Indole-3-carbinol inhibits CDK6 expression in human MCF-7 breast cancer cells by disrupting Sp1 transcription factor interactions with a composite element in the CDK6 gene promoter

Indole-3-carbinol (I3C), a compound naturally occurring in Brassica vegetab les, can induce a G(1) cell cycle arrest of human MCF-7 breast cancer cells that is accompanied by the selective inhibition of cyclin-dependent kinase 6 (CDK6) expression. Reverse transcriptase-polymerase chain reaction analy sis of CDK6 mRNA de cay rates revealed that I3C had no effect on CDK6 trans cript stability. We report the first identification and functional characte rization of the CDK6 promoter in order to determine whether I3C inhibits CD K6 transcription. In MCF-7 cells stably transfected with CDK6 promoter-link ed luciferase reporter plasmids, I3C inhibited CDK6 promoter activity in an I3C specific response that was not a consequence of the growth-arrested st ate of the cells. Deletion analysis revealed a 167-base pair I3C-responsive region of the CDK6 promoter between -805 and -638, Site-specific mutations within this region revealed that both Sp1 and Ets-like sites, which are sp aced 5 base pairs apart, were necessary for I3C responsiveness in the conte xt of the CDK6 promoter. Electrophoretic mobility shift analysis of protein -DNA complexes formed with nuclear proteins isolated from I3C-treated and - untreated cells, in combination with supershift assays using Spl antibodies , demonstrated that the Sp1-binding site in the CDK6 promoter forms a speci fic I3C-responsive DNA-protein complex that contains the Sp1 transcription factor. Taken together, our results suggest that I3C down-regulates CDKG tr anscription by targeting Sp1 at a composite DNA site in the CDK6 promoter.