Effect of caffeine on the ATR/Chk1 pathway in the epidermis of UVB-irradiated mice

Abstract

Administration of caffeine was shown in earlier studies to enhance UVB-induced apoptosis and inhibit UVB-induced carcinogenesis in hairless SKH-1 mice. Here, we describe a potential mechanism for these in vivo effects. A single irradiation of mouse skin with UVB activated the ataxia-telangiectasia mutated- and Rad3-related (ATR) pathway, causing a severalfold increase in keratinocytes with phospho-Chk1 (Ser(345)) and a marked decrease in mitotic keratinocytes with cyclin B1 compared with baseline. When given in the drinking water for 1 to 2 weeks before UVB, caffeine (0.4 mg/mL) markedly inhibited the UVB-induced phosphorylation of Chk1 on Ser(345) and caused premature expression of cyclin B1 in the epidermis. Normal keratinocytes had delayed mitotic entry for >10 h following UVB. Caffeine administration reduced this mitotic delay to only 4 h and caused markedly increased apoptosis by 6 to 10 h after UVB. p53 knockout mice were used to determine the role of p53 in these processes. Irradiation with UVB markedly decreased the number of mitotic keratinocytes with cyclin B1 in p53 knockout mice, and topical caffeine immediately after UVB abrogated this response and increased UVB-induced apoptosis severalfold. These effects of caffeine in knockout mice were substantially greater than in wild-type mice. The ability of caffeine to promote the deletion of p53(-/-) keratinocytes may be relevant to its inhibitory effect on UVB-induced skin cancer. Our studies indicate that administration of caffeine enhances the removal of DNA-damaged cells by inhibiting the ATR-mediated phosphorylation of Chk1 and prematurely increasing the number of cyclin B1-containing cells that undergo lethal mitosis.

Figure 1

Proposed effects of UVB and caffeine on the ATR/Chk1/cyclin B1 pathway and premature chromatin condensation. The signal that DNA synthesis is not complete (and that chromatin condensation and mitosis should not start) is sent by ATR after it is recruited to stalled replication forks that have been coated by replication protein A. ATR-mediated phosphorylation of Chk1 results in a decreased level and function of cyclin B1, which delays mitotic entry and prevents premature chromatin condensation and mitosis. Inhibition of the ATR/Chk1 pathway in UVB-treated cells should result in a premature increase in cyclin B1 and premature chromatin condensation, mitosis, and cell death, probably by mitotic catastrophe followed by apoptosis, as suggested by Brown and Attardi (20). Adapted with permission from Nghiem et al. (13).

Figure 2

Effects of p.o. administration of caffeine or caffeine sodium benzoate on the levels of phospho-Chk1 (Ser³⁴⁵) and cyclin B1 in UVB-treated epidermis of SKH-1 mice: Western blot studies. Female SKH-1 mice were treated p.o. with caffeine (0.4 mg/mL, 2.1 mmol/L) or caffeine sodium benzoate (Caffeine SB; 2.1 mmol/L) as their sole source of drinking fluid for 1 wk. The animals were then treated with UVB (30 mJ/cm²) and killed 6 h later. A, phospho-Chk1 (Ser³⁴⁵), phospho-Chk2 (Thr⁶⁸), cyclin B1, and phospho-cyclin B1 (Ser¹⁴⁷) were determined by Western blot. B, densitometry results from three independent experiments. The antibodies used for the detection of these proteins are described in Materials and Methods.

Figure 3

Time course for the effects of p.o. administration of caffeine on UVB-induced changes in the percentage of apoptotic sunburn cells, phospho-Chk1 (Ser³⁴⁵)-positive cells, cyclin B1-positive cells, and mitotic cells with cyclin B1 in the epidermis of SKH-1 mice: immunohistochemical studies. Female SKH-1 mice were treated p.o. with caffeine (0.4 mg/mL, 2.1 mmol/L) in the drinking water for 2 wk. Control mice received only water. The animals were then irradiated with UVB (30 mJ/cm²) and killed at the indicated times. The percentage of phospho-Chk1 (Ser³⁴⁵) and cyclin B1-positive cells were determined immunohistochemically. The antibodies used for the detection of these proteins are described in Materials and Methods. Cells with both mitosis (measured by morphology) and cyclin B1 (measured by immunohistochemistry) were also determined. Points, mean of five mice; bars, SE. Shaded areas, premature mitotic entry by epidermal cells from caffeine-treated mice. a, P < 0.01.

Figure 4

Illustration of a cyclin B1-positive keratinocyte. This figure shows a representative mitotic cell with cyclin B1 staining taken from a mouse treated with caffeine and UVB as described in Fig. 3.

Figure 5

Time course for the effects of topical application of caffeine on UVB-induced changes in the percentage of apoptotic sunburn cells, cyclin B1-positive cells, and mitotic cells with cyclin B1 in the epidermis of p53 knockout mice: immunohistochemical studies. Male p53^+/+ or p53^-/- mice (five per group) were treated topically with 100 μL of acetone/water (9:1) or with 6.2 μmol of caffeine in 100 μL of acetone/water (9:1) immediately after 60 mJ/cm² of UVB and 0.5 and 2 h later as described earlier (9). The animals were killed at several times after UVB. Stored paraffin blocks from the earlier study (9) were used for determining the percentage of cyclin B1-positive cells and mitotic cells with cyclin B1 in the epidermis as described in Fig. 3. Data for apoptotic sunburn cells were reported earlier (9) and are included here for comparison with the cyclin B1 data. Points, mean; bars, SE. a, P < 0.01; b, P < 0.05.