Circadian regulation of excision repair of UV-induced DNA damage in mouse epidermis. SKH-1 hairless mice kept under an LD 12:12 cycle were irradiated with 300 J/m² of UVB at the indicated ZT times (either ZT 21 or ZT 09) and skin patches were collected 0–12 h after irradiation to measure UV photoproduct repair by slot blot. (A) Repair of (6-4) PP. A representative slot blot is shown. (Left) Immunoblot analysis of α-(6-4) PP signal. (Right) SYBR-GOLD staining of total DNA. (B) Quantitative analysis of (6-4) PP repair. Error bars indicate means ± SD (n = 2 mice at each time point). (C) Repair of CPDs analyzed by slot blot. (D) Quantitative analysis of CPD repair (n = 2 mice at each time point). (E) Slot blot analysis of (6-4) PP and CPD repair in Cry1^−/−Cry2^−/− mice. Skin was harvested at 0 and 6 h after irradiation and photoproduct levels were probed by immunoblotting. (F) Quantitative analysis of repair in the skin of Cry1^−/−Cry2^−/− mice irradiated at ZT 21 (4:00 AM) and ZT 09 (4:00 PM). The varied kinetics of repair in wild-type mice (A–D) presumably reflect the higher inherent rate of repair of the (6-4) PP and the cyclic expression of XPA. Error bars indicate means ± SD (n = 2 mice at each time point).

Effect of time of day of exposure to UVB on skin carcinogenesis (visual diagnosis) in SKH-1 hairless mice. (A) Physical appearance of representatives of the three experimental groups 23 wk after initiation of irradiation. (B) Kaplan–Meier analysis of tumor-free survival. (C) Average number of tumors per mouse following initiation of UV treatments. (D) Average maximal tumor area per mouse in the AM and PM groups.

Model for the role of the circadian clock in UV-induced skin carcinogenesis. The higher level of DNA replication combined with the nadir of DNA repair capacity in the early morning hours compared with afternoon/evening renders mice more susceptible to mutation and cancer when exposed to sunlight in the morning.

Circadian oscillation of XPA in mouse skin. (A) Mice kept under an LD 12:12 cycle were killed at the indicated zeitgeber times (ZT 0 = light on) and the levels of the indicated proteins were determined from whole skin by immunoblotting. Light and dark boxes indicate the light-on and light-off phases, respectively. Note the robust and antiphase oscillations of the XPA and CRY1 proteins. The core clock protein, Clock, as expected (14), does not exhibit rhythmicity. Actin was used as a loading control. ST, subjective time, with light on at 7:00 AM and off at 7:00 PM. (B) Quantitative analysis of XPA and CRY1 oscillation in the mouse skin. Error bars represent means ± SD (n = 2 mice at each time point). (C) XPA expression in the epidermis of SKH-1 hairless mice. The mice were exposed to UVR at ZT 21 (subjective 4:00 AM) or ZT 9 (subjective 4:00 PM) and epidermis was harvested at 0, 1, 2, 4, 6, and 12 h postirradiation and analyzed for expression of the indicated proteins by immunoblotting. Note that XPA and CRY1 exhibit antiphase rhythms, whereas the oxidative base damage repair enzyme 8-oxo-guanine DNA glycosylase (OGG-1), implicated in repair of oxidative base damage caused by UVR, shows no rhythmicity. GAPDH is a loading control. A representative experiment is shown only with 0, 1, and 2 h postirradiation for clarity and both AM and PM samples were run on the same gel. This experiment was repeated twice with two mice at each time point. (D) Immunohistochemical (IHC) analysis of XPA expression in SKH-1 epidermis at two circadian time points, ZT 22 (subjective 5:00 AM) and ZT 10 (subjective 5:00 PM). (Left) Representative IHC images (blue, nuclei; brown, XPA). Red dashed lines follow the basement membrane, located between the dermis (D) and the epidermis (E). (Right) Quantitative analysis of IHC data. Error bars indicate means ± SD (n = 2 mice at each time point). XPA positive cells were recorded manually using the Aperio ImageScope software, counting at least 10 fields (each field consists of ∼80 cells) per mouse. P value is based on 3-way ANOVA.

Circadian rhythm of DNA replication in mouse tissues. SKH-1 hairless mice were kept under an LD 12:12 cycle. BrdU or vehicle alone (“mock”) was injected intraperitoneally at either ZT 21 (4:00 AM) or ZT 09 (4:00 PM); 90 min later epidermis, intestine, and kidney tissues were harvested and the level of DNA replication was examined by slot blot. (A and B) Results obtained with wild-type SKH-1 hairless mice. (A) Representative slot blot. (B) Quantitative analysis of slot blots. Error bars indicate means ± SD (n = 3 mice at each time point). (C and D) Results obtained with clock-deficient (Cry1^−/−Cry2^−/−) mice. (C) Representative slot blot. (D) Quantitative analysis of the slot blots. Error bars indicate means ± SD (n = 3 mice at each time point).

Quantitative summary of histopathological analysis performed on skin harvested 25 wk after initiation of the experiment. Note that there are significantly more invasive SCC tumors in the AM group than in the PM group of mice (t = 0.00015). The t value for SCC basement membrane invasive is 0.0002, and for SCC muscle invasive it is 0.023.