Down-regulation of p21WAF1/CIP1 or p27Kip1 abrogates antiestrogen-mediated cell cycle arrest in human breast cancer cells - PubMed (original) (raw)

Down-regulation of p21WAF1/CIP1 or p27Kip1 abrogates antiestrogen-mediated cell cycle arrest in human breast cancer cells

S Cariou et al. Proc Natl Acad Sci U S A. 2000.

Abstract

Estrogens and antiestrogens influence the G(1) 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 G(1) 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.

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Figures

Figure 1

Figure 1

Losses of p21 and p27 during estradiol stimulation of quiescent MCF-7 cells. Quiescent, estradiol-depleted MCF-7 cells were stimulated by readdition of 10 nM estradiol, and samples were taken at intervals thereafter. (A) Cell cycle synchrony was determined by dual BrdUrd/propidium iodide pulse labeling and flow cytometric analysis. (B) p21 and p27 immunoblots revealed levels of these proteins during cell cycle progression. (C) p27 protein was assayed by immunohistochemistry in asynchronous cultures at the indicated times after estradiol stimulation of quiescent MCF-7 as described (25).

Figure 2

Figure 2

Different patterns of KIP binding during cyclin D1–cdk4 and cyclin E–cdk2 activation. Cdk4 (A) and cyclin E (C) immunoprecipitates (IP) from cell lysates recovered at intervals after readdition of estradiol to steroid-depleted MCF-7 cells were resolved and analyzed by immunoblotting with the indicated antibodies. Cyclin D1 (B) and cyclin E (D) immunoprecipitates were assayed for kinase activity (36) at intervals after estradiol stimulation of quiescent MCF-7.

Figure 3

Figure 3

p21 and p27 proteins increase during G0/G1 arrest by ER blockade. Asynchronously growing MCF-7 cells were treated with the ER-blocking drug ICI 182780 (Faslodex) at time 0 h, and samples were collected for flow cytometry or protein analysis at times indicated. (A) Cell cycle distribution before and 48 h after drug treatment. (B) Lysates were analyzed by immunoblotting with the indicated antibodies. Cyclin E immunoprecipitates were immunoblotted for associated p21 or p27 or analyzed for associated histone H1 kinase activity as described in ref. . Similar results were obtained for arrests with 4-OH TAM or after transfer to estradiol-depleted, charcoal-stripped serum.

Figure 4

Figure 4

Requirement of p27 for cell cycle arrest by estradiol depletion. (A) Estradiol-depleted MCF-7 cells were lysed before (left lane) or 1 h after exposure to lipid only (L), ASp27 (AS), or MSMp27 (MSM) and were immunoblotted for p21 and p27. Before and at 1 and 21 h after ASp27 transfection, cells were metabolically pulse labeled with [35S]methionine, and p21 and p27 were immunoprecipitated from lysates containing equal trichloroacetic acid incorporation. The positions of metabolically labeled p27 are indicated by arrows. A nonspecific band, migrating close to p27 was present in all lanes, including the control nonspecific IgG lane. (B) Immunoblotting shows cell cycle regulatory protein levels before (left lane) or 21 h after transfection of lipid alone (L), ASp27, or MSMp27. Cyclin E immunoprecipitates (IP) were recovered from the same lysates as in B above and immunoblotted to detect associated proteins (C) or assayed for cyclin E-associated histone H1 kinase activity (D). (E) Flow cytometry 21 h after transfection with ASp27, lipid alone, or MSMp27.

Figure 5

Figure 5

Requirement for p21 and p27 in G1 arrest by ER-blocking drugs or estradiol-depletion. MCF-7 cells were arrested by estradiol depletion or by treatment with 4-OH TAM or ICI 182780. The graph indicates the percentage of S phase cells after a 21-h exposure to lipid only (black bars), antisense (white bars), or mismatch (hatched bars) oligonucleotides to either p21 or p27.

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