INDUCIBLE EXPRESSION OF CYCLIN D1 IN T-47D HUMAN BREAST-CANCER CELLS IS SUFFICIENT FOR CDK2 ACTIVATION AND PRB HYPERPHOSPHORYLATION

The sequential transcriptional activation of cyclins, the regulatory s ubunits of cell cycle specific kinases, regulates progress through the cell cycle. In mitogen-stimulated cells cyclin D1 induction in early G(1) is followed by induction of cyclin E, activation of the cyclin-de pendent kinase Cdk2, and hyperphosphorylation of the retinoblastoma ge ne product (pRB) in mid-to-late G(1) phase. T-47D breast cancer cells expressing cyclin D1 under the control of a metal-responsive metalloth ionein promoter were used to determine whether Cdk2 activation and pRB hyperphosphorylation are consequences of cyclin D1 induction. A 4-5-f old increase in cyclin D1 protein abundance was followed by approximat ely 2-fold increases in cyclin E protein abundance and Cdk2 activity a nd by hyperphosphorylation of pRB. These responses were apparent simil ar to 3 h after the increase in cyclin D1 protein, and similar to 3 h prior to the entry of cyclin D1-stimulated cells into S phase 12 h aft er zinc treatment. Cyclin D1 immunoprecipitates contained Cdk4 but no detectable Cdk2 and displayed pRb but not histone H1 kinase activity. Cdk2 activation was therefore likely to be due to increased abundance of cyclin E/Cdk2 complexes rather than formation of active cyclin D1/C dk2 complexes. The sequence of events following zinc induction of cycl in D1 thus mimicked that following mitogen induction of cyclin D1. The se data show that cyclin D1 induction is sufficient for Cdk2 activatio n and pRB hyperphosphorylation in T-47D human breast cancer cells, pro viding evidence that cyclin D1 induction is a critical event in G(1) p hase progression. (C) 1996 Wiley-Liss, Inc.