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Autocrine regulation of cell proliferation by estrogen receptor-alpha in estrogen receptor-alpha-positive breast cancer cell lines.

Tan H, Zhong Y, Pan Z - BMC Cancer (2009)

Bottom Line: E2 stimulation of ICI182780 treated cells, however, promotes the expression and colocalization of ERalpha and Ki-67 as well as the cell cycle progressing through the S and G2/M phases.Inhibition of EGFR signaling does not inhibit the autocrine action of ERalpha.All of the three ERalpha-positive cell lines used in our study showed colocalization of ERalpha and Ki-67, indicating that these cell lines might be originated from primary tumor cells with autocrine regulation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Animal Science, Vermont Cancer Center, University of Vermont, Burlington, VT 05405, USA. htan1@uvm.edu

ABSTRACT

Background: Estrogen receptor-alpha (ERalpha) is essential for mammary gland development and is a major oncogene in breast cancer. Since ERalpha is not colocalized with the cell proliferation marker Ki-67 in the normal mammary glands and the majority of primary breast tumors, it is generally believed that paracrine regulation is involved in ERalpha mediated cell proliferation. In the paracrine model, ERalpha-positive cells don't proliferate but will release some paracrine growth factors to stimulate the neighboring cells to proliferate. In a subpopulation of cancer cells in some primary breast tumors, however, ERalpha does colocalize with the cell proliferation marker Ki-67, suggesting an autocrine regulation by ERalpha in some primary breast tumors.

Methods: Colocalization of ERalpha with Ki-67 in ERalpha-positive breast cancer cell lines (MCF-7, T47D, and ZR75-1) was evaluated by immunofluorescent staining. Cell cycle phase dependent expression of ERalpha was determined by co-immunofluorescent staining of ERalpha and the major cyclins (D, E, A, B), and by flow cytometry analysis of ERalphahigh cells. To further confirm the autocrine action of ERalpha, MCF-7 cells were growth arrested by ICI182780 treatment, followed by treatment with EGFR inhibitor, before estrogen stimulation and analyses for colocalization of Ki-67 and ERalpha and cell cycle progression.

Results: Colocalization of ERalpha with Ki-67 was present in all three ERalpha-positive breast cancer cell lines. Unlike that in the normal mammary glands and the majority of primary breast tumors, ERalpha is highly expressed throughout the cell cycle in MCF-7 cells. Without E2 stimulation, MCF-7 cells released from ICI182780 treatment remain at G1 phase. E2 stimulation of ICI182780 treated cells, however, promotes the expression and colocalization of ERalpha and Ki-67 as well as the cell cycle progressing through the S and G2/M phases. Inhibition of EGFR signaling does not inhibit the autocrine action of ERalpha.

Conclusion: Our data indicate that ERalpha can mediate estrogen-induced cell proliferation in an autocrine mode in ERalpha-positive breast cancer cell lines. All of the three ERalpha-positive cell lines used in our study showed colocalization of ERalpha and Ki-67, indicating that these cell lines might be originated from primary tumor cells with autocrine regulation.

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E2 stimulation of ICI182780 treated cells induced the expression and colocalization of ERα and Ki-67. A, E2 stimulation of cells released from ICI182780 treatment. MCF-7 cells were treated with ICI182780 for 72–96 hr, then ICI182780 was removed by washing with serum-free medium. After removal of ICI182780, cells were treated by 5 nM E2 for 24 hr; control cells were replaced with medium without E2. Magnification, 200×. B, stimulation of E2 by adding E2 directly to cells under ICI182780 treatment. Cells were treated with 10 nM ICI182780 for 72–96 hr, then 100 nM E2 was added directly to the cells without removing ICI182780 for estrogen stimulation. The expression of ERα and Ki-67 was evaluated by immunofluorescent staining. Overlay of the ERα-staining and Ki-67 staining showed the colocalization (Merge). DAPI was used for nuclear staining. Magnification, 200×.
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Figure 4: E2 stimulation of ICI182780 treated cells induced the expression and colocalization of ERα and Ki-67. A, E2 stimulation of cells released from ICI182780 treatment. MCF-7 cells were treated with ICI182780 for 72–96 hr, then ICI182780 was removed by washing with serum-free medium. After removal of ICI182780, cells were treated by 5 nM E2 for 24 hr; control cells were replaced with medium without E2. Magnification, 200×. B, stimulation of E2 by adding E2 directly to cells under ICI182780 treatment. Cells were treated with 10 nM ICI182780 for 72–96 hr, then 100 nM E2 was added directly to the cells without removing ICI182780 for estrogen stimulation. The expression of ERα and Ki-67 was evaluated by immunofluorescent staining. Overlay of the ERα-staining and Ki-67 staining showed the colocalization (Merge). DAPI was used for nuclear staining. Magnification, 200×.

Mentions: To evaluate the effect of estrogen stimulation, cells were stimulated by adding E2 either after removal of ICI182780 or in the presence of ICI182780. We first evaluated the effect of estrogen stimulation after removal of ICI182780. MCF-7 cells were treated for ICI182780 for 72–96 hr and the treatment was stopped by washing with fresh serum-free medium to remove ICI182780. The cells were then stimulated with estrogen by adding medium containing 5 nM estrogen but without ICI182780. The control group of cells was replaced with medium without estrogen. In the cells stimulated with estrogen, both ERα and Ki-67 were induced with ERα and Ki-67 colocalized in most cells (Fig. 4A). In the control group of cells without estrogen stimulation, no high level of ERα or Ki-67 was observed although the cells were released from ICI182780 treatment. We also determined the effect of estrogen stimulation by adding estrogen directly to the cells still under ICI182780 treatment (Fig. 4B). The continuous presence of ICI182780 was to help minimize the secondary factors that might promote cell proliferation. Cells were treated with 10 nM ICI182780 for 72–96 hrs before the estrogen was added to the cells for a final concentration of 100 nM. Similar to the result shown in Fig. 4A, excessive estrogen stimulation in the presence of ICI182780 also induced the expression of high levels of ERα and Ki-67 with the two proteins colocalized in most cells (Fig. 4B). Collectively, these data indicate that activation of ERα by estrogen in ICI182780 arrested cells induces the expression of high levels of ERα and Ki-67 and their colocalization, supporting the autocrine mode of ERα action.


Autocrine regulation of cell proliferation by estrogen receptor-alpha in estrogen receptor-alpha-positive breast cancer cell lines.

Tan H, Zhong Y, Pan Z - BMC Cancer (2009)

E2 stimulation of ICI182780 treated cells induced the expression and colocalization of ERα and Ki-67. A, E2 stimulation of cells released from ICI182780 treatment. MCF-7 cells were treated with ICI182780 for 72–96 hr, then ICI182780 was removed by washing with serum-free medium. After removal of ICI182780, cells were treated by 5 nM E2 for 24 hr; control cells were replaced with medium without E2. Magnification, 200×. B, stimulation of E2 by adding E2 directly to cells under ICI182780 treatment. Cells were treated with 10 nM ICI182780 for 72–96 hr, then 100 nM E2 was added directly to the cells without removing ICI182780 for estrogen stimulation. The expression of ERα and Ki-67 was evaluated by immunofluorescent staining. Overlay of the ERα-staining and Ki-67 staining showed the colocalization (Merge). DAPI was used for nuclear staining. Magnification, 200×.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2636826&req=5

Figure 4: E2 stimulation of ICI182780 treated cells induced the expression and colocalization of ERα and Ki-67. A, E2 stimulation of cells released from ICI182780 treatment. MCF-7 cells were treated with ICI182780 for 72–96 hr, then ICI182780 was removed by washing with serum-free medium. After removal of ICI182780, cells were treated by 5 nM E2 for 24 hr; control cells were replaced with medium without E2. Magnification, 200×. B, stimulation of E2 by adding E2 directly to cells under ICI182780 treatment. Cells were treated with 10 nM ICI182780 for 72–96 hr, then 100 nM E2 was added directly to the cells without removing ICI182780 for estrogen stimulation. The expression of ERα and Ki-67 was evaluated by immunofluorescent staining. Overlay of the ERα-staining and Ki-67 staining showed the colocalization (Merge). DAPI was used for nuclear staining. Magnification, 200×.
Mentions: To evaluate the effect of estrogen stimulation, cells were stimulated by adding E2 either after removal of ICI182780 or in the presence of ICI182780. We first evaluated the effect of estrogen stimulation after removal of ICI182780. MCF-7 cells were treated for ICI182780 for 72–96 hr and the treatment was stopped by washing with fresh serum-free medium to remove ICI182780. The cells were then stimulated with estrogen by adding medium containing 5 nM estrogen but without ICI182780. The control group of cells was replaced with medium without estrogen. In the cells stimulated with estrogen, both ERα and Ki-67 were induced with ERα and Ki-67 colocalized in most cells (Fig. 4A). In the control group of cells without estrogen stimulation, no high level of ERα or Ki-67 was observed although the cells were released from ICI182780 treatment. We also determined the effect of estrogen stimulation by adding estrogen directly to the cells still under ICI182780 treatment (Fig. 4B). The continuous presence of ICI182780 was to help minimize the secondary factors that might promote cell proliferation. Cells were treated with 10 nM ICI182780 for 72–96 hrs before the estrogen was added to the cells for a final concentration of 100 nM. Similar to the result shown in Fig. 4A, excessive estrogen stimulation in the presence of ICI182780 also induced the expression of high levels of ERα and Ki-67 with the two proteins colocalized in most cells (Fig. 4B). Collectively, these data indicate that activation of ERα by estrogen in ICI182780 arrested cells induces the expression of high levels of ERα and Ki-67 and their colocalization, supporting the autocrine mode of ERα action.

Bottom Line: E2 stimulation of ICI182780 treated cells, however, promotes the expression and colocalization of ERalpha and Ki-67 as well as the cell cycle progressing through the S and G2/M phases.Inhibition of EGFR signaling does not inhibit the autocrine action of ERalpha.All of the three ERalpha-positive cell lines used in our study showed colocalization of ERalpha and Ki-67, indicating that these cell lines might be originated from primary tumor cells with autocrine regulation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Animal Science, Vermont Cancer Center, University of Vermont, Burlington, VT 05405, USA. htan1@uvm.edu

ABSTRACT

Background: Estrogen receptor-alpha (ERalpha) is essential for mammary gland development and is a major oncogene in breast cancer. Since ERalpha is not colocalized with the cell proliferation marker Ki-67 in the normal mammary glands and the majority of primary breast tumors, it is generally believed that paracrine regulation is involved in ERalpha mediated cell proliferation. In the paracrine model, ERalpha-positive cells don't proliferate but will release some paracrine growth factors to stimulate the neighboring cells to proliferate. In a subpopulation of cancer cells in some primary breast tumors, however, ERalpha does colocalize with the cell proliferation marker Ki-67, suggesting an autocrine regulation by ERalpha in some primary breast tumors.

Methods: Colocalization of ERalpha with Ki-67 in ERalpha-positive breast cancer cell lines (MCF-7, T47D, and ZR75-1) was evaluated by immunofluorescent staining. Cell cycle phase dependent expression of ERalpha was determined by co-immunofluorescent staining of ERalpha and the major cyclins (D, E, A, B), and by flow cytometry analysis of ERalphahigh cells. To further confirm the autocrine action of ERalpha, MCF-7 cells were growth arrested by ICI182780 treatment, followed by treatment with EGFR inhibitor, before estrogen stimulation and analyses for colocalization of Ki-67 and ERalpha and cell cycle progression.

Results: Colocalization of ERalpha with Ki-67 was present in all three ERalpha-positive breast cancer cell lines. Unlike that in the normal mammary glands and the majority of primary breast tumors, ERalpha is highly expressed throughout the cell cycle in MCF-7 cells. Without E2 stimulation, MCF-7 cells released from ICI182780 treatment remain at G1 phase. E2 stimulation of ICI182780 treated cells, however, promotes the expression and colocalization of ERalpha and Ki-67 as well as the cell cycle progressing through the S and G2/M phases. Inhibition of EGFR signaling does not inhibit the autocrine action of ERalpha.

Conclusion: Our data indicate that ERalpha can mediate estrogen-induced cell proliferation in an autocrine mode in ERalpha-positive breast cancer cell lines. All of the three ERalpha-positive cell lines used in our study showed colocalization of ERalpha and Ki-67, indicating that these cell lines might be originated from primary tumor cells with autocrine regulation.

Show MeSH
Related in: MedlinePlus