Cell cycle and anti-estrogen effects synergize to regulate cell proliferation and ER target gene expression

Abstract

Antiestrogens are designed to antagonize hormone induced proliferation and ERalpha target gene expression in mammary tumor cells. Commonly used drugs such as OH-Tamoxifen and ICI 182780 (Fulvestrant) block cell cycle progression in G0/G1. Inversely, the effect of cell cycle stage on ER regulated gene expression has not been tested directly. We show that in ERalpha-positive breast cancer cells (MCF-7) the estrogen receptor gene and downstream target genes are cell cycle regulated with expression levels varying as much as three-fold between phases of the cell cycle. Steroid free culture conditions commonly used to assess the effect of hormones or antiestrogens on gene expression also block MCF-7 cells in G1-phase when several ERalpha target genes are overexpressed. Thus, cell cycle effects have to be taken into account when analyzing the impact of hormonal treatments on gene transcription. We found that antiestrogens repress transcription of several ERalpha target genes specifically in S phase. This observation corroborates the more rapid and strong impact of antiestrogen treatments on cell proliferation in thymidine, hydroxyurea or aphidicolin arrested cells and correlates with an increase of apoptosis compared to similar treatments in lovastatin or nocodazol treated cells. Hence, cell cycle effects synergize with the action of antiestrogens. An interesting therapeutic perspective could be to enhance the action of anti-estrogens by associating hormone-therapy with specific cell cycle drugs.

Figure 1. ERα target gene expression is cell cycle regulated in MCF-7 cells.

A) FACS analysis after propidium iodide staining on asynchronous untreated cells (1), cells blocked in G1 by lovastatin treatment 20 µM for 32 h (2), in S phase by thymidine treatment 3 mM for 24 h (3) or in G2/M by nocodazol 50 ng/ml for 24 h (4). B) Real-time PCR analysis of ESR1, PGR, CCND1 and H2AFZ gene expression in G1 (lovastatin), S (thymidine) and G2/M (nocodazol) cell cycle phases. 2×10⁶ of MCF-7 cells were seeded in 10 cm dishes and after 24 h submitted to specific cell cycle arrest drugs as in (A). Total RNA was extracted and reverse transcribed. The amount of analyzed genes cDNA was measured RT-qPCR divided by the amount of RLP0 cDNA. (n = 3) one representative experiment is shown. C) Real-time PCR analysis of ESR1, PGR, CCND1 and H2AFZ gene expression. For lovastatin treatment cells were treated as in (B). For nocodazol/check-off treatment, 12×10⁶ cells were splited into two 140 cm dishes. After 24 h of nocodazol treatment (25 ng/ml), G2/M arrested cells are harvested by check-off.and seeded in a clean dish. After 7 hours in complete medium total RNA was extracted and reverse transcribed (n = 2).

Figure 2. Steroid free medium induces an E2 reversible G1 block in ERα-positive MCF-7 cells.

1.5×10⁶ ERα-positive MCF-7 cells were cultivated in red medium (R) for 24 h, then medium was removed and cells were cultivated in red or white medium A) FACS analysis of MCF-7 cells after 4 days in red medium only (R), 3 days in red medium complemented with E2 10⁻⁸ M for 24 h, in white medium only for 4 days (W) or 3 days in white medium complemented with E2 10⁻⁸ M for 24 h (W+E2). For growth curves, 1×10⁶ ERα-positive MCF-7 cells were cultivated in red medium (R) for 24 h, then medium was removed and cells were cultivated in red or white medium. Cells were counted by trypan blue exclusion at different time points. B) ESR1, PGR and CCND1 gene expression was analyzed by RT-qPCR.

Figure 3. OH-TAM and ICI induced G1 cell cycle block and decreased ERα target gene expression.

2×10⁶ MCF-7 cells were cultivated in red medium for 24 h. OH-TAM or ICI were added to the medium at a final concentration of 1 µM. After 24 h cell cycle was analyzed by FACS and expression of ERα target genes by RT-qPCR.

Figure 4. Effect of OH-TAM and ICI on gene expression is cell cycle dependent.

2×10⁶ cells were cultivated in red medium for 24 h. Cells were blocked in G1 by lovastatin 20 µM, 32 h (1), in S by thymidine 3 mM 24 h (2) and in G2/M by nocodazol 50 ng/ml, 24 h (3). Cell cycle arrested cells were treated by Tamoxifen (OH-TAM 1 µM, 24 h) or ICI 182.780 (ICI 1 µM, 24 h). (n = 2) one representative experiment is shown. Relative mRNA expression in G1, S or G2/M phases of ERα target with or without anti-estrogen treatment was analyzed by RT-qPCR.

Figure 5. Effect of OH-TAM and ICI on cell proliferation is enhanced in S-phase.

3×10⁵ MCF-7 cells were split in 60 cm dishes and cultivated in red medium for 12 h. A) Cells were treated by lovastatin (20 µM, 32 h), thymidine (3 mM, 24 h) or nocodazol (50 ng/ml, 24 h) until T0. Cell proliferation was monitored for untreated (NT) or for OH-TAM (1 µM) or ICI (1 µM) treated cells. Cells were counted by trypan blue exclusion in triplicate at different time points. Number of cells at T0 was set to 1 and the doubling time was calculated. B-C-D). Cells were treated by lovastatin 20 µM for 32 h (B), by thymidine 3 mM, 24 h (C) or nocodazol 50 ng/ml, 24 h (D) until T0. Then, OH-TAM 1 µM or ICI 1 µM added to the medium and cells were counted by trypan blue exclusion in triplicate at different time points.

Figure 6. OH-TAM and ICI induced apoptosis specifically in S-phase in MCF-7 arrested cells.

A) 3×10⁵ MCF-7 cells were split in 60 cm dishes and cultivated in red medium for 12 h. Cells were treated by lovastatin (20 µM, 32 h), thymidine (3 mM, 24 h) or nocodazol (50 ng/ml, 24 h) until T0. Accumulation of cleaved PARP (black arrow) was monitored by western blotting for untreated (NT) or for OH-TAM (1 µM) or ICI (1 µM) treated cells. B) Quantification analysis was performed using the ImageGauge 4.0 software. * increase >50% of cleaved PARP. C) 3×10⁶ cells were seeded in 10 cm dishes. After 12 h, cells were treated or not (NT) with 3 mM thymidine for 24 h. Cells were treated with thymidine (Th) or Tamoxifen 1 µM (Th + TAM), ICI 100 nM or 1 µM (Th + ICI) for 24 h. After trypsinisation, cells were counted by trypan blue staining. Total cell number was set to 100% (n = 2). D) 3×10⁶ cells were splited in 10 cm dishes. At T0 cells were treated with hydroxyl urea 1,5 mM or aphidicolin 1 µg/ml for 24 h. Then, cells were treated or not with Tamoxifen or ICI 1 µM for another 24 h. Cells were counted by trypan blue exclusion. Number of cells at T0 was set to 1 and the doubling time was calculated. E) Cells were treated as in (D). Accumulation of cleaved PARP (black arrow) was monitored by western blotting. Quantification analysis was performed using the ImageGauge 4.0 software. * increase >50% of cleaved PARP.