CYCLIN D2 ACTIVATES CDK2 IN PREFERENCE TO CDK4 IN HUMAN BREAST EPITHELIAL-CELLS

To investigate the possibility of differing roles for cyclins D1 and D 2 in breast epithelial cells, we examined the expression, cell cycle r egulation and activity of these two G(1) cyclins in both 184 normal br east epithelial cells and T-47D breast cancer cells. Synchromisation s tudies in 184 cells demonstrated that cyclin D1 and cyclin D2 were dif ferentially regulated during G(1), with cyclin D2 abundance increasing by 3.7-fold but only small changes in cyclin D1 abundance observed. T he functional consequences of increased cyclin D2 expression were exam ined in T-47D cells, which express no detectable cyclin D2, Induced ex pression of cyclin D2 resulted in increases in cyclin E expression, pR B phosphorylation and the percentage of cells in S-phase, while consti tutive expression resulted in a consistent trend toward reduced depend ence on serum for continued proliferation. Thus, cyclin D2 is a positi ve regulator of G(1) progression in breast cells analogous to the well -documented effects of cyclin D1. Indeed, equimolar concentrations of inducible cyclin D1 and D2 resulted in quantitatively similar cell cyc le effects. Marked divergence was found, however, in the CDKs activate d by the two cyclins in breast epithelial cells. Cyclin D2 complexes c ontained a higher Cdk2/Cdk4 ratio than cyclin D1 complexes. The cyclin D2-associated kinase activity was largely inhibited by Cdk2-specific inhibitors and could phosphorylate histone H1, a substrate for Cdk2 bu t not for Cdk4 and Cdk6. Therefore, cyclin D2 preferentially activated Cdk2 in breast epithelial cells. In contrast, Cdk4 and Cdk6 were pred ominantly responsible for cyclin D1-associated kinase activity as prev iously reported. Thus, although cyclins D1 and D2 elicited similar eff ects on breast epithelial cell cycle progression they appeared to achi eve this end via activation of different CDKs. This is the first evide nce of cyclin D2 activating Cdk2 in mammalian cells thus providing fur ther evidence that D-type cyclins are not necessarily redundant.