ANTIESTROGEN INHIBITION OF CELL-CYCLE PROGRESSION IN BREAST-CANCER CELLS IS ASSOCIATED WITH INHIBITION OF CYCLIN-DEPENDENT KINASE-ACTIVITY AND DECREASED RETINOBLASTOMA PROTEIN-PHOSPHORYLATION

To define the mechanisms by which antiestrogens inhibit breast cancer cell proliferation, the effects of the antiestrogen ICI 182780 on G(1) cyclins and their cyclin-dependent kinase (CDK) partners were investi gated in MCF-7 cells. Inhibition of entry into S phase became evident 9 h after treatment, with the proportion of cells in S phase reaching a minimum by 24 h. ICI 182780 increased the proportion of the hypophos phorylated, growth inhibitory form of the retinoblastoma protein (pRB) . This change began at 4-6 h, preceding effects on S phase. This sugge sts that there are early effects on the activities of CDKs that target pRB that are not merely a consequence of changes in cell cycle progre ssion. The kinase activity of Cdk2 decreased to low levels at 18-24 h when changes in S phase and pRB phosphorylation were well advanced. An earlier effect was seen on kinase activity associated with immunoprec ipitated cyclin D1, which was reduced approximately 40% by 12 h, with further decreases at 18-24 h. Cdk2 and Cdk4 protein levels remained co nstant over 24 h. Cyclin D1 messenger RNA and protein were down-regula ted by ICI 182780 from 2 h, with levels halved at 8 h. ICI 182780 also increased the expression of the CDK inhibitors p27(KIP1) and p21(WAF1 /CIP1) af later times. These observations are compatible with the hypo thesis that antiestrogens block entry of cells into S phase and inhibi t cell proliferation as the consequence of an early decline in pRB pho sphorylation contributed to by reduced cyclin D1/Cdk4 activity. At lat er times, increased CDK inhibitor abundance may act to repress Cdk2 an d Cdk4 activities, causing additional reductions in pRB phosphorylatio n, thus maintaining the antiestrogen blockade of cell cycle progressio n.