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Cancer Functional Genomics Unit, Murdoch Children’s Research Institute, 10th Floor Royal Children’s Hospital, Parkville, Victoria 3052, Australia [T. B., D. J. V.]; Department of Pathology, The University of Melbourne, Victoria 3010, Australia [M. C. S., J. E. A., D. J. V.]; Molecular Pathology Laboratory, Victorian Breast Cancer Research Consortium, Australia [J. E. A.]; Department of Surgery, St Vincent’s Hospital, Fitzroy, Victoria 3065, Australia [M. A. H.]; Children’s Medical Research Institute, Westmead, Sydney, NSW 2145, Australia [A. C. C., R. R. R.]; and Eos Biotechnology, South San Francisco, California 94080 [D. W., R. G.]
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ABSTRACT |
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10,000 known genes and 25,000 expressed sequence tag clusters was performed to identify genes induced by estrogen and repressed by the pure antiestrogen ICI 182 780 in vitro that correlated with estrogen receptor (ER) expression in primary breast carcinomas in vivo. Stanniocalcin (STC) 2 was identified as one of the genes that fulfilled these criteria. DNA microarray hybridization showed a 3-fold induction of STC2 mRNA expression in MCF-7 cells in 
3 h of estrogen exposure and a 3-fold repression in the presence of antiestrogen (one-way ANOVA, P < 0.0005). In 13 ER-positive and 12 ER-negative breast carcinomas, the microarray-derived mRNA levels observed for STC2 correlated with tumor ER mRNA (Pearson’s correlation, r = 0.85; P < 0.0001) and ER protein status (Spearman’s rank correlation, r = 0.73; P < 0.0001). The expression profile of STC2 was further confirmed by in situ hybridization and immunohistochemistry on a larger cohort of 236 unselected breast carcinomas using tissue microarrays. STC2 mRNA and protein expression were found to be associated with tumor ER status (Fisher’s exact test, P < 0.005). The related gene, STC1, was also examined and shown to be associated with ER status in breast carcinomas (Fisher’s exact test, P < 0.05). This study demonstrates the feasibility of using global gene expression data derived from an in vitro model to pinpoint novel estrogen-responsive genes of potential clinical relevance. |
Introduction |
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Recent cDNA microarray analyses have identified widespread differences in gene expression between ER-positive and ER-negative breast cancer clinical samples. Specifically, in two independent studies representing a total of 83 breast carcinomas, the clustering of global gene expression patterns divided tumors into two major groups distinguished by ER status (3
, 4)
. As yet, no systematic analysis has been described to unify the observed changes and determine which genes represent novel estrogen-responsive genes. We undertook a comprehensive analysis of genes responsive to estrogen and the pure antiestrogen ICI 182 780 in the well-studied, ER-positive breast cancer cell line model, MCF-7 (5)
, using a high-density Affymetrix array capable of measuring 35,000 genes and expressed sequence tag clusters. To further pinpoint estrogen target genes with potential clinical relevance, these in vitro data were combined with the expression profiles of a cohort of 13 ER-positive and 12 ER-negative primary breast carcinomas, and the results were evaluated on an independent panel of 236 clinical breast cancer samples. Using this approach, STC2, a homologue of a glycoprotein hormone originally found to regulate calcium/phosphate homeostasis in bony fish (6)
, was identified as an estrogen-responsive gene that was also differentially expressed between ER-positive and ER-negative breast carcinomas.
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Materials and Methods |
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Tumor Specimens.
mRNA was extracted from a total of 25 archival fresh frozen breast carcinomas obtained from patients undergoing surgery at the Peter MacCallum Cancer Institute. A pathologist grossly dissected histologically verified tumor tissue from normal adjacent tissue. All mRNA was extracted by the standard guanidinium thiocyanate methodology. The ER protein status of the tumors was determined clinically by immunohistochemistry. Tumors were deemed ER positive if 
10% of cells stained at an intensity of weak or above. Of the 25 tumors, 13 were classified as ER positive and 12 ER negative. Of the 13 ER-positive cases, 6 were weakly staining, 5 moderately, and 2 strongly. Appropriate institutional approval was obtained for all tumor material used in this study.
Microarray Hybridization.
Oligonucleotide arrays (Eos Biotechnology-specified Affymetrix 43K GeneChip Set) composed of 10,000 human genes and 25,000 human expressed sequence tag clusters were used for hybridization. The protocols used for poly(A)+ mRNA purification, cDNA synthesis, in vitro transcription, chip hybridization, and statistical analysis are described by Glynne et al. (7)
.
Tumor Specimens and Tissue Arrays.
Archived formalin-fixed, paraffin-embedded specimens of primary breast carcinoma were retrieved from files at the Department of Pathology at the Peter MacCallum Cancer Institute and the Department of Surgery at St. Vincent’s Hospital. Cases used from the Peter MacCallum Institute represented women undergoing surgery from 1996 to 1998 and from St. Vincent’s Hospital from 1991 to 1994. All specimens were obtained surgically from patients and in 1 h fixed in 10% buffered formalin for 24 h and then embedded in paraffin wax in routine manner. Storage of paraffin blocks was at room temperature. A total of 236 primary breast tumors was used to construct tissue arrays, representing primary invasive breast carcinoma. The Institutional Ethics Committees of the Peter MacCallum Cancer Institute and St Vincent’s Hospital approved the use of archival tumor tissue for this study.
Probe Labeling by in Vitro Transcription of DNA with DIG.
Linearized cDNA probe (1 µg) was in vitro transcribed and labeled with DIG, according to the manufacturer’s instructions (Roche Diagnostics Australia Pty. Ltd., Melbourne, Victoria, Australia). Labeled probe (5 µl) was electrophoresed on a 1.5% agarose gel to check for riboprobe integrity. Serial dilutions were used to estimate the concentration of labeled probe against DIG-labeled control mRNA (Roche Diagnostics Australia Pty. Ltd.), according to the manufacturer’s instructions.
Slide Preparation and Probe Hybridization.
STC1 and STC2 mRNA was localized by in situ hybridization. For each gene, separate tissue array-derived slides were probed with an in vitro-transcribed, DIG-labeled antisense and sense probe (negative control). In addition, a cytokeratin-19 probe was used to verify the integrity of tissue mRNA.
In situ hybridization was performed in RNase-free conditions on 5-µm sections of tissue arrays mounted on APES (3-aminopropyltriethoxysilane)-coated slides and dried for 2 h at 60°C. Sections were dewaxed and then placed into 0.2 M HCl, shaken at room temperature for 20 min, and then washed twice in diethyl pyrocarbonate-treated sterile water for 5 min with shaking at room temperature. Sections were then digested with 1 µg/ml proteinase K in prewarmed digest buffer [100 mM Tris (pH 8.0) and 50 mM EDTA (pH 8.0)] for 30 min at 37°C. The digest was stopped by washing slides in 0.2% glycine/PBS for 10 min at 4°C. Sections were then washed briefly in 0.1 M triethanolamine before adding acetic anhydride for 5 min to a final concentration of 0.25%.
Prehybridization was performed by adding 100 µl of prehybridization solution [50% deionized formamide, 24 mM Tris (pH 7.4), 1 M EDTA, 375 mM NaCl, 10% Dextran Sulfate, 1 x Denharts Solution, 250 µg/ml yeast total mRNA, 100 µg/ml single-stranded DNA, and 250 µg/ml tRNA] to each section. Sections were then covered with a coverslip and incubated for 2 h at 50°C (STC1 probe) or at 60°C (STC2 and CK19 probes). For the hybridization reaction, DIG-labeled probe was denatured for 5 min at 65°C and added to hybridization solution (same as prehybridization solution with 0.1% SDS, 0.1% sodium thiosulfate, and 0.1 mM DTT) to a final probe concentration of 200 ng/ml. This probe was thoroughly mixed with the hybridization solution by vortexing for 1 min and then denatured for 15 min at 65°C. Hybridization solution (150 µl) with probe was then added to each slide and incubated at 50°C (STC1 probe) or at 60°C (STC2 and CK19 probes).
Slide Washes and Probe Visualization.
Slides were then washed for 15 min in 2 x SSC at room temperature, followed by decreasing concentrations of SSC (x2, x1, and x0.1) for 15 min. To digest, unbound probe sections were treated with RNase A (final concentration: 20 µg/ml) for 1 h at 37°C, followed by two washes in PBS. Slides were then incubated in blocking solution [Roche Blocking Reagent 1% (w/v) in buffer 1, 100 mM maleic acid, and 150 mM NaCl (pH 7.5)] for 30 min. Blocking solution (100 µl) containing 0.75 units/ml Anti-DIG[Fab]-AP (Roche Diagnostics Australia Pty. Ltd.) was added to each section, coverslipped, and incubated for 1 h at room temperature. Probe localization was visualized by adding nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Roche Diagnostics Australia Pty. Ltd.) color reagent, according to the manufacturer’s instructions. Sections were then rinsed in water to stop color development, counterstained with 0.1% methyl-green, and mounted using Kaiser’s glycerol gelatin (Merck, Whitehouse Station, NJ).
Scoring of in Situ Hybridization.
Each arrayed tissue sample was probed with a sense and antisense probe for STC1 and STC2. Staining of normal human endometrium with STC1/STC2 probes served as a positive and internal negative tissue control. For each tumor array, two independent hybridization experiments were performed. Tumors were deemed to express STC1/STC2 mRNA if there was positive staining in the two independent experiments that was greater than staining with the cognate sense strand probe and negative control probe. Positive staining ranged from moderate to strong staining in a small percentage of scattered tumor cells (5–10%), to 10% of tumor cells staining at an intensity of weak to strong compared with the sense control.
Immunohistochemistry.
Polyclonal antibodies for STC1 (rabbit) and STC2 (sheep) were generated as described previously (8)
. Staining was performed on 3-µm sections of formalin-fixed, paraffin wax-embedded tissue rehydrated through graded alcohols. Antigen retrieval was used for the ER, PR, STC1, and STC2 antibodies, which consisted of 2 min of heating under pressure in a pressure cooker in 10 mM sodium citrate (pH 6.0). Sections were stained using a DAKO Autostainer (DAKO Corp., Carpinteria, CA) using established protocols as described by Armes et al. (9)
. The primary antibody was applied at the following dilutions in 10% FCS made up in 50 mM Tris-HCl (pH 7.6) and 0.05% Tween 20 for 30 min at room temperature: ER (1:200; DAKO), PR (1:800; DAKO), pS2 (1:100; Novocastra Laboratories), STC1 (1:3000), and STC2 (1:50; diluted in background reducing diluent, instead of FCS; DAKO). Negative controls were incubated in 10% FCS minus the primary antibody. Biotinylated secondary antibodies were detected with streptavidin peroxidase by using the LSAB 2 kit (pS2) or the LSAB+ kit (ER, PR, STC1, and STC2; DAKO). The final color reaction was carried out using aminoethylcarbazole as a chromogen (AEC+; DAKO) and counterstained with hematoxylin.
For all of the antibodies, the intensity of staining and proportion of positive cells were determined for each case, according to a method described by Armes et al. (9)
. Briefly, a semiquantitative estimate of expression levels of the antigen was based on the combined score for the proportion of staining cells and the intensity of staining. The proportion score represented the estimated percentage of positive cells as a fraction of tumor cells (0, <10%; 1, 11–25%; 2, 26–50%; 3, 51–75%; 4, 76–90%; and 5, >91%.). The intensity score represented the average staining intensity for positive cells (0, none; 1, weak; 2, moderate; and 3, strong). Levels of staining were derived as follows: samples with an intensity score of 0 or having <10% of cells staining were designated negative; samples with intensity score of 1 in 10% of cells were designated weak. For intensity levels 2 and 3, combined scores of 2–3 were designated as weak, 4–6 as moderate, and 7 or 8 as strong expression. For analysis purposes, staining of all antibodies was considered positive if 10% of cells stained unequivocally at an intensity of weak, moderate, or strong compared with the no primary antibody control. For STC1, STC2, and pS2, an additional, reproducible staining pattern was also observed in a subset of the cases. This consisted of moderate-to-strong unequivocal staining in scattered tumor cells (5–10%). For analysis purposes, this pattern was also considered indicative of positive staining for these antigens. Results were obtained for two independent immunohistochemistry experiments.
Statistics.
Statistical comparisons between groups were assessed by standard contingency table analysis using two-tailed Fisher’s exact test. Data were analyzed using the program StatXact 4 (Cytel Software Corp., Cambridge, MA). Pearson’s and Spearman’s rank correlation were performed using GraphPad InStat version 3.00 for Windows 95 (GraphPad Software, San Diego CA).5
Nominal Ps are given without adjustment for multiple comparisons. A cutoff P of 0.05 was taken to indicate significance. An agglomerative hierarchical clustering method was used to investigate the relationship among the mRNA expression patterns of the tumor samples. Data were imported into the Spotfire Decision Site 6.2 for Functional Genomics program and reordered in similarity in expression patterns over the 25 samples using the agglomerative clustering algorithm for hierarchical clustering. For the in vitro MCF-7 experiments, expression profiles were analyzed by one-way ANOVA for statistical significance specifying time, treatment (estrogen or vector; ICI 182 780 or vector), experiment repeat, and treatment by time interaction as factors in the model. The probability that treatment with estrogen had no effect on the expression level of STC2 in MCF7 cells was 3 x 10-4; the probability that treatment with ICI 182 780 had no effect on the expression level of STC2 in MCF7 cells was 5 x 10-5.
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Results |
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35,000 genes in each of the samples. Through this analysis, 299 genes were found to be regulated by estrogen and/or ICI 182 780 in MCF-7 cells, with a significance value cutoff of P 0.0005. To focus on genes that were also associated with ER expression in breast carcinomas, the expression profile of the 299 estrogen and/or ICI 182 780-responsive genes found in MCF-7 cells were compared between 13 ER-positive and 12 ER-negative breast tumors. STC2 was identified as one of the genes that fulfilled these criteria. The expression profile of the top 10 differentially expressed genes (including STC2) between ER-positive and ER-negative tumors that were also found to be estrogen and/or ICI 182 780 regulated in MCF-7 cells (although not the subject of further discussion in this study) is shown in Fig. 1
. STC2 was differentially expressed between ER-positive and ER-negative tumors (ER status by immunohistochemistry, unpaired t test, P = 0.001) and had mRNA levels that correlated with ER mRNA levels (Pearson’s correlation, n = 25, r = 0.85; P < 0.0001) and ER protein status (Spearman’s rank correlation, n = 25, r = 0.73; P < 0.0001). STC2 mRNA expression in estrogen-stimulated MCF-7 cells showed a 3-fold increase in expression 3 h of treatment and remained elevated at 24 and 48 h (ANOVA, P = 0.0003). After treatment of actively growing MCF-7 cells with the pure antiestrogen ICI 182 780, STC2 mRNA levels decreased 3-fold in 6 h (ANOVA, P = 0.00005). Taken together, these results suggest that ER signaling is sufficient to induce high levels of STC2 mRNA in this in vitro experiment and that this pathway involving ER and STC2 is active in breast cancers in vivo. Interestingly, the closely related family member STC1 was found by microarray analysis to be 2–6-fold higher in 3 of the 13 ER-positive tumors and also in the ER-positive cell line MCF-7, relative to the average expression in 12 ER-negative tumors and 3 ER-negative cell lines (MDA-MB- 231/453/435), respectively, warranting further investigation in breast carcinomas in vivo.
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In Situ Analysis of STC2 mRNA and Protein Levels in Primary Breast Carcinomas and Relationship to ER, PR, and pS2.
For STC2 to be a useful clinical marker of ER activity, we reasoned that STC2 mRNA should be transcribed in transformed epithelial cells and that expression of the message in ER-positive tumors should be accompanied by an increased frequency of STC2 protein expression in ER-positive tumors. To answer these questions with a high level of statistical confidence, we used tissue microarrays of 236 invasive breast cancer samples from different patients (Table 1)
and examined the relationship between STC2 mRNA and protein levels with the protein status of ER and its target genes PR and pS2. Staining for STC2 transcripts after hybridization of breast tumor sections with digioxenin-labeled riboprobes revealed specific expression of STC2 in the tumor cells. The expression pattern for STC2 mRNA in tumors was variable, ranging from: (a) negative to (b) scattered cells staining moderate to strong and to (c) the majority of tumor cells staining strongly. A positive STC2 mRNA staining pattern was observed in 75 of 216 (35%) tumors. When tumors were divided into two subgroups according to their mRNA expression pattern for STC2 as either positive or negative, an association was observed for protein staining of the ER (Fisher’s exact test, P < 0.0001) and PR (Fisher’s exact test, P = 0.0001; Table 1
). A trend was also observed between STC2 mRNA status and pS2 (Fisher’s exact test, P = 0.08).
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. Consistent with STC2 mRNA expression, STC2 immunoreactivity was also observed in breast tumor cells. Infiltrating lymphocytes and stromal cells were negative. Staining was observed in the cytoplasm of cells, as expected for a secreted glycoprotein hormone. Again, the patterns of immunohistochemical staining observed for STC2 were similar to that observed for STC2 mRNA by in situ hybridization. STC2 immunoreactivity ranged from intensely staining isolated cells to positive staining in a majority of tumor cells. For tumors showing STC2 mRNA positivity, 83% also displayed protein expression by STC2 antibody. The minority of discordant cases may reflect the numerous post-transcriptional mechanisms that regulate the stability and turnover of RNA and protein. For the STC2 mRNA-negative cases, about half was observed to be immunoreactive for STC2 protein, perhaps reflecting increased sensitivity for protein detection over mRNA detection. A significant association was observed between immunohistochemical staining for STC2 protein and positivity for ER (Fisher’s exact test, P = 0.003) and pS2 immunoreactivity (Fisher’s exact test, P = 0.0002; Table 1
). A trend was observed between STC2 and PR protein status, although this did not quite reach statistical significance (Fisher’s exact test, P = 0.07).
On the basis of the microarray results of high STC1 mRNA levels in a subset of the ER-positive tumors, in situ hybridization and immunohistochemistry on tissue arrays were also performed for STC1. mRNA positivity for STC1 was identified in 82 of 169 (49%) tumors. For STC1 mRNA expression status, a positive association was observed with ER (Fisher’s exact test, P = 0.0002) and PR immunoreactivity (Fisher’s exact test, P = 0.01; Table 2
). The relationship between mRNA and protein expression of both STC1 and STC2 with ER status in breast carcinomas is shown in Fig. 2
. No association was observed between STC1 mRNA and pS2 protein expression (Fisher’s exact test, P = 0.7). For tumors showing STC1 mRNA expression, 75% also displayed protein expression by the STC1 antibody. A positive association was observed between immunohistochemical staining for STC1 protein and positivity for ER (Fisher’s exact test, P = 0.01) and PR (Fisher’s exact test, P = 0.007; Tables 2
). No association was observed between STC1 and pS2 immunoreactivity (Fisher’s exact test, P = 0.2)
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Discussion |
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Further insight was provided by the examination of two ER target genes, PR and pS2. STC1 and STC2 mRNA levels were both correlated with PR protein expression. STC2 protein levels were also correlated positively with the expression of another ER target gene, pS2. Although STC2 mRNA levels did show a trend for a positive association with pS2 positivity, this relationship was not statistically significant. This may relate to an increased sensitivity for detection of STC2 protein over mRNA. Alternatively, tumor cells that are immunoreactive to STC2 may not necessarily transcribe the gene but may represent the site of action because of the active sequestration of the hormone. Indeed, others have observed this expression pattern for STC1 mRNA versus protein in mammalian kidney and ovary (15 , 16) . In addition, STC1/2 protein levels may be high, although mRNA expression is nondetectable because of low-level transcription coupled to a low rate of protein turnover.
The estrogen responsiveness of STC2 in vitro is consistent with the study by Charpentier et al. (17) , who identified STC2 as a novel estrogen target gene in MCF-7 cells using Serial Analysis of Gene Expression. In addition, we identified three potential ER/Sp1-binding elements in the 5' upstream region of the human STC2 gene, comparable with the estrogen-responsive enhancer elements found in numerous other ER target genes (11, 12, 13) . Functional studies are now needed to determine whether any of these three ER/Sp1 sites identified in the 5' genomic sequence of the human STC2 gene bind ER, Sp1, or ER/Sp1 complexes and confer estrogen responsiveness. The absence of such ER/Sp1 motifs in the 5' upstream region of the STC1 gene was consistent with the lack of transcriptional regulation of STC1 mRNA by estrogen in MCF-7 cells. However, the presence of an imperfect HRE for the androgen/glucocorticoid/progesterone/mineralocorticoid receptor subfamily suggests that STC1 may be under regulation by alternate steroid receptors. This may explain the observed correlation between STC1 with PR and, hence, also to ER in the breast carcinomas studied.
STC was originally identified as an antihypercalcemic hormone in bony fish produced by the corpuscles of Stannius, a specialized endocrine gland adjacent to the kidney (18) . In fish, rising plasma calcium levels stimulate STC synthesis and secretion (19) . STC counteracts hypercalcemia by slowing calcium uptake in the gills, increasing phosphate reabsorption in the renal proximal tubules, and inhibiting intestinal calcium transport (20 , 21) . To date, most studies to delineate the role of mammalian STC have focused on STC1. Consistent with its role in fish, STC1 has been observed to regulate mineral homeostasis in mammals. Infusion of recombinant human STC1 into rats increases renal phosphate reabsorption (22) . Furthermore, application of STC1 to the serosal surface of rat or pig duodenal mucosa decreases calcium absorption and increases phosphate uptake (23) .
Additional information concerning the role of mammalian STC1/2 has come from cellular localization studies. Of particular interest are expression studies performed in the mouse ovary during different reproductive states. STC1 is highly expressed in mouse and human ovary, where it is produced in the theca-interstitial cells and thought to act in a paracrine manner to regulate corpus luteum function (15) . In this recent study by Deol et al. (15) , the expression of mouse STC1 was shown to be dramatically up-regulated in the ovary during pregnancy and lactation. This increase in ovarian STC1 was accompanied by the detection of STC1 immunoreactivity in the mouse serum, suggesting a role for ovarian-derived STC1 as an endocrine regulator of mammary gland morphogenesis or milk production (15) . Taken together with our finding of the estrogenic regulation of STC2 expression in breast cancer cells, the published data suggest that STC genes may also be subject to dynamic hormonal regulation in the breast. Additionally of interest, in light of STC’s well-documented role in Ca2+ regulation, are the findings that other calcium-mobilizing proteins, including parathyroid hormone-related protein and also osteoprotogenin ligand (also known as RANKL), play a role in breast physiology, the organ required for transmission of maternal calcium to neonates in mammals (24 , 25) . Knockout studies of STC genes are now needed to more precisely delineate their role in mammary gland physiology.
In conclusion, global transcript analysis of an experimental breast cancer cell line model of ER activation and inhibition can predict genes that may be of importance to clinical disease. STC2 was identified as a novel estrogen target gene, which when tested by in situ hybridization and immunohistochemistry in a larger cohort of breast carcinomas, correlated positively with ER and PR status, as did the related STC1 gene. The coexpression of STC1 and STC2 with ER protein in a subset of ER-positive breast carcinomas suggests that the two genes may play a role in the biology of some estrogen-responsive tumors and that STC1/2 expression data may provide additional information from standard practice regarding ER pathway activity. To potentially exploit this information in the management of breast cancer, additional investigation is needed to address the physiological function of STC genes in the mammary gland and determine how STC1/2 expression patterns in breast carcinomas relate to patient response with antiestrogen treatment.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the University of Melbourne, the Peter MacCallum Cancer Institute, and the Victorian Breast Cancer Research Consortium. 
2 To whom requests for reprints should be addressed, at Cancer Functional Genomics Unit, Murdoch Children’s Research Institute, 10th Floor, Royal Children’s Hospital, Parkville, Victoria 3052, Australia. Phone: 61-3-8341-6231; Fax: 61-3-9348-1391; E-mail: bourast{at}murdoch.rch.unimelb.edu.au.
3 The abbreviations used are: ER, estrogen receptor; ERE, estrogen-response element; HRE, hormone response element; PR, progesterone receptor; DIG, digoxigenin; STC, stanniocalcin.
4 Internet address: http://www.atcc.org.
5 Internet address: http://www.graphpad.com.
Received 10/ 2/01. Accepted 1/18/02.
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S. Amatschek, U. Koenig, H. Auer, P. Steinlein, M. Pacher, A. Gruenfelder, G. Dekan, S. Vogl, E. Kubista, K.-H. Heider, et al. Tissue-Wide Expression Profiling Using cDNA Subtraction and Microarrays to Identify Tumor-Specific Genes Cancer Res., February 1, 2004; 64(3): 844 - 856. [Abstract] [Full Text] [PDF]
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D.-Y. Wang, R. Fulthorpe, S. N. Liss, and E. A. Edwards Identification of Estrogen-Responsive Genes by Complementary Deoxyribonucleic Acid Microarray and Characterization of a Novel Early Estrogen-Induced Gene: EEIG1 Mol. Endocrinol., February 1, 2004; 18(2): 402 - 411. [Abstract] [Full Text] [PDF]
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N. Yadav, J. Lee, J. Kim, J. Shen, M. C.-T. Hu, C. M. Aldaz, and M. T. Bedford Specific protein methylation defects and gene expression perturbations in coactivator-associated arginine methyltransferase 1-deficient mice PNAS, May 27, 2003; 100(11): 6464 - 6468. [Abstract] [Full Text] [PDF]
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