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A novel androgen receptor-binding element modulates Cdc6 transcription in prostate cancer cells during cell-cycle progression.

Jin F, Fondell JD - Nucleic Acids Res. (2009)

Bottom Line: AR binds at a distinct androgen-response element (ARE) in the Cdc6 promoter that is functionally required for androgen-dependent Cdc6 transcription.We found that peak AR occupancy at the novel ARE occurs during the G1/S phase concomitant with peak Cdc6 mRNA expression.We also identified several of the coactivators and corepressors involved in AR-dependent Cdc6 transcriptional regulation in vivo and further characterized ligand-induced alterations in histone acetylation and methylation at the Cdc6 promoter.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Robert Wood Johnson Medical School, UMDNJ, Piscataway, NJ 08854, USA.

ABSTRACT
The androgen receptor (AR) plays a pivotal role in the onset and progression of prostate cancer by promoting cellular proliferation. Recent studies suggest AR is a master regulator of G1-S progression and possibly a licensing factor for DNA replication yet the mechanisms remain poorly defined. Here we report that AR targets the human Cdc6 gene for transcriptional regulation. Cdc6 is an essential regulator of DNA replication in eukaryotic cells and its mRNA expression is inversely modulated by androgen or antiandrogen treatment in androgen-sensitive prostate cancer cells. AR binds at a distinct androgen-response element (ARE) in the Cdc6 promoter that is functionally required for androgen-dependent Cdc6 transcription. We found that peak AR occupancy at the novel ARE occurs during the G1/S phase concomitant with peak Cdc6 mRNA expression. We also identified several of the coactivators and corepressors involved in AR-dependent Cdc6 transcriptional regulation in vivo and further characterized ligand-induced alterations in histone acetylation and methylation at the Cdc6 promoter. Significantly, AR silencing in prostate cancer cells markedly decreases Cdc6 expression and androgen-dependent cellular proliferation. Collectively, our results suggest that Cdc6 is a key regulatory target for AR and provide new insights into the mechanisms of prostate cancer cell proliferation.

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The ARE in Cdc6 promoter confers androgen-dependent transcriptional activation. (A) Schematic representation of human Cdc6 promoter-luciferase constructs. (B) Seventy-two hours post-androgen starvation, LNCaP cells were transfected with the pGL3-Cdc6-1.7 kb reporter construct and then treated with or without 10 nM R1881 for 24 h. (C) LNCaP cells cultured in 10% FBS were transfected with pGL3-Cdc6-1.7 kb and then treated with 30 µM Casodex for 10 h. (D) Seventy-two hours post-androgen starvation, LNCaP cells were transfected with the indicated reporter constructs and then treated with or without 10 nM R1881 for 24 h. (E–G) DU145 cells cultured in 10% FBS were cotransfected with pGL2-Cdc6-ARE and the indicated nuclear receptor expression constructs and then treated with or without 10 nM R1881 (panel E) 10 nM dexamethasone (panel F) or 100 nM T3 (panel G) for 24 h. Whole cell extract were prepared and assayed for luciferase activity. Luciferase values are presented as the mean ± SE of triplicate transfections.
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Figure 4: The ARE in Cdc6 promoter confers androgen-dependent transcriptional activation. (A) Schematic representation of human Cdc6 promoter-luciferase constructs. (B) Seventy-two hours post-androgen starvation, LNCaP cells were transfected with the pGL3-Cdc6-1.7 kb reporter construct and then treated with or without 10 nM R1881 for 24 h. (C) LNCaP cells cultured in 10% FBS were transfected with pGL3-Cdc6-1.7 kb and then treated with 30 µM Casodex for 10 h. (D) Seventy-two hours post-androgen starvation, LNCaP cells were transfected with the indicated reporter constructs and then treated with or without 10 nM R1881 for 24 h. (E–G) DU145 cells cultured in 10% FBS were cotransfected with pGL2-Cdc6-ARE and the indicated nuclear receptor expression constructs and then treated with or without 10 nM R1881 (panel E) 10 nM dexamethasone (panel F) or 100 nM T3 (panel G) for 24 h. Whole cell extract were prepared and assayed for luciferase activity. Luciferase values are presented as the mean ± SE of triplicate transfections.

Mentions: We next sought to determine whether the ARE in the Cdc6 gene promoter is responsible for the androgen-induced increase in Cdc6 mRNA observed in androgen-sensitive prostate cancer cells (Figure 1). To address this question, we first measured transcription from a Cdc6 promoter-luciferase reporter gene (pGL3-Cdc6-1.7 kb) containing 1.7 kb of the native human Cdc6 promoter region (−1700 to +7) (20). Androgen-starved LNCaP cells were transfected with either pGL3-Cdc6-1.7 kb or an empty pGL3 control and then cultured with or without the synthetic androgen R1881 (Figure 4B). Basal expression from the pGL3-Cdc6-1.7 kb construct (minus R1881) was considerably higher than that observed with the pGL3 control, presumably due to the presence of Sp1 and E2F sites in the Cdc6 promoter construct near the transcription start site. Consistent with the idea that the ARE region is required for androgen-dependent Cdc6 transcription, addition of R1881 stimulated transcription from pGL3-Cdc6-1.7 kb greater than 2-fold (Figure 4B), whereas addition of the antiandrogen Casodex reduced transcription by nearly 2-fold (Figure 4C). Moreover, androgen-dependent transcription from a Cdc6 promoter reporter construct (pGL2-Cdc6-ARE) containing only the ARE and GATA region (bps −781 to −575) was stimulated nearly 4-fold in LNCaP cells (Figure 4D) and in prostate cancer DU145 cells transiently transfected with AR (Figure 4E).Figure 4.


A novel androgen receptor-binding element modulates Cdc6 transcription in prostate cancer cells during cell-cycle progression.

Jin F, Fondell JD - Nucleic Acids Res. (2009)

The ARE in Cdc6 promoter confers androgen-dependent transcriptional activation. (A) Schematic representation of human Cdc6 promoter-luciferase constructs. (B) Seventy-two hours post-androgen starvation, LNCaP cells were transfected with the pGL3-Cdc6-1.7 kb reporter construct and then treated with or without 10 nM R1881 for 24 h. (C) LNCaP cells cultured in 10% FBS were transfected with pGL3-Cdc6-1.7 kb and then treated with 30 µM Casodex for 10 h. (D) Seventy-two hours post-androgen starvation, LNCaP cells were transfected with the indicated reporter constructs and then treated with or without 10 nM R1881 for 24 h. (E–G) DU145 cells cultured in 10% FBS were cotransfected with pGL2-Cdc6-ARE and the indicated nuclear receptor expression constructs and then treated with or without 10 nM R1881 (panel E) 10 nM dexamethasone (panel F) or 100 nM T3 (panel G) for 24 h. Whole cell extract were prepared and assayed for luciferase activity. Luciferase values are presented as the mean ± SE of triplicate transfections.
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Figure 4: The ARE in Cdc6 promoter confers androgen-dependent transcriptional activation. (A) Schematic representation of human Cdc6 promoter-luciferase constructs. (B) Seventy-two hours post-androgen starvation, LNCaP cells were transfected with the pGL3-Cdc6-1.7 kb reporter construct and then treated with or without 10 nM R1881 for 24 h. (C) LNCaP cells cultured in 10% FBS were transfected with pGL3-Cdc6-1.7 kb and then treated with 30 µM Casodex for 10 h. (D) Seventy-two hours post-androgen starvation, LNCaP cells were transfected with the indicated reporter constructs and then treated with or without 10 nM R1881 for 24 h. (E–G) DU145 cells cultured in 10% FBS were cotransfected with pGL2-Cdc6-ARE and the indicated nuclear receptor expression constructs and then treated with or without 10 nM R1881 (panel E) 10 nM dexamethasone (panel F) or 100 nM T3 (panel G) for 24 h. Whole cell extract were prepared and assayed for luciferase activity. Luciferase values are presented as the mean ± SE of triplicate transfections.
Mentions: We next sought to determine whether the ARE in the Cdc6 gene promoter is responsible for the androgen-induced increase in Cdc6 mRNA observed in androgen-sensitive prostate cancer cells (Figure 1). To address this question, we first measured transcription from a Cdc6 promoter-luciferase reporter gene (pGL3-Cdc6-1.7 kb) containing 1.7 kb of the native human Cdc6 promoter region (−1700 to +7) (20). Androgen-starved LNCaP cells were transfected with either pGL3-Cdc6-1.7 kb or an empty pGL3 control and then cultured with or without the synthetic androgen R1881 (Figure 4B). Basal expression from the pGL3-Cdc6-1.7 kb construct (minus R1881) was considerably higher than that observed with the pGL3 control, presumably due to the presence of Sp1 and E2F sites in the Cdc6 promoter construct near the transcription start site. Consistent with the idea that the ARE region is required for androgen-dependent Cdc6 transcription, addition of R1881 stimulated transcription from pGL3-Cdc6-1.7 kb greater than 2-fold (Figure 4B), whereas addition of the antiandrogen Casodex reduced transcription by nearly 2-fold (Figure 4C). Moreover, androgen-dependent transcription from a Cdc6 promoter reporter construct (pGL2-Cdc6-ARE) containing only the ARE and GATA region (bps −781 to −575) was stimulated nearly 4-fold in LNCaP cells (Figure 4D) and in prostate cancer DU145 cells transiently transfected with AR (Figure 4E).Figure 4.

Bottom Line: AR binds at a distinct androgen-response element (ARE) in the Cdc6 promoter that is functionally required for androgen-dependent Cdc6 transcription.We found that peak AR occupancy at the novel ARE occurs during the G1/S phase concomitant with peak Cdc6 mRNA expression.We also identified several of the coactivators and corepressors involved in AR-dependent Cdc6 transcriptional regulation in vivo and further characterized ligand-induced alterations in histone acetylation and methylation at the Cdc6 promoter.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Robert Wood Johnson Medical School, UMDNJ, Piscataway, NJ 08854, USA.

ABSTRACT
The androgen receptor (AR) plays a pivotal role in the onset and progression of prostate cancer by promoting cellular proliferation. Recent studies suggest AR is a master regulator of G1-S progression and possibly a licensing factor for DNA replication yet the mechanisms remain poorly defined. Here we report that AR targets the human Cdc6 gene for transcriptional regulation. Cdc6 is an essential regulator of DNA replication in eukaryotic cells and its mRNA expression is inversely modulated by androgen or antiandrogen treatment in androgen-sensitive prostate cancer cells. AR binds at a distinct androgen-response element (ARE) in the Cdc6 promoter that is functionally required for androgen-dependent Cdc6 transcription. We found that peak AR occupancy at the novel ARE occurs during the G1/S phase concomitant with peak Cdc6 mRNA expression. We also identified several of the coactivators and corepressors involved in AR-dependent Cdc6 transcriptional regulation in vivo and further characterized ligand-induced alterations in histone acetylation and methylation at the Cdc6 promoter. Significantly, AR silencing in prostate cancer cells markedly decreases Cdc6 expression and androgen-dependent cellular proliferation. Collectively, our results suggest that Cdc6 is a key regulatory target for AR and provide new insights into the mechanisms of prostate cancer cell proliferation.

Show MeSH
Related in: MedlinePlus