J Breast Cancer.  2008 Jun;11(2):56-63. 10.4048/jbc.2008.11.2.56.

Changes of Coregulators, MAP Kinase Activity and p27/kip1 with Estrogen or Antiestrogen Treatment in Breast Cancer Cell Line

• Park S
• Heo MK
• Lee MJ
• Kim JH
• Park BW

• KMID: 2241932
• DOI: http://doi.org/10.4048/jbc.2008.11.2.56

Abstract

PURPOSE: Estrogen, various polypeptide hormones and growth factors are associated with the development and progression of breast cancer. Coregulatory proteins are also associated with estrogen receptor (ER) transcriptional activity and tamoxifen resistance. Therefore, it is necessary to investigate the change of coregulator mRNAs and various cell proliferation proteins and cell cycle-related proteins after treatment with estrogen or antiestrogen.
METHODS
MCF-7 cells were maintained in dextran-coated charcoal stripped 10% Dulbecco's Modified Eagle Medium (DMEM). To measure the change of the coactivators' (src-1, P/CAF, CBP, AIB1) mRNAs and corepressors' (SMRT, N-coR) mRNAs, multiple PCR was carried out using specific primers. In addition, intracellular proteins related to cell proliferation and cell cycle regulation were measured by performing Western blotting after treatment with estrogen or tamoxifen. The change of mitogen activated protein kinases was also measured by performing Western after tamoxifen treatment for 4 weeks.
RESULTS
Coactivator mRNAs expression rapidly decreased in 15 min after estrogen treatment but this recovered to the initial level in 3 hr. The pattern was similar for the case of tamoxifen treatment. Corepressor mRNAs expression rapidly decreased in 15 min after estrogen treatment and it remained at a lower level until 24 hr after estrogen treatment. With tamoxifen treatment, the initial response was similar to the cases of estrogen treatment, but the xpression gradually increased 3 hr after tamoxifen treatment. Treatment of estrogen induced intracellular concentrations of c-myc and Ki-67 and it increased nuclear translocation of NF-kappaB and phosphor-ERK and it decreased the intracellular cell cycle suppressor p27/kip1. Tamoxifen treatment increased nuclear p27/kip1 but it decreased c-myc, NF-kappaB and phosphor-ERK. Long-term (4 weeks) treatment of tamoxifen was associated with decrease of activated ERK and p38 but there was no change in phospho-Akt level.
CONCLUSION
Estrogen induced cell proliferation and the survival pathway-related factors, but it decreased the cell cycle suppressor p27/kip1. Long-term treatment with antiestrogen tamoxifen might decrease the MAPK activities in ERalpha-expressing tumor cells.

Keyword

Estrogen receptor; Coregulatory protein; Tamoxifen; p27/kip1; MAP kinase

MeSH Terms

Blotting, Western
Breast
Breast Neoplasms
Cell Cycle
Cell Line
Cell Proliferation
Charcoal
Eagles
Estrogen Receptor Modulators
Estrogens
Intercellular Signaling Peptides and Proteins
MCF-7 Cells
Mitogen-Activated Protein Kinases
NF-kappa B
Peptide Hormones
Phosphotransferases
Polymerase Chain Reaction
Proteins
RNA, Messenger
Tamoxifen
Charcoal
Estrogen Receptor Modulators
Estrogens
Intercellular Signaling Peptides and Proteins
Mitogen-Activated Protein Kinases
NF-kappa B
Peptide Hormones
Phosphotransferases
Proteins
RNA, Messenger
Tamoxifen

Figure

• 

  Fig 1 Sequential change of the mRNA expression of coactivators in MCF-7 cells after treatment of estrogen or tamoxifen. Coactivator mRNAs expression rapidly decreased in 15-30 min after estrogen treatment, but recovered to initial level in 3 hr. With tamoxifen treatment, the pattern was similar as in case of estrogen treatment.

• 

  Fig 2 Sequential change of the mRNA expression of corepressors in MCF-7 cells after treatment of estrogen or tamoxifen. Corepressor mRNA expression rapidly decreased in 15-30 min after estrogen treatment and remained lower level until 24 hr after estrogen treatment. With tamoxifen treatment, initial response was similar as in cases of estrogen treatment, but the expression gradually increased since 3 hr after tamoxifen treatment.

• 

  Fig 3 Expression of various proteins in MCF-7 cells after 24 hrtreatment of estrogen (10 nM) or tamoxifen (1 µM) or DMSO. (A) Nuclear expression of Ki-67, c-Myc, and cytosolic expression of phospho-ERK increased but both nuclear and cytosolic expression of p27/kip1 decreased after estrogen treatment. There was no significant change after tamoxifen treatment. (B) ERαexpression was downregulated with treatment of estrogen, but there was no change of ERαexpression with additional TGFβ1 treatment.

• 

  Fig 4 The change of MAP kinases activities in MCF-7 cells after 4 weeks-treatment with tamoxifen (1 µM) or DMSO. The activity of ERK and p38 was decreased after 4 weeks-treatment with tamoxifen but the change of Akt activity was not remarkable.

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