated with faster DNA repair in skin type IV. The Journal of investigative dermatology. 2002;118(5):825–9. Epub 2002/05/02. pmid:11982760
- 31. Mallet JD, Dorr MM, Drigeard Desgarnier MC, Bastien N, Gendron SP, Rochette PJ. Faster DNA Repair of Ultraviolet-Induced Cyclobutane Pyrimidine Dimers and Lower Sensitivity to Apoptosis in Human Corneal Epithelial Cells than in Epidermal Keratinocytes. PloS one. 2016;11(9):e0162212. Epub 2016/09/10. pmid:27611318
- 32. Lo HL, Nakajima S, Ma L, Walter B, Yasui A, Ethell DW, et al. Differential biologic effects of CPD and 6-4PP UV-induced DNA damage on the induction of apoptosis and cell-cycle arrest. BMC Cancer. 2005;5:135. Epub 2005/10/21. pmid:16236176
- 33. Courdavault S, Baudouin C, Sauvaigo S, Mouret S, Candeias S, Charveron M, et al. Unrepaired cyclobutane pyrimidine dimers do not prevent proliferation of UV-B-irradiated cultured human fibroblasts. Photochemistry and photobiology. 2004;79(2):145–51. Epub 2004/04/08. pmid:15068027
- 34. D'Errico M, Teson M, Calcagnile A, Proietti De Santis L, Nikaido O, Botta E, et al. Apoptosis and efficient repair of DNA damage protect human keratinocytes against UVB. Cell Death Differ. 2003;10(6):754–6. Epub 2003/05/23. pmid:12761584
- 35. Norbury CJ, Zhivotovsky B. DNA damage-induced apoptosis. Oncogene. 2004;23(16):2797–808. Epub 2004/04/13. pmid:15077143
- 36. Kulms D, Schwarz T. Molecular mechanisms involved in UV-induced apoptotic cell death. Skin Pharmacol Appl Skin Physiol. 2002;15(5):342–7. Epub 2002/09/20 pmid:12239429
- 37. Weinkauf B, Rukwied R, Quiding H, Dahllund L, Johansson P, Schmelz M. Local gene expression changes after UV-irradiation of human skin. PloS one. 2012;7(6):e39411. Epub 2012/07/05. pmid:22761785
- 38. McKay BC, Stubbert LJ, Fowler CC, Smith JM, Cardamore RA, Spronck JC. Regulation of ultraviolet light-induced gene expression by gene size. Proceedings of the National Academy of Sciences of the United States of America. 2004;101(17):6582–6. Epub 2004/04/17. pmid:15087501
- 39. Lee S, Jo M, Lee J, Koh SS, Kim S. Identification of novel universal housekeeping genes by statistical analysis of microarray data. Journal of biochemistry and molecular biology. 2007;40(2):226–31. Epub 2007/03/31. pmid:17394773
- 40. Pastila R. Effects of ultraviolet radiation on skin cell proteome. Adv Exp Med Biol. 2013;990:121–7. Epub 2013/02/05. pmid:23378008
- 41. Wu CL, Chou HC, Cheng CS, Li JM, Lin ST, Chen YW, et al. Proteomic analysis of UVB-induced protein expression- and redox-dependent changes in skin fibroblasts using lysine- and cysteine-labeling two-dimensional difference gel electrophoresis. J Proteomics. 2012;75(7):1991–2014. Epub 2012/01/25. pmid:22270008
- 42. Yan Y, Xu H, Peng S, Zhao W, Wang B. Proteome analysis of ultraviolet-B-induced protein expression in vitro human dermal fibroblasts. Photodermatol Photoimmunol Photomed. 2010;26(6):318–26. Epub 2010/11/26. pmid:21091790
- 43. Oksenych V, Zhovmer A, Ziani S, Mari PO, Eberova J, Nardo T, et al. Histone methyltransferase DOT1L drives recovery of gene expression after a genotoxic attack. PLoS genetics. 2013;9(7):e1003611. Epub 2013/07/19. pmid:23861670
- 44. Tornaletti S, Rozek D, Pfeifer GP. The distribution of UV photoproducts along the human p53 gene and its relation to mutations in skin cancer. Oncogene. 1993;8(8):2051–7. Epub 1993/08/01. pmid:8336934
- 45. Strozyk E, Kulms D. The role of AKT/mTOR pathway in stress response to UV-irradiation: implication in skin carcinogenesis by regulation of apoptosis, autophagy and senescence. Int J Mol Sci. 2013;14(8):15260–85. Epub 2013/07/28. pmid:23887651
- 46. Zhao H, Traganos F, Darzynkiewicz Z. Kinetics of the UV-induced DNA damage response in relation to cell cycle phase. Correlation with DNA replication. Cytometry A. 2010;77(3):285–93. Epub 2009/12/17. pmid:20014310
- 47. Salucci S, Burattini S, Battistelli M, Baldassarri V, Maltarello MC, Falcieri E. Ultraviolet B (UVB) irradiation-induced apoptosis in various cell lineages in vitro. Int J Mol Sci. 2012;14(1):532–46. Epub 2012/12/29. pmid:23271369
- 48. Yoshikawa T, Rae V, Bruins-Slot W, Van den Berg JW, Taylor JR, Streilein JW. Susceptibility to effects of UVB radiation on induction of contact hypersensitivity as a risk factor for skin cancer in humans. The Journal of investigative dermatology. 1990;95(5):530–6. Epub 1990/11/01. pmid:2230216
- 49. Haruta N, Kubota Y, Hishida T. Chronic low-dose ultraviolet-induced mutagenesis in nucleotide excision repair-deficient cells. Nucleic Acids Res. 2012;40(17):8406–15. Epub 2012/06/30. pmid:22743272
- 50. Steerenberg PA, Daamen F, Weesendorp E, Van Loveren H. No adaptation to UV-induced immunosuppression and DNA damage following exposure of mice to chronic UV-exposure. Journal of photochemistry and photobiology B, Biology. 2006;84(1):28–37. Epub 2006/03/01. pmid:16504533
- 51. Pascucci B, Versteegh A, van Hoffen A, van Zeeland AA, Mullenders LH, Dogliotti E. DNA repair of UV photoproducts and mutagenesis in human mitochondrial DNA. J Mol Biol. 1997;273(2):417–27. Epub 1997/11/05. pmid:9344749
- 52. Rochette PJ, Brash DE. Human telomeres are hypersensitive to UV-induced DNA Damage and refractory to repair. PLoS genetics. 2010;6(4):e1000926. Epub 2010/05/06. pmid:20442874
- 53. Mouret S, Bogdanowicz P, Haure MJ, Castex-Rizzi N, Cadet J, Favier A, et al. Assessment of the photoprotection properties of sunscreens by chromatographic measurement of DNA damage in skin explants. Photochemistry and photobiology. 2011;87(1):109–16. Epub 2010/11/26. pmid:21091484
- 54. Pfeifer GP, Besaratinia A. UV wavelength-dependent DNA damage and human non-melanoma and melanoma skin cancer. Photochemical & photobiological sciences: Official journal of the European Photochemistry Association and the European Society for Photobiology. 2012;11(1):90–7.
- 55. Branzei D, Foiani M. Regulation of DNA repair throughout the cell cycle. Nature reviews Molecular cell biology. 2008;9(4):297–308. Epub 2008/02/21. pmid:18285803
- 56. Pope-Varsalona H, Liu FJ, Guzik L, Opresko PL. Polymerase eta suppresses telomere defects induced by DNA damaging agents. Nucleic Acids Res. 2014;42(21):13096–109. Epub 2014/10/31. pmid:25355508
- 57. Ljungman M, Zhang F. Blockage of RNA polymerase as a possible trigger for u.v. light-induced apoptosis. Oncogene. 1996;13(4):823–31. Epub 1996/08/15. pmid:8761304
- 58. Sale JE, Lehmann AR, Woodgate R. Y-family DNA polymerases and their role in tolerance of cellular DNA damage. Nature reviews Molecular cell biology. 2012;13(3):141–52. Epub 2012/02/24. pmid:22358330
- 59. Xu X, Blackwell S, Lin A, Li F, Qin Z, Xiao W. Error-free DNA-damage tolerance in Saccharomyces cerevisiae. Mutat Res Rev Mutat Res. 2015;764:43–50. Epub 2015/06/05. pmid:26041265
- 60. Waters LS, Minesinger BK, Wiltrout ME, D'Souza S, Woodruff RV, Walker GC. Eukaryotic translesion polymerases and their roles and regulation in DNA damage tolerance. Microbiol Mol Biol Rev. 2009;73(1):134–54. Epub 2009/03/05. pmid:19258535
- 61. Prakash S, Johnson RE, Prakash L. Eukaryotic translesion synthesis DNA polymerases: specificity of structure and function. Annu Rev Biochem. 2005;74:317–53. Epub 2005/06/15. pmid:15952890
- 62. Ford JM, Hanawalt PC. Li-Fraumeni syndrome fibroblasts homozygous for p53 mutations are deficient in global DNA repair but exhibit normal transcription-coupled repair and enhanced UV resistance. Proceedings of the National Academy of Sciences of the United States of America. 1995;92(19):8876–80. Epub 1995/09/12. pmid:7568035
- 63. Brash DE, Wikonkal NM, Remenyik E, van der Horst GT, Friedberg EC, Cheo DL, et al. The DNA damage signal for Mdm2 regulation, Trp53 induction, and sunburn cell formation in vivo originates from actively transcribed genes. The Journal of investigative dermatology. 2001;117(5):1234–40. Epub 2001/11/17. pmid:11710938
- 64. Roos WP, Thomas AD, Kaina B. DNA damage and the balance between survival and death in cancer biology. Nat Rev Cancer. 2016;16(1):20–33. Epub 2015/12/19. pmid:26678314
- 65. Garinis GA, Mitchell JR, Moorhouse MJ, Hanada K, de Waard H, Vandeputte D, et al. Transcriptome analysis reveals cyclobutane pyrimidine dimers as a major source of UV-induced DNA breaks. Embo j. 2005;24(22):3952–62. Epub 2005/10/28. pmid:16252008
- 66. Greussing R, Hackl M, Charoentong P, Pauck A, Monteforte R, Cavinato M, et al. Identification of microRNA-mRNA functional interactions in UVB-induced senescence of human diploid fibroblasts. BMC Genomics. 2013;14:224. Epub 2013/04/06. pmid:23557329
- 67. Johnson RD, Liu N, Jasin M. Mammalian XRCC2 promotes the repair of DNA double-strand breaks by homologous recombination. Nature. 1999;401(6751):397–9. Epub 1999/10/12. pmid:10517641
- 68. Miller AM. Role of IL-33 in inflammation and disease. Journal of Inflammation (London, England). 2011;8:22.
- 69. Mantymaa P, Siitonen T, Guttorm T, Saily M, Kinnula V, Savolainen ER, et al. Induction of mitochondrial manganese superoxide dismutase confers resistance to apoptosis in acute myeloblastic leukaemia cells exposed to etoposide. British journal of haematology. 2000;108(3):574–81. Epub 2000/04/12. pmid:10759716
- 70. Xu H, Yan Y, Li L, Peng S, Qu T, Wang B. Ultraviolet B-induced apoptosis of human skin fibroblasts involves activation of caspase-8 and -3 with increased expression of vimentin. Photodermatol Photoimmunol Photomed. 2010;26(4):198–204. Epub 2010/07/16. pmid:20626822
- 71. Tatum D, Li S. Evidence that the histone methyltransferase Dot1 mediates global genomic repair by methylating histone H3 on lysine 79. J Biol Chem. 2011;286(20):17530–5. Epub 2011/04/05. pmid:21460225
- 72. Kim W, Choi M, Kim JE. The histone methyltransferase Dot1/DOT1L as a critical regulator of the cell cycle. Cell Cycle. 2014;13(5):726–38. Epub 2014/02/15. pmid:24526115
- 73. Conde F, Refolio E, Cordon-Preciado V, Cortes-Ledesma F, Aragon L, Aguilera A, et al. The Dot1 histone methyltransferase and the Rad9 checkpoint adaptor contribute to cohesin-dependent double-strand break repair by sister chromatid recombination in Saccharomyces cerevisiae. Genetics. 2009;182(2):437–46. Epub 2009/04/01. pmid:19332880
- 74. Schwarz T. Mechanisms of UV-induced immunosuppression. Keio J Med. 2005;54(4):165–71. Epub 2006/02/03. pmid:16452825
- 75. Rochette PJ, Brash DE. Progressive apoptosis resistance prior to senescence and control by the anti-apoptotic protein BCL-xL. Mechanisms of ageing and development. 2008;129(4):207–14. Epub 2008/02/12. pmid:18262222
- 76. Mallet JD, Rochette PJ. Ultraviolet light-induced cyclobutane pyrimidine dimers in rabbit eyes. Photochemistry and photobiology. 2011;87(6):1363–8. Epub 2011/07/21. pmid:21770949
- 77. Rochette PJ, Bastien N, McKay BC, Therrien JP, Drobetsky EA, Drouin R. Human cells bearing homozygous mutations in the DNA mismatch repair genes hMLH1 or hMSH2 are fully proficient in transcription-coupled nucleotide excision repair. Oncogene. 2002;21(37):5743–52. Epub 2002/08/13. pmid:12173044
- 78. Kuluncsics Z, Perdiz D, Brulay E, Muel B, Sage E. Wavelength dependence of ultraviolet-induced DNA damage distribution: involvement of direct or indirect mechanisms and possible artefacts. Journal of photochemistry and photobiology B, Biology. 1999;49(1):71–80. Epub 1999/06/12. pmid:10365447
- 79. Ichihashi M, Ando H. The maximal cumulative solar UVB dose allowed to maintain healthy and young skin and prevent premature photoaging. Experimental dermatology. 2014;23 Suppl 1:43–6. Epub 2014/09/23.
- 80. Gendron SP, Rochette PJ. Modifications in stromal extracellular matrix of aged corneas can be induced by ultraviolet A irradiation. Aging Cell. 2015;14(3):433–42. Epub 2015/03/03. pmid:25728164
- 81. Maere S, Heymans K, Kuiper M. BiNGO: a Cytoscape plugin to assess overrepresentation of gene ontology categories in biological networks. Bioinformatics. 2005;21(16):3448–9. Epub 2005/06/24. pmid:15972284
- 82. Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2003;13(11):2498–504. Epub 2003/11/05. pmid:14597658
- 83. Lamore SD, Qiao S, Horn D, Wondrak GT. Proteomic identification of cathepsin B and nucleophosmin as novel UVA-targets in human skin fibroblasts. Photochemistry and photobiology. 2010;86(6):1307–17. Epub 2010/10/16. pmid:20946361
- 84. Croft D, Mundo AF, Haw R, Milacic M, Weiser J, Wu G, et al. The Reactome pathway knowledgebase. Nucleic Acids Res. 2014;42(Database issue):D472–7. Epub 2013/11/19. pmid:24243840
- 85. Milacic M, Haw R, Rothfels K, Wu G, Croft D, Hermjakob H, et al. Annotating cancer variants and anti-cancer therapeutics in reactome. Cancers (Basel). 2012;4(4):1180–211. Epub 2012/01/01.