AIB1 enhances estrogen-dependent induction of cyclin D1 expression.

AIB1 was isolated as a gene amplified in breast cancer and encodes a protein that acts as a steroid receptor coactivator. The role of steroid receptor coactivators such as AIB1 in breast cancer development is not clear. It is possible that AIB1 cooperates with estrogen receptor alpha in regulating estrogen-dependent cell proliferation. Ectopic expression of the estrogen receptor alpha in different cell lines does not confer estrogen-induced proliferation. This inability of the estrogen receptor to drive proliferation has been recently correlated with a lack of estrogen-dependent cyclin D1 expression in cells engineered to express the estrogen receptor. In this study, we evaluated whether high levels of AIB1 enable the estrogen receptor to direct the transcription of cyclin D1. We show here that AIB1 and other steroid receptor coactivators can enhance the functional interaction of the estrogen receptor with the cyclin D1 promoter. Increases of AIB1 levels in breast cancer cells by amplification and/or overexpression may represent one way to confer estrogen-dependent mitogenic stimulation to breast cancer cells.

Functional activity of ectopically expressed estrogen receptor is not sufficient for estrogen-mediated cyclin D1 expression.

Estrogen receptor function can drive cyclin D1 expression and proliferation in human breast cancer cells (MCF-7). Recent studies showing that estrogen receptor-positive epithelial cells in the human mammary gland are nonproliferative suggest that the direct mitogenic effect of estrogen on mammary epithelial cells may be acquired during breast cancer development. Because estrogen-dependent cyclin D1 expression has been linked to its mitogenicity, we characterized the ability of estrogen to regulate cyclin D1 expression in estrogen receptor-negative breast cancer cells (MDA-MB-231) and nontransformed human keratinocytes (HaCaT) stably expressing the estrogen receptor. In both cases, estrogen receptor function did not induce cyclin D1 expression. Although MCF-7 cells respond to estrogen by inducing the AP-1 family components c-Fos and c-Jun, HaCaT cells expressing estrogen receptor do not. These results may explain the lack of estrogen-dependent cyclin D1 expression and proliferation in cells ectopically expressing the estrogen receptor. Therefore, estrogen receptor function alone is not sufficient for estrogen-dependent cyclin D1 expression and proliferation. Other transcriptional cofactors that allow estrogen receptor to induce expression of AP-1 may be required for estrogen to act as a mitogen.

Estrogen-dependent cyclin E-cdk2 activation through p21 redistribution.

In order to elucidate the mechanisms by which estrogens and antiestrogens modulate the growth of breast cancer cells, we have characterized the changes induced by estradiol that occur during the G1 phase of the cell cycle of MCF-7 human mammary carcinoma cells. Addition of estradiol relieves the cell cycle block created by tamoxifen treatment, leading to marked activation of cyclin E-cdk2 complexes and phosphorylation of the retinoblastoma protein within 6 h. Cyclin D1 levels increase significantly while the levels of cyclin E, cdk2, and the p21 and p27 cdk inhibitors are relatively constant. However, the p21 cdk inhibitor shifts from its association with cyclin E-cdk2 to cyclin D1-cdk4, providing an explanation for the observed activation of the cyclin E-cdk2 complexes. These results support the notion that cyclin D1 has an important role in steroid-dependent cell proliferation and that estrogen, by regulating the activities of G1 cyclin-dependent kinases, can control the proliferation of breast cancer cells.

The restriction point and control of cell proliferation.

Research over the past two decades has defined a window of time in the early/mid G1 phase of the cell cycle during which mammalian cells are responsive to extracellular signals. Recent evidence indicates that this period ends with the phosphorylation of the retinoblastoma protein, enabling the cells to pass through the restriction point at the end of mid G1 phase and to commit to completing the remaining phases of the growth cycle.

Expression of a constitutive form of calcium/calmodulin dependent protein kinase II leads to arrest of the cell cycle in G2.

Calcium/calmodulin dependent protein kinase II (CaMKII) is a multifunctional serine/threonine protein kinase. We have created a calcium/calmodulin independent form of this enzyme by truncation. Expression of this enzyme fragment in a rabbit reticulocyte lysate yields a constitutive enzyme with specific activity similar to the activated native enzyme. We have established mammalian cell lines that transiently express this constitutive enzyme using the glucocorticoid-inducible mouse mammary tumor virus long terminal repeat. The transient increase in kinase activity results in a complete cessation of cell cycle progression. This block develops as a consequence of a specific arrest of the cell cycle in G2. During the block, increases in histone H1 kinase activity present in p13 beads or anti-cdc2 immunoprecipitates are seen in parallel with the accumulation of cells at G2, arguing that the arrest is not due to a failure to activate cdc2 as a histone H1 kinase. These results suggest that other changes in serine/threonine protein phosphorylation besides those involved in activation of cdc2 as a histone H1 kinase may be necessary for proper G2-M transition.