Induction of transforming growth factor-β receptor type II expression in estrogen receptor-positive breast cancer cells through SP1 activation by 5-aza-2'-deoxycytidine

S. Ammanamanchi, S. J. Kim, L. Z. Sun, M. G. Brattain

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Abstract

Previous studies suggest that estrogen receptor-positive (ER+) breast cancer cells acquire resistance to transforming growth factor-β (TGF-β) because of reduced expression levels of TGF-β receptor type H (RII). We now report that treatment of ER+ breast cancer cells with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-2'-dC) leads to accumulation of RII transcript and protein in three different cell lines. RII induction restored TGF-β response in MCF-7L breast cancer cells as indicated by the enhanced activity of a TGF-β responsive promoter-reporter construct (p3TP-Lux). A transiently transfected RII promoter-reporter element (RII- chloramphenicol acetyltransferase) showed an increase in activity in the 5- aza-2'-dC-treated MCF-7L cells compared with untreated cells, suggesting the activation of a transactivator of RII transcription. Using electrophoretic mobility shift assays, the enhanced binding of proteins from 5-aza-2'-dC- treated MCF-7L nuclear extracts to radiolabeled Sp1 oligonucleotides was demonstrated. An RII promoter-chloramphenicol acetyltransferase construct containing a mutation in the Sp1 site was not expressed in the 5-aza-2'-dC- treated MCF-7L cells, further demonstrating that induction of Sp1 activity by 5-aza-2'-dC in the MCF-7L cells was critical to RII expression. Northern analysis indicated that 5-aza-2'-dC treatment did not affect the Sp1 transcript levels. Western blot analysis revealed an increase of Sp1 protein in the 5-aza-2'-dC-treated MCF-7L cells, but there was no change in the c- Jun levels. Studies after cyclohexamide treatment suggested an increase in the Sp1 protein stability from the 5-aza-2'-dC-treated MCF-7L extracts compared with untreated control extracts. These results indicate that the transcriptional repression of RII in the ER+ breast cancer cells is caused by suboptimal activity of Sp1, whereas treatment with 5-aza2'-dC stabilizes the protein thus increasing steady-state Sp1 levels and thereby leads to enhanced RII transcription and subsequent restoration of TGF-β sensitivity.

Original languageEnglish (US)
Pages (from-to)16527-16534
Number of pages8
JournalJournal of Biological Chemistry
Volume273
Issue number26
DOIs
StatePublished - Jun 26 1998

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decitabine
Growth Factor Receptors
Transforming Growth Factors
Estrogen Receptors
Chemical activation
Cells
Breast Neoplasms
Chloramphenicol O-Acetyltransferase
Transcription
Proteins
Electrophoretic mobility
Trans-Activators
Methyltransferases
Oligonucleotides
Restoration
Assays
Carrier Proteins
Protein Stability
Electrophoretic Mobility Shift Assay
DNA

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Induction of transforming growth factor-β receptor type II expression in estrogen receptor-positive breast cancer cells through SP1 activation by 5-aza-2'-deoxycytidine. / Ammanamanchi, S.; Kim, S. J.; Sun, L. Z.; Brattain, M. G.

In: Journal of Biological Chemistry, Vol. 273, No. 26, 26.06.1998, p. 16527-16534.

Research output: Contribution to journalArticle

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abstract = "Previous studies suggest that estrogen receptor-positive (ER+) breast cancer cells acquire resistance to transforming growth factor-β (TGF-β) because of reduced expression levels of TGF-β receptor type H (RII). We now report that treatment of ER+ breast cancer cells with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-2'-dC) leads to accumulation of RII transcript and protein in three different cell lines. RII induction restored TGF-β response in MCF-7L breast cancer cells as indicated by the enhanced activity of a TGF-β responsive promoter-reporter construct (p3TP-Lux). A transiently transfected RII promoter-reporter element (RII- chloramphenicol acetyltransferase) showed an increase in activity in the 5- aza-2'-dC-treated MCF-7L cells compared with untreated cells, suggesting the activation of a transactivator of RII transcription. Using electrophoretic mobility shift assays, the enhanced binding of proteins from 5-aza-2'-dC- treated MCF-7L nuclear extracts to radiolabeled Sp1 oligonucleotides was demonstrated. An RII promoter-chloramphenicol acetyltransferase construct containing a mutation in the Sp1 site was not expressed in the 5-aza-2'-dC- treated MCF-7L cells, further demonstrating that induction of Sp1 activity by 5-aza-2'-dC in the MCF-7L cells was critical to RII expression. Northern analysis indicated that 5-aza-2'-dC treatment did not affect the Sp1 transcript levels. Western blot analysis revealed an increase of Sp1 protein in the 5-aza-2'-dC-treated MCF-7L cells, but there was no change in the c- Jun levels. Studies after cyclohexamide treatment suggested an increase in the Sp1 protein stability from the 5-aza-2'-dC-treated MCF-7L extracts compared with untreated control extracts. These results indicate that the transcriptional repression of RII in the ER+ breast cancer cells is caused by suboptimal activity of Sp1, whereas treatment with 5-aza2'-dC stabilizes the protein thus increasing steady-state Sp1 levels and thereby leads to enhanced RII transcription and subsequent restoration of TGF-β sensitivity.",
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N2 - Previous studies suggest that estrogen receptor-positive (ER+) breast cancer cells acquire resistance to transforming growth factor-β (TGF-β) because of reduced expression levels of TGF-β receptor type H (RII). We now report that treatment of ER+ breast cancer cells with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-2'-dC) leads to accumulation of RII transcript and protein in three different cell lines. RII induction restored TGF-β response in MCF-7L breast cancer cells as indicated by the enhanced activity of a TGF-β responsive promoter-reporter construct (p3TP-Lux). A transiently transfected RII promoter-reporter element (RII- chloramphenicol acetyltransferase) showed an increase in activity in the 5- aza-2'-dC-treated MCF-7L cells compared with untreated cells, suggesting the activation of a transactivator of RII transcription. Using electrophoretic mobility shift assays, the enhanced binding of proteins from 5-aza-2'-dC- treated MCF-7L nuclear extracts to radiolabeled Sp1 oligonucleotides was demonstrated. An RII promoter-chloramphenicol acetyltransferase construct containing a mutation in the Sp1 site was not expressed in the 5-aza-2'-dC- treated MCF-7L cells, further demonstrating that induction of Sp1 activity by 5-aza-2'-dC in the MCF-7L cells was critical to RII expression. Northern analysis indicated that 5-aza-2'-dC treatment did not affect the Sp1 transcript levels. Western blot analysis revealed an increase of Sp1 protein in the 5-aza-2'-dC-treated MCF-7L cells, but there was no change in the c- Jun levels. Studies after cyclohexamide treatment suggested an increase in the Sp1 protein stability from the 5-aza-2'-dC-treated MCF-7L extracts compared with untreated control extracts. These results indicate that the transcriptional repression of RII in the ER+ breast cancer cells is caused by suboptimal activity of Sp1, whereas treatment with 5-aza2'-dC stabilizes the protein thus increasing steady-state Sp1 levels and thereby leads to enhanced RII transcription and subsequent restoration of TGF-β sensitivity.

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