Overlapping but Distinct Gene Regulation Profiles by Glucocorticoids and Progestins in Human Breast Cancer Cells
Yihong Wan and
Steven K. Nordeen
Department of Pathology and Program in Molecular Biology, University of Colorado Health Sciences Center, Denver, Colorado 80262
Address all correspondence and requests for reprints to: Steven K. Nordeen, Department of Pathology B216, University of Colorado Health Sciences Center, 4200 East 9th Avenue, Denver, Colorado 80262. E-mail: steve.nordeen{at}uchsc.edu.
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ABSTRACT
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Glucocorticoids and progestins bind to receptors that share many structural and functional similarities, including virtually identical DNA recognition specificity. Nonetheless, the two hormones mediate very distinct biological functions. For example, progestins are associated with the incidence and progression of breast cancer, whereas glucocorticoids are growth suppressive in mammary cancer cells. To understand the mechanisms that engender biological specificity, it is necessary to identify genes that are differentially regulated by the two receptors. Here we employ Affymetrix oligonucleotide arrays to compare glucocorticoid- and progestin-regulated gene expression in a human breast cancer cell line. This global analysis reveals that the two hormones regulate overlapping but distinct sets of genes, including 31 genes that are differentially regulated. Surprisingly, the set of differentially regulated genes was almost as large as the set of genes regulated by both hormones. Examination of the set of differentially regulated genes suggests mechanisms behind the distinct growth effects of the two hormones in breast cancer. The differential regulation of four genes representing different regulatory patterns was confirmed by RT-PCR and Northern blot analyses. Treatment with cycloheximide or RU486 indicates that the regulation is a primary, receptor-mediated event. Detailed analyses of genes identified in these studies will furnish a mechanistic understanding of differential regulation by glucocorticoids and progestins.
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INTRODUCTION
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TRANSCRIPTION FACTORS ARE grouped into families based on their most conserved domain, the DNA-binding domain. In many cases, family members share similar, if not indistinguishable, DNA sequence recognition properties. Thus, a fundamental question in molecular biology concerns the extent to which related factors are functionally redundant in a given cell or tissue and the mechanisms by which factor-specific gene regulation is accomplished.
GR and PR are closely related members of the steroid receptor family of transcription factors (1). They share many similar structural and functional characteristics, including DNA sequence recognition specificity (2, 3, 4, 5, 6). The two receptors associate with a similar complex of molecular chaperones in the absence of hormone (7) and with a similar set of coactivators in the presence of hormone (8, 9). Despite the similarity of the two receptors, the cognate hormones display a very distinct spectrum of physiological actions. Classic actions of glucocorticoids include regulation of metabolism, inhibition of inflammation and the immune system, and suppression of bone formation (10). The major physiological role of progestins in the mammal are to establish and maintain pregnancy; to promote lobular-alveolar development in the mammary gland, and to suppress milk protein synthesis before parturition (11). Even in tissues that express both GR and PR, these two hormones may exert opposite biological actions. For example, in bone, glucocorticoids stimulate bone resorption (12, 13, 14), whereas progestins prevent bone loss (15, 16). In mammary gland, glucocorticoids promote milk protein synthesis and lactation (17, 18, 19), whereas progestins inhibit milk production and secretion (11). Furthermore, there is an association of progestins with the incidence and progression of breast cancer (20), whereas glucocorticoids are growth suppressive in mammary cancer cells (21, 22).
How can two receptors with such remarkable similarity mediate such dramatically different biological functions? Only a handful of cellular promoters regulated by GR or PR have been identified. Many of the studies on the mechanisms of GR and PR function have used the mouse mammary tumor virus promoter. This promoter is induced by both steroids under most circumstances, although chromatin environment may differentially influence mouse mammary tumor virus induction by the two hormones by mechanisms as yet poorly understood (23). Understanding the basis of the distinct physiology of glucocorticoids and progestins is severely limited by the paucity of genes and promoters identified to be differentially regulated by the two receptors. In this study, we have performed a global analysis of the gene regulation by glucocorticoids and progestins in a human breast cancer cell line using Affymetrix oligonucleotide microarrays. The results demonstrate that, in addition to genes regulated by both receptors, there is a set of genes that are differentially regulated. The systematic identification of such genes reveals potential avenues of differential regulation of cell growth by the two hormones and opens a new avenue for future studies on the molecular mechanisms underlying hormone-specific gene regulation.
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RESULTS AND DISCUSSION
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Overlapping but Distinct Gene Regulation Profiles by Glucocorticoids and Progestins
To understand the basis of distinct actions of glucocorticoids and progestins in a tissue such as the mammary gland in which both receptors are expressed, and especially to identify genes that are differentially regulated by the two hormones, we performed microarray analysis of gene expression in the human breast cancer cell line T47D/A1-2. T47D/A1-2 cells express comparable levels of both GR and PR (24). Total RNA was isolated from cells treated with vehicle, dexamethasone (Dex, 100 nM), or R5020 (10 nM) for 2 h or 6 h. Probes generated from this RNA were hybridized to Affymetrix HuGeneFL arrays to analyze expression of 5,600 full-length human genes.
In each single array hybridized with cRNAs from T47D/A1-2 cells, 3040% of the genes exhibit detectable expression. Figure 1
shows the fluorescent images of the probe sets in the arrays hybridized to cRNAs for 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) and ß-actin. 11ß-HSD2 has been shown to be inducible by both glucocorticoids and progestins (25) and served as a positive control for the array analysis. As shown in Fig. 1A
and Table 1
, both Dex and R5020 treatment led to a dramatic increase in gene-specific hybridization. As an RNA loading control, ß-actin hybridization signals did not change after either hormone treatment (Fig. 1B
and Table 3
).

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Figure 1. Transcript Monitoring by Hybridization to Oligonucleotide Arrays
The figure shows the fluorescent images of the probe sets hybridized to the cRNA for 11ß-HSD2 (A) and ß-actin (B). Each gene is detected by 20 vertical pairs of 25-mer oligos. The top oligo is a perfect match (PM), and the bottom oligo has a mismatch (MM) at the central position and serves as an internal control for hybridization specificity. The average difference in hybridization intensity between the PM and MM oligos is used to determine transcript level. Different oligos hybridize at different efficiency depending on sequence composition. The use of 20 pairs increases the accuracy of the measurement. A, Array results for a hormone-regulated gene. Expression of 11ß-HSD2 is induced by both Dex and R5020 at 2 and 6 h. B, Array results for a constitutive gene. Expression of ß-actin is unchanged with either hormone treatment, indicating equal RNA loading. Veh, Vehicle; D-2 and D-6, Dex (100 nM) for 2 h and 6 h; R-2 and R-6, R5020 (10 nM) for 2 h and 6 h.
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Of 5,600 genes analyzed, 70 were up-regulated by glucocorticoids by more than 3-fold at either 2 h or 6 h treatment according to the criteria described in Materials and Methods, and another 33 were down-regulated by more than 3-fold. The number of genes that are up-regulated and down-regulated by progestins by more than 3-fold are 47 and 34, respectively. The majority of the genes (>90%) identified have not been previously described to be regulated by either glucocorticoids or progestins and therefore represent novel hormone-regulated targets. Among these genes, 25 were up-regulated by both hormones (Table 1
), and 12 were down-regulated by both hormones by more than 3-fold (Table 2
). Of particular interest for this work, 31 genes have been identified to be differentially regulated by the two hormones by more than 3-fold (Table 3
). These results demonstrate that glucocorticoids and progestins regulate overlapping but distinct sets of genes. In light of the paucity of genes described to be differentially regulated by the two hormones, it was surprising that the number of genes differentially regulated by more than 3-fold, 31, approached the number regulated by both by more than 3-fold, 37. These represented about 1.52% of the genes whose expression could be detected. Novel hormone-regulated targets identified in this study will enhance our understanding of the role of these two hormones as both physiological regulators and pharmacological agents. (Please refer to the 10 Tables published as supplemental data on The Endocrine Societys Journals Online web site, http://mend.endojournals.org/.)
Confirmation of Differential Regulation with RT-PCR and Northern Blot Analyses
Four differentially regulated genes identified by the array analysis as representing different patterns of regulation were selected for further analysis. The first of these genes, G0S8 (also known as RGS2, regulator of G protein signaling 2) is specifically induced by Dex but not by R5020 (Table 3
). G0S8/RGS2 encodes a basic helix-loop-helix phosphoprotein (26) and specifically inhibits the function of Gq
as a GTPase activating protein (27). Knockout mice show reduced T cell proliferation and antiviral immunity, increased anxiety responses, and decreased male aggression (28). The selective glucocorticoid induction was confirmed by both semiquantitative RT-PCR and Northern blot (Fig. 2A
). Quantitation of the Northern blot showed that Dex treatment led to a more than 20-fold induction, whereas R5020 treatment had minimal effect. The hormone-specific induction of G0S8/RGS2 suggests a selective coupling between glucocorticoid and G protein signaling. Previous studies have shown that glucocorticoids suppress the growth of Con8 rat mammary tumor cells by inducing a G1/G0 cell cycle arrest (21). Interestingly, G0S8/RGS2 has been shown to be induced in growth-arrested cells and to promote adipocyte differentiation (29). The induction of G0S8/RGS2 may also play a role in the growth-suppressive effect of glucocorticoids in mammary carcinoma cells.

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Figure 2. Expression Analyses of Selected, Differentially Regulated Genes
T47D/A1-2 cells were treated with vehicle (V or Veh), Dex (D, 100 nM), or R5020 (R, 10 nM) for 2 h (D2 or R2) or 6 h (D6 or R6). Total RNA was isolated and used for semiquantitative RT-PCR and Northern blot analyses. Quantitation of the Northern blot is shown on the right in each panel. A, G0S8, a gene specifically induced by glucocorticoids; B, PLZF, a gene preferentially induced by glucocorticoids; C, INHBB, a gene specifically induced by progestins; D, IEX-1, a gene preferentially down-regulated by glucocorticoids.
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The second gene analyzed, the promyelocytic leukemia zinc finger protein (PLZF), was originally identified as the fusion partner of the RAR
gene in a variant chromosomal translocation in acute promyelocytic leukemia (30). It is a transcription repressor with a kruppel-like zinc finger domain and a BTB/POZ domain (30, 31). PLZF represses transcription by recruiting a histone deacetylase through the silencing mediator of retinoid and thyroid hormone receptor-mSin3-histone deacetylase corepressor complex (32). Like G0S8/RGS2, RT-PCR and Northern blot analyses confirm that PLZF is strongly induced by Dex (Table 3
and Fig. 2B
) but unlike G0S8/RGS2, PLZF is also weakly induced by R5020. In addition, Northern blot showed that PLZF is expressed as two transcripts, 11 kb and 9 kb. Both transcripts are induced 3-fold higher by Dex than by R5020. PLZF has been shown to suppress the growth of myeloid cells by inducing G0/G1 arrest and apoptosis, partly through the binding and repression of the cyclin A2 promoter (33, 34). It is likely that the growth-suppressive effect of glucocorticoids in mammary carcinoma cells is also mediated, in part, through the robust induction of PLZF.
In contrast to PLZF and G0S8/RGS2, the array analysis suggests that the gene for the ßB-subunit of inhibins and activins (INHBB for inhibin ßB) is specifically induced by progestins but not glucocorticoids (Table 3
). Inhibins and activins are members of the TGFß superfamily, which are potent mediators of proliferation or antiproliferation and differentiation in different cell types. RT-PCR and Northern blot confirmed the array result (Fig. 2C
). Northern blot showed that there are two transcripts for INHBB as seen previously for rat INHBB (35). R5020 treatment induced the expression of both transcripts, whereas Dex treatment had no effect on the 4-kb transcript and slightly down-regulated the 3-kb transcript. Progesterone is a mammogenic hormone essential for lobuloalveolar morphogenesis (36). Interestingly, female mice in which both alleles for INHBB have been deleted have lactation failure due to retarded ductal elongation and alveolar morphogenesis during puberty, pregnancy, and parturition (37). Our results suggest that the mammogenic effect of progesterone may be mediated, in part, by the specific induction of INHBB.
Unlike the previous three genes, the differential regulation of the IEX-1/Dif-2 gene involves a hormone-specific down-regulation. Northern blot demonstrated that Dex treatment led to a dramatic 80% down- regulation by 6 h, whereas R5020 treatment resulted in a weak, transient down-regulation with expression returning to control levels by 6 h (Fig. 2D
). IEX-1 was identified as a radiation-inducible immediate-early gene in human squamous carcinoma cells (38). It is also known as Dif-2 and is down-regulated during monocyte differentiation (39). The IEX-1/Dif-2 gene is induced by multiple signals, many of which are associated with proliferation, e.g. lipopolysaccharide, C2ceramide, lysophosphatidylcholine, phorbol esters, serum, or growth factors (39, 40). The association of IEX-1/Dif-2 with proliferation suggests that the growth-suppressive effect of glucocorticoids in mammary carcinoma cells is mediated, in part, through the down-regulation of IEX-1/Dif-2. The connection between additional genes differentially regulated by glucocorticoids and progestins and differential actions on mammary cell proliferation are discussed further below.
Previous studies have shown that the Affymetrix oligo array technology produces highly reliable results, many of which have been confirmed by conventional approaches (41, 42, 43). In this study, for all four genes tested individually, there is excellent agreement between the microarray results and the Northern blot analyses. The analysis of the promoters of these genes will provide a mechanistic understanding for differential regulation. Preliminary studies have mapped the glucocorticoid-mediated inhibition of TNF
-induced IEX-1/Dif-2 promoter activity to a small proximal promoter region containing a nuclear factor-
B element and juxtaposed SP-1-CCAAT enhancer binding protein elements (Wan, Y., unpublished data). The mutually inhibitory actions of GR and nuclear factor-
B have been well documented (44).
Hormone Regulation Is Direct and Receptor Mediated
The 2- and 6-h treatment times used for array analysis were chosen so that primary, receptor-mediated regulatory events, not secondary events, would be identified. To confirm that the differential regulation of the four genes above was mediated by the cognate receptors and direct, T47D/A1-2 cells were treated with protein synthesis inhibitor cycloheximide or the GR/PR antagonist RU486 along with Dex or R5020. Total RNA was isolated, and the expression of each gene was determined by semiquantitative RT-PCR (Fig. 3
). Although cycloheximide itself can have an effect on mRNA levels, the hormone regulation pattern is maintained after cycloheximide treatment for all four genes, indicating that the hormone regulation is a direct effect that does not require de novo protein synthesis. In contrast, the hormone regulation is completely abolished after RU486 treatment, indicating that hormonal regulation is mediated through GR and/or PR.

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Figure 3. Hormone Regulation Is Receptor Dependent and Protein Synthesis Independent
T47D/A1-2 cells were treated with Dex (D, 100 nM), R5020 (R, 10 nM), cycloheximide (CHX, 40 µg/ml), or RU486 (100 nM) for 6 h. Total RNA was isolated and semiquantitative RT-PCR performed with gene-specific primers.
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Differential Gene Regulation by Glucocorticoids and Progestins Mediate Hormone- Specific Effects
Progestins are associated with the incidence and progression of breast cancer (20). Recent work suggests that progestins may prime mammary cells to respond to growth factors (45). In contrast, glucocorticoids are growth suppressive in mammary cancer cells (21, 22). To test whether glucocorticoids and progestins have different effects on the proliferation of T47D/A1-2 cells, we assessed cell growth by monitoring the total DNA content of hormone-treated cell populations (Fig. 4A
). The results demonstrated that Dex treatment inhibited cell growth throughout the time course, whereas R5020 treatment initially stimulated cell growth and then became inhibitory. To examine further the effect of glucocorticoids and progestins on the cell cycle progression of T47D/A1-2 cells within the first 2448 h after hormone treatment, we also performed flow cytometry of propidium iodide-stained cells (Fig. 4B
). Typical of a nonsynchronous population of proliferating cells, approximately 30% of untreated cells are in S or G2/M phases. Dex treatment decreased the fraction of cells in S+G2/M throughout the time course. By 48 h, the fraction of cells in S+G2/M had declined from 30% to 13%, suggesting that Dex treatment led to a cell cycle arrest in G1. This supports the observation in a previous study that Dex suppresses the growth of Con8 rat mammary tumor cells by inducing a G1/G0 cell cycle arrest (21). In contrast, R5020 treatment increased the fraction of cells in S+G2/M initially. At the peak (around 18 h), almost half of the cell population was in S+G2/M, a 63% increase over control. Together with the initial increase in DNA content, this indicates that progestins are growth promoting in the early hours of the treatment. By 3036 h after R5020 treatment, the fraction of cells in S+G2/M dropped below controls to a level similar to Dex-treated cells, indicating that progestins are growth suppressive in the late hours of the treatment. This biphasic effect of progestins on the cell cycle progression of T47D cells has been previously described (46, 47). The current hypothesis is that progestin treatment initially drives cells to go through the first cell cycle to a decision point at the G1/S boundary; secondly, it induces cellular changes that permit other factors to influence the ultimate proliferative or differentiative state of the cells (45). For example, only progestin-primed T47D cells become highly sensitive to the proliferative effects of epidermal growth factor (46). In summary, the T47D/A1-2 cell growth and cell cycle studies clearly demonstrated that during the first 24 h of hormone treatment, glucocorticoids are growth suppressive, whereas progestins are growth stimulating. The early time points (2 h and 6 h) used in our microarray analysis allowed us to assess the early molecular changes that may account for the differential growth effect of the two hormones on the T47D/A1-2 cells.

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Figure 4. Glucocorticoids and Progestins Mediate Different Effects on the Growth of the T47D/A1-2 Cells
A, T47D/A1-2 cells were treated with vehicle, Dex (100 nM), or R5020 (10 nM) for the indicated number of days. Cell proliferation was determined by measuring DNA content in each well using the Hoechst DNA assay. The experiment shown is representative of three independent experiments each done in triplicate ± SE. B, T47D/A1-2 cells were treated with vehicle, Dex (100 nM), or R5020 (10 nM) for the indicated number of hours. The fraction of cells in S+G2/M was measured by flow cytometry. The ordinate indicates the difference between the fraction of cells in S+G2/M in the hormone-treated sets and the fraction of cells in S+G2/M in the vehicle-treated controls. In this study, approximately 2535% of control cells were in S+G2/M.
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From the examination of the differentially regulated genes identified, a pattern emerged that gives insight into the differential effects of the two hormones on cell proliferation. A number of the genes that are preferentially induced by glucocorticoids or suppressed by progestins are growth suppressive (Table 3
, top). G0S8/RGS2 and PLZF have been discussed above. Also of note are genes preferentially suppressed by R5020, including RAR
1 and vitamin D3 up-regulated protein 1 (VDUP1). RAR
inhibits proliferation and activates apoptosis in breast cancer cells (48, 49). VDUP1 suppresses cell proliferation by inhibiting the reducing potential of the disulfide reducing protein thioredoxin and down-regulating thioredoxin expression (50, 51, 52). Expression of VDUP1 is down-regulated in chemically induced rat mammary tumors (53). Thus, in mammary cancer cells, the growth-suppressive effect of glucocorticoids may be mediated through the induction of G0S8/RGS2 and PLZF, whereas the growth-promoting effect of progestins may be mediated through the down-regulation of RAR
and VDUP1.
In contrast, many of the genes that are preferentially induced by progestins or suppressed by glucocorticoids are potentially proliferation-related (Table 3
, bottom). Mac-2 Binding Protein/90K is a tumor-derived antigen and is expressed at elevated levels in the serum of patients with breast cancer and other types of cancer (54).
-N-Acetylgalactosaminidase (NaGalase) is an extracellular matrix-degrading enzyme that is produced exclusively by cancer cells (55). The NaGalase levels in mice bearing squamous cell carcinoma increased with time of tumor growth and were directly proportional to tumor burden (56). INHBB promotes mammary gland ductal elongation and alveolar morphogenesis (37). MAPK phosphatase 2 can be induced by growth factors and is up-regulated in cells transformed by v-Jun or mutated K-ras (57, 58). Pim-2 is a protooncogene that induces lymphoid tumors synergistically with c-myc in mice (59, 60). IEX-1/Dif-2 gene can be induced by serum or growth factors (40) and is expressed in proliferating monocytes but significantly down-regulated during differentiation (39). In mammary cancer cells, the growth-suppressive effect of glucocorticoids may be mediated through the down-regulation of NaGalase, MAPK phosphatase 2, and IEX-1/Dif-2, whereas the growth-promoting effect of progestins may be mediated through the induction of Mac-2 BP/90K, INHBB, and Pim-2 h.
In addition to the effects on mammary cancer cell growth, glucocorticoids promote milk protein synthesis and lactation (17, 18, 19), whereas progestins inhibit milk production and secretion (11). Vasoactive intestinal polypeptide (VIP) receptor-related protein is the long isoform of VIP receptor. VIP has been shown to be a physiological mediator of PRL release in the rat (61). It is likely that glucocorticoid-specific stimulation of lactation is, in part, mediated by glucocorticoid-specific induction of VIP receptors (Table 3
).
In summary, the genes identified to be differentially regulated by glucocorticoids and progestins provide potential mechanisms through which the two hormones exert different or opposite biological effects. With a better understanding of the functionally uncharacterized genes identified here, more potential mechanisms will emerge. Future studies exploring these potential mechanisms will assist in developing tumor markers and therapeutic agents for cancer. In addition, genes identified in this study can be used as model systems to investigate the molecular mechanisms underlying differential gene regulation by glucocorticoids and progestins. The fruits of these investigations will enhance our appreciation of the larger question of how related transcription factors mediate distinct, even opposing, biological actions.
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MATERIALS AND METHODS
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Cell Culture and RNA Extraction
The T47D/A1-2 cell line was maintained in MEM (Life Technologies, Inc., Gaithersburg, MD) supplemented with 5% FBS (HyClone Laboratories, Inc., Logan, UT), 10 mM HEPES, nonessential amino acids, and 200 µg/ml of G418. For RNA preparations, cells were grown until 80% confluence and fed with fresh medium the day before treatment and RNA extraction. Steroids and inhibitors were used at the following concentration: dexamethasone (100 nM), R5020 (10 nM), RU486 (100 nM), cycloheximide (40 µg/ml). RNA was prepared using a Purescript RNA isolation kit (Gentra Systems, Minneapolis, MN). RNA was treated with ribonuclease (RNase)-free deoxyribonuclease I (DNaseI) (Ambion, Inc., Austin, TX) to remove all trace of genomic DNA, and the DNaseI was inactivated by 20 mM EDTA and heating at 75 C for 15 min. For preparing labeled cRNA for array hybridization, total RNA was also extracted with phenol/chloroform (Ambion, Inc.) and precipitated with ethanol.
Preparation of Labeled cRNA and Array Hybridization
All procedures were performed according to the instructions from Affymetrix (Santa Clara, CA). Total RNA (10 µg) was converted into double-stranded cDNA by using an oligo-dT primer with a T7 promoter at the 5'-end and the SuperScript Choice system for cDNA synthesis (Life Technologies, Inc.). Double-stranded cDNA was extracted with phenol/chloroform, precipitated with ethanol, and resuspended in 12 µl RNase-free dH2O. Half of the cDNA was used for in vitro transcription with a T7 RNA polymerase Megascript system (Ambion, Inc.) in the presence of biotinylated UTP and CTP (Enzo, Farmingdale, NY). The labeled cRNA was purified with RNeasy columns (QIAGEN, Chatsworth, CA), fragmented, and used to make up the hybridization cocktail containing control oligonucleotide B2 and four control bacterial and phage cRNAs (BioB, BioC, BioD, cre).
An aliquot of each sample was first hybridized to an Affymetrix Test 2 Array to determine sample quality according to manufacturers criteria. All samples passed the test and were hybridized to a set of five Affymetrix GeneChip HuGeneFL Arrays, each carrying probes for about 5600 full-length human genes. After washing and staining, the arrays were scanned using a laser scanner controlled by the GeneChip 3.3 software (Affymetrix). To amplify the staining signal so that weakly expressed genes can also be detected, the cartridges were subjected to antibody amplification, second staining, and scanning.
Data Analysis and Criteria for Selecting Regulated Genes
The scanned data were analyzed using GeneChip Expression Analysis Software (version 3.3, Affymetrix). To correct for minor differences in overall chip fluorescence, intensity values were scaled to 2500 so that the overall fluorescence intensity of each chip was equivalent (62).
It has been established that a change of 2-fold or greater is both significant and accurate using oligonucleotide array technology (63). In this study, a more stringent cut-off, a change of at least 3-fold at either of the two treatment time points, was used for selecting genes that are hormonally regulated. A 3-fold or greater change is deemed valid only when at least one sample in the comparison pair is scored as "present" and the change is also scored as "increase" or "decrease" by the GeneChip analysis software. The criteria for selecting genes that are differentially regulated by two hormones are: 1) a fold difference of at least 3 when comparing a Dex-treated sample with a R5020-treated sample at either time point; 2) a 3-fold or greater difference is deemed valid only when the change is scored as increase or decrease; 3) at least one of the four comparison pairs between hormone-treated sample and vehicle-treated sample has a valid change of at least 3-fold according to the criteria above.
Semiquantitative RT-PCR
DNaseI-treated total RNA was reverse transcribed into single-stranded cDNA using random primers and SuperScript II or Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.). For semiquantitative PCR, the best cycle number for linear amplification of the cDNA using each gene-specific primer pair was determined by amplifying serially diluted cDNA templates. The cDNA from each experiment sample was PCR amplified at this cycle, and the products were run on agarose gel and visualized by ethidium bromide staining.
Northern Blot Analyses
DNaseI-treated total RNA (30 µg) was separated on formaldehyde agarose gel and transferred to Hybond N+ nylon membrane (Amersham Pharmacia Biotech, Arlington Heights, IL). cDNA probes for G0S8, PLZF, and IEX-1 represent the entire coding sequences and were generated by RT-PCR and subcloning into vectors. A cDNA probe for INHBB was isolated from plasmid pSP65 (provided by Genentech, Inc., South San Francisco, CA). cDNA probes were labeled with a RediPrime II kit (Pharmacia Biotech, Piscataway, NJ) and hybridized with the membrane at 42 C overnight. After washing, the blot was quantitated by PhosphorImager with the STORM 860 system (Molecular Dynamics, Inc., Sunnyvale, CA).
Cell Growth Assay
T47D/A1-2 cells (5 x 104) were plated into triplicate wells in 12-well plates with 2 ml of serum-containing medium. After 2024 h, the cells were treated with 1/1,000 volume of vehicle (95% ethanol), Dex (100 nM), or R5020 (10 nM). Cells were harvested every 24 h after hormone addition, and the DNA content in each well was measured using a Hoechst DNA assay as described previously (64). The culture medium was removed and 300 µl of 0.5 M NaOH were added to each well and pipetted up and down. The 12-well plates were frozen immediately at -20 C until all time points were collected. After thawing, samples were pipetted to dissolve DNA and were neutralized with an equal volume of 0.5 M HCl + 200 mM phosphate buffer. Two aliquots of each sample were mixed with equal volumes of Hoechst stain (0.2 µg/ml Hoechst 33258 in phosphate buffer containing 4.6 M NaCl) and were incubated in the dark at room temperature for 45 min. The relative fluorescence of each sample was measured using a MicroFLUOR plate reader (Dynatech Corp., Chantilly, VA). Five known concentrations of calf thymus DNA were used to develop a standard curve from which relative fluorescence units could be converted to micrograms per milliliter DNA for each sample.
Flow Cytometry
T47D/A1-2 cells (3 x 105) were plated into duplicate wells in six-well plates with 3 ml of serum-containing medium. After 2830 h, the cells were treated with 1/10,000 volume of vehicle (95% ethanol), Dex (100 nM), or R5020 (10 nM). Cells were harvested at the start of treatment (0 h) and every 6 or 12 h after hormone addition with trypsin-EDTA. The cell suspension was pelleted, washed with cold PBS, and resuspended in 1 ml of Krishans stain (65) containing propidium iodide and ribonuclease (RNase A). Samples were stained at 4 C for at least overnight before flow cytometry. For each sample, 10,000 cells were analyzed on an Epics 752 flow cytometer (Coulter Electronics, Hialeah, FL) using an incident beam on forward angle vs. 90° light scatter to eliminate cellular debris and doublets. Red fluorescence, corresponding to DNA, was collected through a 590-nm long pass filter, and histograms of DNA content vs. cell number were constructed. Cell cycle analyses of the DNA histograms were performed using the ModFIt Analysis Program (Veritey Software House, Topsham, ME), which provides fits for the G0/G1, S, and G2/M fractions of the population. The S- and G2/M-phase fractions were combined into a single growth fraction. In Fig. 4
, the ordinate shows the difference between the fraction of cells in S+G2/M in the hormone-treated sets and the fraction of cells in S+G2/M in the vehicle-treated controls.
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ACKNOWLEDGMENTS
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We thank the Affymetrix Microarray Core Facility in the University of Colorado Health Sciences Center for making this study possible and the Flow Cytometry Core Lab in the University of Colorado Health Sciences Center for the assistance in flow cytometry studies. We also thank Widya Johannes for her aid in the Hoechst DNA assay.
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FOOTNOTES
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This work was supported by NIH Grant DK-37061 (to S.K.N.) and Predoctoral Fellowship Award DAMD17-99-1-9445 from the Department of Defense (to Y.W.).
Abbreviations: Dex, Dexamethasone; DNase, deoxyribonuclease; 11ß-HSD2, 11ß-hydroxysteroid dehydrogenase type 2; INHBB, inhibin ßB; NaGalase, N-acetylgalactosaminidase; PLZF, promyelocytic leukemia zinc finger protein; RNase, ribonuclease; VDUP1, vitamin D3 up-regulated protein 1; VIP, vasoactive intestinal polypeptide.
Received for publication August 24, 2001.
Accepted for publication December 19, 2001.
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REFERENCES
|
---|
-
Thornton JW 2001 Evolution of vertebrate steroid receptors from an ancestral estrogen receptor by ligand exploitation and serial genome expansions. Proc Natl Acad Sci USA 98:56715676[Abstract/Free Full Text]
-
Cato AC, Miksicek R, Schutz G, Arnemann J, Beato M 1986 The hormone regulatory element of mouse mammary tumour virus mediates progesterone induction. EMBO J 5:22372240[Abstract]
-
Hynes N, van Ooyen AJ, Kennedy N, Herrlich P, Ponta H, Groner B 1983 Subfragments of the large terminal repeat cause glucocorticoid-responsive expression of mouse mammary tumor virus and of an adjacent gene. Proc Natl Acad Sci USA 80:36373641[Abstract]
-
Lieberman BA, Bona BJ, Edwards DP, Nordeen SK 1993 The constitution of a progesterone response element. Mol Endocrinol 7:515527[Abstract]
-
Payvar F, DeFranco D, Firestone GL, Edgar B, Wrange O, Okret S, Gustafsson JA, Yamamoto, KR 1983 Sequence-specific binding of glucocorticoid receptor to MTV DNA at sites within and upstream of the transcribed region. Cell 35:381392[Medline]
-
Scheidereit C, Geisse S, Westphal HM, Beato M 1983 The glucocorticoid receptor binds to defined nucleotide sequences near the promoter of mouse mammary tumour virus. Nature 304:749752[Medline]
-
Pratt WB, Toft DO 1997 Steroid receptor interactions with heat shock protein and immunophilin chaperones. Endocr Rev 18:306360[Abstract/Free Full Text]
-
McKenna NJ, Lanz RB, OMalley BW 1999 Nuclear receptor coregulators: cellular and molecular biology. Endocr Rev 20:321344[Abstract/Free Full Text]
-
Westin S, Rosenfeld MG, Glass CK 2000 Nuclear receptor coactivators. Adv Pharmacol 47:89112[Medline]
-
Porterfield SP 1996 Adrenal gland. In: Endocrine physiology. St. Louis: Mosby-Year Book, Inc.; Chap 7:139146
-
Graham JD, Clarke CL 1997 Physiological action of progesterone in target tissues. Endocr Rev 18:502519[Abstract/Free Full Text]
-
Lane NE, Lukert B 1998 The science and therapy of glucocorticoid-induced bone loss. Endocrinol Metab Clin North Am 27:465483[Medline]
-
Rackoff PJ, Rosen CJ 1998 Pathogenesis and treatment of glucocorticoid-induced osteoporosis. Drugs Aging 12:477484[Medline]
-
Ziegler R, Kasperk C 1998 Glucocorticoid-induced osteoporosis: prevention and treatment. Steroids 63:344348[CrossRef][Medline]
-
Nomura S, Hashmi S, McVey JH, Ham J, Parker M, Hogan BL 1989 Evidence for positive and negative regulatory elements in the 5'-flanking sequence of the mouse sparc (osteonectin) gene. J Biol Chem 264:1220112207[Abstract/Free Full Text]
-
Prior JC 1990 Progesterone as a bone-trophic hormone. Endocr Rev 11:386398[Abstract]
-
Doppler W, Groner B, Ball RK 1989 Prolactin and glucocorticoid hormones synergistically induce expression of transfected rat ß-casein gene promoter constructs in a mammary epithelial cell line. Proc Natl Acad Sci USA 86:104108[Abstract]
-
Groner B, Altiok S, Meier V 1994 Hormonal regulation of transcription factor activity in mammary epithelial cells. Mol Cell Endocrinol 100:109114[CrossRef][Medline]
-
Groner B, Gouilleux F 1995 Prolactin-mediated gene activation in mammary epithelial cells. Curr Opin Genet Dev 5:587594[CrossRef][Medline]
-
Horwitz KB 1992 The molecular biology of RU486. Is there a role for antiprogestins in the treatment of breast cancer? Endocr Rev 13:146163[Medline]
-
Goya L, Maiyar AC, Ge Y, Firestone GL 1993 Glucocorticoids induce a G1/G0 cell cycle arrest of Con8 rat mammary tumor cells that is synchronously reversed by steroid withdrawal or addition of transforming growth factor-
. Mol Endocrinol 7:11211132[Abstract]
-
Lippman M, Bolan G, Huff K 1976 The effects of glucocorticoids and progesterone on hormone-responsive human breast cancer in long-term tissue culture. Cancer Res 36:46024609[Abstract]
-
Lambert JR, Nordeen SK 1998 Steroid-selective initiation of chromatin remodeling and transcriptional activation of the mouse mammary tumor virus promoter is controlled by the site of promoter integration. J Biol Chem 273:3270832714[Abstract/Free Full Text]
-
Nordeen SK, Kuhnel B, Lawler-Heavner J, Barber DA, Edwards DP 1989 A quantitative comparison of dual control of a hormone response element by progestins and glucocorticoids in the same cell line. Mol Endocrinol 3:12701278[Abstract]
-
Darnel AD, Archer TK, Yang K 1999 Regulation of 11ß-hydroxysteroid dehydrogenase type 2 by steroid hormones and epidermal growth factor in the Ishikawa human endometrial cell line. J Steroid Biochem Mol Biol 70:203210[CrossRef][Medline]
-
Siderovski DP, Heximer SP, Forsdyke DR 1994 A human gene encoding a putative basic helix-loop-helix phosphoprotein whose mRNA increases rapidly in cycloheximide-treated blood mononuclear cells. DNA Cell Biol 13:125147[Medline]
-
Heximer SP, Watson N, Linder ME, Blumer KJ, Hepler JR 1997 RGS2/G0S8 is a selective inhibitor of Gq
function. Proc Natl Acad Sci USA 94:1438914393[Abstract/Free Full Text]
-
Oliveira-Dos-Santos AJ, Matsumoto G, Snow BE, Bai D, Houston FP, Whishaw IQ, Mariathasan S, Sasaki T, Wakeham A, Ohashi PS, Roder JC, Barnes CA, Siderovski DP, Penninger JM 2000 Regulation of T cell activation, anxiety, and male aggression by RGS2. Proc Natl Acad Sci USA 97:1227212277[Abstract/Free Full Text]
-
Nishizuka M, Honda K, Tsuchiya T, Nishihara T, Imagawa M 2001 RGS2 promotes adipocyte differentiation in the presence of ligand for peroxisome proliferator-activated receptor
. J Biol Chem 276:2962529627[Abstract/Free Full Text]
-
Chen Z, Brand NJ, Chen A, Chen SJ, Tong JH, Wang ZY, Waxman S, Zelent A 1993 Fusion between a novel Kruppel-like zinc finger gene and the retinoic acid receptor-
locus due to a variant t(11;17) translocation associated with acute promyelocytic leukaemia. EMBO J 12:11611167[Abstract]
-
Dong S, Zhu J, Reid A, Strutt P, Guidez F, Zhong HJ, Wang ZY, Licht J, Waxman S, Chomienne C, Chen Z, Zelent A, Chen SJ 1996 Amino-terminal protein-protein interaction motif (POZ-domain) is responsible for activities of the promyelocytic leukemia zinc finger-retinoic acid receptor-
fusion protein. Proc Natl Acad Sci USA 93:36243629[Abstract/Free Full Text]
-
David G, Alland L, Hong SH, Wong CW, DePinho RA, Dejean A 1998 Histone deacetylase associated with mSin3A mediates repression by the acute promyelocytic leukemia-associated PLZF protein. Oncogene 16:25492556[CrossRef][Medline]
-
Shaknovich R, Yeyati PL, Ivins S, Melnick A, Lempert C, Waxman S, Zelent A, Licht JD 1998 The promyelocytic leukemia zinc finger protein affects myeloid cell growth, differentiation, and apoptosis. Mol Cell Biol 18:55335545[Abstract/Free Full Text]
-
Yeyati PL, Shaknovich R, Boterashvili S, Li J, Ball HJ, Waxman S, Nason-Burchenal K, Dmitrovsky E, Zelent A, Licht JD 1999 Leukemia translocation protein PLZF inhibits cell growth and expression of cyclin A. Oncogene 18:925934[CrossRef][Medline]
-
Feng ZM, Bardin CW, Chen CL 1989 Characterization and regulation of testicular inhibin ß-subunit mRNA. Mol Endocrinol 3:939948[Abstract]
-
Shyamala G 1999 Progesterone signaling and mammary gland morphogenesis. J Mammary Gland Biol Neoplasia 4:89104[CrossRef][Medline]
-
Robinson GW, Hennighausen L 1997 Inhibins and activins regulate mammary epithelial cell differentiation through mesenchymal-epithelial interactions. Development 124:27012708[Abstract/Free Full Text]
-
Kondratyev AD, Chung KN, Jung MO 1996 Identification and characterization of a radiation-inducible glycosylated human early-response gene. Cancer Res 56:14981502[Abstract]
-
Pietzsch A, Buchler C, Aslanidis C, Schmitz G 1997 Identification and characterization of a novel monocyte/macrophage differentiation-dependent gene that is responsive to lipopolysaccharide, ceramide, and lysophosphatidylcholine. Biochem Biophys Res Commun 235:49[CrossRef][Medline]
-
Schafer H, Lettau P, Trauzold A, Banasch M, Schmidt WE 1999 Human PACAP response gene 1 (p22/PRG1): proliferation-associated expression in pancreatic carcinoma cells. Pancreas 18:378384[Medline]
-
Jelinsky SA, Samson LD 1999 Global response of Saccharomyces cerevisiae to an alkylating agent. Proc Natl Acad Sci USA 96:14861491[Abstract/Free Full Text]
-
Harkin DP, Bean JM, Miklos D, Song YH, Truong VB, Englert C, Christians FC, Ellisen LW, Maheswaran S, Oliner JD, Haber DA 1999 Induction of GADD45 and JNK/SAPK-dependent apoptosis following inducible expression of BRCA1. Cell 97:575586[Medline]
-
Fambrough D, McClure K, Kazlauskas A, Lander ES 1999 Diverse signaling pathways activated by growth factor receptors induce broadly overlapping, rather than independent, sets of genes. Cell 97:727741[Medline]
-
McKay LI, Cidlowski JA 1999 Molecular control of immune/inflammatory responses: interactions between nuclear factor-
B and steroid receptor-signaling pathways. Endocr Rev 20:435459[Abstract/Free Full Text]
-
Lange CA, Richer JK, Horwitz KB 1999 Hypothesis: progesterone primes breast cancer cells for cross-talk with proliferative or antiproliferative signals. Mol Endocrinol 13:829836[Free Full Text]
-
Groshong SD, Owen GI, Grimison B, Schauer IE, Todd MC, Langan TA, Sclafani RA, Lange CA, Horwitz KB 1997 Biphasic regulation of breast cancer cell growth by progesterone: role of the cyclin-dependent kinase inhibitors, p21 and p27(Kip1). Mol Endocrinol 11:15931607[Abstract/Free Full Text]
-
Musgrove EA, Lee CS, Sutherland RL 1991 Progestins both stimulate and inhibit breast cancer cell cycle progression while increasing expression of transforming growth factor
, epidermal growth factor receptor, c-fos, and c-myc genes. Mol Cell Biol 11:50325043[Medline]
-
Fanjul AN, Bouterfa H, Dawson M, Pfahl M 1996 Potential role for retinoic acid receptor-
in the inhibition of breast cancer cells by selective retinoids and interferons. Cancer Res 56:15711577[Abstract]
-
Raffo P, Emionite L, Colucci L, Belmondo F, Moro MG, Bollag W, Toma S 2000 Retinoid receptors: pathways of proliferation inhibition and apoptosis induction in breast cancer cell lines. Anticancer Res 20:15351543[Medline]
-
Nishiyama A, Matsui M, Iwata S, Hirota K, Masutani H, Nakamura H, Takagi Y, Sono H, Gon Y, Yodoi J 1999 Identification of thioredoxin-binding protein-2/vitamin D(3) up-regulated protein 1 as a negative regulator of thioredoxin function and expression. J Biol Chem 274:2164521650[Abstract/Free Full Text]
-
Nakamura H, Masutani H, Tagaya Y, Yamauchi A, Inamoto T, Nanbu Y, Fujii S, Ozawa K, Yodoi J 1992 Expression and growth-promoting effect of adult T-cell leukemia-derived factor. A human thioredoxin homologue in hepatocellular carcinoma. Cancer 69:20912097[Medline]
-
Nakamura H, Nakamura K, Yodoi J 1997 Redox regulation of cellular activation. Annu Rev Immunol 15:351369[CrossRef][Medline]
-
Yang X, Young LH, Voigt JM 1998 Expression of a vitamin D-regulated gene (VDUP-1) in untreated- and MNU-treated rat mammary tissue. Breast Cancer Res Treat 48:3344[CrossRef][Medline]
-
Fusco O, Querzoli P, Nenci I, Natoli C, Brakebush C, Ullrich A, Iacobelli S 1998 90K (MAC-2 BP) gene expression in breast cancer and evidence for the production of 90K by peripheral-blood mononuclear cells. Int J Cancer 79:2326[CrossRef][Medline]
-
Yamamoto N, Naraparaju VR, Asbell SO 1996 Deglycosylation of serum vitamin D3-binding protein leads to immunosuppression in cancer patients. Cancer Res 56: 827831
-
Korbelik M, Naraparaju VR, Yamamoto N 1998 The value of serum
-N-acetylgalactosaminidase measurement for the assessment of tumour response to radio- and photodynamic therapy. Br J Cancer 77:10091014[Medline]
-
Fu SL, Waha A, Vogt PK 2000 Identification and characterization of genes upregulated in cells transformed by v-Jun. Oncogene 19:35373545[CrossRef][Medline]
-
Yip-Schneider MT, Lin A, Marshall MS 2001 Pancreatic tumor cells with mutant K-ras suppress ERK activity by MEK-dependent induction of MAP kinase phosphatase-2. Biochem Biophys Res Commun 280:992997[CrossRef][Medline]
-
Allen JD, Verhoeven E, Domen J, van der Valk M, Berns A 1997 Pim-2 transgene induces lymphoid tumors, exhibiting potent synergy with c-myc. Oncogene 15:11331141[CrossRef][Medline]
-
Baytel D, Shalo, S, Madgar I, Weissenberg R, Don J 1998 The human Pim-2 proto-oncogene and its testicular expression. Biochim Biophys Acta 1442:274285[Medline]
-
Abe H, Engler D, Molitch ME, Bollinger-Gruber J, Reichlin S 1985 Vasoactive intestinal peptide is a physiological mediator of prolactin release in the rat. Endocrinology 116:13831390[Abstract]
-
Tamayo P, Slonim D, Mesirov J, Zhu Q, Kitareewan S, Dmitrovsky E, Lander ES, Golub TR 1999 Interpreting patterns of gene expression with self-organizing maps: methods and application to hematopoietic differentiation. Proc Natl Acad Sci USA 96:29072912[Abstract/Free Full Text]
-
Wodicka L, Dong H, Mittmann M, Ho MH, Lockhart DJ 1997 Genome-wide expression monitoring in Saccharomyces cerevisiae. Nat Biotechnol 15: 13591367
-
Hedlund TE, Miller GJ 1994 A serum-free defined medium capable of supporting growth of four established human prostatic carcinoma cell lines. Prostate 24:221228[Medline]
-
Krishan A 1975 Rapid flow cytofluorometric analysis of mammalian cell cycle by propidium iodide staining. J Cell Biol 66:188193[Abstract]