Effect of transient expression of the oestrogen receptor on constitutive and inducible CYP1A1 in Hs578T human breast cancer cells

Abstract

Summary Hs578T human breast cancer cells are an oestrogen receptor (ER)-negative cell line. Treatment of these cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in formation of a 6.9 S nuclear aryl hydrocarbon (Ah) receptor complex, which bound to a [32P]dioxin-responsive element in a gel electrophoretic mobility shift assay. However, TCDD does not induce CYPlAl gene expression or chloramphenicol acetyl transferase (CAT) activity in cells transiently transfected with pRNHllc or pMCAT5.12, which are Ahresponsive plasmids derived from the 5'-flanking region of the human and murine CYP1A1 genes respectively. Restoration of Ah responsiveness was investigated by co-transfecting Hs578T cells with pRNH1 lc or pMCAT5.12 and plasmids that express the ER (hER), Ah receptor (AhR) and AhR nuclear translocator (Arnt) proteins. ER expression resulted in significantly increased basal CAT activity; however, TCDD did not induce CAT activity in the transiently transfected cells. Expression of the AhR or Arnt proteins did not alter basal or inducible CAT activity. Expression of N-or C-terminal truncated ER in Hs578T resulted in differential regulation of Ah responsiveness. In Hs578T cells transiently expressing the ER, which contains Cterminal deletions (amino acids 282-595), basal CAT activity was also increased; however, Ah responsiveness was not restored. In contrast, transient expression of N-terminal-deleted (amino acids 1 -178) ER resulted in a marked decrease in basal CAT activity but a restoration of Ah responsiveness. These results suggest that basal and inducible CAT activity in Hs578T cells transiently transfected with pRNHl lc is modulated differentially by ER domains that are present in the N-and C-terminal regions of the ER.

The aryl hydrocarbon receptor constitutively represses c-myc transcription in human mammary tumor cells

“…(Previous reports indicated some difficulty in inducing CYP1A1 promoter-driven CAT reporter activity in human tumor cells ( (Thomsen et al, 1994;Wang et al, 1996). However, we have noted that the ease with which AhR-dependent reporter activity is induced is highly sensitive to culture conditions, with confluent or near-confluent monolayers downregulating their AhR levels and CYP1A1 inducibility.)…”

Activation of telomerase in BeWo cells by estrogen and 2,3,7,8-tetrachlorodibenzo-p-dioxin in co-operation with c-Myc

“…It has also been previously demonstrated that CYP1A1 induction is influenced by the estrogen receptor (ER) and that a functional ER is required for this activation (13)(14)(15). In more recent studies, it was demonstrated that the presence of 17-ß estradiol (E2) in ovariectomized rats could significantly influence the induction of CYP1A1 by TCDD (16).…”

Interaction between Dioxin Signaling and Sex Steroid Hormones

“…How- tent, 24) and Ah responsiveness is dependent not only on the expression of AhR but also ER-α levels. [24][25][26] ER-negative breast cancer cell lines such as MDA-MB-231 and Hs578T are normally not responsive to Ah, but transient transfection of ER-α into these cells restores their Ah responsiveness. 25,26) Detailed sequential reporter gene assays have revealed that both the N-and C-terminal transactivation domains of ER-α are responsible for AhR responsiveness.…”

“…[24][25][26] ER-negative breast cancer cell lines such as MDA-MB-231 and Hs578T are normally not responsive to Ah, but transient transfection of ER-α into these cells restores their Ah responsiveness. 25,26) Detailed sequential reporter gene assays have revealed that both the N-and C-terminal transactivation domains of ER-α are responsible for AhR responsiveness. 26) The mechanisms involved are unclear, but several possibilities can be speculated: 1) ER-α might interact with liganded AhR-ARNT complexes directly or through some bridging factors; and 2) ER-α might displace negative regulatory factors or facilitate the binding of critical transcription factors to the upstream promoter region.…”

“…25,26) Detailed sequential reporter gene assays have revealed that both the N-and C-terminal transactivation domains of ER-α are responsible for AhR responsiveness. 26) The mechanisms involved are unclear, but several possibilities can be speculated: 1) ER-α might interact with liganded AhR-ARNT complexes directly or through some bridging factors; and 2) ER-α might displace negative regulatory factors or facilitate the binding of critical transcription factors to the upstream promoter region. One report has indicated that ER-α does not interact directly with the liganded AhR-ARNT complex, 27) but both of these can physically interact with Sp1 protein.…”