The 5′ flanking region of the human pS2 gene mediates its transcriptional activation by estrogen in MCF-7 cells (original) (raw)
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PNAS
We have shown previously that an increase in the level of accumulated pS2 mRNA is first detectable in MCF-7 cells after 3 hr of estradiol treatment. Using in vitro nuclear run-on transcription with nuclei prepared from MCF-7 cells grown in the presence of estradiol or in estradiol-stripped medium, we demonstrate here that expression of the pS2 gene is controlled by estrogen at the transcriptional level, Induction of transcription is a very early event that is already apparent within 15 min after addition of estradiol to the culture medium. In addition, pretreatment of the cells with the protein synthesis inhibitor cycloheximide does not prevent induction of pS2 gene transcription, indicating that it corresponds to a primary effect of estrogen. The pS2 gene in MCF-7 cells represents a unique example of a human gene whose transcription is directly controlled by estrogen.
Regulation of the estrogen-responsive pS2 gene in MCF-7 human breast cancer cells
2000
To understand how hormones and antihormones regulate transcription of estrogen-responsive genes, in vivo footprinting was used to examine the endogenous pS2 gene in MCF-7 cells. While the consensus pS2 estrogen response element (ERE) half site was protected in the absence of hormone, both the consensus and imperfect ERE half sites were protected in the presence of estrogen. 4-Hydroxytamoxifen and ICI 182,780 elicited distinct footprinting patterns, which differed from those observed with vehicle-or with estrogen-treated cells suggesting that the partial agonist/antagonist and antagonist properties of 4-hydroxytamoxifen or ICI 182,780, respectively, may be partially explained by modulation of protein-DNA interactions. Footprinting patterns in and around the TATA and CAAT sequences were identical in the presence and in the absence of estrogen suggesting that the basal promoter is accessible and poised for transcription even in the absence of hormone. In vitro DNase I footprinting experiments demonstrated that the estrogen receptor bound to the pS2 ERE and that adjacent nucleotides were protected by MCF-7 nuclear proteins. These findings indicate that transcription of the pS2 gene is modulated by alterations in protein binding to multiple sites upstream of the basal promoter, but not by changes in protein-DNA interactions in the basal promoter.
Proceedings of the National Academy of Sciences, 1984
We have shown previously that an increase in the level of accumulated pS2 mRNA is first detectable in MCF-7 cells after 3 hr of estradiol treatment. Using in vitro nuclear run-on transcription with nuclei prepared from MCF-7 cells grown in the presence of estradiol or in estradiol-stripped medium, we demonstrate here that expression of the pS2 gene is controlled by estrogen at the transcriptional level. Induction of transcription is a very early event that is already apparent within 15 min after addition of estradiol to the culture medium. In addition, pretreatment of the cells with the protein synthesis inhibitor cycloheximide does not prevent induction of pS2 gene transcription, indicating that it corresponds to a primary effect of estrogen. The pS2 gene in MCF-7 cells represents a unique example of a human gene whose transcription is directly controlled by estrogen.
The Journal of Steroid Biochemistry and Molecular Biology, 2007
Oestrogen receptors (ER)␣ and  modify the expression of genes involved in cell growth, proliferation and differentiation through binding to oestrogen response elements (EREs) located in a number of gene promoters. Transient transfection of different luciferase reporter vectors 3xEREs-Vit, 2xEREs-tk and ERE-C3 showed that the transactivation capacity of both ER subtypes was influenced by 1) the nature of the inducer (oestradiol (E 2 ), phyto-and anti-oestrogen (AE)), 2) the structure of the promoter (nucleotidic sequence, number of ERE, length of the promoter sequence) and 3) the cell line (containing endogenous ER (MCF-7) or in which ER was stably expressed (MDA-MB-231-HE-5 (ER␣+) or MDA-MB-231-HERB (ER+)). ER subtype did not display the same efficacy on the different constructions in the presence of E 2 and of AE according to the cell (e.g. in MCF-7 cells: tk Vit C3∼0 while in MDA-MB-231 cells: Vit tk∼C3). E 2 response was higher in MCF-7 cells, probably due to higher ER expression level (maximal at 10 −10 M instead of 10 −8 M for E 2 in HE-5 cells). Finally, the same ligand could exert opposite activities on the same promoter according to the ER isoform expressed: in the MDA-MB-231 cells, AE acted as inducers of the C3 promoter via ER whereas ER␣/AE complexes down-regulated this promoter.
Expression via Two Estrogen-Responsive Elements Present in the Coding Sequence
1999
We have found that 17-estradiol induces bcl-2 transcription in human breast cancer MCF-7 cells. To identify cis-acting elements involved in this regulation, we have analyzed hormone responsiveness of transiently transfected reporter constructs containing the bcl-2 major promoter (P 1). Hormone inducibility was observed only when either of two sequences, located within the bcl-2 coding region and showing one and two mutations with respect to the consensus estrogen-responsive element, were inserted downstream from the P 1 promoter. Both sequences behaved as enhancers exclusively in cells expressing the estrogen receptor and were able to bind this receptor in in vitro assays. Transfections into MCF-7 cells of plasmids carrying a bcl-2 cDNA fragment which included these two elements revealed that their simultaneous presence resulted in an additive effect on reporter gene activity, whose size resembled the increase of endogenous bcl-2 mRNA level observed in untransfected cells after hormone treatment. Moreover, the identified elements were able to mediate up-regulation of bcl-2 expression by 17-estradiol, since exogenous bcl-2 mRNA was induced by hormone challenge of MCF-7 cells transiently transfected with a vector containing the bcl-2 coding sequence cloned under the control of a non-estrogen-responsive promoter. Finally, we show that hormone prevention of apoptosis, induced by incubating MCF-7 cells with hydrogen peroxide, was strictly related to bcl-2 up-regulation. Our results indicate that the bcl-2 major promoter does not contain cis-acting elements directly involved in transcriptional control by 17-estradiol and that hormone treatment inhibits programmed cell death in MCF-7 cells, inducing bcl-2 expression via two estrogen-responsive elements located within its coding region.
British Journal of Cancer, 1997
We have cloned and characterized a 77-kDa oestrogen receptor (ER) from an oestrogen-independent subclone of the MCF-7 human breast cancer cell line. This receptor contains an in-frame, tandem duplication of exons 6 and 7, located in the steroid-binding domain of the ER. This mutation has abrogated ligand binding, but not DNA binding, in this mutant ER. We previously described the partial structure of a unique oestrogen receptor (ER) that is expressed in an oestrogen-independent MCF-7:2A subclone of the breast cancer cell line MCF-7 (Pink JJ, Wu SQ, Wolf DM, Bilimoria MM, Jordan VC 1 996a, Nucleic Acids Res 24 962-969). Sequence analyses determined the molecular weight of this 80-kDa ER to be 77 kDa, and hereafter this protein will be designated as ER77. Examination of the entire coding sequence of the ER77 mRNA indicates that it contains a tandem duplication of exons 6 and 7. Using a coupled transcription/translation system, a 77-kDa ER, which corresponds to the protein observed in the MCF-7:2A cells, was expressed. The ER77 protein does not bind the ligands [3H] oestradiol or [3H]tamoxifen aziridine. In DNA binding gel shift assays, the in vitro synthesized ER77 binds to a consensus vitellogenin A2 oestrogenresponse element. In transient transfection experiments, the mutant ER, alone or in combination with the wild-type ER, does not induce expression of an oestrogen-responsive luciferase reporter construct. In fact, expression of the ER77 in the ER-positive T47D:A18 cell line inhibits E2-induced luciferase expression. Overexpression of wild-type ER in T47D:A1 8 cells leads to elevated constitutive expression of the luciferase reporter, which was inhibited by co-transfection with ERR7. These data suggest that the ER77 can interfere with normal ER activity and does not act as a constitutive activator of oestrogen-independent growth in MCF-7:2A cells. Consequently, the constitutive growth observed in MCF-7:2A cells is probably the result of other ER-mediated pathways.
The Journal of Biological Chemistry, 1984
The effects of estradiol and antiestrogens on two estrogen-regulated proteins (52 and 160 kDa) and an estrogen-inducible mRNA (pS2) have been measured in the MCF7 human breast cancer cell line and two tamoxifen-resistant sublines. In MCF7 cells, tamoxifen (or nafoxidine) alone does not significantly affect pS2 mRNA levels nor transcription of the pS2 gene but completely antagonizes the ability of estradiol to increase pS2 RNA levels. The estrogen-mediated increase in pS2 mRNA levels resembles that of two estrogen-regulated proteins (52 and 160 kDa) that are released from MCF, cells into the culture medium. This is distinct from the estrogen-mediated increase in progesterone receptor which is also obtained by tamoxifen treatment. In the two tamoxifen-resistant sublines (R27 and RTxG), tamoxifen does not increase pS2 mRNA levels or 160-kDa protein production but increases the production of the 52-kDa protein. These results show that in these tamoxifen-resistant cells, tamoxifen has become fully estrogenic for one, but not all estrogen-regulated responses.