The E2-responsiveness of the three MCF-7 strains was measured and compared to their response to IGF-I in a DNA-synthesis assay. The cells were seeded in 24-well plates in steroid- and serum-free medium. After 26 h, 1 nM E2, 20 ng/ml of IGF-I (I₍₂₀₎), 2 ng/ml of IGF-I (I₍₂₎) or the combination of 1 nM E2 and 2 ng/ml of IGF-I (I₍₂₎E2) were added. Twenty-four h later, ³H-thymidine (³H-TdR) was added for a 6 h period, after which the cells were harvested. Figure 1 shows the ³H-TdR incorporation in the MCF-7 cell lines, normalized to ³H-TdR incorporation induced by 20 ng/ml of IGF-I. A small amount of ³H-TdR is incorporated in untreated serum-starved MCF-7S cells, approximately 1% of the incorporation induced by 20 ng/ml of IGF-I. The MCF-7 ATCC and the MCF-7 NKI cells show higher ³H-TdR incorporation values in untreated serum-starved cells, 6 and 12% of the 20 ng/ml IGF-I value, respectively. This indicates that these cells are less well synchronized by serum- and steroid-deprivation and may need additional treatment to reach quiescence. In MCF-7S cells, only a moderate increase in ³H-TdR incorporation is induced by stimulation with either 2 ng/ml of IGF-I or 1 nM E2 in MCF-7S cells, to 3 and 9% of the IGF-I (20 ng/ml) value respectively. When both IGF-I (2 ng/ml) and E2 are added, a strong increase in ³H-TdR incorporation is observed to 88% of the incorporation level of 20 ng/ml of IGF-I. Thus, a clear synergism of E2 and IGF-I on the induction of DNA-synthesis in MCF-7S cells is demonstrated. In MCF-7 ATCC cells, the ³H-TdR incorporation induced by 2 ng/ml of IGF-I is much higher (26% of the incorporation seen with 20 ng/ml of IGF-I) than in MCF-7S cells. Also 1 nM E2 induces a much larger increase in incorporation (40%) when compared to the incorporation in MCF-7S cells. However, the combination of the two still is synergistic, resulting in a higher incorporation level (115%) than the added effect of the two hormones individually (66%). In MCF-7 NKI cells, E2 is an even stronger inducer of ³H-TdR incorporation. In this MCF-7 strain, addition of E2 results in a higher incorporation level than observed with mitogenic amounts of IGF-I. No synergy between IGF-I and E2 can be observed in MCF-7 NKI, since ³H-TdR incorporation induced by 1 nM E2 by itself is as high as by the combination of IGF-I (2 ng/ml) and 1 nM E2.

To investigate whether MCF-7 ATCC and MCF-7 NKI express higher levels of ERα than MCF-7S, we analyzed the ERα content of serum-starved MCF-7S, MCF-7 ATCC, and MCF-7 NKI cells by Western blotting. We find that the level of ERα expression in MCF-7S and MCF-7 NKI are comparable, whereas the MCF-7 ATCC express slightly higher levels of ERα (figure 2A). The observed difference in expression levels does not correlate with E2 inducible proliferation in the three strains of MCF-7.

In order to detect differences in functionality of the ER, we analyzed the expression level of an E2-responsive gene, cyclin D1, in untreated serum-starved and in E2-treated cells. Like in MCF-7S cells, E2 treatment induces a strong increase in cyclin D1 levels in both MCF-7 ATCC and MCF-7 NKI cells. No significant differences in cyclin D1 induction were observed in the three MCF-7 strains (figure 2B). Taken together, these results indicate that the differences in growth response upon E2 treatment of the MCF-7 strains are not caused by their ER expression level or functionality.

To investigate whether E2 can induce cell cycle progression in MCF-7 ATCC and NKI cells without subsequent activation of the IGF type I receptor, a DNA synthesis assay was performed in which 200 μg/ml suramin was added to the cells one hour prior to stimulation with IGF-I, E2 or the combination of both hormones. Suramin is an anti-tumor agent inhibiting the proliferation of a variety of human cancer cell lines cultured in vitro 12131415. Suramin acts extracellularly by interfering with the binding of growth factors to their receptors 16, including several which are known to be involved in tumor growth or development, like IGF-I 17, and TGFβ 18.

We find that IGF-I-induced incorporation of ³H-TdR is completely inhibited in all three MCF-7 cell lines, when the cells are preincubated with suramin (figure 3). Surprisingly, preincubation with suramin also completely abolishes E2-induced DNA synthesis in all three MCF-7 laboratory strains. These results suggest that activation of a suramin-sensitive receptor is essential for E2-induced cell cycle progression.

To test whether E2-responsiveness is dependent on IGF-signaling, the IGF-RI was blocked by the addition of 1 μg/ml αIR3 antibody to the culture medium, which prevents binding of IGF-I and -II to the receptor. Figure 4 shows that IGF-I-induced ³H-TdR incorporation is completely inhibited in all three strains of MCF-7 treated with αIR3. However, also the effect of E2 on ³H-TdR incorporation is reduced to levels comparable to the incorporation in untreated serum-starved cells. This shows that all three MCF-7 strains loose their E2-responsiveness when IGF-RI signaling is blocked.

The observed inhibition of DNA synthesis theoretically might be due to toxicity of the antibodies added to the cells. To exclude this possibility, the specificity of inhibition was examined by determining whether other signaling pathways are still intact in antibody treated cells. To this end, the TPA-induced synthesis of the CKI p21^cip1/waf1 protein was studied on Western blot. TPA induces the synthesis of p21^cip1/waf1 via activation of the ERK pathway independent of IGF-RI 19. A moderate elevation of p21^cip1/waf1 levels is also observed when MCF-7 cells are treated with mitogenic amounts of IGF-I 20.

Figure 5 shows that the addition of 100 ng/ml of TPA to the medium induces expression of p21^cip1/waf1. IGF-I (20 ng/ml) also clearly results in higher p21^cip1/waf1 levels. αIR3 is able to totally block the upregulation of p21^cip1/waf1 induced by IGF-I, but does not inhibit the stimulation of p21^cip1/waf1 expression by TPA. These results show that the inhibition of E2-induced DNA synthesis in E2-responsive MCF-7 cells by αIR3 is a result of specific inhibition of IGF-RI signaling.

In view of the fact that activation of the IGF-RI is important for E2-induced cell cycle progression in MCF-7 ATCC and MCF-7 NKI cells, we investigated the possibility of autocrine stimulation of the IGF-RI in these MCF-7 strains. To this end, we studied the effect of conditioned media on the E2-responsiveness of the MCF-7 strains in a DNA-synthesis assay. MCF-7S, ATCC and NKI cells were seeded in 162 cm² culture flasks and incubated in serum-free, estrogen-free medium for two days, after which the conditioned media were collected. For the DNA-synthesis assay, MCF-7S, MCF-7 ATCC and MCF-7 NKI cells were seeded in 24 wells plates, and serum-starved for 24 h. Prior to stimulation the serum-free medium on the three MCF-7 strains was replaced by the conditioned media, or by fresh serum-free medium as a control. After 15 minutes, no mitogens, IGF-I (2 or 20 ng/ml), 1 nM E2 or the combination of 1 nM E2 and 2 ng/ml IGF-I were added to the media. 24 h later, ³H-TdR was added for a 6 h period, after which the cells were harvested.

Figure 6 shows the effect of the conditioned media on the MCF-7S cells. In untreated steroid- and serum-free medium, MCF-7S cells are not triggered to progress through the cell cycle by addition of E2. Culturing the cells in conditioned medium from the MCF-7S cell line has no significant effect on the ³H-TdR incorporation induced by any of the hormone treatments. However, when E2 is administered together with conditioned medium from MCF-7 ATCC or MCF-7 NKI, the cells progress through S-phase as is demonstrated by elevated incorporation of ³H-TdR in these cells.

Also in the MCF-7 ATCC cell line, the conditioned media from MCF-7 ATCC and MCF-7 NKI, but not the conditioned medium of MCF-7S, induce additional ³H-TdR incorporation in cells treated with E2 when compared to cells incubated in fresh serum-free medium with E2. In MCF-7 ATCC, incubation in the conditioned media from the two E2-inducible cell lines leads to a higher incorporation in untreated and in IGF-I (2 ng/ml) treated cells. This increase is less pronounced in the MCF-7S cell line (figure 6).

In MCF-7 NKI cells, the conditioned media have no significant effect on the amount of incorporation induced by E2, but they do stimulate ³H-TdR incorporation in untreated and in IGF-I (2 ng/ml) stimulated cells (figure 6). These results suggest that the MCF-7 ATCC and MCF-7 NKI cells secrete a factor into the medium that activates the IGF-RI and synergizes with E2 to induce cell cycle progression in all three strains of MCF-7.

Anti-p21^CIP1/WAF1 (H-164) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). IGF-I was from GroPep (Adelaide, Australia). Aprotinin, E2, sodium selenite, suramin, and TPA were purchased from Sigma (St. Louis, MO). Albumin, bovine fraction V (BSA, Catalogue number 840533), Na₃VO₄ and transferrin were from ICN Biomedicals, Inc. (Aurora, OH). The αIR3 antibody was a kind gift of Dr. S. van Buul-Offers from the Department of Pediatric Endocrinology, UMC Utrecht, The Netherlands.

The MCF-7 human breast cancer cell lines were cultured in Dulbecco's Minimal Essential Medium/Ham's F12 medium (1:1), supplemented with 10% fetal calf serum, 300 μg/ml of glutamine, 100 IU/ml of penicillin and 100 μg/ml of streptomycin. Cell cultures of 40–60% confluence were used for experiments. Prior to experiments, MCF-7 cells were cultured for 24 h in phenol red-free medium containing 5% dextran-coated charcoal-treated serum, followed by an incubation in phenol red-free, serum-free medium, supplemented with 0.2% bovine serum albumin (BSA), 10 μg/ml of transferrin and 30 nM sodium selenite for at least 24 h.

Assays were performed in 24-well plates. Serum-starved cells were stimulated with IGF-I, E2, or a combination of the two and after 24 h of incubation ³H-labeled thymidine (2 μCi/ml) was added. 30 h after the addition of the stimuli, cells were fixed with 10% trichloroacetic acid, washed in PBS and lysed in 0.1 M NaOH, followed by liquid scintillation counting.

Experiments were performed in 6-well plates. After stimulation, cells were washed with ice-cold PBS and lysed in lysis buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol). Lysates were boiled for 5 min and protein concentrations were determined using the BCA protein assay (Pierce, Rockford, IL). After addition of 0.1% bromophenol blue and 5% β-mercaptoethanol to the samples, equal amounts of protein (5–50 μg) were size-separated on a SDS-polyacrylamide gel and transferred to nitrocellulose. The membrane was stained with Ponceau S (Sigma) to check for equal loading and blocked with 2–3% non-fat milk or 0.5–2% BSA in TBST (150 mM NaCl, 10 mM Tris-HCl pH 8.0, 0.2% Tween 20) for 1 h. Incubation with the primary antibody was performed overnight at 4°C in 0.1 × blocking solution. The membrane was washed in TBST and exposed to horseradish peroxidase-coupled secondary antibody for 1 h in TBST. For detection the Western Blot Chemiluminescence Reagent Plus system (NEN Life Science Products Inc., Boston, MA) was used.