We have addressed the question of rapid, nongenomic mechanisms that may be involved in the mitogenic action of estrogens in hormone-dependent breast cancer cells. In quiescent, estrogen-deprived MCF-7 cells, estradiol did not induce a rapid activation of either the MAPK/ERK or phosphatidylinositol-3 kinase (PI-3K)/Akt pathway, whereas the entry into the cell cycle was documented by the successive inductions of cyclin D1 expression, hyperphosphorylation of the retinoblastoma protein (Rb), activity of the promoter of the cyclin A gene, and DNA synthesis. However, pharmacological inhibitors of the src family kinases, 4-amino-5-(4-methylphenyl)-7-(t-butyl) pyrazolo[3,4-d] pyrimidine (PP1) or of the PI-3K (LY294002) did prevent the entry of the cells into the cell cycle and inhibited the late G1 phase progression, whereas the inhibitor of MAPK/ERK activation (U0126) had only a partial inhibitory effect in the early G1 phase. In agreement with these results, small interfering RNA targeting Akt strongly inhibited the estradiolinduced cell cycle progression monitored by the activation of the promoter of the cyclin A gene. The expression of small interfering RNA targeting MAPK 1 and 2 also had a clear inhibitory effect on the estradiol-induced activation of the cyclin A promoter and also antagonized the estradiol-induced transcription directed by the estrogen response element. Finally, transfection of the estrogen receptor into NIH3T3 fibroblasts did not confer to the cells sensitivity to a mitogenic action of estradiol. We conclude that the induction of the cell cycle by estradiol does not require a direct activation of MAPK/ERK or PI-3K signaling protein kinase cascades, but that these kinases appear to have a permissive role in the cell cycle progression.

The effects of GA, an ansamycin antibiotic in development as a lead anticancer drug, were studied in mouse BP-A31 fibroblasts and in human cancer-derived cell lines. GA and related molecules act by inhibiting the chaperone function of the Hsp90 protein through competition for ATP binding. The antiproliferative effects of GA have been attributed to destabilization of the Raf-1 protein, one of the targets of Hsp90, and to the resulting inhibition of MAPK. Addition of GA to BP-A31 cells, synchronously progressing through the G 1 phase, inhibited Rb hyperphosphorylation and G 1 /S transition irrespective of the time of addition. The G 1 arrest was accompanied by a progressive decrease in Raf-1 content, especially of the phosphorylated form; however, GA caused only partial inhibition of MAPK phosphorylation. We show that GA triggers a rapid and marked decrease in the kinase activity of the cyclin E/cdk2 complex coupled with a decline in both total and cdk2-associated cyclin E. In transient transfection experiments, inhibition of cyclin E expression by GA was correlated with inhibition of the transcriptional activity of the cyclin E gene promoter. Transduction of mitogenic signals from the plasma membrane to the nucleus is relayed by the GTP-binding Ras protein and the Raf-1 protein kinase. 1,2 Activated Raf-1 phosphorylates MAPK/ ERK kinases (MEKs), 3,4 which in turn activate MAPKs/ERKs, 5 favoring their nuclear translocation 6 and consequently the activation of transcription factors. 7 Thereafter, the sequential activation of several cyclin/cdk complexes 8 controls progression through the G 1 phase toward the S phase. Cdk activity is regulated by both the cyclin abundance 9,10 and the interplay of kinases and phosphatases. [11][12][13] Cyclin D expression and assembly with cdk4/cdk6 is promoted by MAPK, thus providing a link between the 2 cascades. 14 -17 Cdk4 and cdk6 associated with D-type cyclins are activated from mid-to late G 1 phase, while cdk2 associated with cyclin E is activated in late G 1 phase. 18 Subsequently, cdk2 and cdk1 assemble with A and B cyclins and are active during the S/G 2 and M phases. Two families of specific cki proteins also modulate the activity of cyclin/cdk complexes. The INK4 family (p15, p16, p18 and p19) specifically targets the kinases associated with the D-type cyclins, whereas the Cip/Kip family (p21Kip1 , p57 Kip2 ) interacts with all cdks. 19,20 ckis of the Cip/Kip family also function as adapters, allowing the assembly and subcellular addressing of the cyclin D/cdk complexes. 17,21 Sequestration of Cip/Kip proteins by cyclin D/cdk complexes lowers the constraint of these ckis on cyclin E/cdk2 complexes. 20 The retinoblastoma gene product, Rb, and related proteins, p107 and p130, are among the key cdk substrates. Hypophosphorylated Rb is a negative regulator of the E2F family of transcription factors. 21,22 Phosphorylation of Rb occurs in an orderly manner: first, cyclin D-activated cdk4 phosphorylates Rb on specific residues (including Ser 780 ). [23][24][25] This releases t...

Rapamycin, a bacterial macrolide antibiotic, is a potent immunosuppressant agent that blocks cell proliferation by inhibiting the G1/S transition in several cell types. In sensitive cells, rapamycin inhibits the phosphorylation of p70 S6K and of Rb; however, the precise mechanisms involved have not been elucidated. In the mouse BP-A31 fibroblasts, synchronised in G0/G1 phase by serum starvation and induced to reinitiate the G1-phase progression, rapamycin inhibited the entry into S phase. The effect of rapamycin was situated in early G1 phase. The assembly of the cyclin D1/cdk4 complexes that phosphorylate Rb early in the G1 phase was not modified by the drug. Nevertheless, an inhibition of the activation of cyclin D1/cdk4 and cyclin E/cdk2 as well as of Rb phosphorylation accompanied the cell cycle arrest. Remarkably, rapamycin reduced the level of total p21 WAF1/CIP1 as well as that of p21 WAF1/CIP1 associated with the cyclin D1/ cdk4 complexes. Besides its inhibitory activity toward cdk, p21 WAF1/CIP1 has been recently found to participate in the formation/stabilisation/nuclear translocation of cyclin D1/ cdk4 complexes. We propose that the inhibition of the expression of p21 WAF1/CIP1 is a mechanism by which rapamycin inhibits the triggering of the cdk cascade in the BP-A31 cells. Stimulation of quiescent eukaryotic cells by different growth factors triggers a cascade of events leading to the reinitiation of the cell cycle. The activation of the cascade of cyclin-dependent protein kinases (cdks) is a common feature of all the different signalling pathways and is situated in time after the immediate early events, among which the ras/mitogen-activated protein kinase (MAP kinase) is considered to be essential. The activation of cdks requires the association with the appropriate cyclins as well as the phosphorylation/dephosphorylation of specific amino acid residues. 1,2 The activity of cdk is further subject to modulation by cdk inhibitory proteins (cki), members of 2 groups by sequence homology: INK4 proteins (p15, p16, p18, p19), which specifically interact with cdk4 and cdk6, and Cip/Kip proteins (p21, p27 and p57), which do not display a marked target specificity. 3 Cdk4 (or cdk6 in certain cell types) associated with D-type cyclins is activated in mid-G1 phase, followed by cdk2 bound to cyclin E in late G1 phase. Sequential cdk4 and cdk2 activation leads to phosphorylation of Rb and related proteins on specific serine/threonine residues. Phosphorylation of Rb during the G1 phase of the cell cycle reverses its growth-suppressive effects by releasing the transcription factors E2F-DP. 4,5 These factors then trigger the expression of genes that enable the cells to progress into late G1 and S phase. 6,7 Rapamycin, a bacterial macrolide antibiotic, is a potent immunosuppressive drug that blocks the proliferation of T and B lymphocytes 8 -10 but is also an inhibitor of the mitogenic response in other cell types such as osteosarcoma, 11 hepatoma, 12 3T3 fibroblasts, 13 vascular endothelial cells 14 and MCF7 breast...