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

Wl Wang,W Porter,S Safe,Js Thomsen,M Moore

doi:10.1038/bjc.1996.55

Abstract

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 CYP1A1 gene expression or chloramphenicol acetyl transferase (CAT) activity in cells transiently transfected with pRNH11c or pMCAT5.12, which are Ah-responsive 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 pRNH11c 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 C-terminal 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 pRNH11c is modulated differentially by ER domains that are present in the N- and C-terminal regions of the ER.

Highlights

The results show that TCDD did not induce CYPlAI in this cell line; the cells expressed the aryl hydrocarbons (Ahs) receptor (AhR) and TCDD induced formation of a 6.9 S nuclear AhR complex, which bound to a DRE in a gel electrophoretic mobility shift assay
Both of these plasmids were kindly provided by Dr R Hines (Wayne State University, Detroit, MI, USA). pMCAT5.12 is a construct containing the mouse DRE2 fused to the mouse mammary tumour virus (MMTV) promoter driving the chloramphenicol acetyl transferase (CAT) gene and was provided by Dr JP Whitlock (Stanford University)
The effects of human ER (hER) expression on restoration of Ah responsiveness in Hs578T cells was investigated in cells cotransfected with pRNHllc+hER

Summary

Methods

Chemicals and biochemicalsTCDD and 2,3,7,8-tetrachlorodibenzofuran (TCDF) (>99% pure) were prepared in this laboratory. [3H]TCDD (37 Ci mmol-') was prepared in this laboratory and purified by high-pressure liquid chromatography (>98% pure). The plasmid pRNHIlc contains the regulatory human CYPlAl region from the Taql site at -1142 to the BcII site at + 2434 fused to the bacterial CAT reporter gene (Hines et al, 1988). PRNH21c was derived from pRNHIlc and is deleted from -831 to -560 (Hines et al, 1988) Both of these plasmids were kindly provided by Dr R Hines (Wayne State University, Detroit, MI, USA). The hER plasmid was a generous gift from Dr Ming Jer Tsai (Baylor College of Medicine). This plasmid contains the human ER cDNA. Arnt and AhR cDNAs were kindly provided by Drs Bradfield and Hankinson (Burbach et al, 1992; Reyes et al, 1992) and constructed into pcDNAI and pcDNA3 vectors respectively

Results

Discussion

Conclusion