Abstract
Indole-3-carbinol (I3C), a dietary compound found in cruciferous vegetables, induces a robust inhibition of CDK2 specific kinase activity as part of a G1 cell cycle arrest of human breast cancer cells. Treatment with I3C causes a significant shift in the size distribution of the CDK2 protein complex from an enzymatically active 90 kDa complex to a larger 200 kDa complex with significantly reduced kinase activity. Co-immunoprecipitations revealed an increased association of both a 50 kDa cyclin E and a 75 kDa cyclin E immunoreactive protein with the CDK2 protein complex under I3C-treated conditions, whereas the 90 kDa CDK2 protein complexes detected in proliferating control cells contain the lower molecular mass forms of cyclin E. I3C treatment caused no change in the level of CDK2 inhibitors (p21, p27) or in the inhibitory phosphorylation states of CDK2. The effects of I3C are specific for this indole and not a consequence of the cell cycle arrest because treatment of MCF-7 breast cancer cells with either the I3C dimerization product DIM or the anti-estrogen tamoxifen induced a G1 cell cycle arrest with no changes in the associated cyclin E or subcellular localization of the CDK2 protein complex. Taken together, our results have uncovered a unique effect of I3C on cell cycle control in which the inhibition of CDK2 kinase activity is accompanied by selective alterations in cyclin E composition, size distribution, and subcellular localization of the CDK2 protein complex.
Highlights
Considerable epidemiological evidence show that diets high in vegetable and fiber lead to low cancer risks and confer protection from various forms of cancers, including breast cancer [1, 2]
CDK2 protein complexes were immunoprecipitated from total cell lysates by using a CDK2-specific antibody, and half of each sample was assayed in vitro for kinase activity and the other half analyzed for CDK2 protein by Western blot analysis
CDK2 protein kinase activity was monitored by the ability of the immunoprecipitated CDK2-cyclin protein complexes to phosphorylate the C-terminal domain of retinoblastoma tumor suppressor protein (Rb) fused to GST
Summary
I3C, indole-3-carbinol; CDK, cyclin-dependent kinase; Rb, retinoblastoma; DIM, 3–3Ј-diindolylmethane; CDC, cell division cycle; GST, glutathione S-transferase; CAK, CDKactivating kinase; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; PBS, phosphate-buffered saline; Me2SO, dimethyl sulfoxide. In this study we demonstrate for the first time that I3C selectively controls the size distribution of the CDK2-cyclin E protein complex with concomitant alterations in cyclin E interactions and subcellular localization that results in an inhibition of CDK2 enzymatic activity in human breast cancer cells
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