Apigenin is a plant flavonoid which has been shown to possess significant chemopreventive activity in inhibiting chemically- induced and ultraviolet light-induced skin cancer in the mouse skin model. Although apigenin shows promise as a chemopreventive agent, little is known about its molecular mechanism of action. The investigators' preliminary results indicate that apigenin treatment of cultured keratinocytes produces substantial G2/M cell cycle arrest and inhibition of p34cdc2 kinase activity, the cyclin dependent kinase which regulates G2/M transition in mammalian cells. Additional experiments indicated that apigenin had no effect on steady-state protein levels of p34cdc2 Kinase but may effect the p34cdc2 phosphorylation status. In the present application the investigators propose to test the hypothesis that apigenin acts as a chemopreventive agent in skin by inducing cell cycle arrest at G2/M via inhibition of p34cdc2 kinase, the cyclin dependent kinase which is responsible for the G2/M transition in mammalian cells. The following Aims will test this hypothesis both in vitro and in vivo, in epidermal cells at various stages of tumorigenesis: (1) Aim 1 will investigate whether apigenin arrest keratinocytes in G2/M by affecting the phosphorylation state of p34cdc2 kinase, thereby inhibiting its activity. (2) Aim #2 will study whether apigenin is arresting cells at G2/M by acting at the level of cyclin B, either by (a) inhibiting the accumulation of cyclin B during G2, (b) inhibiting the association of cyclin B with the cyclin-dependent kinase p34cdc2, or (c) promoting the association of a cyclin inhibitor with the cyclin/cyclin-dependent kinase complex. (3) Aim #3 will investigate whether apigenin is able to induce G2/M arrest in keratinocyte cell lines at various stages of tumorigenesis. (4) Aim #4 will employ the UV-B carcinogenesis model in SKH-1 mice to examine whether apigenin treatment of mouse skin can inhibit the growth of skin tumor cells in vivo even after foci of tumor cells are present.
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