1. Persistence and Tolerance of DNA Damage Induced by Chronic UVB Irradiation of the Human Genome 
Roxanne Bérubé, Marie-Catherine Drigeard Desgarnier, Thierry Douki, Ariane Lechasseur, Patrick J. Rochette 
Journal of Investigative Dermatology 
Volume 138, Issue 2, Pages (February 2018)
DOI: /j.jid
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2. Figure 1
Residual CPDs induced by CLUV irradiation and impact on cellular division. NHDFs were irradiated with the CLUV protocol or a single acute 400 J/m2 UVB. Cells were harvested and DNA was purified 12 hours after the last irradiation of the CLUV treatment or immediately after the acute UVB irradiation. (a) Representative image of the immuno-slot blot of CPDs using an anti-CPD antibody. Anti–single-strand DNA antibody was used to control the amount of DNA. (b) Quantification of the immuno-slot blot shows that the signal level of residual CPDs induced by CLUV treatment is equivalent to approximately 50% of those induced by acute irradiation with 400 J/m2 UVB. This indicates that residual CPDs induced by the CLUV irradiation represent the amount of CPDs induced by 200 J/m2. Because the cumulative dose induced by the CLUV is 1,125 J/m2, we can conclude that approximately 18% of the CLUV-induced CPDs remain on the DNA. (c) Cellular division after CLUV irradiation and compared with a nonirradiated control. Twelve hours after the last irradiation of the CLUV treatment, cells were reseeded at lower density and counted every day for 7 days. Cellular count is compared with the amount of cells at day 1. CLUV-irradiated cells divided at a slower rate than unirradiated cells. It takes 2–3 days to reach a population doubling in unirradiated cells, whereas it takes 5 days to achieve the same replication in CLUV-treated cells. Each experiment has been performed on three different NHDF cell strains (N = 3), performed in triplicate (n = 3). CLUV, chronic low-dose UVB; CPD, cyclobutane pyrimidine dimer; NHDF, normal human diploid fibroblast.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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3. Figure 2
Dilution of residual CPDs through DNA replication. NHDFs were CLUV-irradiated, harvested 12 hours after the last irradiation, and reseeded at lower density to allow cellular replication. Cells were then harvested every 24 hours for 7 days, metaphase spreads were prepared, and CPDs were quantified on chromosomes by immunocytofluorescence using an anti-CPD antibody. (a) Representative CPD immunocytofluorescence on metaphase shows three different CPD labelings on chromosomes. In the first, both chromatids of each chromosome contain CPDs (top panel). In the second, only one of the two chromatids contains CPDs (middle panel). In the third, none of the chromatids contains CPDs (bottom panel). (b) Quantification of chromosomes containing two, one, or no damaged chromatids shows that both chromatids containing CPDs are found when cells have low levels of cellular replication. Chromosomes containing no detectable CPDs are found when cells accumulate cellular replication. Chromosomes containing only one damaged chromatid represent an intermediate event. (c) Explanatory schema of the CPD distribution in chromatids according to the semiconservative replication mode. When irradiated, all strands of the DNA contain CPDs. After the first DNA replication, one strand of each chromatid contains CPDs. After the second DNA replication cycle, one chromatin will contain a damaged DNA strand, but the other will be free of DNA damage. As the DNA replication accumulates, DNA strands containing CPDs will be diluted, and we will observe an increased amount of undamaged chromosomes. This theory is in accordance with the counted chromosomes containing two, one, or no damaged chromatids and cellular replication measured (Figure 1c). Each experiment has been performed on three different NHDF cell strains (N = 3), and at least 120 chromosomes were counted for each N and each condition. Scale bar = 5 μm. CLUV, chronic low-dose UVB; CPD, cyclobutane pyrimidine dimer; NHDF, normal human diploid fibroblast.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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4. Figure 3
Localization of residual CPDs. (a) Heterochromatin and euchromatin were isolated by ChIP from NHDFs irradiated with the CLUV regimen (CLUV) or a single acute 400 J/m2 UVB (Acute). CPDs in each fraction were shown by ELISA. Distribution of CPDs is similar in euchromatin and heterochromatin after acute UVB irradiation. However, residual CPDs induced by the CLUV treatment are concentrated in the heterochromatin. This indicates that the residual CPDs are less accessible DNA damage that are refractory to repair. (b) Using HPLC/MS-MS technique, we have determined CPD distribution in each dipyrimidine site in DNA of NHDFs irradiated with the CLUV regimen (CLUV) or a single acute 400 J/m2 UVB (Acute). After acute UVB irradiation, CPDs are concentrated in TT, followed by TC and CT. CC CPDs were not detectable using this technique. Residual CPDs induced by the CLUV treatment follow the same distribution of TT > TC > CT, but the level of TT is significantly increased. This indicates that residual CPDs are concentrated in TT dipyrimidine sites. Each experiment has been performed on 3–4 different NHDF cell strains (N = 3). Standard error of the mean, ∗P < 0.05, Student t test. C, cytosine; ChIP, chromatin immunoprecipitation; CLUV, chronic low-dose UVB; CPD, cyclobutane pyrimidine dimer; HPLC/MS-MS, high-performance liquid chromatography/tandem mass spectrometry; NHDF, normal human diploid fibroblast; T, thymine.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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5. Figure 4
Consequences of CLUV-induced residual CPDs on the accumulation of SCE. Consequences of CLUV-induced residual CPDs on genome stability was evaluated by measuring the accumulation of SCE. Twelve hours after the last irradiation of the CLUV regimen, cells were harvested and reseeded at lower density in presence of BrdU to allow replication and BrdU incorporation. Cells were harvested 3 days after the reseeding. (a) Representative immunocytofluorescence of BrdU on metaphase chromosomes from CLUV-irradiated cells. We can observe many SCEs in chromosomes (white arrows). (b) Quantification of SCE in NHDFs, CLUV-irradiated (CLUV) or not (NoUV). We can observe a significant increase in SCE after a CLUV irradiation. Because we began BrdU incorporation 12 hours after the last CLUV irradiation, the observed SCE increase is most likely due to residual CPDs rather than UV irradiation itself. Each experiment has been performed on three different NHDF cell strains (N = 3), and at least 120 chromosomes were counted for each N and each condition ∗P < Scale bar = 5 μm. CLUV, chronic low-dose UVB; CPD, cyclobutane pyrimidine dimer; NHDF, normal human diploid fibroblast; SCE, sister chromatin exchange.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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