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The circadian clock controls sunburn apoptosis and erythema in mouse skin.

Gaddameedhi S, Selby CP, Kemp MG, Ye R, Sancar A - J. Invest. Dermatol. (2014)

Bottom Line: Early-morning exposure to UV also produced maximal activation of ataxia telangiectasia mutated and Rad3-related (Atr)-mediated DNA damage checkpoint signaling, including activation of the tumor suppressor p53, which is known to control the process of sunburn apoptosis.These data provide early evidence that the circadian clock has an important role in the erythemal response in UV-irradiated skin.The early morning is when DNA repair is at a minimum, and thus the acute responses likely are associated with unrepaired DNA damage.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA [2] Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA.

ABSTRACT
Epidemiological studies of humans and experimental studies with mouse models suggest that sunburn resulting from exposure to excessive UV light and damage to DNA confers an increased risk for melanoma and non-melanoma skin cancer. Previous reports have shown that both nucleotide excision repair, which is the sole pathway in humans for removing UV photoproducts, and DNA replication are regulated by the circadian clock in mouse skin. Furthermore, the timing of UV exposure during the circadian cycle has been shown to affect skin carcinogenesis in mice. Because sunburn and skin cancer are causally related, we investigated UV-induced sunburn apoptosis and erythema in mouse skin as a function of circadian time. Interestingly, we observed that sunburn apoptosis, inflammatory cytokine induction, and erythema were maximal following an acute early-morning exposure to UV and minimal following an afternoon exposure. Early-morning exposure to UV also produced maximal activation of ataxia telangiectasia mutated and Rad3-related (Atr)-mediated DNA damage checkpoint signaling, including activation of the tumor suppressor p53, which is known to control the process of sunburn apoptosis. These data provide early evidence that the circadian clock has an important role in the erythemal response in UV-irradiated skin. The early morning is when DNA repair is at a minimum, and thus the acute responses likely are associated with unrepaired DNA damage. The prior report that mice are more susceptible to skin cancer induction following chronic irradiation in the AM, when p53 levels are maximally induced, is discussed in terms of the mutational inactivation of p53 during chronic irradiation.

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Circadian regulation of UV-induced sunburn apoptosis in mouse skin. (A) Representative immunofluorescence images of TUNEL assay for the detection of apoptotic cells in the skin of UV-irradiated mice with the C57BL/6 background. Mice were maintained in an LD12:12 cycle and shaved 1 day before irradiation. Both wild-type and Cry1/2−/−mice were irradiated with UV (4000 J/m2) either at ZT21 (4AM) or ZT09 (4PM), and skin tissues were collected at 0, 6, and 12 hr post-UV for analysis with TUNEL assays. TUNEL-positive cells are green and nuclei are stained blue with DAPI. E, epidermis; D, dermis. (B) TUNEL-positive (apoptotic) cells in wild-type and Cry1/2−/− mouse epidermis as a result of UVR at ZT21 (4AM) and ZT09 (4PM). Positive cells were calculated as the percentage of the total number of DAPI-positive cells. All of the values were then normalized to 100 relative to the 12 hr, AM wild-type response sample (which actually had a value of 20% TUNEL-positive cells). (C) Representative immunofluorescence images of TUNEL assay from wild-type and Per1/2−/− mouse skin. Samples were processed as described in B. (D) Quantification of C. AM wild-type response sample contained 10% TUNEL-positive cells but was normalized to 100 for quantification. n=2 or 3 mice at each time point. Error bars represent means ± standard deviation (SD). NS= Not Significant.
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Figure 2: Circadian regulation of UV-induced sunburn apoptosis in mouse skin. (A) Representative immunofluorescence images of TUNEL assay for the detection of apoptotic cells in the skin of UV-irradiated mice with the C57BL/6 background. Mice were maintained in an LD12:12 cycle and shaved 1 day before irradiation. Both wild-type and Cry1/2−/−mice were irradiated with UV (4000 J/m2) either at ZT21 (4AM) or ZT09 (4PM), and skin tissues were collected at 0, 6, and 12 hr post-UV for analysis with TUNEL assays. TUNEL-positive cells are green and nuclei are stained blue with DAPI. E, epidermis; D, dermis. (B) TUNEL-positive (apoptotic) cells in wild-type and Cry1/2−/− mouse epidermis as a result of UVR at ZT21 (4AM) and ZT09 (4PM). Positive cells were calculated as the percentage of the total number of DAPI-positive cells. All of the values were then normalized to 100 relative to the 12 hr, AM wild-type response sample (which actually had a value of 20% TUNEL-positive cells). (C) Representative immunofluorescence images of TUNEL assay from wild-type and Per1/2−/− mouse skin. Samples were processed as described in B. (D) Quantification of C. AM wild-type response sample contained 10% TUNEL-positive cells but was normalized to 100 for quantification. n=2 or 3 mice at each time point. Error bars represent means ± standard deviation (SD). NS= Not Significant.

Mentions: At the cellular level, erythemogenic doses of UVR are associated with apoptosis (also called “sunburn apoptosis”) (Ziegler et al., 1994). To determine the effect of the circadian clock on sunburn apoptosis, we irradiated C57BL/6 mice with UVR either in the early morning or the late afternoon and then harvested mouse skin at 0, 6, and 12 hrs after UVR. Apoptosis was measured using the fluorometric TUNEL assay (TdT-mediated dUTP Nick-End Labeling) in which fragmented DNA from apoptotic cells is end-labeled with fluorophore. Figure 2 shows a greater apoptotic response in the AM group compared to the PM group. To determine if sunburn apoptosis is controlled by the circadian clock, we used mice in which the clock was disrupted by mutating essential clock genes. In both Cry1, Cry2-double knockout mice (Cry1/2−/− in Figure 2A, B) and Per1, Per2 double knockout mice (Per1/2−/− in Figure 2C, D), the effect of the time of the day on sunburn apoptosis was abolished. The elevated apoptosis in wild-type mice irradiated in the AM was found to be correlated with reduced repair of UV-induced DNA photoproducts (Gaddameedhi et al., 2011). Although it has been reported that p53 contributes to excision repair and UV survival in human cells (Ferguson and Oh, 2005; Ford and Hanawalt, 1997), mouse p53 protein doesn’t seems to have a role in excision repair or UV survival (Ishizaki et al., 1994), which could be due to the fact that the mouse XPE gene, which encodes the DDB2 protein, doesn’t have a p53 recognition domain (Tan and Chu, 2002). Therefore, circadian oscillation of p53 in mouse skin may not have a significant role in regulating excision repair capacity.


The circadian clock controls sunburn apoptosis and erythema in mouse skin.

Gaddameedhi S, Selby CP, Kemp MG, Ye R, Sancar A - J. Invest. Dermatol. (2014)

Circadian regulation of UV-induced sunburn apoptosis in mouse skin. (A) Representative immunofluorescence images of TUNEL assay for the detection of apoptotic cells in the skin of UV-irradiated mice with the C57BL/6 background. Mice were maintained in an LD12:12 cycle and shaved 1 day before irradiation. Both wild-type and Cry1/2−/−mice were irradiated with UV (4000 J/m2) either at ZT21 (4AM) or ZT09 (4PM), and skin tissues were collected at 0, 6, and 12 hr post-UV for analysis with TUNEL assays. TUNEL-positive cells are green and nuclei are stained blue with DAPI. E, epidermis; D, dermis. (B) TUNEL-positive (apoptotic) cells in wild-type and Cry1/2−/− mouse epidermis as a result of UVR at ZT21 (4AM) and ZT09 (4PM). Positive cells were calculated as the percentage of the total number of DAPI-positive cells. All of the values were then normalized to 100 relative to the 12 hr, AM wild-type response sample (which actually had a value of 20% TUNEL-positive cells). (C) Representative immunofluorescence images of TUNEL assay from wild-type and Per1/2−/− mouse skin. Samples were processed as described in B. (D) Quantification of C. AM wild-type response sample contained 10% TUNEL-positive cells but was normalized to 100 for quantification. n=2 or 3 mice at each time point. Error bars represent means ± standard deviation (SD). NS= Not Significant.
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Figure 2: Circadian regulation of UV-induced sunburn apoptosis in mouse skin. (A) Representative immunofluorescence images of TUNEL assay for the detection of apoptotic cells in the skin of UV-irradiated mice with the C57BL/6 background. Mice were maintained in an LD12:12 cycle and shaved 1 day before irradiation. Both wild-type and Cry1/2−/−mice were irradiated with UV (4000 J/m2) either at ZT21 (4AM) or ZT09 (4PM), and skin tissues were collected at 0, 6, and 12 hr post-UV for analysis with TUNEL assays. TUNEL-positive cells are green and nuclei are stained blue with DAPI. E, epidermis; D, dermis. (B) TUNEL-positive (apoptotic) cells in wild-type and Cry1/2−/− mouse epidermis as a result of UVR at ZT21 (4AM) and ZT09 (4PM). Positive cells were calculated as the percentage of the total number of DAPI-positive cells. All of the values were then normalized to 100 relative to the 12 hr, AM wild-type response sample (which actually had a value of 20% TUNEL-positive cells). (C) Representative immunofluorescence images of TUNEL assay from wild-type and Per1/2−/− mouse skin. Samples were processed as described in B. (D) Quantification of C. AM wild-type response sample contained 10% TUNEL-positive cells but was normalized to 100 for quantification. n=2 or 3 mice at each time point. Error bars represent means ± standard deviation (SD). NS= Not Significant.
Mentions: At the cellular level, erythemogenic doses of UVR are associated with apoptosis (also called “sunburn apoptosis”) (Ziegler et al., 1994). To determine the effect of the circadian clock on sunburn apoptosis, we irradiated C57BL/6 mice with UVR either in the early morning or the late afternoon and then harvested mouse skin at 0, 6, and 12 hrs after UVR. Apoptosis was measured using the fluorometric TUNEL assay (TdT-mediated dUTP Nick-End Labeling) in which fragmented DNA from apoptotic cells is end-labeled with fluorophore. Figure 2 shows a greater apoptotic response in the AM group compared to the PM group. To determine if sunburn apoptosis is controlled by the circadian clock, we used mice in which the clock was disrupted by mutating essential clock genes. In both Cry1, Cry2-double knockout mice (Cry1/2−/− in Figure 2A, B) and Per1, Per2 double knockout mice (Per1/2−/− in Figure 2C, D), the effect of the time of the day on sunburn apoptosis was abolished. The elevated apoptosis in wild-type mice irradiated in the AM was found to be correlated with reduced repair of UV-induced DNA photoproducts (Gaddameedhi et al., 2011). Although it has been reported that p53 contributes to excision repair and UV survival in human cells (Ferguson and Oh, 2005; Ford and Hanawalt, 1997), mouse p53 protein doesn’t seems to have a role in excision repair or UV survival (Ishizaki et al., 1994), which could be due to the fact that the mouse XPE gene, which encodes the DDB2 protein, doesn’t have a p53 recognition domain (Tan and Chu, 2002). Therefore, circadian oscillation of p53 in mouse skin may not have a significant role in regulating excision repair capacity.

Bottom Line: Early-morning exposure to UV also produced maximal activation of ataxia telangiectasia mutated and Rad3-related (Atr)-mediated DNA damage checkpoint signaling, including activation of the tumor suppressor p53, which is known to control the process of sunburn apoptosis.These data provide early evidence that the circadian clock has an important role in the erythemal response in UV-irradiated skin.The early morning is when DNA repair is at a minimum, and thus the acute responses likely are associated with unrepaired DNA damage.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA [2] Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA.

ABSTRACT
Epidemiological studies of humans and experimental studies with mouse models suggest that sunburn resulting from exposure to excessive UV light and damage to DNA confers an increased risk for melanoma and non-melanoma skin cancer. Previous reports have shown that both nucleotide excision repair, which is the sole pathway in humans for removing UV photoproducts, and DNA replication are regulated by the circadian clock in mouse skin. Furthermore, the timing of UV exposure during the circadian cycle has been shown to affect skin carcinogenesis in mice. Because sunburn and skin cancer are causally related, we investigated UV-induced sunburn apoptosis and erythema in mouse skin as a function of circadian time. Interestingly, we observed that sunburn apoptosis, inflammatory cytokine induction, and erythema were maximal following an acute early-morning exposure to UV and minimal following an afternoon exposure. Early-morning exposure to UV also produced maximal activation of ataxia telangiectasia mutated and Rad3-related (Atr)-mediated DNA damage checkpoint signaling, including activation of the tumor suppressor p53, which is known to control the process of sunburn apoptosis. These data provide early evidence that the circadian clock has an important role in the erythemal response in UV-irradiated skin. The early morning is when DNA repair is at a minimum, and thus the acute responses likely are associated with unrepaired DNA damage. The prior report that mice are more susceptible to skin cancer induction following chronic irradiation in the AM, when p53 levels are maximally induced, is discussed in terms of the mutational inactivation of p53 during chronic irradiation.

Show MeSH
Related in: MedlinePlus