To obtain comprehensive information on 17␤-estradiol (E2) sensitivity of genes that are inducible or suppressible by this hormone, we designed a method that determines ligand sensitivities of large numbers of genes by using DNA microarray and a set of simple Perl computer scripts implementing the standard metric statistics. We used it to characterize effects of low (0 -100 pM) concentrations of E2 on the transcriptome profile of MCF7͞BUS human breast cancer cells, whose E2 dose-dependent growth curve saturated with 100 pM E2. Evaluation of changes in mRNA expression for all genes covered by the DNA microarray indicated that, at a very low concentration (10 pM), E2 suppressed Ϸ3-5 times larger numbers of genes than it induced, whereas at higher concentrations (30 -100 pM) it induced Ϸ1.5-2 times more genes than it suppressed. Using clearly defined statistical criteria, E2-inducible genes were categorized into several classes based on their E2 sensitivities. This approach of hormone sensitivity analysis revealed that expression of two previously reported E2-inducible autocrine growth factors, transforming growth factor ␣ and stromal cell-derived factor 1, was not affected by 100 pM and lower concentrations of E2 but strongly enhanced by 10 nM E2, which was far higher than the concentration that saturated the E2 dose-dependent growth curve of MCF7͞BUS cells. These observations suggested that biological actions of E2 are derived from expression of multiple genes whose E2 sensitivities differ significantly and, hence, depend on the E2 concentration, especially when it is lower than the saturating level, emphasizing the importance of characterizing the ligand dosedependent aspects of E2 actions. E strogens bind to estrogen receptors (ERs), which belong to the steroid receptor family of transcription factors, and the liganded ERs activate or suppress transcription of genes by recruiting coactivator or corepressor proteins. ERs bind directly to genomic DNA sequences that are known as estrogen response elements, or they interact with genomic DNA indirectly through other DNA-binding proteins such as AP-1 or Sp1. ERs also interact with key components of other signal transduction pathways such as the Src tyrosine kinase or phosphatidyl inositol 3-kinase, affecting gene expression indirectly through these pathways (1). Moreover, changes in gene expression that occur as primary responses to estrogens may exert secondary influences on expression of other genes. Thus, the majority of the biological actions of estrogens are derived from, or at least associated with, induction or suppression of certain sets of genes, even when such actions are considered indirect or nongenomic.To characterize estrogen effects on gene expression, several laboratories have used DNA microarrays and determined the transcriptome profiles of estrogen-dependent human breast cancer cells cultured in the presence or absence of 17␤-estradiol (E2) (2-4). In these experiments, cells were subjected to hormone starvation for up to 5 days and then stimulated wi...

CITED1, a CBP/p300-binding nuclear protein that does not bind directly to DNA, is a transcriptional coregulator. Here, we show evidence that CITED1 functions as a selective coactivator for estrogen-dependent transcription. When transfected, CITED1 enhanced transcriptional activation by the ligand-binding/AF2 domain of both estrogen receptor-␣ (ER␣) and ER␤ in an estrogen-dependent manner, but it affected transcriptional activities of other nuclear receptors only marginally. CITED1 bound directly to ER␣ in an estrogen-dependent manner through its transactivating domain, and this binding activity was separable from its p300-binding activity. CITED1 was strongly expressed in nulliparous mouse mammary epithelial cells and, when expressed in ER-positive MCF-7 breast cancer cells by transduction, exogenous CITED1 enhanced sensitivity of MCF-7 cells to estrogen, stabilizing the estrogen-dependent interaction between p300 and ER␣. The estrogen-induced expression of the transforming growth factor-␣ (TGF-␣) mRNA transcript was enhanced in the CITED1-expressing MCF-7 cells, whereas estrogen-induced expression of the mRNA transcripts for progesterone receptor or pS2 was not affected. Chromatin immunoprecipitation assay revealed that endogenous CITED1 is recruited to the chromosomal TGF-␣ promoter in MCF-7 cells in an estrogen-dependent manner but not to the pS2 promoter. These results suggest that CITED1 may play roles in regulation of estrogen sensitivity in a gene-specific manner.

Evidence has been accumulating that some estrogen-dependent human breast cancers require estrogen for not only proliferation but also survival. To obtain insights into the molecular mechanisms of apoptosis of breast cancer cells subjected to estrogen starvation or exposed to antiestrogens, we characterized changes in the gene expression profile of MCF-7͞BUS human breast cancer cells and revealed a strong induction of Bik, a member of the BH3-only proapoptotic proteins. The Bik mRNA transcript and protein were strongly induced by estrogen starvation or exposure to fulvestrant, a pure antiestrogen that competes with the natural estrogens for binding to the estrogen receptors. This Bik induction preceded apoptotic cell death, which was blocked by zVAD-fmk, a pancaspase inhibitor. Amounts of the Bcl-2-related proteins, such as Bcl-2, Bcl-XL, or Bax, showed only marginal changes in the presence or absence of estrogens or antiestrogens. Suppression of Bik expression by using the small interfering RNA effectively blocked the fulvestrant-induced breast cancer cell apoptosis. These results indicate that Bik is induced in MCF-7͞BUS cells in the absence of estrogen signaling and plays a critical role in the antiestrogenprovoked breast cancer cell apoptosis.A t least 60% of human breast cancers express estrogen receptor ␣ (ER␣), and about half of these ER␣-positive tumors require estrogen for their growth (1). Although the requirement of estrogen for proliferation of such hormonedependent tumors has been widely accepted, evidence is accumulating that this steroid hormone is also necessary for survival of breast cancer cells (2-5). The importance of the mitochondriadependent apoptotic pathway involving the Bcl-2 family apoptosis regulator proteins in estrogen-regulated breast cancer cell apoptosis has been supported by a number of studies (6-16). The antiapoptotic members of the Bcl-2 family, such as Bcl-2, share three or four conserved domains known as Bcl-2 Homology (BH) regions. Proapoptotic members such as Bax share two or three BH regions. The antiapoptotic members suppress the release of cytochrome c from mitochondria, whereas the proapoptotic members facilitate this process. When released to the cytosol, cytochrome c activates Apaf-1, which in turn activates the apoptosis-initiator caspase (caspase-9). Another group of apoptosis regulators, often referred as BH3-only proteins, only share the 9-aa BH3 region and, through this domain, bind directly to the antiapoptotic members of the Bcl-2 family to inhibit their apoptosis-suppressing function. It has been reported that expression of Bcl-2 in MCF-7 human breast cancer cells is enhanced by 17␤-estradiol (E2) and decreased by antiestrogens, whereas expression of Bax is not affected by E2 or antiestrogens (7-14). However, reported changes in the amount of Bcl-2 with estrogen starvation or exposure to antiestrogens have been relatively small, typically Ͻ2-fold, suggesting possible involvement of other hormonally controlled factors that coordinately regulate apoptosis with B...

Induction of mRNA for BIK proapoptotic protein by doxorubicin or ;-irradiation requires the DNA-binding transcription factor activity of p53. In MCF7 cells, pure antiestrogen fulvestrant also induces BIK mRNA and apoptosis. Here, we provide evidence that, in contrast to doxorubicin or ;-irradiation, fulvestrant induction of BIK mRNA is not a direct effect of the transcriptional activity of p53, although p53 is necessary for this induction. It is known that p53 up-regulated modulator of apoptosis (PUMA) mRNA is induced directly by the transcriptional activity of p53. Whereas ;-irradiation induced both BIK and PUMA mRNA, only BIK mRNA was induced by fulvestrant. Whereas both fulvestrant and doxorubicin induced BIK mRNA, only doxorubicin enhanced the DNA-binding activity of p53 and induced PUMA mRNA. Small interfering RNA (siRNA) suppression of p53 expression as well as overexpression of dominant-negative p53 effectively inhibited the fulvestrant induction of BIK mRNA, protein, and apoptosis. Transcriptional activity of a 2-kb BIK promoter, which contained an incomplete p53-binding sequence, was not affected by fulvestrant when tested by reporter assay. Fulvestrant neither affected the stability of the BIK mRNA transcripts. Interestingly, other human breast cancer cells, such as ZR75-1, constitutively expressed BIK mRNA even without fulvestrant. In these cells, however, BIK protein seemed to be rapidly degraded by proteasome, and siRNA suppression of BIK in ZR75-1 cells inhibited apoptosis induced by MG132 proteasome inhibitor. These results suggest that expression of BIK in human breast cancer cells is regulated at the mRNA level by a mechanism involving a nontranscriptional activity of p53 and by proteasomal degradation of BIK protein. (Cancer Res 2006; 66(20): 10153-61)