Estrogen regulation of cyclin D1 gene expression in ZR-75 breast cancer cells involves multiple enhancer elements.

Cyclin D1 gene expression is induced by 17beta-estradiol (E2) in human breast cancer cells and is important for progression of cells through the G(1) phase of the cell cycle. The mechanism of activation of cyclin D1 is mitogen- and cell context-dependent, and this study describes the role of multiple promoter elements required for induction of cyclin D1 by E2 in estrogen receptor (ER)-positive ZR-75 breast cancer cells. Transcriptional activation of cyclin D1 by E2 was dependent, in part, on a proximal cAMP-response element at -66, and this was linked to induction of protein kinase A-dependent pathways. These results contrasted to a recent report showing that induction of cyclin D1 by E2 in ER-positive MCF-7 and HeLa cells was due to up-regulation of c-jun and subsequent interaction of c-Jun-ATF-2 with the CRE. Moreover, further examination of the proximal region of the cyclin D1 promoter showed that three GC-rich Sp1-binding sites at -143 to -110 were also E2-responsive, and interaction of ERalpha and Sp1 proteins at these sites was confirmed by electromobility shift and chromatin immunoprecipitation assays. Thus, induction of cyclin D1 by E2 in ZR-75 cells is regulated through nuclear ERalpha/Sp1 and epigenetic protein kinase A activation pathways, and our results suggest that this mechanism may be cell context-dependent even among ER-positive breast cancer cell lines.

Transcriptional activation of heat shock protein 27 gene expression by 17beta-estradiol and modulation by antiestrogens and aryl hydrocarbon receptor agonists.

Heat shock protein 27 (Hsp 27) is expressed in mammary tumors and may play a role in tumor growth and response to anti-neoplastic drug therapy. 17beta-Estradiol (E2) induces Hsp 27 mRNA levels in MCF-7 human breast cancer cells, and we have investigated the comparative inhibitory mechanisms using the aryl hydrocarbon receptor (AhR) agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the direct-acting antiestrogen ICI 164,384. TCDD inhibited E2-induced Hsp 27 gene expression and analysis of the Hsp 27 gene promoter showed that the inhibitory response was associated with AhR interactions with a pentanucleotide motif at -3 to +2 in the promoter that corresponded to the core sequence of a dioxin responsive element. In contrast, ICI 164,384 induced Hsp 27 gene expression and reporter gene activity in MCF-7 cells and this represents one of the few examples of the estrogen receptor-alpha (ERalpha) agonist activity of the 'pure' antiestrogen ICI 164,384.

Estrogen and aryl hydrocarbon receptor expression and crosstalk in human Ishikawa endometrial cancer cells.

Ishikawa endometrial cancer cells express the estrogen receptor (ER), and this study investigates aryl hydrocarbon receptor (AhR) expression and inhibitory AhR-ER crosstalk in this cell line. Treatment of Ishikawa cells with the AhR agonist [3H]2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) gave a radiolabeled nuclear complex that sedimented at 6.0 S in sucrose density gradients, and Western blot analysis confirmed that Ishikawa cells expressed human AhR and AhR nuclear translocator (Arnt) proteins. Treatment of Ishikawa cells with 10 nM TCDD induced a 9.7-fold increase in CYP1A1-dependent ethoxyresorufin O-deethylase (EROD) activity and a 10.5-fold increase in chloramphenicol acetyltransferase (CAT) activity in cells transfected with pRNH11c containing an Ah-responsive human CYP1A1 gene promoter insert (-1142 to +2434). Inhibitory AhR-ER crosstalk was investigated in Ishikawa cells using E2-induced cell proliferation and transcriptional activation assays in cells transfected with E2-responsive constructs containing promoter inserts from the progesterone receptor and vitellogenin A2 genes. AhR agonists including TCDD, benzo[a]pyrene (BaP) and 6-methyl-1,3,8-trichlorodibenzofuran, inhibited 32-47% of the E2-induced responses. In contrast, neither estrogen nor progesterone inhibited EROD activity induced by TCDD in Ishikawa cells, whereas inhibitory ER-AhR crosstalk was observed in ECC-1 endometrial cells suggesting that these interactions were cell context-dependent.

Estrogen and aryl hydrocarbon responsiveness of ECC-1 endometrial cancer cells.

ECC-1 endometrial cancer cells express estrogen receptor alpha (ER(alpha)), and 17beta-estradiol (E2) induces cell proliferation, cathepsin D mRNA levels, and reporter gene activity in cells transiently transfected with constructs derived from the human cathepsin D and creatine kinase B (pCD and pCKB, respectively) gene promoters. The comparative antiestrogenic activity of aryl hydrocarbon receptor (AhR) agonists and ER(alpha) antagonists were also determined in these endometrial cancer cells. A functional AhR was expressed in ECC-1 cells and AhR agonists including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) inhibited E2-induced cell proliferation and transactivation. This was comparable to inhibitory AhR-ER crosstalk in breast cancer cell lines. The pure ER antagonist ICI 182,780 also exhibited antiestrogenic activity in ECC-1 cells; however, the results obtained for 4'-hydroxytamoxifen were response-specific. 4'-Hydroxytamoxifen alone did not induce ECC-1 cell proliferation but completely inhibited E2-induced cell proliferation. 4'-Hydroxytamoxifen primarily exhibited ER antagonist activities in transactivation assays and this contrasted to the predominant ER agonist responses observed in other endometrial cancer cell lines. The unique cellular context of ECC-1 cells was confirmed using pCKB and constructs expressing wild-type ER or ER variants expressing activation function 1 (AF1) or AF2 (ER-AF1 and ER-AF2, respectively). 4'-Hydroxytamoxifen did not induce reporter gene activity in cells cotransfected with pCKB and ER-AF1 or ER-AF2; however, in cotreatment studies (4'-hydroxytamoxifen plus E2), 4'-hydroxytamoxifen inhibited E2-induced transcriptional activation by ER-AF1 or ER-AF2. Thus, the primarily antiestrogenic activity observed for 4'-hydroxytamoxifen in ECC-1 cells may be related to the inability to activate gene expression through AF1-dependent pathways.

Estrogen- and antiestrogen-responsiveness of HEC1A endometrial adenocarcinoma cells in culture.

HEC1A endometrial cancer cells express the wild-type form of the estrogen receptor (ER) and 17beta-estradiol (E2) induces proliferation of these cells. In contrast, tamoxifen only causes a minimal increase (<20%) in cell proliferation. In HEC1A cells transiently transfected with the C3-Luc plasmid derived from the complement C3 gene, both E2 and tamoxifen exhibited ER agonist activity and tamoxifen was also a partial antagonist for this response. The relative ER agonist/antagonist activities of E2, tamoxifen and ICI 182,780 were also investigated in HEC1A1 cells transiently transfected with two E2-responsive plasmids, pCATHD-CAT and pCKB-CAT which contain 5'-promoter inserts from the cathepsin D and creatine kinase B genes, respectively. The results showed that E2 and tamoxifen induced reporter gene activity in cells transiently transfected with both constructs. ICI 182,780 exhibited partial ER agonist activity only in cells transiently transfected with pCKB-CAT and antagonized E2-induced reporter gene activity using both the CKB- and CATHD-derived constructs. These results demonstrate that HEC1A endometrial cancer cells are E2-responsive and represent a useful cell culture model for understanding hormone/antihormone-induced endometrial cell responses.