Indole-3-carbinol (I3C), a naturally occurring compound in vegetables such as cabbage, broccoli and Brussel's sprouts, and its dimerization product 3-3'-diindolylmethane (DIM) are promising chemotherapeutic and chemopreventative agents for human reproductive cancers. We have demonstrated that the direct exposure of human breast cancer cells to I3C or DIM induces a stringent G1 cell cycle arrest through a multistep signaling cascade that controls expression, activity and cellular utilization of key cell cycle components. I3C, but not DIM, down-regulates cyclin dependent kinase-6 (CDK6) expression and promoter activity, whereas, DIM selectively and rapidly stimulates expression and promoter activity of p21Waf1/Cip1, a critical inhibitor of certain CDKs. The indole regulation of cell cycle gene expression result from specific changes in Sp1 transcription factor-promoter interactions. We have recently discovered that I3C, but not DIM, alters the size, composition and subcellular localization (nuclear to cytoplasm) of the CDK2 protein complex, and a novel 85 kDa protein was uncovered that associates with the CDK2 protein complex in an I3C dependent manner. Thus, the transcriptional control of CDK6 and p21Waf1/Cip1, and the posttanslational regulation of CDK2 protein complex utilization are newly defined down-stream targets of the anti-proliferative indole signaling pathway in human breast cancer cells. Our hypothesis is that I3C and DIM induce a G1 cell cycle arrest of human reproductive cancer cells by activating complementary and distinct cascades that that subsequently control the transcription and posttranslational utilization of key cell cycle components. The indole regulated transcription factors, in addition to Spl, that target the CDK6 and p21Waf1/Cip1 promoters will be identified by interactions with indole responsive regions of cell cycle gene promoters, and functionally tested in a cellular text by manipulation of their expression and/or activity. The novel 85 kDa protein that interacts with the CDK2 protein complex in an I3C dependent manner will be defined and characterized for its role in the I3C disruption of the CDK2 protein complex. The actions of the I3C regulated cell cycle components on cancer cell invasion properties, and in the formation of human breast cancer cell-derived tumors will be assessed using in vitro and in vivo strategies. Our collaborative studies represent the first experimental steps necessary to understand the transcriptional and posttranslational mechanisms by which natural indoles control the cell cycle of human breast cancer cells. This information will be particularly valuable to develop new classes of I3C-based therapeutics for reproductive cancers.
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