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Department of Chemical Engineering and Applied Chemistry (D.-Y.W., E.A.E.) and Banting and Best Department of Medical Research (D.-Y.W.), University of Toronto, Toronto, Ontario M5S 3E5; Department of Physical and Environmental Sciences (R.F.), University of Toronto at Scarborough, Scarborough, Ontario M1C 1A4; and Department of Chemistry, Biology and Chemical Engineering (S.N.L.), Ryerson University, Toronto, Ontario M5B 2K3 Canada
Address all correspondence and requests for reprints to: Elizabeth A. Edwards, Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, Ontario M5S 3E5, Canada. E-mail: edwards{at}chem-eng.utoronto.ca.
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 TOP ABSTRACT INTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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To elucidate the mechanisms of estrogen function, it is necessary to identify the estrogen target genes. These estrogen targets may serve as biomarkers to monitor EDCs and estrogen-related diseases. Although many estrogen-responsive genes have been characterized, the complete set of estrogen-regulated genes is unknown. DNA microarray technology permits the transcriptional analysis of a large number of genes and gene products simultaneously. Knowledge of gene-expression differences in large numbers of patients with breast cancer will ultimately result in the definition of global gene-expression patterns associated with ER status (5). Microarray studies have helped to define global gene expression data derived from an in vitro model to pinpoint novel estrogen-responsive genes of potential clinical relevance (6, 7) and to monitor possible environmental EDCs (8). In this study our goal was to identify genes the expression of which was regulated early in response to 17ß-estradiol (E2) treatment in MCF7 cells, which is a hormone-responsive human breast cancer cell line. We also examined which genes were responsive to putative EDCs, because of the potential use of gene-expression patterns as a broad screening tool in the analysis of estrogenicity in environmental samples. Using microarrays, we identified seven early estrogen-responsive genes, including one new gene, which we named EEIG1. EEIG1 was responsive to a group of estrogenic chemicals. Moreover, it was only expressed in ER-positive breast cancer cell lines and not in ER-negative breast cancer cell lines. Expression of EEIG1 was repressed by antiestrogens but not by protein synthesis inhibitors.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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, left column). The most highly expressed 25 of the 55 up-regulated EST fragments (Fig. 1, bar a, expanded in right column) were further analyzed and were found to comprise 19 human genes and two unknown ESTs. In addition, a cluster of genes that responded very quickly to estrogen was identified (Fig. 1, bar b).
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, identified in blue). These known estrogen-regulated genes included trefoil factor 1 (TFF1/pS2) (10, 11), keratin 8 (KRT8) (12), heat shock 60-kDa protein 1 (HSPD1) (11), heat shock 70-kDa protein 8/10 (HSPA8/10) (11), and NM23A protein expressed in nonmetastatic cells 1 (NME1) (11, 13). Five of the known estrogen-regulated genes were found in the cluster of genes that responded early to E2 (Fig. 1, bar b), including estrogen-induced gene 4 (E2IG4) (11), IGF binding protein 4 (IGFBP4) (7), ß 1,4-galactosyltransferase, polypeptide 1 (B4GALT1) (14), solute carrier family 2 member 1/glucose transporter 1 (SLC2A1/GLUT1) (15, 16), and three fragments of X-box binding protein 1 (XBP1) (6, 7). TFF1/pS2 is a well-known estrogen-induced gene in human breast cancer (10). KRT8 was isolated by differential screening, and its different expression between ER-positive breast cancer cell line MCF7 and ER-negative cell line MDA-MB-231 was demonstrated by Northern blot hybridization (12). E2-induced expression of TFF1, E2IG4, NME1, HSPD1, and HSPA8/10 was identified by SAGE and further confirmed by Northern blot hybridization (11). Very recently, estrogen-induced expression of XBP1, IGFBP4, NME1, and B4GALT1 has been reported in microarray experiments (6, 7, 13). Custodia et al. (8) found E2 induction of two members of the Caenorhabditis elegans HSP70 protein family (Hsp-1 and Hsp-2) using microarrays. The independent confirmation of these known estrogen-responsive genes provides additional support for these previous results and validates our microarray data.
Eight of the 19 candidate genes found to be up-regulated by estrogen in this study have never been shown to be estrogen responsive and may be new targets of estrogen activity (Fig. 1, identified in black). Of these genes, cytochrome c (HCS) and heterogeneous nuclear ribonucleoprotein A/B (HNRPAB) were induced in MCF-7 after 4–8 h of E2 treatment. Moreover, two fragments corresponding to each of these genes were present on the microarray, and each pair showed the same expression pattern. To confirm that up-regulated genes identified by microarray experiments were truly estrogen regulated, the expression of HCS and HNRPAB was monitored by semiquantitative RT-PCR (Fig. 2). The RT-PCR results and microarray data were consistent.
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, bar b) included 10 ESTs corresponding to six known human genes, one gene for a hypothetical human protein (that we have subsequently called EEIG1, see below) and one unknown EST. Of the six known human genes, five (E2IG4, IGFBP4, B4GALT1, SLC2A1/GLUT1, and XBP1) are known to be estrogen regulated, and one corresponded to a known human gene, ATPase family gene 3-like 1 (AFG3L1), which has never previously been shown to be estrogen responsive.
Induction of E2IG4 and IGFBP4 after exposure to E2 for 2–3 h was previously reported (7, 11). We observed a similar time response in our microarray experiments (Fig. 1). Elevated levels of SLC2A1/GLUT1 mRNA were reported in immature rat uteri (15) and monkey brain parenchyma (16) treated with E2. Here, our experimental data showed that SLC2A1was also a target for E2 in a human breast cancer cell line (Figs. 1 and 2). XBP1 is a transcription factor (17) and was shown to be differentially expressed in ER
-positive and -negative human breast cancers (5, 18). In microarray experiments by Finlin et al. (6) and Bouras et al. (7), XBP1 was also identified as estrogen regulated. Three EST fragments corresponding to XBP1 were printed on our microarrays, and each was found to cluster within the early up-regulated ESTs (Fig. 1). RT-PCR confirmed that XBP1 was estrogen induced as early as 2 h after E2 treatment of MCF-7 cells (Fig. 2).
B4GALT1 belongs to a family of at least six related ß-1,4-galactosyltransferases, and its deficiency causes a disease termed congenital disorder of glycosylation type IId (19). B4GALT1 was identified together with PDZK1 and ER from a human breast cancer cDNA library subtracted from normal tissues (20), which suggests a strong relationship between B4GALT1 and ER status. Very recently, Inoue et al. (14) reported that B4GALT1 was induced by estrogen after 72 h of treatment in both MCF-7 and T-47D breast cancer cells but not in other cancer cell lines derived from breast, ovary, endometrium, stomach, brain, renal, or melanoma cells in microarray experiments. Using RT-PCR, we showed that the expression of B4GALT1 increased as early as 2 h after exposure of MCF-7 to E2 (Fig. 2). These data confirm that B4GALT1 is also an early estrogen-regulated gene. Sotiriou et al. (21) reported that the expression of B4GALT1 in human breast cancers appeared stronger in patients who responded poorly to chemotherapy compared with those who responded well, suggesting that B4GALT1 might play a role in breast cancer development.
AFG3L1 is a human homolog of the yeast ATPase family gene 3-like 1. Its mouse ortholog, Afg3l1, encodes an ATP-dependent zinc metalloprotease that is targeted to the mitochondria. However, AFG3L1 does not seem to be translated in humans (22). In our microarray experiments, AFG3L1 was induced 2 h after E2 treatment in MCF-7 cells. RT-PCR confirmed this E2-regulated expression of AFG3L1 although the effect was not as strong as with B4GALT1 (Fig. 2). This result may provide new evidence for AFG3L1 activity in human breast cancer cells and suggest that AFG3L1 maybe a target of estrogen.
EEIG1Is a Novel Early Estrogen-Induced Gene
An up-regulated EST corresponding to the gene of human hypothetical protein, LOC90676 (GenBank accession no. XM_033421) was found in the cluster of early E2 responsive genes. We called this gene EEIG1 for early estrogen-induced gene 1 (Fig. 1, bar b, identified in red). According to the chromosome 9 working draft sequence (GenBank accession no. NT_029366), EEIG1 is located at 9q34.13 and consists of eight exons and seven introns. Exon 2 contains the partial 5'-untranslated regions (UTRs) and the ATG initiation codon. The open reading frame (ORF) sequence of 726 bp shares 83.5% identity with its 750-bp mouse homolog (accession no. BC031157). The 242-amino acid sequence of EEIG1 shares 92% identity with the 255-amino acid sequence in the mouse homolog. No functional information is available for EEIG1. To confirm that EEIG1 is a novel early estrogen-induced gene, its expression in response to estrogen was analyzed by quantitative multiplex RT-PCR using a specific primer pair, EEIG (see Table 1 and Fig. 3 for sequence and location), located at the 5'-UTR of LOC90676 and covering the microarray EST fragment. Detection of EEIG1 transcripts was clearly observed in MCF-7 cells exposed to E2 compared with the carrier-only controls (Fig. 2). The pattern of gene expression was similar to what we detected in microarray experiments (Fig. 1).
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). To determine to which mRNA sequence (LOC90676 or FLJ00179), EEIG1 corresponded to in our experiments, we designed and synthesized specific primers for both mRNA sequences and tested these with RT-PCR. Amplification of cDNA occurred only in the reactions with the LOC90676-specific primer pairs but not with the FLJ00179-specific primer pairs (Fig. 3B). The LOC90676-specific primers also successfully amplified cDNA from other ER-positive cell lines exposed to E2, but not from ER-negative breast cancer cell lines (Fig. 3C). A new human mRNA AI426465 (GenBank accession no. BC047949) and a mouse mammary tumor mRNA C230093N12Rik clone MGC:38572 (GenBank accession no. NM_153560) are also similar to LOC90676 but not to FLJ00179 (Fig. 3A). These results confirm the identity of EEIG1 as the gene for hypothetical protein LOC90676, and that EEIG1 is a novel early estrogen-induced gene.
Effect of E2 Concentration on EEIG1Expression
To determine the dose response and the lowest concentration of E2 that results in a detectable response, MCF-7 cells were exposed to different concentrations of E2, ranging from 10-7 nM to 100 nM for 4 h. Expression of B4GALT1, EEIG1, and PDZK1detected by RT-PCR demonstrated a dose-dependent response in these experiments (Fig. 4A). Induction of gene expression was clearly detectable down to 10-3 nM E2. The highest levels of expression were detected at 10 nM E2, which was the working concentration used in this study.
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). Because many chemicals released into the environment potentially disrupt the endocrine system in wildlife and in humans, these results have many implications for the connection between chemicals in the environment and cancer or other ecosystem or human health effects (23, 24, 25). B4GALT1, EEIG1, and other responsive genes could be used as indicators of exposure to exogenous estrogenic compounds such as possible environmentally relevant endocrine disruptors. Moreover, the response to individual putative estrogenic compounds was different for the different genes, PDZK1, B4GALT1, and EEIG1 (Fig. 4B), indicating that gene-expression patterns might be specific for each compound or group of compounds. These patterns of gene expression may provide a means to identify the source of estrogenic activity in complex environmental samples.
Expression of EEIG1 and PDZK1 appeared to be specifically related to the presence of estrogenic compounds, because these genes were not significantly induced in MCF-7 cells exposed to progesterone, to the androgens testosterone (Tes) or dihydrotestosterone (DHT), or to cholesterol (Cho), a biochemical precursor to steroid hormones (Fig. 4C). These results suggest that genes such as EEIG1 and PDZK1 are specifically responsive only to estrogenic compounds in MCF-7 cells.
EEIG1Expression Depends on ER But Not on New Protein Synthesis
Tamoxifen (OHT) acts as an inhibitor of estrogen-induced responses and inhibits the effect of E2 both on cell proliferation and on the regulation of specific genes. For example, expression of the E2-responsive gene PDZK1 was found to be repressed by OHT (6). ICI 182,780 (ICI) is also widely used as an antiestrogen (14). Therefore, we compared the expression of EEIG1 and PDZK1 in the presence and absence of OHT and ICI to determine whether the expression of EEIG1 depends on ER activity, as is the case with PDZK1. In our experiments, OHT and ICI significantly blocked the E2-induced expression of these two genes (Fig. 4C). The inhibitive effect of OHT and ICI further confirms specific E2-induced expression of EEIG1. Furthermore, we observed that the E2-mediated induction of EEIG1 and PDZK1 was not prevented by pretreatment of the cell with two protein synthesis inhibitors, cycloheximide (CHX) and puromycin (Pur) (26, 27) (Fig. 4C). These results suggest that these genes are directly regulated by E2.
EEIG1 and B4GALT1 Expression Is Related to Cell ER Status
The microarray data and RT-PCR analysis revealed that both B4GALT1 and EEIG1 responded early to E2 treatment. This early effect may be expected to be more reproducible and indicative that these genes may be direct targets of activated ER. We therefore focused additional experiments on these two genes. To investigate the correlation of the estrogen responsiveness of the B4GALT1 and EEIG1 to ER status in human breast carcinomas, we examined the expression after exposure to E2 of these two genes in three ER-positive and three ER-negative human breast carcinoma cell lines by RT-PCR. B4GALT1 and EEIG1 were expressed in ER-positive cell lines MCF-7, T47-D, and ZR-75–1 but were not expressed in ER-negative breast carcinoma cell lines MDA-MB-231, MDA-MB-435, and Hs578T (Fig. 4D). The expression patterns of ER and PDZK1 under the same conditions are shown for comparison. The selective expression in ER-positive cell lines for B4GALT1 and EEIG1 shows their expression is dependent on the presence of a functional ER. The pattern of expression suggests an important role for these two genes in the physiological response of breast tumors to estrogen.
In summary, using microarray and RT-PCR techniques, 19 estrogen-regulated genes were confirmed in the human breast cancer cell line MCF-7. Seven of these genes, including a newly identified human gene EEIG1, were induced by E2 within 2 h. We also found that some estrogenic compounds and environmental contaminants differentially enhance the transcription of estrogen-regulated genes, suggesting that assays of multiple gene expression profiles could be useful in identifying potential endocrine-disrupting chemicals.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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-ethinylestradiol (EE2) and diethylstilbestrol (DES); xeno-estrogens: 4-nonylphenol (4NP) and 4-octylphenol (4OP); phyto-estrogens: ß-sitosterol (Sit) and genistein (Gen); pesticides: 1,1,1-trichloro-2-(o-chlorophenyl)-2-(p-chlorophenyl)-ethane (DDT) and alachlor (Ala); androgens: Tes and DHT; progesterone (Pro); cholesterol (Cho); antiestrogens: OHT and ICI (Tocris Cookson, Inc., Ellisville, MO); protein synthesis inhibitors: CHX and Pur; and solvent and carriers: ethanol (EtOH, solvent for most of the endocrine compounds), 2-hydroxypropyl-ß-cyclodextrin (HC, carrier for water-soluble E2), and methyl-ß-cyclodextrin (MC, carrier for water-soluble Cho). Cells treated with only EtOH, HC, or MC were used as controls for different endocrine compounds. Cell culture media and supplements and microarray hybridization and RT-PCR reagents were products of Invitrogen (Carlsbad, CA) except where noted.
Cell Lines and Cell Culture
Human breast cancer cell lines including ER-positive cell lines MCF-7, T47-D, and ZR-75–1 and ER-negative cell lines MDA-MB-231, MDA-MB-435, and Hs578T were obtained from American Type Culture Collection (Manassas, VA). All cell lines were maintained in RPMI 1640 medium supplemented by 10% fetal bovine serum, penicillin, and streptomycin and incubated at 37 C in 5% CO2. Before estrogen exposure, the cells were transferred into phenol red-free RPMI 1640 medium supplemented with 10% charcoal/dextran treated (hormone-free) fetal bovine serum (Hyclone Laboratories, Inc., Logan, UT) and subsequently grown for 6 d. The cells were then challenged with estrogen or estrogen-like compounds at specific concentrations for different time intervals as described below.
Experiments with Estrogen
To study the temporal response to estrogen stimulation, MCF7 cells were first grown in estrogen-free medium for 6 d, after which the medium was amended with 10 nM water-soluble E2 in HC. In microarray experiments, a water-soluble E2 bound to HC was used to treat the cells, rather than E2 in ethanol, to avoid possible interactions between ethanol and ER signaling (28), A similar set of MCF7 cells amended with an equal volume of HC but without E2 was used as a control. Cells were harvested from both the estrogen-treated samples and the control samples 2, 4, 8, 12, 24, 48, 72, or 94 h after exposure. To compare the different responses in cell lines with and without ER, six different human breast cancer cell lines were grown in the absence of estrogen for 6 d and then were exposed to 10 nM E2 for 4 h. To determine the effects of concentration, MCF7 cells were exposed to E2 concentrations of 0 (carrier only) 10-7, 10-5, 10-3, 10-1, 10, and 103 nM. Cells were harvested after 4 h exposure to E2.
Experiments with Putative EDCs
To study the effects of different endocrine compounds or possible EDCs, MCF7 cells that were pregrown in estrogen-free medium for 6 d were amended with either 10 nM E2, 100 nM E3, 8 nM EE2, 4 nM DES, 10 µM 4NP, 10 µM 4OP, 100 µM Gen, 100 µM Sit, 1 µM DDT or 10 µM Ala; or 10 nM Cho, Tes, DHT, and Pro. These chemicals are relatively water insoluble. In these experiments, E2 was solubilized in HC, Cho was solubilized in MC, and all other chemicals were dissolved in ethanol. Concentrations of putative environmental EDCs were chosen based on their relative potencies in cell proliferation or ER binding in MCF-7 cells compared with E2 (29) and concentrations used in other studies (9, 11, 14). Cells were harvested after 4 h exposure. MCF7 cells amended with an equal volume of carrier (HC, MC, or ethanol) were used as controls in these experiments.
Experiments with Antiestrogens and Protein Synthesis Inhibitors
To understand the effects of estrogen antagonists on the E2-induced gene expression, MCF-7 cells were treated with 10 nM E2 and either antiestrogens OHT (100 nM) or ICI (50 nM) for 4 h. To determine whether E2-induced gene expression depended on new protein synthesis, MCF-7 cells were pretreated for 1 h with 50 µM of the protein synthesis inhibitors CHX or Pur, and then cultured in the presence of 10 nM E2 for 4 h.
RNA Preparation
All cell pellets were immediately frozen in liquid nitrogen and stored at -70 C. QIAshredder spin columns (QIAGEN, Valencia, CA) were used to homogenize the samples. Total RNA was extracted from cell pellets using the QIAGEN RNeasy kit according to the manufacturer’s instructions, and then frozen at -70 C until used.
Microarray Hybridizations
For comparative microarray hybridization, cDNAs were synthesized from E2-treated test RNA or HC reference RNA and labeled according to the manufacturer’s protocols of the Microarray Centre at the Ontario Cancer Institute, Toronto, Canada (http://www.microarray.ca/). Briefly, 10 µg of total RNA were reverse transcribed with Superscript II reverse transcriptase, AncT primer (5'-T20VN-3') and aminoallyl-dUTP (Sigma) at 42 C for 2 h. cDNA from test and reference samples were labeled with Cy5 and Cy3 dyes, respectively (Amersham Pharmacia Biotech, Arlington Heights, IL) at room temperature for 1 h, following recommended protocols of the Microarray Centre. The two separately labeled cDNA probes from test and reference samples were purified and combined, and then hybridized to a pair of cDNA microarray slides containing 19,008 characterized and unknown human ESTs. Hybridization was conducted at 37 C overnight in DIG Easy Hyb solution (Roche Clinical Laboratories, Indianapolis, IN). The slides were washed in 0.1x standard saline citrate and 0.1% sodium dodecyl sulfate at 50 C and then scanned using a GenePix 4000 scanner (Axon Instruments, Inc., Union City, CA). Microarray image analysis was performed using Axon’s GenePix Pro 3.0.
Data Analysis
The data files produced by GenePix Pro 3.0 were further analyzed using in-house software developed by Goryachev et al. (30). This DOS-based software was used to normalize Cy5 and Cy3 channels by setting the average ratio of Cy5:Cy3 equal to 1 (assuming the method of constant majority), and to calculate normalized expression ratios as test sample divided by control sample for each EST on the microarray. The software was also used to combine normalized data files from several data sets and to compare and average results obtained from replicate microarray experiments. To minimize false-positive results, replicate DNA microarray experiments (two to four arrays per experimental condition) were performed. Only those genes that had similar expression levels in at least two independent experiments and showed at least a 2-fold increase or decrease in expression were selected as estrogen-regulated candidate genes for further analysis. Hierarchical clustering was performed on all time course experiments, starting from normalized average data for candidate genes for each time point, using Cluster and TreeView software (http://rana.lbl.gov/EisenSoftware.htm).
RT-PCR
RT-PCR was used to ensure that the cells were responding to estrogen and to confirm that the genes identified by cDNA microarray screening were indeed estrogen responsive. RT-PCR was conducted according to the protocol of Invitrogen with slight modifications. Briefly, total RNA (10 µg) was reverse transcribed with the oligo(dT)20 primer described above in a total reaction volume of 40 µl. PCR was carried out using 1 µl of reverse transcription product for amplification of target genes in a total reaction volume of 25 µl. PCR was performed using a GeneAmp PCR System 2400 (PerkinElmer Corp., Norwalk, CT). In semiquantitative RT-PCR, target genes and the reference gene ß-actin were amplified from the same sample in individual reactions. The PCR conditions were as follows: 94 C for 30 sec, 57 C for 30 sec, and 72 C for 1 min repeated over 25 cycles. In quantitative multiplex RT-PCR, target genes and the reference gene GAPDH were amplified simultaneously in a single reaction. In this case, 30 PCR cycles were used. PCR products were visualized by electrophoresis on 1.2% agarose gels. The reference genes ß-actin and GAPDH were used to confirm the success of RT reaction and to verify that an approximately equal cDNA template was added to each reaction. The sequences of the primers and their amplified cDNA fragments sizes are listed in Table 1. To confirm that the cells were indeed responding to added estrogen, PDZK1, a gene known to be expressed in response to estrogen (9), was used as a biomarker.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: Ala, Alachlor; B4GALT1, ß 1,4-galactosyltransferase, polypeptide 1; Cho, cholesterol; CHX, cycloheximide; DDT, 1,1,1-trichloro-2-(o-chlorophenyl)-2-(p-chlorophenyl)-ethane; DES, diethylstilbestrol; DHT, dihydrotestosterone; E2, 17ß-estradiol; E3, estriol; EDCs, endocrine disrupting chemicals; EEIG1, early estrogen-induced gene 1; EE2, 17--ethinylestradiol; ERs, estrogen receptors; EST, expressed sequence tag; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Gen, genistein; HC, 2-hydroxypropyl-ß-cyclodextrin; ICI, ICI 182780; IGFBP4, IGF binding protein 4; MC, methyl-ß-cyclodextrin; 4NP, 4-nonylphenol; OHT, 4-OH-tamoxifen; 4OP, 4-octylphenol; ORF, open reading frame; Pro, Progesterone; Pur, puromycin; Sit, ß-sitosterol; Tes, testosterone; UTR, untranslated region
Received for publication June 1, 2003. Accepted for publication October 27, 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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-Estradiol-induced VEGF-A expression in rat pituitary tumor cells is mediated through ER independent but PI3K-Akt dependent signaling pathway. Biochem Biophys Res Commun 300:209–215[CrossRef][Medline]
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