| Budget Amount *help |
¥3,980,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥480,000)
Fiscal Year 2007: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
Fiscal Year 2006: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
P21 was originally identified as a target protein of p53 and then found to inhibit the activity of Cdk-cyclin complexes, thereby regulating cell cycle as a brake. The p21 expression is mainly controlled by diverse mechanisms in a p53-dependent manner. Due to accumulating evidence that p53 is mutated in many human cancer cells, the mutation of p53 has been recognized as one of the major events in carcinogenesis. However, it appears p21 is rarely mutated in human tumors. Therefore, the agents that induce p21 expression through a p53-independent pathway might contribute to cancer prevention or treatment.
Recently, we established a bioassay method using p53-negative human osteosarcoma MG63 cells to search for compounds that activate the p21 promoter in a p53-independent manner. On the guidance of this bioassay, we isolated aaptamine, a marine alkaloid, cryptolepine, a plant alkaloid and secaronic acid D, a bacterial polyketide as active components. Aaptamine activates p21 promoter stably transfected in MG63 cells at the concentrations of 20-50 μg/ml. Expression of p21 in wild-type MG63 cells also increased with aaptamine treatment. The 48 hr treatment of aaptamine induced G2/M arrest of cell cycle in MG63 cells. Furthermore, responsive elements in p21 promote of aaptamine were analyzed. The full length p21 promoter and a series of deleted or mutated constructs fused with luciferase reporter were transiently expressed in MG63 cells, and the cells were treated with aaptamine, showing that Sp1-3, Sp1-4, and Sp1-5, -6 play important roles in the activation of p21 promoter by aaptamine. In conclusion, our investigation indicated that aaptamine activates the p21 promoter through Sp 1 sites between -82 and -50 by and therefore induces p21 expression in a p53-independent manner.
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