c-Myc or Cyclin D1 Mimics Estrogen Effects on Cyclin E-Cdk2 Activation and Cell Cycle Reentry

Abstract

Estrogen-induced progression through G 1 phase of the cell cycle is preceded by increased expression of the G 1 -phase regulatory proteins c-Myc and cyclin D1. To investigate the potential contribution of these proteins to estrogen action, we derived clonal MCF-7 breast cancer cell lines in which c-Myc or cyclin D1 was expressed under the control of the metal-inducible metallothionein promoter. Inducible expression of either c-Myc or cyclin D1 was sufficient for S-phase entry in cells previously arrested in G 1 phase by pretreatment with ICI 182780, a potent estrogen antagonist. c-Myc expression was not accompanied by increased cyclin D1 expression or Cdk4 activation, nor was cyclin D1 induction accompanied by increases in c-Myc. Expression of c-Myc or cyclin D1 was sufficient to activate cyclin E-Cdk2 by promoting the formation of high-molecular-weight complexes lacking the cyclin-dependent kinase inhibitor p21, as has been described, following estrogen treatment. Interestingly, this was accompanied by an association between active cyclin E-Cdk2 complexes and hyperphosphorylated p130, identifying a previously undefined role for p130 in estrogen action. These data provide evidence for distinct c-Myc and cyclin D1 pathways in estrogen-induced mitogenesis which converge on or prior to the formation of active cyclin E-Cdk2-p130 complexes and loss of inactive cyclin E-Cdk2-p21 complexes, indicating a physiologically relevant role for the cyclin E binding motifs shared by p130 and p21.Estrogenic steroids elicit mitogenic responses in a variety of cell types, particularly those of female reproductive tissues, including uterus and mammary gland tissues. In addition, estrogens have well-described mitogenic actions on neoplastic breast epithelial cells both in vivo (55) and in vitro (25), and this effect has been linked to the established role of estrogens in the development and progression of the majority of human breast cancers (16). Estrogenic steroids, e.g., 17␤-estradiol (E 2 ), stimulate resting (G 0 -phase) cells to enter the cell cycle and accelerate G 1 -S-phase progression (23,58). Advances in the understanding of molecular mechanisms controlling cell cycle progression (31,50,51,66) have identified cyclin-dependent kinases (CDKs) as potential targets of E 2 -induced mitogenesis (2,12,39,42).Sensitivity to mitogenic stimulation is limited to G 1 phase of the cell cycle, transit through which is regulated by the activities of Cdk4, Cdk6, and Cdk2. These CDKs are activated by cyclin binding: Cdk4 and Cdk6 by D-type cyclins (50) and Cdk2 by cyclin E (22). Additional control of cyclin-CDK activity is achieved by phosphorylation/dephosphorylation of specific residues conserved among CDKs and by interaction with two families of CDK inhibitors: the INK4 family, of which p16 INK4A is prototypic, and the p21 WAF1,CIP1,SDI1 /p27 KIP1 /p57 KIP2 family (reviewed in references 31 and 51). Other factors, such as the activity of Cdc25 phosphatases that catalyze the removal of inhibitory phosphates on CDKs (31), identify a furt...

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

“…ND, c-myc mRNA levels too low to be quantitated accurately.-2 Significant differences (p Ͻ 0.05) from control clone "#"18c. The present study confirmed and extended the study of Prall et al 34 who, by using an MCF-7 cell model that harbored a heavy metal-inducible metallothionein promoter-driven c-myc gene construct, reported that zinc induced expression of c-myc was sufficient for S-phase entry of cells arrested in G 1 by treatment of cells with IC1. Neither cell growth kinetics nor apoptosis, however, was measured in this cited study.…”

“…Neither cell growth kinetics nor apoptosis, however, was measured in this cited study. As well, the data published by Prall et al 34 has revealed that a considerable amount of c-Myc protein was present in cells before the addition of zinc, indicating that c-myc expression directed by the metallothionein promoter was less stringent, presumably due to the presence of other heavy metallic ions in the growth medium. In comparison, our reverse tetracycline-inducible c-myc cell model offers rapid, specific and stringent control of c-myc expression; the levels of c-myc mRNA and protein were barely discernible in the presence of ICI before the addition of doxycycline.…”

Cell cycle basis for the onset and progression of c-Myc-induced, TGFα-enhanced mouse mammary gland carcinogenesis

“…This is surprising, as suppression of cyclin E by c-Myc has not been reported, and relevant literature suggests that c-Myc can activate expression of cyclin E in vitro (Amati et al, 1998;Obaya et al, 1999). Several studies have suggested that cyclin D1/cdk4 should be activated prior to the onset of cyclin E/cdk2 activity in order to ensure an orderly transition to S phase (Obaya et al, 1999;Prall et al, 1998). Thus, it is possible that the lack of a su cient amount of cyclin D1 may hamper the expression of cyclin E. It is even possible that prevention of expression of cyclin E may facilitate the cell growth during the early stages of the carcinogenic process in c-myc animals, as it has been shown that stable overexpression of cyclin E, rendered by cDNA transfection, inhibits growth of mammary epithelial cells (Sgambato et al, 1996).…”

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

“…Thus, pRb is an important target of estrogens in regulating MCF-7 cell proliferation, and its inactivation can contribute to the development of antiestrogen resistance. The fact that both pRb inactivation and overexpression of cyclin D1 (Pacilio et al, 1998;Prall et al, 1998) promote MCF-7 cell proliferation in the presence of antiestrogens con®rms an important role for the cyclin D/pRb pathway in estrogen dependence. It also suggests that the primary mechanism by which cyclin D1 overcomes an antiestrogen-mediated arrest is by activating CDK 4/6, which in turn phosphorylate pRb.…”

“…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).…”