3451

In earlier studies, we demonstrated that administration of caffeine to SKH-1 mice inhibited UVB-induced skin carcinogenesis and stimulated UVB-induced apoptosis by p53-dependent and p53-independent mechanisms, and the maximum effect of caffeine on UVB-induced apoptosis was at 6-10 hr post-UVB.

In the present study, we found that oral administration of caffeine (0.4 mg/ml in the drinking water) to SKH-1 mice for one week prior to UVB irradiation markedly inhibited the UVB-induced increase in the level of p-Chk1 (Ser345) and increased the level of cyclin B1 at 6 hr post-UVB when compared with measurements after UVB alone, but there was little or no effect of caffeine administration on p-Chk2 (Thr68) or p-cyclin B1 (Ser147) as measured by Western blots. Administration of caffeine had little or no effect on these biomarkers in the absence of UVB. A time course study using immunohistochemical assays indicated that irradiation of SKH-1 mice with UVB increased the number of epidermal cells with p-Chk1 (Ser345) and decreased the number of mitotic cells with cyclin B1 between 6-10 hr post-UVB. Oral administration of caffeine for one week stimulated UVB-induced apoptosis, inhibited the UVB-induced increase in p-Chk1 (Ser345) and abrogated the decrease in mitotic cells with cyclin B1 at 6-10 hr post-UVB. Although the caffeine-induced increase in apoptosis after UVB was maximal between 6-10 hr post-UVB and then declined, caffeine continued to affect p-Chk1 (Ser345) and cyclin B1 beyond 10 hr. Topical application of caffeine immediately after UVB irradiation also prematurely increased the number of cyclin B1 positive cells but had little or no effect on p-cyclin B1 (Ser147) at 6 hr post-UVB when compared with measurements after UVB alone. A time course study indicated a dramatic UVB-induced decrease in the percentage of mitotic cells with cyclin B1 in the epidermis of p53 knockout mice between 2-16 hr after UVB. This decrease was abrogated between 6-16 hr post-UVB by topical application of caffeine indicating that caffeine exerts its effect by a p53-independent mechanism. The time course for the stimulatory effect of caffeine for increasing mitotic cells with cyclin B1 in the epidermis of mice treated with UVB paralleled the time course for the formation of apoptotic sunburn cells.

Our studies provide the first evidence that administration of caffeine enhances DNA damage-induced cell killing in vivo in animals by inhibiting the ATR-mediated phosphorylation of Chk1 and increasing the number of epidermal cells undergoing premature mitosis in the presence of cyclin B1. This effect of caffeine results in an early and inappropriate increase in mitotic cells with cyclin B1 which is consistent with the known role for the ATR-Chk1 pathway in preventing premature chromatin condensation. (Supported in part by NIH grant CA114442.)

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA