Estrogen-induced Activation of Cdk4 and Cdk2 during G1-S Phase Progression Is Accompanied by Increased Cyclin D1 Expression and Decreased Cyclin-dependent Kinase Inhibitor Association with Cyclin E-Cdk2

Abstract

Estrogens induce cell proliferation in target tissues by stimulating progression through G 1 phase of the cell cycle, but the underlying molecular targets remain undefined. To determine the role of the cyclin/cyclin-dependent kinase (CDK)/retinoblastoma protein (pRB) pathway in this response we treated MCF-7 breast cancer cells with the pure estrogen antagonist ICI 182780 to inhibit estrogen-induced gene expression and induce G 1 phase arrest. Subsequent treatment with 17␤-estradiol resulted in the synchronous entry of cells into S phase commencing at 12 h. The proportion of cells in S phase reached a maximum of 60% at 21-24 h. Cells subsequently completed mitosis and entered a second semisynchronous round of replication. Entry into S phase was preceded by increased activity of both Cdk4 and cyclin E-Cdk2 and hyperphosphorylation of pRB, all within the first 3-6 h of estradiol treatment. The increase in Cdk4 activity was accompanied by increases in cyclin D1 mRNA and protein, indicating that an initiating event in the activation of Cdk4 was increased cyclin D1 gene expression. In contrast, the levels of Cdk2 and the CDK inhibitors p21 (WAF1/CIP1/SDI1) and p27 (KIP1) in total cell lysates and in cyclin E immunoprecipitates were unaltered at these early time points. However, an inhibitory activity was present in antiestrogenpretreated cell lysates toward recombinant cyclin E-Cdk2 and was relieved by estradiol treatment. This activity was attributable predominantly to p21. These apparently conflicting data were resolved by performing gel filtration chromatography, which revealed that only a minority of cyclin E-Cdk2 complexes were active following estradiol treatment. Active complexes eluted at a higher molecular weight than inactive complexes, were relatively deficient in both p21 and p27, and contained Cdk2 with increased threonine 160 phosphorylation, consistent with a mechanism of activation of cyclin E-Cdk2 involving both reduced CDK inhibitor association and CDK-activating kinase-mediated phosphorylation of Cdk2. These results provide an explanation for the early activation of both cyclin D1-Cdk4 and cyclin E-Cdk2 complexes that accompany G 1 -S phase progression in response to estradiol.

Association between farnesoid X receptor expression and cell proliferation in estrogen receptor-positive luminal-like breast cancer from postmenopausal patients

“…5a, CDCA and E 2 exerted opposite effects on the cdk inhibitor p27, whereas these drugs induced an increase of the cell cycle mediator cyclin D1, suggesting that they somehow differ in their action on G1 phase progression. Results obtained here with E 2 are consistent with previous work showing that MCF-7 cell exposure to estrogen leads to a decrease of p27 [23] due to enhanced proteasomal degradation [24], and a concomitant activation of cyclin E-cdk2. On the other hand, the fact that CDCA induces both a proliferative response (Fig.…”

C‐myc gene expression alone is sufficient to confer resistance to antiestrogen in human breast cancer cells

“…43 In addition, antiestrogens inhibit the expression of cyclin D1 44 and stimulate that of cdk inhibitors, p27 Kip1 and p21 Waf1/Cip1 . 45 Therefore, it is probable that ICI has impaired the activities of many proteins that, in conjunction with c-myc, regulate cell proliferation, so that restoration of c-myc alone by dox induction was insufficient to restore cell growth to the fullest extent.…”

Reversal of an antiestrogen-mediated cell cycle arrest of MCF-7 cells by viral tumor antigens requires the retinoblastoma protein-binding domain

“…Proliferation of MCF-7 cells is estrogen-dependent and antiestrogen sensitive both in vitro and in vivo (Osborne et al, 1985;Soule and McGrath, 1980;Sutherland et al, 1983a), and in the presence of antiestrogens these cells accumulate in G 1 . Estrogen treatment of G 1 -arrested MCF-7 cells induces S phase entry, and this induction is associated with increases in cyclin D1 protein levels, cyclin D1/CDK4 kinase activity, CDK2 kinase activity, and pRb phosphorylation (Foster and Wimalasena, 1996;Planas-Silva and Weinberg, 1997;Prall et al, 1997;Watts et al, 1995). In addition, overexpression of cyclin D1 in the presence of antiestrogens leads to pRb phosphorylation, and is su cient to promote proliferation for at least one cell cycle (Pacilio et al, 1998;Prall et al, 1998).…”

“…For example, regulation of cyclin E/CKD2 kinase activity by estrogen and antiestrogens occurs in the absence of changes in cyclin E or CDK2 protein levels. To account for this observation, it has been proposed that induced or overexpressed cyclin D1 protein binds and sequesters the CDK inhibitor p21 cip1 , leading to activation of cyclin E/CDK2 complexes (Planas-Silva and Weinberg, 1997;Prall et al, 1997). Another potential mechanism of cyclin D1 action is via its interactions with cellular transcription factors.…”