Estrogens and antiestrogens influence the G1 phase of the cell cycle. In MCF-7 breast cancer cells, estrogen stimulated cell cycle progression through loss of the kinase inhibitor proteins (KIPs) p27 and p21 and through G1 cyclin-dependent kinase (cdk) activation. Treatment with antiestrogen drugs, Tamoxifen or ICI 182780, caused cell cycle arrest, with up-regulation of both p21 and p27 levels, an increase in their binding to cyclin E-cdk2, and kinase inhibition. The requirement for these KIPs in the arrests induced by estradiol depletion or by antiestrogens was investigated with antisense. Antisense inhibition of p21 or p27 expression in estradiol-depleted or antiestrogenarrested MCF-7 led to abrogation of cell cycle arrest, with loss of cyclin E-associated KIPs, activation of cyclin E-cdk2, and S phase entrance. These data demonstrate that depletion of either p21 or p27 can mimic estrogen-stimulated cell cycle activation and indicate that both of these KIPs are critical mediators of the therapeutic effects of antiestrogens in breast cancer. E stradiol is mitogenic in up to 50% of de novo breast cancers, causing recruitment of quiescent cells into G 1 and shortening the G 1 -to-S phase interval (1, 2). Although 70% of breast cancers express the estrogen receptor (ER), only two-thirds of these will respond to antiestrogens, of which, Tamoxifen is the most widely used (3, 4). Antiestrogens, such as Tamoxifen, its active metabolite, 4-hydroxytamoxifen (4-OH TAM), and the more potent steroidal antiestrogen ICI 182780 (Faslodex) lead to a G 0 ͞G 1 arrest in susceptible ER-positive breast cancer cells (5-8). Unfortunately, hormonally responsive breast cancers invariably develop resistance to antiestrogens despite the continued expression of wild-type ER in most cases (9-12). Estrogens induce conformational changes in the ER, which promote its nuclear localization, dimerization, and function as a ligand-activated transcription factor (13-15). In addition, ligand binding to the ER can rapidly and transiently activate signal transduction pathways, notably the mitogen-activated protein kinase in breast cancer and in other cell types (16,17). Because antiestrogen resistance usually develops in the presence of an intact ER, the mechanisms by which ER modulates the cell cycle may be altered during breast cancer progression. The evolution of prostate cancer to hormone independence also occurs without loss of the androgen receptor (18,19) and may reflect a common mechanism of cell cycle misregulation.Progression through the cell cycle is governed by a family of cyclin-dependent kinases (cdks), whose activity is regulated by phosphorylation (20), activated by cyclin binding (21,22), and inhibited by the cdk inhibitors of the inhibitor of cdk4 (INK4) family (p16 INK4A , p15 INK4B , p18, and p19) and kinase inhibitor protein (KIP) family (p21 WAF-1͞CIP-1 , p27 Kip1 , and p57 KIP2 ;. Passage through G 1 into S phase is regulated by the activities of cyclin D-, cyclin E-, and cyclin A-associated kinases. Although p27 protein is st...
We show that p27 localization is cell cycle regulated and we suggest that active CRM1/RanGTPmediated nuclear export of p27 may be linked to cytoplasmic p27 proteolysis in early G1. p27 is nuclear in G0 and early G1 and appears transiently in the cytoplasm at the G1/S transition. Association of p27 with the exportin CRM1 was minimal in G0 and increased markedly during G1-to-S phase progression. Proteasome inhibition in mid-G1 did not impair nuclear import of p27, but led to accumulation of p27 in the cytoplasm, suggesting that export precedes degradation for at least part of the cellular p27 pool. p27-CRM1 binding and nuclear export were inhibited by S10A mutation but not by T187A mutation. A putative nuclear export sequence in p27 is identified whose mutation reduced p27-CRM1 interaction, nuclear export, and p27 degradation. Leptomycin B (LMB) did not inhibit p27-CRM1 binding, nor did it prevent p27 export in vitro or in heterokaryon assays. Prebinding of CRM1 to the HIV-1 Rev nuclear export sequence did not inhibit p27-CRM1 interaction, suggesting that p27 binds CRM1 at a non-LMB-sensitive motif. LMB increased total cellular p27 and may do so indirectly, through effects on other p27 regulatory proteins. These data suggest a model in which p27 undergoes active, CRM1-dependent nuclear export and cytoplasmic degradation in early G1. This would permit the incremental activation of cyclin E-Cdk2 leading to cyclin E-Cdk2-mediated T187 phosphorylation and p27 proteolysis in late G1 and S phase. INTRODUCTIONThe Cdk inhibitor p27 is an important regulator of G1 progression. It is highly expressed in G0, where it binds tightly and inhibits cyclin E-Cdk 2 Polyak et al., 1994;Slingerland et al., 1994). In mid-G1, p27 also plays a role in the assembly and nuclear import of D-type cyclinCdk complexes (LaBaer et al., 1997;Cheng et al., 1999). p27 levels are regulated by translational controls and by proteolysis, and decrease as cells progress from G1 to S phase (Hengst and Reed, 1996;Millard et al., 1997;Slingerland and Pagano, 2000). The ubiquitin-dependent proteolysis of p27 (Pagano et al., 1995) is regulated by its phosphorylation at threonine 187 (T187) by cyclin E-Cdk 2 in late G1 and S phase (Sheaff et al., 1997;Vlach et al., 1997;Montagnoli et al., 1999). T187 phosphorylation allows recognition of p27 by its SCF-type E3 ligase, comprised of Skp1, Cul1, and the F-box protein, Skp2 and Roc1 and the Cks1 cofactor Ohta et al., 1999;Sutterluty et al., 1999;Tsvetkov et al., 1999;Ganoth et al., 2001;Spruck et al., 2001). Recent evidence suggests that p27 proteolysis is regulated by at least two distinct mechanisms, with mitogenic signaling conditioning p27 for degradation in early G1 in a manner independent of T187 phosphorylation (Hara et al., 2001;Malek et al., 2001), whereas Skp2-dependent cyclin E-Cdk 2-mediated degradation occurs in S phase after T187 phosphorylation (Malek et al., 2001). Although p27 is detected in the nuclei of most normal quiescent cells (Slingerland and Pagano, 2000), the relationship between its int...
We report here the cloning and characterization of human and mouse cyclin E2, which de®ne a new subfamily within the vertebrate E-type cyclins, while all previously identi®ed family-members belong to the cyclin E1 subfamily. Cyclin E2/CKD2 and cyclin E/CDK2 complexes phosphorylate histone H1 in vitro with similar speci®c activities and both are inhibited by p27 Kip1 . Cyclin E2 mRNA levels in human cells oscillate throughout the cell cycle and peak at the G1/S boundary, in parallel with the cyclin E mRNA. In cells, cyclin E2 is complexed with CDK2, p27 and p21. Like cyclin E, cyclin E2 is an unstable protein in vivo and is stabilized by proteasome inhibitors. Cyclin E2-associated kinase activity rises in late G1 and peaks very close to cyclin E activity. In two malignantly transformed cell lines, cyclin E2 activity is sustained throughout S phase, while cyclin E activity has already declined and cyclin A activity is only beginning to rise. We speculate that cyclin E2 is not simply redundant with cyclin E, but may regulate distinct rate-limiting pathway(s) in G1-S control.
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