Gene Expression Profiling Identifies Lobe-Specific and Common Disruptions of Multiple Gene Networks in Testosterone-Supported, 17β-Estradiol- or Diethylstilbestrol-Induced Prostate Dysplasia in Noble Rats

Neville N C Tam; Carol Ying-Ying Szeto; Maureen A Sartor; Mario Medvedovic; Shuk-Mei Ho

doi:10.1593/neo.07889

. 2008 Jan;10(1):20–40. doi: 10.1593/neo.07889

Gene Expression Profiling Identifies Lobe-Specific and Common Disruptions of Multiple Gene Networks in Testosterone-Supported, 17β-Estradiol- or Diethylstilbestrol-Induced Prostate Dysplasia in Noble Rats¹²

Neville N C Tam ^*, Carol Ying-Ying Szeto ^*, Maureen A Sartor ^†, Mario Medvedovic ^†, Shuk-Mei Ho ^*

PMCID: PMC2216049 PMID: 18231636

Abstract

The xenoestrogen diethylstilbestrol (DES) is commonly believed to mimic the action of the natural estrogen 17β-estradiol (E₂). To determine if these two estrogens exert similar actions in prostate carcinogenesis, we elevated circulating levels of estrogen in Noble (NBL) rats with E₂/DES-filled implants, while maintaining physiological levels of testosterone (T) in the animals with T-filled implants. The two estrogens induced dysplasia in a lobe-specific manner, with E₂ targeting only the lateral prostate (LP) and DES impacting only the ventral prostate (VP). Gene expression profiling identified distinct and common E₂-disrupted versus DES-disrupted gene networks in each lobe. More importantly, hierarchical clustering analyses revealed that T + E₂ treatment primarily affected the gene expression pattern in the LP, whereas T + DES treatment primarily affected the gene expression profile in the VP. Gene ontology analyses and pathway mapping suggest that the two hormone treatments disrupt unique and/or common cellular processes, including cell development, proliferation, motility, apoptosis, and estrogen signaling, which may be linked to dysplasia development in the rat prostate. These findings suggest that the effects of xenoestrogens and natural estrogens on the rat prostate are more divergent than previously suspected and that these differences may explain the lobe-specific carcinogenic actions of the hormones.

Introduction

Androgens are essential to the growth and functioning of the normal prostate and undoubtedly play a key role in prostate carcinogenesis [1]. Emerging evidence shows that estrogens also have a critical role in prostatic diseases, including prostate cancer (PCa) [2,3]. The incidence of PCa increases dramatically with age in human males, whose testosterone (T) levels in both the circulation and in prostate decline, whereas those of 17β-estradiol (E₂) remain relatively stable [2–4]. Thus, the age-associated alterations in the sex hormone milieu toward an estrogen predominance have been proposed as an endogenous risk factor for prostate carcinogenesis. Dynamic changes in the expression of estrogen receptor-β during PCa progression in humans [5,6] also suggest the involvement of estrogen signaling in the malignant transformation of the prostate. Moreover, the findings of aberrant activation of aromatase expression in benign prostatic hyperplasia and PCa [3,7] further advance the concept that local production of estrogens is pivotal in the pathobiology of this gland.

Humans are exposed to xenoestrogens from various sources, including food, drugs, and other exogenous venues. Xenoestrogens are believed to cause endocrine disruptions that lead to pathogenesis in reproductive end organs [8,9]. One agent of public health concern is diethylstilbestrol (DES), a synthetic estrogen, which was widely used as a growth-promoting animal feed additive [10]. Epidemiological studies have established a strong association between maternal exposure to DES and increased risk of vaginal, cervical, and perhaps breast cancers in DES daughters [11,12]. It remains uncertain, however, whether prenatal exposure to DES elevates cancer risk in men [13]. Transient exposure of neonatal rodents to estrogen increases susceptibility of the prostate to inflammation and cancer development later in life [14]. Together, these findings strongly suggest that natural or synthetic estrogen-mediated endocrine disruption leads to the evolution of prostatic diseases, including PCa.

We have established the T plus estrogen-induced dysplasia/carcinoma model in Noble (NBL) rats as a robust study system for deciphering the contributions of estrogens, in adulthood, on the pathogenesis of dysplasia, a precancerous lesion, and adenocarcinoma of the prostate [15,16]. In this model, estrogens are administered through subcutaneous implants of hormone-filled Silastic capsules with the coimplantation of T-filled capsules to maintain physiological levels of this androgen in the circulation [17]. It is well known that increased exposure to estrogens leads to a decline in circulating levels of T through the hypothalamic-pituitary-gonadal axis. The topographical localization of premalignant/malignant lesions within the rat prostate gland has been found to be dependent on the type of estrogens used. Under the same exogenous androgen support, E₂ induces epithelial dysplasia and adenocarcinoma in the lateral prostate (LP) but not in the ventral prostate (VP) [16–19], whereas synthetic DES specifically targets the VP but not the LP [19,20]. Why two different estrogens induce lobe-specific carcinogenic actions in the prostate remains unclear. To address this issue, we conducted gene expression profiling analyses on the LP and VP of NBL rats exposed to T + E₂ or T + DES. The experimental design and the application of a bioinformatic tool of gene network mapping enabled us to identify lobe-specific and common estrogen-mediated disruptions in multiple biologic networks/pathways that may be linked to prostate carcinogenesis and explain the distinctive action of the two estrogens.

Materials and Methods

Animals and Hormonal Treatment

The animal usage protocol was approved by the Institutional Animal Care Committee at the University of Cincinnati. Male NBL rats (5–6 weeks old) were purchased from Charles River Laboratories (Kingston, NY), kept under standard conditions, and treated as previously reported [16,19,20]. Briefly, animals were randomized into three groups (n = 5 for each group). Rats in the T + E₂ treatment group received subcutaneous implants of two pieces of 2-cm-long Silastic capsules containing T (Sigma, St Louis, MO) and one piece of 1-cm-long capsules packed with E₂ (Sigma), whereas the T + DES treatment group received the same number of Silastic capsules of the same length filled with T and DES (Sigma). Age-matched untreated control rats were implanted with empty capsules. At the end of a 16-week treatment period, animals were sacrificed with an overdose of isoflurane, and VPs and LPs were excised. One half of each lobe was processed for histologic examination, and the other half was snap-frozen for RNA extraction.

Microarray Hybridization

The Atlas Glass Rat 3.8 I Microarray (Clontech, Palo Alto, CA) carrying 3800 named rat genes (spotted oligonucleotides) were used as the gene chip platform. The Atlas Glass Fluorescent Labeling Kit (Clontech) was used for synthesizing and purifying fluorescent-labeled cDNA probes for hybridization to glass microarrays. The labeling and hybridization procedures were performed in accordance with the manufacturer's instruction manual. In brief, amino-modified first-strand cDNA probes were synthesized with aminoallyl. 2′-deoxyuridine 5′-triphosphate incorporation. Then Cy3 fluorescent dye was coupled to the cDNAs derived from individual prostatic lobes, whereas Cy5 dye was conjugated to the universal rat reference RNA obtained from Stratagene (La Jolla, CA). Equal quantities of two labeled probes were mixed and hybridized in a Corning microarray hybridization chamber (Corning, Corning, NY) at 50°C overnight (≥16 hours). Spikes of positive control probes were also included as an internal control for the process of cDNA probe synthesis and the dye-coupled reaction. Finally, the signal was obtained using a microarray scanner (GenePix 4000B; Axon Instruments, Foster City, CA). A probe coverage of >90% was achieved for all arrays. Five animal replicates for each treatment/tissue group (T + E₂-treated VP or LP, T + DES-treated VP and LP, and untreated VP and LP) were used to conduct a 30-microarray analysis to assess changes in the gene expression pattern due to treatment and lobe specificity.

Microarray Data Normalization and Analysis

The data were analyzed to identify differentially expressed genes in 1) the T + E₂-treated LPs and VPs compared with untreated controls and 2) the T + DES-treated LPs and VPs compared with untreated controls. Five biologic replicate arrays for each experimental condition, all versus universal reference, were performed. R statistical software and the limma Bioconductor package [21] were used for analysis. Data normalization was performed in two steps separately for each microarray [22–24]. First, background-adjusted intensities were log-transformed, and the differences (M) and averages (A) of log-transformed values were calculated as M = log₂(X₁) - log₂(X2) and A = [log₂(X₁) + log₂(X₂)]/2, where X₁ and X₂ denote the Cy5 and Cy3 intensities, respectively. Second, normalization was performed by fitting the array-specific local regression model of M as a function of A and obtaining residuals. The statistical analysis was performed for each gene separately by fitting a one-way analysis of variance (ANOVA) model with treatment. Estimated fold changes were calculated from the ANOVA model; an intensity-based empirical Bayes method was used to modify the resulting t-statistics from each comparison [25]. This method obtains more precise estimates of variance by pooling information across genes and by accounting for the dependency of variance on probe intensity level. Genes with a false discovery rate (FDR) <0.05 [26] were considered to be significantly differentially expressed. Clustering was performed using normalized, centered sample ratios. The gene list used for clustering consisted of all genes having an FDR <0.05 for at least one comparison (1063 genes). T + E₂ and T + DES samples were each clustered using the Ward clustering method and Euclidean dissimilarity metric with 1063 genes.

Identification of Estrogen-Regulated Gene Expression

Genes differentially regulated in the different lobes with T + DES and T + E₂ treatment were identified separately in each drug treatment. Gene lists were generated according to their expression signature in different gene expression clusters; each group was described in the Results section.

Pathway and Network Analysis

Biologic relationships between differentially expressed genes were mapped by Ingenuity Pathway Analysis (IPA) 3.1 software (www.ingenuity.com). Gene lists of different patterns of gene expression in response to T + E₂ and T + DES treatments (as described in the Results section) were uploaded to the IPAWeb application in an Excel file format containing expression data and GenBank accession number as identifier. The biologic relationship of uploaded genes was mapped with IPA software into networks according to the published literature in the database. A score was assigned to each network in the data set to estimate the relevance of the network to the uploaded gene list. A higher score means that the network is more relevant to the gene list entered by the user [27]. The two highest score networks were selected in this study, and genes in these two networks were selected for further post hoc analysis.

Reverse Transcription and Real-Time Quantitative Polymerase Chain Reaction

The total RNA from each sample of prostate tissue was reverse-transcribed into cDNA using Superscript III (Invitrogen, Carlsbad, CA). Specific primers were designed using either Primer Express 3 (Applied Biosystems, Foster City, CA) or Primer3 software [28]; the sequence of the primer is listed in Table W1. Real-time quantitative polymerase chain reaction (q-PCR) was performed on the 7900HT Fast Real-Time PCR System (ABI Biosystems) using the Power SYBR Green PCR master mix (Applied Biosystems). Polymerase chain reaction was performed in a total volume of 10 µl containing 50 ng of total cDNA, 1x Power SYBR Green PCR master mix, and a final primer concentration of 800 nM. The relative expression level was analyzed by the ΔΔC_t method [29] and one-way ANOVA followed by Tukey post hoc analysis, where P < .05 was considered statistically significant.

Results

T + E₂ and T + DES Treatments Differentially Induced Dysplasia in LP and VP, Respectively

The expected physiological and histologic changes resulting from T + E₂ and T + DES treatment were observed as reported previously [15,18–20]. Dysplasia was observed in the T + E₂-treated LPs (100% incidence) and T + DES.treated VPs (100% incidence), whereas no preneoplastic lesions were observed in T + E₂-treated VPs and T + DES LPs. In T + E₂-treated LPs, the dysplastic lesions were often accompanied by inflammatory infiltrates [17].

Hierarchical Clustering Identified Differential Action of E₂ and DES in the Two Prostate Lobes

Unsupervised hierarchical clustering was performed for each hormone treatment group (T + E₂ or T + DES) to determine the relatedness of replicate LP and VP samples (n = 5 per group) according to similarity in gene expression patterns among the 1063 genes with a significant difference in expression across samples, without prior knowledge of gene and sample identity.

Hierarchical clustering of samples showed that all LPs treated with T + E₂ formed one cluster distinct from the cluster containing all untreated LPs. The hormone treatment, however, did not partition the hormone-treated and -untreated VPs, which formed a single large cluster (Figure 1A). These results indicate that the T + E₂ treatment induced changes in gene expression mainly in the LP and had little, if any, effect on the gene expression pattern in the VP.

Hierarchical clustering analysis of T + E₂ and T + DES gene expression data set. (A) Dendrogram of T + E₂ expression data set in LP and VP. (B) Dendrogram of T + DES expression data set in LP and VP. (C) Venn diagram showing the number of genes differentially expressed in each treatment group compared with the respective untreated control. (D) Gene interaction network of a subset of differentially expressed genes that are common in both LP and VP dysplasia. Genes bordered with red were validated by quantitative real-time PCR.

In a similar manner, T + DES treatment segregated only the VPs into two distinct clusters (treated and untreated) and did not partition the LPs into distinctive groups (Figure 1B). These findings indicate that the hormone treatment altered primarily the gene expression pattern in the VP and had little effect on LP gene expression. It is interesting that the gene expression pattern in the VP after T + DES treatment appeared to more closely resemble that observed in the LPs, as the T + DES-treated VPs formed a cluster more closely linked to the LP cluster than the one comprising untreated VPs.

Identification of Estrogen-Induced Differentially Expressed Genes Related to Dysplasia in Rat LP or VP

We used the following criteria to identify two panels of dysplasia-related genes: 1) the T + E₂-induced LP dysplasia panel contains genes whose expression changed following T + E₂ treatment in the LPs harboring dysplasia but excludes those whose expression also changed in the VPs with no dysplastic changes (253 genes; Figure 1C, left; Table 1), and 2) the T + DES-induced VP dysplasia panel includes genes whose expression changed after T + DES treatment in the VPs harboring dysplasia but excludes those whose expression also changed in the LPs without dysplasia (198 genes; Figure 1C, right; Table 2).

Table 1.

T + E₂-Induced LP Dysplasia Panel: Genes Whose Expression Selectively Changed following T + E₂ Treatment in the LPs Harboring Dysplasia, but not in the VPs.

Gene Names	Locus ID	Symbol	Fold Changes	Functions
prohibitin^*	25344	PHB	3.42	Cell death; cell signaling; cellular growth and proliferation; gene transcription
nuclease-sensitive element binding protein 1^*	29206	NSEP1	3.08	Cell death; gene transcription
potassium channel, subfamily K, member 9^*	84429	KCNK9	3.08	Cell death
vesicle-associated membrane protein 2^*	24803	VAMP2	2.97	Cell death; cellular assembly and organization; cellular movement
cytochrome c oxidase subunit IV isoform 1^*	29445	COX4I1	2.71	Others/unclassified
PR-Vbeta1^*	498341	PR-Vbeta1	2.68	Others/unclassified
melanocortin 5 receptor^*	25726	MC5R	2.47	Cell signaling
glutamate receptor, ionotropic, kainate 3^*	298521	GRIK3	2.45	Cell signaling
6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4^*	54283	PFKFB4	2.41	Others/unclassified
potassium voltage-gated channel, Shaw-related subfamily, member 1^*	25327	KCNC1	2.39	Molecular transport
actin alpha cardiac 1^*	29275	ACTC1	2.34	Others/unclassified
eukaryotic translation elongation factor 1 alpha 1^*	171361	EEF1A1	2.31	Cell death, protein synthesis
ATPase, Na⁺/K⁺ transporting, alpha 3 polypeptide^*	24213	ATP1A3	2.29	Inflammation; molecular transport
gamma-aminobutyric acid A receptor, rho 1^*	29694	GABRR1	2.25	Cell death; cell signaling
guanylate cyclase 1, soluble, alpha 3^*	25201	GUCY1A3	2.25	Cellular movement
transforming growth factor alpha^*	24827	TGFA	2.24	Biomolecule metabolism; cell cycle; cell death; cell morphology; cell signaling; cellular development; cellular growth and proliferation; cellular movement; gene transcription; molecular transport; protein synthesis
ribosomal protein S12^*	65139	RPS12	2.20	Protein synthesis
tumor protein p53^*	24842	TP53	2.16	Cell cycle; cell death; cell morphology; cell signaling; cellular assembly and organization; cellular development; cellular growth and proliferation; cellular movement; cellular response to therapeutics; DNA replication, recombination and repair; free radical scavenging; gene transcription; inflammation; molecular transport; post-translational modification; protein synthesis; RNA post-transcriptional modification
nuclear receptor subfamily 3, group C, member 2^*	25672	NR3C2	2.13	Biomolecule metabolism; cell signaling; gene transcription; molecular transport; protein synthesis
secretory carrier membrane protein 1^*	29521	SCAMP1	2.09	Cellular assembly and organization
CEA-related cell adhesion molecule 1^*	81613	CEACAM1	2.08	Cell death; cell signaling; cellular growth and proliferation; cellular movement; inflammation
olfactory marker protein^*	24612	OMP	2.08	Cell signaling
guanine nucleotide binding protein, alpha z subunit^*	25740	GNAZ	2.05	Cell signaling; cellular development; cellular movement; molecular transport; biomolecule metabolism
phosphodiesterase 1C^*	81742	PDE1C	2.04	Others/unclassified
ribosomal protein S3a^*	29288	RPS3A	2.04	Cell death; cellular development; cellular growth and proliferation; protein synthesis
insulin-like 6^*	50546	IL1RAP	1.95	Cell signaling
A kinase (PRKA) anchor protein 14^*	60332	AKAP14	1.94	Others/unclassified
CD38 antigen^*	25668	CD38	1.94	Cell cycle; cell death; cell signaling; cellular development; cellular growth and proliferation; cellular movement; biomolecule metabolism; molecular transport; post-translational modification
p21 (CDKN1A)-activated kinase 1^*	29431	PAK1	1.93	Cell death; cell morphology; cell signaling; cellular assembly and organization; cellular development; cellular movement
regenerating islet-derived 3 alpha^*	171162	REG3G	1.93	Others/unclassified
ubiquitin-conjugating enzyme E2I^*	25573	UBE2I	1.93	Cell signaling; gene transcription; protein synthesis
ATPase, H⁺ transporting, V1 subunit F^*	116664	ATP6V1F	1.91	Molecular transport
homeo box A1^*	25607	HOXA1	1.89	Cell death; cellular development; cellular movement; gene transcription
regenerating islet-derived 3 gamma^*	24620	REG3G	1.89	Others/unclassified
Fas apoptotic inhibitory molecule 2^*	246274	FAIM2	1.86	Cell death
barrier to autointegration factor 1^*	114087	BANF1	1.85	DNA replication, recombination, and repair
thyroid hormone receptor alpha^*	81812	THRA	1.85	Biomolecule metabolism; cell death; cell morphology; cellular development; free radical scavenging; gene transcription; protein synthesis
neuromedin B receptor^*	25264	NMBR	1.83	Others/unclassified
ATPase, Na⁺/K⁺ transporting, beta 2 polypeptide^*	24214	ATP1B2	1.82	Others/unclassified
Chondroitin sulfate proteoglycan 5^*	50568	CSPG5	1.81	Cell signaling
MAD homolog 7 (Drosophila)^*	81516	SMAD7	1.78	Cell death; cell morphology; cell signaling; cellular development; cellular growth and proliferation; gene transcription
tachykinin receptor 2^*	25007	TACR2	1.78	Others/unclassified
alanyl (membrane) aminopeptidase^*	81641	ANPEP	1.76	Cell death; cell morphology; cellular development; cellular movement; protein synthesis
growth hormone-releasing hormone^*	29446	GHRH	1.75	Cell morphology; cell signaling; cellular growth and proliferation; biomolecule metabolism; molecular transport
protein kinase N1^*	29355	PKN1	1.75	Cell signaling; cellular growth and proliferation; cellular movement; gene transcription
slit homolog 3 (Drosophila)^*	83467	SLIT3	1.74	Others/unclassified
dopamine receptor 2^*	24318	DRD2	1.72	Cell death; cell signaling; cellular growth and proliferation; cellular movement; gene transcription; inflammation; biomolecule metabolism
glutathione peroxidase 3^*	64317	GPX3	1.72	Cellular growth and Proliferation; Post-translational modification; protein synthesis
vasoactive intestinal peptide receptor 1^*	24875	VIPR1	1.71	Cell death; cell signaling; cellular growth and proliferation; cellular movement; biomolecule metabolism; molecular transport
amphiphysin 1^*	60668	AMPH	1.70	Cell signaling; cellular assembly and organization; cellular movement; gene transcription
G protein beta subunit-like^*	64226	GBL	1.70	Others/unclassified
heat shock 70 kDa protein 5^*	25617	HSPA5	1.69	Cell death; cellular growth and proliferation; inflammation
hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1^*	29632	HSD3B2	1.68	Others/unclassified
POU domain, class 3, transcription factor 2^*	29588	POU3F2	1.68	Cellular development; cellular growth and proliferation; cellular movement; gene transcription
ribosomal protein S9^*	81772	RPS9	1.64	Protein synthesis
glucocorticoid modulatory element binding protein 2^*	83635	GMEB2	1.63	Gene transcription
glutamate receptor, ionotropic, NMDA2B^*	24410	GRIN2B	1.63	Cell signaling
solute carrier family 8 (sodium/calcium exchanger), member 3^*	140448	SLC8A3	1.63	Cell death; cell signaling
ATPase, Ca²⁺ transporting, ubiquitous^*	25391	ATP2A3	1.62	Others/unclassified
CTD-binding SR-like rA1^*	56081	SR-A1	1.60	Others/unclassified
solute carrier family 2 (facilitated glucose transporter), member 2^*	25351	SLC2A2	1.60	Cell death
calcineurin binding protein 1^*	94165	CABIN1	1.59	Cell cycle; cell death; cell morphology; cell signaling; cellular development; cellular growth and proliferation; cellular movement; DNA replication, recombination, and repair; gene transcription; inflammation; biomolecule metabolism; molecular transport
heterogeneous nuclear ribonucleoprotein A1^*	29578	LOC144983	1.59	Others/unclassified
casein kinase 1, gamma 1^*	64086	CSNK1G1	1.57	Others/unclassified
potassium inwardly rectifying channel, subfamily J, member 10^*	29718	KCNJ10	1.56	Cell morphology; cellular development; inflammation; molecular transport
potassium large conductance calcium-activated channel, subfamily M, beta member 1^*	29747	KCNMB1	1.56	Cell signaling; molecular transport
potassium voltage-gated channel, Shaw-related subfamily, member 3^*	117101	KCNC3	1.56	Molecular transport
CCAAT/enhancer binding protein (C/EBP), delta^*	25695	CEBPD	1.55	Cell death; cellular development; cellular growth and proliferation; gene transcription; inflammation
guanine nucleotide binding protein, beta 3^*	60449	GNB3	1.55	Cell signaling
ribosomal protein S19^*	29287	RPS19	1.54	Cellular development; cellular growth and proliferation; cellular movement; protein synthesis
aquaporin 5^*	25241	AQP5	1.53	Inflammation
chloride channel Kb^*	79430	CLCNKB	1.53	Others/unclassified
fatty acid binding protein 3^*	79131	FABP3	1.52	Cellular growth and proliferation; biomolecule metabolism; molecular transport
endothelial differentiation, lysophosphatidic acid G-protein-coupled receptor, 2^*	116744	EDG2	1.51	Cell death; cell morphology; cell signaling; cellular development; cellular growth and proliferation; cellular movement; gene transcription
killer cell lectin-like receptor subfamily B member 1B^*	25192	KLRB1	1.51	Cell death
suppression of tumorigenicity 18^*	266680	ST18	1.50	Gene transcription
pregnancy upregulated nonubiquitously expressed CaM kinase^*	29660	PNCK	1.49	Others/unclassified
Arg/Abl-interacting protein ArgBP2^*	114901	SORBS2	1.48	Cell death; cell morphology; cell signaling
sodium channel, voltage-gated, type IV, alpha polypeptide^*	25722	SCN4A	1.48	Molecular transport
calcitonin/calcitonin-related polypeptide, alpha^*	24241	CALCA	1.47	Cell cycle; cell death; cell morphology; cell signaling; cellular development; cellular growth and proliferation; cellular movement; DNA replication, recombination, and repair; gene transcription; inflammation; biomolecule metabolism; molecular transport
RASD family, member 2^*	171099	RASD2	1.47	Others/unclassified
spondin 1^*	64456	SPON1	1.46	Others/unclassified
sulfotransferase family, cytosolic, 1C, member 2^*	171072	SULT1C2	1.46	Others/unclassified
acyl-CoA synthetase long-chain family member 4^*	113976	ACSL4	1.45	Cell death
aldehyde dehydrogenase family 1, subfamily A2^*	116676	ALDH1A2	1.45	Cell death; cellular development; cellular growth and proliferation; biomolecule metabolism
CD3 antigen, zeta polypeptide^*	25300	CD247	1.45	Cell death; cell morphology; cell signaling; cellular development; cellular growth and proliferation; gene transcription; inflammation; post-translational modification
discs, large homolog 4 (Drosophila)^*	29495	DLG4	1.45	Cell signaling; cellular assembly and organization
calmodulin 3	24244	CALM3	1.43	Cell signaling; cellular growth and proliferation
carboxypeptidase E^*	25669	CPE	1.43	Biomolecule metabolism; molecular transport
phosphofructokinase, muscle^*	65152	PFKM	1.42	Others/unclassified
translocase of outer mitochondrial membrane 20 homology (yeast)^*	266601	TOMM20	1.42	Others/unclassified
complement component 1, q subcomponent, beta polypeptide^*	29687	C1QB	1.40	Others/unclassified
regulator of G-protein signaling 19^*	59293	RGS19	1.40	Cell signaling; cellular development; biomolecule metabolism; protein synthesis
Carcinoembryonic antigen gene family (CGM3)^*	24256	PSG18	1.39	Others/unclassified
gamma-glutamyl hydrolase^*	25455	GGH	1.39	Others/unclassified
N-acetyltransferase 8 (camello like)^*	64570	NAT8	1.37	Others/unclassified
pyrimidinergic receptor P2Y, G-protein-coupled, 6^*	117264	P2RY6	1.37	Others/unclassified
protein tyrosine phosphatase, receptor type, K, extracellular region^*	360302	PTPRK	1.36	Cellular growth and proliferation
cytochrome P450, 4a12^*	266674	CYP4A22	1.35	Others/unclassified
gamma-aminobutyric acid A receptor, alpha 1^*	29705	GABRA1	1.35	Cell morphology; cell signaling
ferritin, heavy polypeptide 1^*	25319	FTH1	1.34	Cell death; cell morphology; cellular growth and proliferation; DNA replication, recombination, and repair; free radical scavenging; cell death; cell morphology; cellular growth and proliferation; free radical scavenging
guanylate cyclase 1, soluble, beta 2^*	25206	GUCY1B2	1.34	Others/unclassified
proteasome (prosome, macropain) 28 subunit, beta^*	29614	PSME2	1.31	Cell signaling; cellular growth and proliferation
small inducible cytokine A4^*	116637	CCL4	1.31	Cell death; cell morphology; cell signaling; cellular development; cellular growth and proliferation; cellular movement; inflammation biomolecule metabolism
ADP-ribosylation factor 6^*	79121	ARF6	1.30	Cell death; cell morphology; cell signaling; cellular assembly and organization; cellular movement; biomolecule metabolism; molecular transport; protein trafficking
mannan-binding lectin serine protease 2	64459	MASP2	1.30	Others/unclassified
dopamine receptor 4^*	25432	DRD4	1.29	Cell signaling; biomolecule metabolism; molecular transport
ATP synthase, H⁺ transporting, mitochondrial F₁ complex, delta subunit^*	245965	ATP5D	1.26	Energy production; molecular transport; biomolecule metabolism
calponin 1	65204	CNN1	1.26	Cellular assembly and organization; cellular growth and proliferation; cellular movement
hydroxyacid oxidase 2 (long chain)	84029	HAO2	1.25	Others/unclassified
cytotoxic T-lymphocyte-associated protein 4	63835	CTLA4	1.23	Cell cycle; cell death; cell signaling; cellular development; cellular growth and proliferation; cellular movement; inflammation
potassium voltage-gated channel, subfamily H (eag-related), member 7^*	170739	KCNH7	1.23	Others/unclassified
Ras association (RalGDS/AF-6) domain family 5	54355	RASSF5	1.23	Cell cycle; cell death; cellular growth and proliferation
thymopoietin	25359	TMPO	1.22	Cell cycle; cellular assembly and organization; DNA replication, recombination, and repair; gene transcription
polypyrimidine tract binding protein 1	29497	PTBP1	1.19	Protein synthesis
protein kinase C and casein kinase substrate in neurons 2	124461	PACSIN2	1.16	Cell morphology; cell signaling; cellular assembly and organization
leptin receptor overlapping transcript	56766	LEPROT	1.15	Others/unclassified
phospholipase C, delta 1	24655	PLCD1	1.15	Biomolecule metabolism; cellular growth and proliferation
synaptonemal complex protein SC65	59101	SC65	1.15	Others/unclassified
ADP-ribosylation factor 5	79117	ARF5	1.14	Molecular transport; protein trafficking
crystallin, beta B₂	25422	CRYBB2	1.11	Others/unclassified
prolactin-like protein L	171556	PRLPL	1.10	Others/unclassified
protein phosphatase 3, catalytic subunit, beta isoform	24675	PPP3CB	1.09	Cell death; cellular development; inflammation
calpain 3	29155	CAPN3	1.07	Protein synthesis
cathepsin D	171293	CTSD	1.07	Cell death; cellular growth and proliferation; free radical scavenging; inflammation; molecular transport; protein synthesis
eukaryotic translation initiation factor 2B, subunit 2 beta	84005	EIF2B2	1.07	Cellular development; cellular growth and proliferation; protein synthesis
calcitonin/calcitonin-related polypeptide, alpha	24241	CALCA	-1.13	Cell cycle; cell death; cell morphology; cell signaling; cellular development; cellular growth and proliferation; cellular movement; DNA replication, recombination, and repair; gene transcription; inflammation; biomolecule metabolism; molecular transport
forkhead box M1	58921	FOXM1	-1.13	Cell cycle; cell death; cell morphology; cellular growth and proliferation; gene transcription
adrenal secretory serine protease precursor	64565	TMPRSS11D	-1.14	Others/unclassified
MAP-kinase activating death domain	94193	MADD	-1.19	Cell death; cellular growth and proliferation
calmodulin 3	24244	CALM3	-1.22	Cell signaling; cellular growth and proliferation
neural visinin-like Ca²⁺-binding protein type 3^*	50871	HPCAL1	-1.28	Others/unclassified
fertility protein SP22	117287	PARK7	-1.30	Cell death; cell signaling; cellular growth and proliferation
actin, beta^*	81822	ACTB	-1.31	Cellular growth and proliferation; cellular movement
G-protein-coupled receptor 37^*	117549	GPR37	-1.33	Cell death; cell signaling
glycogen synthase kinase 3 alpha^*	50686	GSK3A	-1.34	Cellular movement
ribosomal protein L22	81768	RPL22	-1.34	Protein synthesis
actinin alpha 4^*	63836	ACTN4	-1.35	Cell death; cellular growth and proliferation; cellular movement
midline 1^*	54252	MID1	-1.35	Cellular assembly and organization
dipeptidase 1 (renal)^*	94199	DPEP1	-1.36	Others/unclassified
polymeric immunoglobulin receptor^*	25046	PIGR	-1.36	Cell signaling; cellular growth and proliferation; cellular movement
adducin 2 (beta)^*	24171	ADD2	-1.38	Cellular development; cellular growth and proliferation; inflammation; molecular transport
c-mer protooncogene tyrosine kinase^*	65037	MERTK	-1.38	Cell death; cell morphology; cell signaling; cellular development; cellular growth and proliferation
caspase 7^*	64026	CASP7	-1.39	Cell death; protein synthesis
ddx5 gene	287765	DDX5	-1.40	Cell death; cellular growth and proliferation; gene transcription
vesicle docking protein^*	56042	VDP	-1.40	Cellular assembly and organization; molecular transport; protein trafficking
acetylcholinesterase^*	83817	ACHE	-1.41	Cell death; cell morphology; cell signaling; cellular development; cellular growth and proliferation; DNA replication, recombination, and repair; post-translational modification; protein synthesis
phenylalkylamine Ca²⁺ antagonist (emopamil) binding protein^*	117278	EBP	-1.41	Cellular development
phosphodiesterase 4C, cAMP-specific^*	290646	PDE4C	-1.41	Inflammation
transition protein 2^*	24840	TNP2	-1.44	Others/unclassified
casein kinase 1, alpha 1^*	113927	CSNK1A1	-1.45	Cell death
nuclear receptor subfamily 4, group A, member 3	58853	NR4A3	-1.46	Cellular development; cellular growth and proliferation; gene transcription
phospholipase C, beta 3^*	29322	PLCB3	-1.46	Biomolecule metabolism; cellular movement; molecular transport
myelin-associated oligodendrocytic basic protein^*	25037	MOBP	-1.47	Others/unclassified
retinoic acid receptor, alpha^*	24705	RARA	-1.47	Cell death; cell signaling; cellular development; cellular growth and proliferation; cellular movement; biomolecule metabolism; gene transcription; inflammation
putative pheromone receptor Go-VN13C^*	286986	EG665255	-1.48	Others/unclassified
prolyl 4-hydroxylase, beta polypeptide^*	25506	P4HB	-1.49	Cell death
protein kinase, lysine-deficient 1^*	116477	WNK1	-1.49	Molecular transport
Pyruvate carboxylase^*	25104	PC	-1.49	Others/unclassified
ribosomal protein S15^*	29285	RPS15	-1.49	Others/unclassified
ATP synthase, H⁺ transporting, mitochondrial F1 complex, O subunit^*	192241	ATP5O	-1.50	Others/unclassified
granzyme M (lymphocyte met-ase 1)^*	29252	GZMM	-1.50	Cell death
metastasis-associated 1^*	64520	MTA1	-1.50	Cell morphology; cell signaling; cellular growth and proliferation; cellular movement; gene transcription
Rous sarcoma oncogene^*	83805	SRC	-1.50	Cell death; cell morphology; cell signaling; cellular development; cellular growth and proliferation; cellular movement; gene transcription; biomolecule metabolism; molecular transport; protein synthesis
neurexophilin 3^*	59315	NXPH3	-1.51	Others/unclassified
phosphodiesterase 4A^*	25638	PDE4A	-1.51	Cell death; cell signaling; cellular development; inflammation; molecular transport; biomolecule metabolism
calcium channel, voltage-dependent, alpha 1I subunit^*	56827	CACNA1I	-1.52	Cell signaling
transient receptor potential cation channel, subfamily C, member 4^*	84494	TRPC4	-1.53	Others/unclassified
tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon polypeptide^*	29753	YWHAE	-1.53	Cell cycle; cell signaling; cellular movement
polymerase (RNA) II (DNA-directed) polypeptide G^*	117017	POLR2G	-1.54	Gene transcription
F-box only protein 2^*	85273	FBXO2	-1.55	Cellular growth and proliferation; protein synthesis
apolipoprotein C–I^*	25292	APOC1	-1.56	Biomolecule metabolism; molecular transport
proprotein convertase subtilisin/kexin type 3^*	54281	FURIN	-1.56	Cell signaling; cellular growth and proliferation; cellular movement; protein synthesis
chemokine orphan receptor 1^*	84348	CXCR7	-1.57	Cellular growth and proliferation; cellular movement
heat shock protein, alpha-crystallin-related, B6^*	192245	HSPB6	-1.57	Others/unclassified
interleukin 5^*	24497	IL5	-1.57	Cell cycle; cell death; cell morphology; cell signaling; cellular development; cellular growth and proliferation; cellular movement; gene transcription; molecular transport
interleukin enhancer binding factor 3^*	84472	ILF3	-1.57	Cellular growth and proliferation; gene transcription
protein tyrosine phosphatase, receptor type, F^*	360406	PTPRF	-1.57	Cell cycle; cell death; cell morphology; cell signaling; cellular assembly and organization; cellular growth and proliferation; cellular movement
par-3 (partitioning defective 3) homolog (C. elegans)^*	81918	PARD3	-1.58	Cell morphology; cell signaling; cellular development; gene Transcription
ribosomal protein L27^*	64306	RPL27	-1.58	Others/unclassified
linker for activation of T cells^*	81511	LAT	-1.59	Biomolecule metabolism; cell death; cell morphology; cell signaling; cellular development; gene transcription; molecular transport
nuclear factor I/C^*	29228	NFIC	-1.59	Gene transcription
potassium voltage-gated channel, shaker-related subfamily, beta member 1^*	29737	KCNAB1	-1.59	Molecular transport
Bcl2-associated X protein^*	24887	BAX	-1.60	Cell cycle; cell death; cell morphology; cell signaling; cellular assembly and organization; cellular development; cellular growth and proliferation; cellular response to therapeutics; DNA replication, recombination, and repair; free radical scavenging; inflammation; biomolecule metabolism; molecular transport
carboxylesterase 1^*	29225	ES22	-1.60	Others/unclassified
ribosomal protein L10A^*	81729	RPL10A	-1.60	Others/unclassified
upstream of NRAS^*	117180	CSDE1	-1.60	Others/unclassified
nucleoporin 62^*	65274	NUP62	-1.61	Cell death; cell signaling; cellular growth and proliferation; gene transcription
eukaryotic translation initiation factor 2B, subunit 4 delta^*	117019	EIF2B4	-1.62	Cellular development; protein synthesis
apolipoprotein A–V^*	140638	APOA5	-1.63	Biomolecule metabolism; molecular transport
myosin IE^*	25484	MYO1E	-1.64	Others/unclassified
paired box gene 8^*	81819	PAX8	-1.64	Cellular development; gene transcription
prion protein^*	24686	PRNP	-1.64	Cell death; cellular development; cellular growth and proliferation; cellular movement
amelogenin X chromosome^*	29160	AMELX	-1.67	Others/unclassified
Unc4.1 homeobox (C. elegans)^*	29375	UNCX4.1	-1.67	Cell death
cofilin 1^*	29271	CFL1	-1.68	Cell morphology; cell signaling; cellular assembly and organization; cellular development; cellular growth and proliferation; cellular movement; molecular transport; protein trafficking
caudal type homeo box 1^*	171042	CDX1	-1.69	Others/unclassified
phosphatidylinositol-4-phosphate 5-kinase, type II, alpha^*	116723	PIP5K2A	-1.70	Biomolecule metabolism; molecular transport
platelet-activating factor acetylhydrolase, isoform 1b, alpha1 subunit^*	114113	PAFAH1B3	-1.70	Cell death; cellular development; biomolecule metabolism
protein tyrosine phosphatase, nonreceptor type substrate 1^*	25528	SIRPA	-1.70	Cell death; cell morphology; cell signaling; cellular assembly and organization; cellular development; cellular growth and proliferation; cellular movement; gene transcription; inflammation
acetylcholinesterase^*	83817	ACHE	-1.71	Cell death; cell morphology; cell signaling; cellular development; cellular growth and proliferation; DNA replication, recombination, and repair; post-translational modification; protein synthesis
ribosomal protein L36^*	58927	RPL36	-1.71	Others/unclassified
G-protein-coupled receptor 24^*	83567	MCHR1	-1.72	Cell signaling; molecular transport; biomolecule metabolism
epididymal retinoic acid-binding protein^*	29552	LCN5	-1.73	Biomolecule metabolism
gap junction membrane channel protein beta 4^*	117055	GJB4	-1.75	Others/unclassified
zinc finger protein 111^*	170849	ZNF227	-1.75	Others/unclassified
cleavage and polyadenylation-specific factor 4^*	252943	CPSF4	-1.76	Cellular growth and proliferation
ADP-ribosylation factor 1^*	64310	ARF1	-1.77	Cellular assembly and organization; cellular growth and proliferation; biomolecule metabolism; molecular transport; protein trafficking
coagulation factor X^*	29243	F10	-1.77	Cell signaling; cellular movement; inflammation; biomolecule metabolism; molecular transport
ephrin A1^*	94268	EFNA1	-1.77	Cell death; cell morphology; cell signaling; cellular assembly and organization; cellular development; cellular growth and proliferation; cellular movement
rabaptin 5^*	54190	RABEP1	-1.78	Others/unclassified
allograft inflammatory factor 1^*	29427	AIF1	-1.79	Cell death; cell morphology; cellular development; cellular growth and proliferation
Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 1^*	64466	CITED1	-1.79	Cellular development; cellular growth and proliferation; gene transcription
testis enhanced gene transcript^*	24822	TEGT	-1.79	Cell death
neurogenic differentiation 2^*	54276	NEUROD2	-1.80	Cellular development; gene transcription
heat shock 10 kDa protein 1^*	25462	HSPE1	-1.83	Cell death
hsp70-interacting protein^*	246146	HSPBP1	-1.83	Others/unclassified
calreticulin^*	64202	CALR	-1.84	Cell death; cellular assembly and organization; cellular development; cellular growth and proliferation; cellular movement; gene transcription; molecular transport; protein trafficking
discoidin domain receptor family, member 1^*	25678	DDR1	-1.84	Cell death; cellular development; cellular growth and proliferation; cellular movement
myosin ID^*	25485	MYO1D	-1.84	Others/unclassified
ribosomal protein L28^*	64638	RPL28	-1.84	Others/unclassified
gap junction membrane channel protein alpha 3^*	79217	GJA3	-1.85	Cell morphology; cell signaling; cellular development
hairy and enhancer of split 3 (Drosophila)^*	64628	HES3	-1.85	Cellular development; gene transcription
ornithine decarboxylase antizyme 1^*	25502	OAZ1	-1.87	Cell death; cellular growth and proliferation
a disintegrin and metallopeptidase domain 1a^*	56777	ADAM1A	-1.89	Cellular movement
mucosal vascular addressin cell adhesion molecule 1^*	54266	MADCAM1	-1.89	Cell signaling; cellular development; cellular movement; inflammation
membrane protein, palmitoylated 3 (MAGUK p55 subfamily member 3)^*	114202	MPP3	-1.90	Others/unclassified
sulfotransferase family 4A, member 1^*	58953	SULT4A1	-1.90	Others/unclassified
CCAAT/enhancer binding protein (C/EBP), alpha^*	24252	CEBPA	-1.91	Cell cycle; cell death; cellular development; cellular growth and proliferation; cellular response to therapeutics; gene transcription; biomolecule metabolism; molecular transport
SNF-related kinase^*	170837	SNRK	-1.92	Cellular development
ADP-ribosylation factor 3^*	140940	ARF3	-1.94	Molecular transport; protein trafficking
aspartyl-tRNA synthetase^*	116483	DARS	-1.94	Cell cycle; cell signaling; protein synthesis
glycine cleavage system protein H (aminomethyl carrier)^*	171133	GCSH	-1.94	Biomolecule metabolism; post-translational modification
3-hydroxyisobutyrate dehydrogenase^*	63938	HIBADH	-1.96	Others/unclassified
preoptic regulatory factor-2^*	286903	KIAA1688	-1.96	Others/unclassified
amino-terminal enhancer of split^*	29466	AES	-1.97	Cell death; gene transcription
ribosomal protein L29^*	29283	RPL29	-1.99	Others/unclassified
quinoid dihydropteridine reductase^*	64192	QDPR	-2.01	Others/unclassified
complexin 2^*	116657	CPLX2	-2.02	Cellular assembly and organization; cellular movement
hairy and enhancer of split 2 (Drosophila)^*	29567	HES2	-2.02	Gene transcription
transmembrane 4 superfamily member 11^*	64364	PLLP	-2.02	Molecular transport
ferritin light chain 1^*	29292	FTL	-2.03	Cellular growth and proliferation
growth hormone-releasing hormone receptor^*	25321	GHRHR	-2.04	Cell signaling; cellular growth and proliferation; molecular transport; biomolecule metabolism
membrane protein, palmitoylated 2 (MAGUK p55 subfamily member 2)^*	85275	MPP2	-2.04	Cell signaling
ribosomal protein S27^*	94266	RPS27	-2.05	Cell signaling; cellular growth and proliferation; protein synthesis
translocase of inner mitochondrial membrane 22 homolog (yeast)^*	79463	TIMM22	-2.06	Others/unclassified
sec22 homolog^*	117513	SEC22A	-2.08	Molecular transport; protein trafficking
transmembrane protein with EGF-like and two follistatin-like domains 1^*	63845	TMEFF1	-2.11	Cellular growth and proliferation
paired-like homeodomain transcription factor 3^*	29609	PITX3	-2.12	Cellular development; gene transcription
fibrinogen, gamma polypeptide^*	24367	FGG	-2.25	Cell signaling
neurogenic differentiation 1^*	29458	NEUROD1	-2.26	Cell death; cell morphology; cell signaling; cellular development; cellular growth and proliferation; cellular movement; gene transcription
steroid-sensitive gene 1^*	64387	CCDC80	-2.31	Others/unclassified
ubiquitin A-52 residue ribosomal protein fusion product 1^*	64156	UBA52	-2.33	Gene transcription; protein synthesis
synuclein, gamma^*	64347	SNCG	-2.42	Cell death; cell morphology; cellular growth and proliferation; cellular movement

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The change in expression was significant compared with untreated control (P < .05). Gene names in italics are common in both panels of T + E₂-treated LP and T + DES-treated VP.

Table 2.

T + DES-Induced VP Dysplasia Panel: Genes Whose Expression Selectively Changed following T + DES Treatment in the VPs Harboring Dysplasia, but not in the LPs.

Gene Names	Locus ID	Symbol	Fold Changes	Functions
glutathione peroxidase 3^*	64317	GPX3	17.188	Cellular development; cellular growth and proliferation; post-translational modification; protein synthesis
ferritin, heavy polypeptide 1^*	25319	FTH1	6.304	Cell death; cell morphology; cellular growth and proliferation; DNA replication, recombination, and repair; free radical scavenging; post-translational modification
protein phosphatase 2a, catalytic subunit, alpha isoform^*	24672	PPP2CA	3.388	Biomolecule metabolism; cell death; cellular growth and proliferation; post-translational modification
monocarboxylate transporter^*	80878	SLC16A3	2.263	Others/unclassified
nuclease-sensitive element binding protein 1^*	29206	NSEP1	1.898	Cell death; gene transcription
thymopoietin^*	25359	TMPO	1.814	Cell cycle; DNA replication, recombination, and repair; gene transcription
homeo box A1^*	25607	HOXA1	1.586	Cell death; cellular development; cellular movement; gene transcription
cystatin C^*	25307	CST3	1.585	Cell death; cellular development; cellular growth and proliferation
thyroid hormone receptor alpha^*	81812	THRA	1.583	Cell death; cell morphology; cellular development; free radical scavenging; gene transcription
tubulin, alpha 1^*	64158	TUBA1A	1.580	Inflammation
phospholipase C, delta 1^*	24655	PLCD1	1.532	Cellular growth and proliferation
glutamate receptor, metabotropic 7^*	81672	GRM7	1.481	Cell signaling
potassium voltage-gated channel, subfamily H (eag-related),member 7^*	170739	KCNH7	1.481	Others/unclassified
plasminogen activator, urokinase^*	25619	PLAU	1.461	Cell cycle; cell death; cell morphology; cell signaling; cellular assembly and organization; cellular development; cellular growth and proliferation; cellular movement; inflammation
vesicle-associated membrane protein 2	24803	VAMP2	1.452	Cell signaling; cellular assembly and organization; cellular movement; molecular transport
glyceraldehyde-3-phosphate dehydrogenase^*	24383	GAPDH	1.401	Others/unclassified
protein phosphatase 1F (PP2C domain containing)^*	287931	PPM1F	1.392	Biomolecule metabolism; cell death; post-translational modification
tumor protein p53^*	24842	TP53	1.379	Cell cycle; cell death; cell morphology; cell signaling; cellular assembly and; organization; cellular development; cellular growth and proliferation; cellular movement; cellular response to therapeutics; DNA replication, recombination, and repair; free radical scavenging; gene transcription; inflammation; post-translational modification; protein synthesis
opioid receptor, mu 1^*	25601	OPRM1	1.375	Cell death; cell signaling; cellular growth and proliferation; cellular movement; inflammation
calcitonin/calcitonin-related polypeptide, alpha^*	24241	CALCA	1.361	Cell cycle; cell death; cell morphology; cell signaling; cellular development; cellular growth and proliferation; cellular movement; DNA replication, recombination, and repair; gene transcription; inflammation
testis-specific protein^*	192229	C3ORF34	1.357	Others/unclassified
eukaryotic translation initiation factor 2B, subunit 2 beta^*	84005	EIF2B2	1.334	Cellular development; cellular growth and proliferation
tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, eta polypeptide^*	25576	YWHAH	1.333	Cellular assembly and organization; gene transcription
glucagon-like peptide 1 receptor	25051	GLP1R	1.328	Cell death; gene transcription; inflammation
transient receptor potential cation channel, subfamily C, member 2^*	64573	TRPC2	1.322	Others/unclassified
dopamine receptor 4^*	25432	DRD4	1.311	Cell signaling
early growth response 3^*	25148	EGR3	1.289	Cell death; cell signaling; cellular development; cellular growth and proliferation; gene transcription
glutamate receptor, ionotropic, NMDA2B^*	24410	GRIN2B	1.248	Cell morphology; cell signaling
chemokine (C-X-C motif) ligand 5^*	60665	CXCL6	1.236	Cell signaling; cellular movement
myosin IC	65261	MYO1C	1.190	Cell morphology
breast cancer anti-estrogen resistance 1	25414	BCAR1	1.190	Cell death; cell morphology; cell signaling; cellular assembly and organization; cellular development; cellular growth and proliferation; cellular movement
aryl hydrocarbon receptor nuclear translocator 2	25243	ARNT2	1.172	Gene transcription
CCAAT/enhancer binding protein (C/EBP), delta	25695	CEBPD	1.151	Cell cycle; cell death; cellular development; cellular growth and proliferation; gene transcription; inflammation; biomolecule metabolism; molecular transport
CTD-binding SR-like rA1	56081	SR-A1	1.148	Others/unclassified
ADP-ribosylation factor 6	79121	ARF6	1.142	Cell death; cell morphology; cellular assembly and organization; cellular movement
calmodulin 3	24244	CALM3	1.138	Cell death; cellular growth and proliferation; post-translational modification
synaptotagmin 3	25731	SYT3	1.115	Cell signaling; cellular assembly and organization; cellular movement; molecular transport
dynein, cytoplasmic, light chain 1	58945	DYNLL1	1.090	Cell death; cell morphology; cellular assembly and organization
MAD homolog 7 (Drosophila)	81516	SMAD7	1.062	Cell cycle; cell death; cell morphology; cellular development; cellular growth and proliferation; cellular movement; gene transcription
v-maf musculoaponeurotic fibrosarcom oncogene family, protein B (avian)	54264	MAFB	1.062	Cell death; cellular development; cellular movement; gene transcription
calmodulin 3	24244	CALM3	1.060	Cellular growth and proliferation; post-translational modification
thymosin, beta 10	50665	TMSB10	1.059	Cell death; cellular assembly and organization; cellular growth and proliferation
aldehyde dehydrogenase family 1, subfamily A2	116676	ALDH1A2	1.035	Cell death; cellular development; cellular growth and proliferation
syntaxin 1B2	24923	STX1B2	1.032	Others/unclassified
aldolase A	24189	ALDOA	-1.013	Others/unclassified
prosaposin	25524	PSAP	-1.031	Cell cycle; cell death; cellular growth and proliferation; biomolecule metabolism; molecular transport; post-translational modification
vesicle-associated membrane protein 2	24803	VAMP2	-1.031	Cell death; cell signaling; cellular assembly and organization; cellular movement; molecular transport
Rous sarcoma oncogene	83805	SRC	-1.070	Biomolecule metabolism; cell cycle; cell death; cell morphology; cell signaling; cellular assembly and organization; cellular development; cellular growth and proliferation; cellular movement; DNA replication, recombination, and repair; gene transcription; molecular transport; post-translational modification
acetylcholinesterase	83817	ACHE	-1.072	Cell death; cell morphology; cell signaling; cellular assembly and organization; cellular development; cellular growth and proliferation; DNA replication, recombination, and repair; post-translational modification; protein synthesis
Max interacting protein 1	25701	MXI1	-1.120	Cell morphology; cellular growth and proliferation; gene transcription
betacellulin	64022	BTC	-1.137	Cell cycle; cell death; cell signaling; cellular development; cellular growth and proliferation; cellular movement; DNA replication, recombination, and repair
argininosuccinate synthetase	25698	ASS1	-1.165	Others/unclassified
nuclear receptor subfamily 1, group D, member 2	259241	NR1D2	-1.170	Gene transcription
stannin	29140	SNN	-1.242	Cell death
phosphatase and tensin homolog^*	50557	PTEN	-1.272	Cell cycle; cell death; cell morphology; cell signaling; cellular assembly and organization; cellular development; cellular growth and proliferation; cellular movement; gene transcription inflammation; biomolecule metabolism; molecular transport; post-translational modification
solute carrier family 17 (sodium-dependent inorganic phosphate cotransporter), member 7^*	116638	SLC17A7	-1.287	Cell signaling
carnitine palmitoyltransferase 1, liver^*	25757	CPT1A	-1.291	Biomolecule metabolism
isopentenyl-diphosphate delta isomerase^*	89784	IDI1	-1.294	Others/unclassified
LIM homeobox protein 5	124451	LHX5	-1.317	Others/unclassified
calpain, small subunit 1^*	29156	CAPNS1	-1.318	Cell death; cellular assembly and organization; cellular growth and proliferation; cellular movement
filaggrin	24641	FLG	-1.325	Cellular assembly and organization
microsomal glutathione S-transferase 1	171341	MGST1	-1.329	Biomolecule metabolism
protein tyrosine phosphatase, nonreceptor type substrate 1^*	25528	SIRPA	-1.330	Cell death; cell morphology; cell signaling; cellular assembly and organization; cellular development; cellular growth and proliferation; cellular movement; gene transcription; inflammation
neurofibromatosis 2	25744	NF2	-1.333	Cell death; cellular growth and proliferation; cellular movement
Notch gene homolog 3 (Drosophila)^*	56761	NOTCH3	-1.335	Cellular development; gene transcription
killer cell lectin-like receptor subfamily B member 1B^*	25192	KLRB1	-1.338	Cell death
basic transcription element binding protein 1^*	117560	KLF9	-1.339	Gene transcription
regulating synaptic membrane exocytosis 1^*	84556	RIMS1	-1.353	Cell morphology; cell signaling; cellular assembly and organization; cellular movement; molecular transport
transglutaminase 1^*	60335	TGM1	-1.376	Biomolecule metabolism; cell death; cellular development; post-translational modification
translocase of inner mitochondrial membrane 23 homolog (yeast)^*	54312	IMM23	-1.393	Others/unclassified
lysozyme	25211	LYZ	-1.399	Others/unclassified
solute carrier family 25 (mitochondrial carrier; oxoglutarate carrier), member 11^*	64201	SLC25A11	-1.407	Others/unclassified
hydroxysteroid 11-beta dehydrogenase 1^*	25116	HSD11B1	-1.407	Cellular development; cellular growth and proliferation
potassium intermediate/small conductance calcium-activated channel, subfamily N, member 3^*	54263	KCNN3	-1.412	Cell signaling
B-cell leukemia/lymphoma 2^*	24224	BCL2	-1.413	Cell cycle; cell death; cell morphology; cell signaling; cellular assembly and organization; cellular development; cellular growth and proliferation; cellular movement; cellular response to therapeutics; DNA replication, recombination, and repair; biomolecule metabolism; gene transcription; inflammation; molecular transport; post-translational modification
hyperpolarization-activated, cyclic nucleotide-gated K⁺ 4^*	59266	HCN4	-1.415	Cell signaling
perforin 1 (pore-forming protein)^*	50669	PRF1	-1.417	Cell death; cell morphology; cell signaling; DNA replication, recombination, and repair; inflammation
calcitonin/calcitonin-related polypeptide, alpha^*	24241	CALCA	-1.433	Cell morphology; cell signaling; cellular development; cellular growth and proliferation; cellular movement; DNA replication, recombination, and repair; gene transcription; inflammation
nuclear RNA export factor 1^*	59087	NXF1	-1.436	Protein synthesis
fibroblast growth factor 14^*	63851	FGF14	-1.439	Others/unclassified
solute carrier family 7 (cationic amino acid transporter,y+ system), member 3^*	29485	SLC7A3	-1.455	Others/unclassified
ATP synthase, H⁺ transporting, mitochondrial F₀ complex, subunit b, isoform 1^*	171375	ATP5F1	-1.456	Cellular growth and proliferation
septin 3^*	56003	SEPT3	-1.468	Others/unclassified
cholinergic receptor, nicotinic, beta polypeptide 2 (neuronal)^*	54239	CHRNB2	-1.469	Cell signaling
wild-type p53-induced gene 1^*	64394	ZMAT3	-1.480	Cell death; cellular growth and proliferation; DNA replication, recombination, and repair
glutamate receptor, ionotropic, kainate 4^*	24406	GRIK4	-1.482	Cell signaling
Kruppel-like factor 15^*	85497	KLF15	-1.488	Gene transcription
aquaporin 6^*	29170	AQP6	-1.496	Others/unclassified
unc-5 homolog A (C. elegans)^*	60629	UNC5A	-1.498	Cell death
integrin beta 4^*	25724	ITGB4	-1.502	Cell death; cell morphology; cellular assembly and organization; cellular development; cellular growth and proliferation; cellular movement; gene transcription
peptidylprolyl isomerase A^*	25518	PPIA	-1.526	Cell death; cellular development; cellular growth and proliferation; cellular movement; DNA replication, recombination, and repair; inflammation; post-translational modification
FXYD domain-containing ion transport regulator 6^*	63847	FXYD6	-1.562	Others/unclassified
ribosomal protein L37^*	81770	RPL37	-1.579	Others/unclassified
RT1 class II, locus Da^*	294269	HLA-DRA	-1.580	Cell signaling; inflammation
FK506 binding protein 12-rapamycin-associated protein 1^*	56718	FRAP1	-1.596	Biomolecule metabolism; cell cycle; cell death; cell morphology; cell signaling; cellular development; cellular growth and proliferation; cellular movement; gene transcription; inflammation; post-translational modification; protein synthesis
eukaryotic translation elongation factor 1 alpha 1^*	171361	EEF1A1	-1.618	Cell death; protein synthesis
glutathione S-transferase theta 1^*	25260	GSTT1	-1.622	Biomolecule metabolism
solute carrier family 4, member 1^*	24779	SLC4A1	-1.630	Cell death; cellular growth and proliferation
fibroblast growth factor 17^*	29368	FGF17	-1.642	Cellular growth and proliferation
forkhead box M1^*	58921	FOXM1	-1.646	Cell cycle; cell death; cell morphology; cellular growth and proliferation; gene transcription
ATPase, class II, type 9A^*	84011	ATP9A	-1.649	Others/unclassified
phosphorylase kinase, gamma 2 (testis)^*	140671	PHKG2	-1.679	Biomolecule metabolism; post-translational modification
arrestin, beta 1^*	25387	ARRB1	-1.683	Cellular movement
rhoB gene^*	64373	RHOB	-1.684	Cell death; cell morphology; cell signaling; cellular assembly and organization; cellular development; cellular growth and proliferation; cellular movement; gene transcription
par-3 (partitioning defective 3) homolog (C. elegans)^*	81918	PARD3	-1.686	Cell morphology; cell signaling; cellular assembly and organization; cellular development; gene transcription
ribosomal protein L21^*	79449	RPL21	-1.687	Others/unclassified
RAS protein-specific guanine nucleotide-releasing factor 1^*	192213	RASGRF1	-1.715	Cell morphology; cell signaling; cellular growth and proliferation; gene transcription
acetylcholinesterase^*	83817	ACHE	-1.741	Cell death; cell morphology; cell signaling; cellular assembly and organization; cellular development; cellular growth and proliferation; DNA replication, recombination, and repair; post-translational modification; protein synthesis
ribosomal protein L18^*	81766	RPL18	-1.741	Others/unclassified
granzyme M (lymphocyte met-ase 1)^*	29252	GZMM	-1.745	Cell death
Bcl2-associated X protein^*	24887	BAX	-1.746	Cell cycle; cell death; cell morphology; cellular assembly and organization; cellular development; cellular growth and proliferation; cellular response to therapeutics; DNA replication, recombination, and repair; free radical scavenging; inflammation; biomolecule metabolism; molecular transport; protein synthesis
legumain^*	63865	LGMN	-1.749	Cellular movement
RAB6A, member RAS oncogene family^*	84379	RAB6A	-1.752	Cellular assembly and organization
matrix metalloproteinase 16^*	65205	MMP16	-1.763	Cellular movement; inflammation
olfactory receptor 226^*	65140	OR6A2	-1.770	Others/unclassified
mitogen-activated protein kinase 14^*	81649	MAPK14	-1.775	Biomolecule metabolism; cell cycle; cell death; cell morphology; cellular development; cellular growth and proliferation; cellular movement; gene transcription; inflammation; post-translational modification
c-mer protooncogene tyrosine kinase^*	65037	MERTK	-1.780	Biomolecule metabolism; cell death; cell morphology; cell signaling; cellular development; cellular growth and proliferation; post-translational modification
clathrin, heavy polypeptide (Hc)^*	54241	CLTC	-1.786	Cell morphology; cellular growth and proliferation
transient receptor potential cation channel, subfamily C, member 4^*	84494	TRPC4	-1.793	Others/unclassified
carnitine palmitoyltransferase 1b^*	25756	CPT1B	-1.795	Biomolecule metabolism
PDZ and LIM domain 1 (elfin)^*	54133	PDLIM1	-1.796	Gene transcription
fatty acid amide hydrolase^*	29347	FAAH	-1.798	Cell death; inflammation
fatty acid binding protein 4, adipocyte^*	79451	FABP4	-1.820	Gene transcription
protein tyrosine phosphatase, nonreceptor type 12^*	117255	PTPN12	-1.826	Biomolecule metabolism; cell morphology; cell signaling; cellular assembly and organization; cellular development; cellular movement; post-translational modification
potassium voltage-gated channel, Shab-related subfamily, member 2^*	117105	KCNB2	-1.834	Others/unclassified
glutathione S-transferase, pi 2^*	29438	GSTP1	-1.856	Cell death; cellular growth and proliferation; biomolecule metabolism; gene transcription
Fc receptor, IgG, alpha chain transporter^*	29558	FCGRT	-1.870	Cell signaling; molecular transport
voltage-gated channel like 1^*	266760	VGCNL1	-1.871	Others/unclassified
prostaglandin D₂ synthase^*	25526	PTGDS	-1.888	Cell death; cell morphology
MAP-kinase activating death domain^*	94193	MADD	-1.934	Cell death; cellular growth and proliferation
neogenin^*	81735	NEO1	-1.935	Cellular growth and proliferation; cellular movement; gene transcription
calbindin 1^*	83839	CALB1	-1.950	Cell death; cell morphology; cell signaling
guanylate cyclase 2e^*	79222	GUCY2D	-1.957	Others/unclassified
adrenal secretory serine protease precursor^*	64565	TMPRSS11D	-1.971	Others/unclassified
ubc2e ubiquitin-conjugating enzyme^*	641452	Ube2d2	-1.986	Others/unclassified
neuroblastoma, suppression of tumorigenicity 1^*	50594	NBL1	-2.003	Cellular movement
collagen, type 1, alpha 1^*	29393	COL1A1	-2.023	Cell morphology; cellular growth and proliferation; cellular movement
homeodomain interacting protein kinase 3^*	83617	HIPK3	-2.034	Biomolecule metabolism; cell signaling; post-translational modification
intercellular adhesion molecule 1^*	25464	ICAM1	-2.052	Cell death; cell morphology; cell signaling; cellular development; cellular growth and proliferation; cellular movement; inflammation
cyclin-dependent kinase 5^*	140908	CDK5	-2.134	Biomolecule metabolism; cell death; cell morphology; cell signaling; cellular assembly and organization; cellular development; cellular growth and proliferation; cellular movement; molecular transport; post-translational modification
ATPase, Cu²⁺ transporting, beta polypeptide^*	24218	ATP7B	-2.137	Cell death
Jun D protooncogene^*	24518	JUND	-2.159	Cell death; cell morphology; cellular growth and proliferation; gene transcription
neurexophilin 3^*	59315	NXPH3	-2.168	Others/unclassified
heterogeneous nuclear ribonucleoprotein methyltransferase-like 3 (S. cerevisiae)^*	89820	PRMT3	-2.171	Biomolecule metabolism; post-translational modification
midline 1^*	54252	MID1	-2.180	Cellular assembly and; organization
ATP-binding cassette, subfamily C (CFTR/MRP), member 1^*	24565	ABCC1	-2.191	Cell death; cellular movement; inflammation; biomolecule metabolism; molecular transport
prothymosin alpha^*	29222	PTMA	-2.313	Cell cycle; cell death; cellular development; cellular growth and proliferation; gene transcription
ubiquilin 1^*	114590	UBQLN1	-2.322	Others/unclassified
similar to Leydig cell tumor 10 kDa protein^*	288913	C19ORF53	-2.346	Others/unclassified
Inhibitor of DNA binding 2, dominant negative helix-loop-helix protein^*	25587	ID2	-2.357	Cell cycle; cell death; cell morphology; cellular development; cellular growth and proliferation; cellular movement; gene transcription
lectin, galactose binding, soluble 1^*	56646	LGALS1	-2.385	Cell cycle; cell death; cell morphology; cellular development; cellular growth and proliferation; cellular movement gene transcription; inflammation; post-translational modification
calreticulin^*	64202	CALR	-2.401	Cell cycle; cell death; cellular assembly and organization; cellular development; cellular growth and proliferation; cellular movement; gene transcription; post-translational modification
carbonic anhydrase 5^*	54233	CA5A	-2.427	Inflammation
mitogen-activated protein kinase 13^*	29513	MAPK13	-2.438	Cell death; cell morphology; gene transcription; inflammation
cytochrome P450, family 19, subfamily a, polypeptide 1^*	25147	CYP19A1	-2.440	Cell death; cell morphology; cellular development; cellular growth and proliferation; cellular movement; gene transcription; biomolecule metabolism
nucleobindin 1^*	84595	NUCB1	-2.513	Others/unclassified
tropomodulin 2^*	58814	TMOD2	-2.563	Cell signaling; cellular assembly and organization
apolipoprotein B editing complex 1^*	25383	APOBEC1	-2.655	Others/unclassified
solute carrier family 7 (cationic amino acid transporter, y+ system), member 7^*	83509	SLC7A7	-2.720	Cell signaling; cellular growth and proliferation
hsp70-interacting protein^*	246146	HSPBP1	-2.772	Post-translational modification
Rsec5 protein^*	171455	EXOC2	-2.779	Cell signaling; molecular transport
MAD homolog 9 (Drosophila)^*	85435	SMAD9	-2.815	Others/unclassified
ERM-binding phosphoprotein^*	59114	SLC9A3R1	-2.851	Cell signaling; cellular growth and proliferation
low-density lipoprotein receptor-related protein 3^*	89787	LRP3	-2.869	Others/unclassified
Rab geranylgeranyl transferase, a subunit^*	58983	RABGGTA	-2.890	Biomolecule metabolism; post-translational modification
cadherin EGF LAG seven-pass G-type receptor 2^*	83465	CELSR2	-2.923	Others/unclassified
tropomyosin 4^*	24852	TPM4	-3.019	Cellular movement
cytochrome P450, family 2, subfamily e, polypeptide 1^*	25086	CYP2E1	-3.161	Cell death; biomolecule metabolism
CEA-related cell adhesion molecule 9^*	116711	CEACAM9	-3.207	Others/unclassified
discoidin domain receptor family, member 2^*	83573	DDR2	-3.253	Others/unclassified
phosphoglucomutase 1^*	24645	PGM1	-3.308	Others/unclassified
gamma-glutamyl carboxylase^*	81716	GGCX	-3.455	Post-translational modification
Cplx1 complexin 1^*	64832	CPLX1	-3.464	Cell signaling; cellular assembly; and organization; cellular movement; molecular transport
septin 9^*	83788	SEPT9	-3.781	Cell cycle; protein synthesis
matrix metallopeptidase 8^*	63849	MMP8	-3.785	Cell death, cellular movement
thyroid hormone receptor interactor 10^*	116717	TRIP10	-4.232	Cell death
carboxylesterase 3^*	113902	CES1	-4.325	Others/unclassified
cyclin-dependent kinase 2^*	362817	CDK2	-4.458	Biomolecule metabolism; cell cycle; cell death; cell morphology; cellular development; cellular growth and proliferation; DNA replication, recombination, and repair; gene transcription; post-translational modification
monoamine oxidase B^*	25750	MAOB	-4.873	Cell death
solute carrier family 6 (neurotransmitter transporter, betaine/GABA), member 12^*	50676	SLC6A12	-5.287	Others/unclassified
peroxiredoxin 1^*	117254	PRDX1	-5.360	Cell death; cell morphology; cellular growth and proliferation; gene transcription; post-translational modification
ADP-ribosylation factor 4^*	79120	ARF4	-5.374	Others/unclassified
glypican 3^*	25236	GPC3	-5.638	Cell death; cellular growth and proliferation
protein kinase C, delta binding protein^*	85332	PRKCDBP	-6.059	Others/unclassified
cd86 antigen^*	56822	CD86	-6.118	Cell signaling; cellular development; cellular growth and proliferation; cellular movement; gene transcription; inflammation
Sjogren syndrome antigen B^*	81783	SSB	-6.131	Gene transcription; protein synthesis
secretoglobin, family 2A, member 1^*	25010	PSBP1	-6.227	Others/unclassified
procollagen, type I, alpha 2^*	84352	COL1A2	-7.076	Cell morphology
A kinase (PRKA) anchor protein 1^*	114124	AKAP1	-8.502	Cell death
methionine adenosyltransferase II, alpha^*	171347	MAT2A	-9.474	Others/unclassified
FXYD domain-containing ion transport regulator 1^*	58971	FXYD1	-9.644	Cellular growth and proliferation; gene transcription
CD24 antigen^*	25145	CD24	-11.299	Cell death; cell morphology; cell signaling; cellular assembly and organization; cellular development; cellular growth and proliferation; cellular movement
nuclear receptor subfamily 1, group I, member 2^*	84385	NR1I2	-11.958	Biomolecule metabolism; gene transcription; inflammation; molecular transport; post-translational modification
glial cell line derived neurotrophic factor family receptor alpha 1^*	25454	GFRA1	-17.708	Cell death; cell morphology; cellular development; cellular; growth and proliferation; cellular movement
cell division cycle 25 homolog A (S. cerevisiae)^*	171102	CDC25A	-19.859	Cell cycle; cell death; cell morphology; cellular growth and proliferation; dna replication, recombination, and repair
5-hydroxytryptamine (serotonin) receptor 5B^*	79247	HTR5B	-22.285	Others/unclassified
NSFL1 (p97) cofactor (p47)^*	83809	NSFL1C	-22.400	Others/unclassified
membrane and microfilament-associated protein p58^*	207121	RGD: 727794	-49.533	Cellular assembly and organization

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The change in expression was significant compared with untreated control (P < .05). Gene names in italics are common in both panels of T + E₂-treated LP and T + DES-treated VP.

Image plots (Figure 2, A, and B1 and B2) showed the up- and downregulated genes in the T + E₂-induced LP dysplasia panel (253 genes), respectively. Although genes in this panel were distributed among 27 IPA networks, two major networks with the highest relevancy scores were identified: one related to cell morphology, cellular growth, proliferation, and movement (Figure 2C), and the other related to apoptosis and cell signaling (Figure 2D). From these two networks, six genes were selected for post hoc confirmation by real-time q-PCR (Figure 3). All six genes showed the predicted expression patterns. Significant differences (P < .01/.05) were observed between the transcript levels in the T + E₂-treated and -untreated LPs, whereas no significant differences (P > .05) in the transcript levels were observed between the treated and untreated VP groups. These genes are the CCAAT/enhancer binding protein-delta (Cebpd), the tumor protein Tp53, MAD homolog 7 (Smad7), homeobox A1 (Hoxa1), killer cell lectin-like receptor subfamily B member 1 (Klrb1), and neurexophilin 3 (Nxph3). Notably, the q-PCR data correlated well with those obtained by microarray analyses (Figure W1A). T + E₂-response genes found in VP, in contrast to those found in the LP, were not mapped to any particular IPA networks (data not shown).

Heat maps and gene interaction networks of differentially expressed genes found exclusively in the LP dysplasia following T + E₂ treatment. Red and green denote upregulated and downregulated expression, respectively, as compared with the overall gene's mean value normalized to the universal rat reference RNA. Columns represent data from a single prostate sample, and rows correspond to a single gene probe. (A) A single cluster of upregulated genes identified in the T + E₂ LP dysplasia, marked with pink (*left panel*); a selected region (blue box) of this cluster is enlarged (*right panel*). (B1 and B2) Two separate downregulated gene clusters observed only in T + E₂ LP dysplasia, marked with pink (*left panels*); selected clusters are magnified (*right panels*). (C and D) Two representative gene interaction networks (with the highest relevancy scores) generated by IPA analysis from the differentially expressed genes in the T + E₂ LP dysplasia panel. Green indicates downregulated; red, upregulated. Genes bordered in red were validated by real-time q-PCR. See Figure 1 for key to IPA network.

Post hoc real-time q-PCR analyses of selected genes in the T + E₂ LP dysplasia panel. Data were normalized to the levels of Rpl19. Bars indicate standard deviations (SD) of three to five animals in each treatment group. *P < .05, **P < .01 by one-way ANOVA with Tukey post hoc analysis.

Image plots illustrated distinctive upregulated (Figure 4A) and downregulated (Figure 4, B1 and B2) genes in the T + DES-induced VP dysplasia panel. In addition, a unique set of genes was exclusively repressed by T + DES in the VP harboring dysplasia (Figure 4B2). These genes were also underexpressed in LPs compared with the untreated VPs. Genes in the T + DES-treated VP panel (198 genes) mapped primarily to two major networks related to 1) apoptosis, cellular development, growth, and proliferation and 2) estrogen signaling, as they exhibited the highest relevancy scores (Figure 4, C and D, respectively). From the two networks, nine genes were selected for post hoc confirmation by q-PCR analyses that correlated well with those obtained by microarrays (Figure W1B). All nine genes showed significant increases in transcript levels (P < .01/.05) in the VP with T + DES treatment and exhibited little or no change in expression (P > .05) in the LPs of the treated group compared with untreated controls (Figure 5). They include Cebpd, Tp53, v-maf musculoaponeurotic fibrosarcoma oncogene homolog B (Mafb), Hoxa1, breast cancer anti-estrogen resistance 1 (Bcar1), plasminogen activator urokinase (Plau), thyroid hormone receptor alpha (Thra), glutathione peroxidase 3 (Gpx3), and v-src sarcoma viral oncogene homolog (Src).

Heat maps and gene interaction networks of differentially expressed genes found uniquely in the VP dysplasia following T + DES treatment. (A) A single cluster of upregulated genes identified only in the T + DES VP dysplasia, marked with pink (*left panel*), and the selected cluster enlarged (*right panel*). (B1 and B2) Clusters of downregulated genes in the VP but not in the LP following T + DES exposure. Interestingly, a single cluster (B2, *left panel*) corresponds to the downregulated genes in the T + DES-treated VP and is also underexpressed in both untreated and treated LPs. A selected region (*blue box*) of this cluster is enlarged (*right panel*). (C and D) Two representative gene interaction networks (with the highest relevancy scores) generated by IPA analysis from the differentially expressed genes in the T + DES VP dysplasia panel. Green indicates downregulated; red, upregulated. Genes bordered with red were validated by real-time q-PCR. See Figure 1 for key to IPA network.

Post hoc validation of selected genes in the T + DES VP dysplasia panel by real-time q-PCR. The data were normalized to the levels of Rpl19. Bars indicated standard deviations (SD) of three to five animals in each treatment group. *P < .05, **P < .01 by one-way ANOVA with Tukey post hoc analysis.

Only 32 genes were found to be common in both dysplasia-related panels (Figure 1C), and 23 (of 32) genes formed a unique gene network when mapped by IPA (Figure 1D). We postulate that this gene network is central to E₂- or DES-induced dysplasia in the LP or VP.

Identification of Estrogen-induced Differentially Expressed Genes That Are Not Related to Dysplasia in Rat LP or VP

We also identified two sets of hormone-induced genes that are not related to dysplasia. These genes exhibited differential expression in the VP following T + E₂ treatment (14 genes; Figure 1C, left bottom) and in the LP following T + DES treatment (190 genes; Figure 1C, right bottom). They are published in Tables W2 and W3.

Genes Insensitive to the T + E₂ and T + DES Treatments

Two sets of 156 and 80 genes were identified as insensitive in the LP and/or VP to T + E₂ or T + DES treatment, respectively (Figure 6, A–C; Tables W4 and W5). Forty-one genes were found in both hormone-insensitive panels and mapped to an IPA network (Figure 6D) that includes the androgen receptor (Ar), a key regulator of prostate function.

Heat maps of genes insensitive to (A) T + E₂ or (B) T + DES. (C) Venn diagram showing the number of genes insensitive to T + E₂ and/or T + DES treatment. Note that 41 genes are found in common in both hormone-insensitive panels and mapped to an IPA network (D) that includes the Ar, a key regulator of prostate function. Gray indicates no change in gene expression levels compared with those in untreated counterparts. See Figure 1 for key to IPA network.

Discussion

Exposure of the human prostate to estrogen may increase as men age [2–4] or with increased exposure to dietary estrogens such as DES or zeranol residues in meat, bisphenol A from food containers, or phytoestrogens [9]. The NBL rat provides a relevant model for elucidating mechanisms underlying carcinogenesis caused by such exposures, such as dysplasia and PCa, that consistently developed in the LP or the VP following chronic T-supported treatment with E2 or DES, respectively [15,18–20]. In the present study, we assessed transcriptional profiles elicited by the xenoestrogen DES in comparison with those of the endogenous hormone E₂ and observed prostatic lobe-specific differential responses to the two estrogens in NBL rats. Hierarchical clustering revealed that the T + E₂ treatment principally affects the LP gene expression profile whereas the T + DES targets that of the VP. Two distinct panels of dysplasia-related genes were identified: the T + E₂ LP panel (253 genes) and the T + DES VP panel (198 genes), with only 32 overlapping genes. These findings indicate that the two estrogens alter gene expression profiles only in the prostatic lobe that develops dysplasia following each of the hormonal treatments. These findings imply a functional divergence of the two estrogens with respect to their oncogenic actions in the rat prostate. Thus, endocrine disruption of gene expression and dysplasia induction of the two estrogens are categorically different [9], although they may share some biologic convergence [30].

The IPA analysis identified distinct gene interaction networks based on differential expression profiles. It classified genes into functional categories and suggested a possible mechanistic linkage among the differentially expressed genes. We also used this tool to help prioritize genes for confirmation. The T + E₂ treatment altered the expression of LP genes primarily in two major networks: one related to cell morphology, cellular growth, proliferation, and movement, and the other related to apoptosis and cell signaling. Exposure to T + DES also affected expression of genes in a similar network associated with apoptosis, cellular development, growth, and proliferation. Intriguingly, it also altered the expression of a set of genes linked to E₂, a finding that may explain why, after T + DES treatment, gene expression profiles in the VP shifted closer to those in the treated and untreated LPs, which are under the influence of natural estrogen. By first mapping genes to the IPA network before selecting genes for confirmation, we were able to validate most of the expression changes in the microarrays with real-time q-PCR. In this regard, we confirmed three genes (Cebpd, Hoxa1, and Tp53) common in the two dysplasia panels. Importantly, these three genes were upregulated only in the prostatic lobes that developed dysplasia, i.e., the T + E₂-treated LP and the T + DES-treated VP. Other lobe-specific genes that were confirmed were Smad7, Klb1, and Nxph3 in the T + E₂-treated LP and Mafb, Src, Bcar1, Gpx3, Plau, and Thra in T + DES-treated VP. Our findings suggest that these gene expression changes induced by T + E₂/DES treatment could be causative factors of dysplasia development. However, it remains possible that these gene expression changes are simply part of the neoplastic transformation process. Future studies employing laser capture-microdissected samples to establish the temporal relationship between change in gene expression pattern and evolution of dysplasia may shed new light on the cause-effect link between the two.

Among all these validated genes, Cebpd, Hoxa1, and Tp53 are the ones upregulated in common in the two dysplasia panels. CCAAT/enhancer binding proteins (C/EBP) are a highly conserved family of basic leucine zipper transcription factors; six members (C/EBP-alpha to -zeta) have been identified to date. This protein family plays a critical role in cell proliferation, apoptosis, and inflammation, depending on the cell type and specific physiological stress [31]. The precise functional role of C/EBP-delta, in particular, in normal prostate biology and PCa remains poorly understood. Cebpd was shown to be an androgen-repressed gene in the normal rat prostate [32,33], supporting its role as an apoptotic mediator and/or a negative regulator of cell proliferation. In a human androgen-dependent PCa xenograft, androgen withdrawal, however, resulted in a decline in the expression of C/EBP-delta [32]. Most published studies focus on the androgenic regulation of C/EBP-delta; this is, therefore, the first report of the association of estrogen-mediated upregulation of C/EBP-delta gene expression with the early phase of prostate carcinogenesis. A recent study showed overexpression of C/EBP-beta, another member of the family, in proliferative inflammatory atrophy in human prostate specimens, suggesting its role in the inflammation-associated carcinogenesis in the prostate [34]. In microarray data, we observed downregulation of C/EBP-alpha (Cebpa) only in T + E₂-treated dysplastic LPs, corroborating its putative suppressive function in epithelial tumorigenesis [35]. Together, these findings raise the possibility that an orchestrated deregulation of C/EBP-family transcripts is involved in the pathogenesis of PCa in humans and rodents.

Homeobox gene (HOX) is a family of transcription factors related to growth and development. In rodents and humans, 39 members of the HOX gene family have been assigned to four gene clusters (A–D) and are numbered according to their expression along the anterior- posterior axis. Hox13 was shown to be involved in cell differentiation during normal prostate development [36], whereas upregulations of HOXC have been observed in PCa cells [37,38]. Knockdown of HOXC6 gene expression led to apoptosis of PCa cells [39], and overexpression of the HOXC8 gene correlated with the loss of tumor differentiation in human PCa [37]. Increased expression of the HOXA1 transcript was detected in human clinical cervical cancer samples and in several cervical cell lines compared with expression in normal tissues [40,41]. Our data suggest that ectopic expression of Hoxa1 is related to the evolution of dysplasia in both LP and VP and that the detailed roles of Hoxa1 in PCa require further study.

Tp53 is a well-known tumor-suppressive transcription factor and acts as a gatekeeper in signaling pathways involved in monitoring cellular stress such as DNA damage and in the determination of congruous responses (DNA repair, cell growth arrest, or apoptosis) to specific physiological stress. Previous studies demonstrated that mutation of Tp53 is not necessarily as late an event in prostate carcinogenesis as previously reported; it is also frequently found in high-grade prostate intraepithelial neoplasia, a precursor lesion of PCa [42,43]. In this regard, we observed upregulation of Tp53 gene expression in dysplastic rat prostate glands treated with sex hormone, implying a role in early prostate tumorigenesis, but whether these glands express wild-type or mutated Tp53 is not known. Alternatively, the induction of Tp53 expression may be a compensatory response to cellular stress imposed by hormonal changes. We recently demonstrated that T + E₂ treatment induced oxidative and nitrosative stress, accompanied by DNA and protein damages, specifically in the LP, which is susceptible to dysplasia/cancer induction [17].

Smad7, an inhibitory Smad, is rapidly induced by TGF-β, thereby creating a negative feedback loop to a variety of TGF-β signaling responses, including proliferation, differentiation, apoptosis, inflammation, tissue remodeling, angiogenesis, and cell adhesion [44]. Disruption of the TGF-β signaling cascade by aberrant overexpression of this negative modulator leads to increased tumorigenicity in colon cancer cells by blocking TGF-β-mediated growth inhibition and apoptosis [45]. In contrast, Smad7 has been shown to mediate apoptosis induced by TGF-β in PCa cells [46], perhaps through crosstalking with other cellular signaling cascades such as the p38 mitogen-activated protein kinase [47] and β-catenin/Wnt [48] pathways. In our study, upregulation of Smad7 in the dysplastic LP (also in the T + DES VP microarray data) suggests perturbations of TGF-β feedback regulation and thus the loss of balance between cell proliferation and apoptosis in the early phase of prostate carcinogenesis.

A particularly interesting gene network found in the T + DES-treated VP (Figure 4, B1 and B2) is related to E₂ signaling. Members include Src, Bcar1, and Plau. Src, a nonreceptor protein tyrosine kinase, triggers the nongenomic estrogen signaling leading to activation of ERK1/2 and cell proliferation in prostate epithelial cells [49,50]. It remains to be determined if the downregulation of Src by DES represents a classic negative feedback loop. The Bcar1 interacts and modulates Src activity [51]. An increase in Bcar1 gene expression, as observed in the rat dysplastic VP, has also been reported in human PCa [52]. The coordinated dysregulation of both Src (downregulation) and Bcar1 (upregulation) may reflect a disruption of the Src-dependent signaling pathway during the early development of PCa, a notion further supported by the altered expression of Plau, a downstream target of Src [53]. Interestingly, both Bcar1 and Plau are involved in cell survival and migration [54,55].

Our group recently demonstrated significant oxidative stress and inflammation in the T + E₂-treated NBL rat LP [17,56]. The microarray data show a relatively small fraction of genes related to redox homeostasis and immune response, possibly due to inherent limitations of the type and coverage size (about 3800 genes) of the array chips we selected. However, T + E₂ treatment was found to upregulate Klrb1 in the LP. This gene, also known as NKR-P1A/CD161 in humans, encodes a natural killer (NK) cell C-type lectin-like receptor that regulates NK cell functions. KLRB1 is expressed not only in NK cells but also in immune cell types, such as T cells, monocytes, and dendritic cells [57]. Thus, it is likely that increased expression of Klrb1 in LP with T + E₂ treatment is due to the infiltration of immune cells [17].

The observation of a dramatic induction of Gpx3 in the VP dysplasia panel, as shown by microarray (>17-fold) and q-PCR (>70-fold), is consistent with our previous report of a marked elevation of GPX enzyme activity and lipid peroxidation in this lobe after T + DES treatment [56]. Hence, the activation in this glutathione-associated detoxification enzyme might be a response to hormone-induced oxidative stress in the gland or represent a cytoprotective mechanism, adapted by dysplastic cells, against oxidative insults. An elevation of GPX3 expression, but not of GPX1 and GPX2, was found in precancerous lesions of NKX3.1 mutant mouse prostate [58]. Taken together, these findings suggest that GPX3 is specifically involved in early prostatic transformation that has a mechanistic link to oxidative stress.

In summary, we showed that genes such as Cebpd, Tp53, and Hoxa1 are at the core of disrupted biologic networks related to hormoneinduced dysplasia. Unbiased gene profiling clearly demonstrated differential susceptibility of the LP and VP to natural and xenoestrogen with regard to alterations in gene expression and induction of dysplasia. Our data suggest more functional divergence between DES and E₂ in the disruption of prostatic functions than that previously suspected. Methodologically, the combined utility of expression profiling and gene network mapping provides an instrumental platform for transcriptome studies.

Supplementary Material

Supplementary Figures and Tables

neo1001_0020SD1.pdf^{(161.1KB, pdf)}

Abbreviations

C/EBP: CCAAT/enhancer binding protein
DES: diethylstilbestrol
E2: 17β-estradiol
LP: lateral prostate
NBL: Noble
PCa: prostate cancer
PCR: polymerase chain reaction
VP: ventral prostate

Footnotes

This article refers to supplementary materials, which are designated by Tables W1, W2, W3, W4, and W5 and Figure W1 and are available online at www.neoplasia.com.

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[R12] 12.Troisi R, Hatch EE, Titus-Ernstoff L, Hyer M, Palmer JR, Robboy SJ, Strohsnitter WC, Kaufman R, Herbst AL, Hoover RN. Cancer risk in women prenatally exposed to diethylstilbestrol. Int J Cancer. 2007;121:356–360. doi: 10.1002/ijc.22631. [DOI] [PubMed] [Google Scholar]

[R13] 13.Strohsnitter WC, Noller KL, Hoover RN, Robboy SJ, Palmer JR, Titus-Ernstoff L, Kaufman RH, Adam E, Herbst AL, Hatch EE. Cancer risk in men exposed in utero to diethylstilbestrol. J Natl Cancer Inst. 2001;93:545–551. doi: 10.1093/jnci/93.7.545. [DOI] [PubMed] [Google Scholar]

[R14] 14.Ho SM, Tang WY, Belmonte de Frausto J, Prins GS. Developmental exposure to estradiol and bisphenol A increases susceptibility to prostate carcinogenesis and epigenetically regulates phosphodiesterase type 4 variant 4. Cancer Res. 2006;66:5624–5632. doi: 10.1158/0008-5472.CAN-06-0516. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R15] 15.Leav I, Ho SM, Ofner P, Merk FB, Kwan PW, Damassa D. Biochemical alterations in sex hormone-induced hyperplasia and dysplasia of the dorsolateral prostates of Noble rats. J Natl Cancer Inst. 1988;80:1045–1053. doi: 10.1093/jnci/80.13.1045. [DOI] [PubMed] [Google Scholar]

[R16] 16.Leav I, Merk FB, Kwan PW, Ho SM. Androgen-supported estrogenenhanced epithelial proliferation in the prostates of intact Noble rats. Prostate. 1989;15:23–40. doi: 10.1002/pros.2990150104. [DOI] [PubMed] [Google Scholar]

[R17] 17.Tam NN, Leav I, Ho SM. Sex hormones induce direct epithelial and inflammation-mediated oxidative/nitrosative stress that favors prostatic carcinogenesis in the Noble rat. Am J Pathol. 2007;171:1334–1341. doi: 10.2353/ajpath.2007.070199. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R18] 18.Thompson CJ, Tam NN, Joyce JM, Leav I, Ho SM. Gene expression profiling of testosterone and estradiol-17β-induced prostatic dysplasia in Noble rats and response to the antiestrogen ICI 182,780. Endocrinology. 2002;143:2093–2105. doi: 10.1210/endo.143.6.8846. [DOI] [PubMed] [Google Scholar]

[R19] 19.Bosland MC, Ford H, Horton L. Induction at high incidence of ductal prostate adenocarcinomas in NBL/Cr and Sprague-Dawley Hsd:SD rats treated with a combination of testosterone and estradiol-17β or diethylstilbestrol. Carcinogenesis. 1995;16:1311–1317. doi: 10.1093/carcin/16.6.1311. [DOI] [PubMed] [Google Scholar]

[R20] 20.Ofner P, Bosland MC, Vena RL. Differential effects of diethylstilbestrol and estradiol-17β in combination with testosterone on rat prostate lobes. Toxicol Appl Pharmacol. 1992;112:300–309. doi: 10.1016/0041-008x(92)90200-c. [DOI] [PubMed] [Google Scholar]

[R21] 21.Smyth GK. Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol. 2004;3 doi: 10.2202/1544-6115.1027. Article 3. [DOI] [PubMed] [Google Scholar]

[R22] 22.Guo J, Sartor M, Karyala S, Medvedovic M, Kann S, Puga A, Ryan P, Tomlinson CR. Expression of genes in the TGF-β signaling pathway is significantly deregulated in smooth muscle cells from aorta of aryl hydrocarbon receptor knockout mice. Toxicol Appl Pharmacol. 2004;194:79–89. doi: 10.1016/j.taap.2003.09.002. [DOI] [PubMed] [Google Scholar]

[R23] 23.Karyala S, Guo J, Sartor M, Medvedovic M, Kann S, Puga A, Ryan P, Tomlinson CR. Different global gene expression profiles in benzo[a] pyrene- and dioxin-treated vascular smooth muscle cells of AHR-knockout and wild-type mice. Cardiovasc Toxicol. 2004;4:47–73. doi: 10.1385/ct:4:1:47. [DOI] [PubMed] [Google Scholar]

[R24] 24.Sartor M, Schwanekamp J, Halbleib D, Mohamed I, Karyala S, Medvedovic M, Tomlinson CR. Microarray results improve significantly as hybridization approaches equilibrium. Biotechniques. 2004;36:790–796. doi: 10.2144/04365ST02. [DOI] [PubMed] [Google Scholar]

[R25] 25.Sartor MA, Tomlinson CR, Wesselkamper SC, Sivaganesan S, Leikauf GD, Medvedovic M. Intensity-based hierarchical Bayes method improves testing for differentially expressed genes in microarray experiments. BMC Bioinformatics. 2006;7:538. doi: 10.1186/1471-2105-7-538. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R26] 26.Benjamini YHY. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc. 1995;57:289–300. [Google Scholar]

[R27] 27.Daly-Burns B, Alam TN, Mackay A, Clark J, Shepherd CJ, Rizzo S, Tatoud R, O'Hare MJ, Masters JR, Hudson DL. A conditionally immortalized cell line model for the study of human prostatic epithelial cell differentiation. Differentiation. 2007;75:35–48. doi: 10.1111/j.1432-0436.2006.00113.x. [DOI] [PubMed] [Google Scholar]

[R28] 28.Rozen S, Skaletsky HJ. Primer3. 1998 Available at: http://www-genome.wi.mit.edu/genome_software/other/primer3.html.

[R29] 29.Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 2001;25:402–408. doi: 10.1006/meth.2001.1262. [DOI] [PubMed] [Google Scholar]

[R30] 30.Buterin T, Koch C, Naegeli H. Convergent transcriptional profiles induced by endogenous estrogen and distinct xenoestrogens in breast cancer cells. Carcinogenesis. 2006;27:1567–1578. doi: 10.1093/carcin/bgi339. [DOI] [PubMed] [Google Scholar]

[R31] 31.Ramji DP, Foka P. CCAAT/enhancer-binding proteins: structure, function and regulation. Biochem J. 2002;365:561–575. doi: 10.1042/BJ20020508. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R32] 32.Yang G, Gregory CW, Shang Q, O'Brien DA, Zhang YL. Differential expression of CCAAT/enhancer-binding protein-delta (C/EBPdelta) in rat androgen-dependent tissues and human prostate cancer. J Androl. 2001;22:471–480. [PubMed] [Google Scholar]

[R33] 33.Desai KV, Michalowska AM, Kondaiah P, Ward JM, Shih JH, Green JE. Gene expression profiling identifies a unique androgen-mediated inflammatory/immune signature and a PTEN (phosphatase and tensin homolog deleted on chromosome 10)-mediated apoptotic response specific to the rat ventral prostate. Mol Endocrinol. 2004;18:2895–2907. doi: 10.1210/me.2004-0033. [DOI] [PubMed] [Google Scholar]

[R34] 34.Wang GL, Shi X, Salisbury E, Sun Y, Albrecht JH, Smith RG, Timchenko NA. Growth hormone corrects proliferation and transcription of phosphoenolpyruvate carboxykinase in livers of old mice via elimination of CCAAT/enhancer-binding protein α-Brm complex. J Biol Chem. 2007;282:1468–1478. doi: 10.1074/jbc.M608226200. [DOI] [PubMed] [Google Scholar]

[R35] 35.Loomis KD, Zhu S, Yoon K, Johnson PF, Smart RC. Genetic ablation of CCAAT/enhancer binding protein α in epidermis reveals its role in suppression of epithelial tumorigenesis. Cancer Res. 2007;67:6768–6776. doi: 10.1158/0008-5472.CAN-07-0139. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R36] 36.Huang L, Pu Y, Hepps D, Danielpour D, Prins GS. Posterior Hox gene expression and differential androgen regulation in the developing and adult rat prostate lobes. Endocrinology. 2007;148:1235–1245. doi: 10.1210/en.2006-1250. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R37] 37.Waltregny D, Alami Y, Clausse N, de Leval J, Castronovo V. Overexpression of the homeobox gene HOXC8 in human prostate cancer correlates with loss of tumor differentiation. Prostate. 2002;50:162–169. doi: 10.1002/pros.10045. [DOI] [PubMed] [Google Scholar]

[R38] 38.Miller GJ, Miller HL, van Bokhoven A, Lambert JR, Werahera PN, Schirripa O, Lucia MS, Nordeen SK. Aberrant HOXC expression accompanies the malignant phenotype in human prostate. Cancer Res. 2003;63:5879–5888. [PubMed] [Google Scholar]

[R39] 39.Ramachandran S, Liu P, Young AN, Yin-Goen Q, Lim SD, Laycock N, Amin MB, Carney JK, Marshall FF, Petros JA, et al. Loss of HOXC6 expression induces apoptosis in prostate cancer cells. Oncogene. 2005;24:188–198. doi: 10.1038/sj.onc.1207906. [DOI] [PubMed] [Google Scholar]

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PERMALINK

Gene Expression Profiling Identifies Lobe-Specific and Common Disruptions of Multiple Gene Networks in Testosterone-Supported, 17β-Estradiol- or Diethylstilbestrol-Induced Prostate Dysplasia in Noble Rats12

Neville N C Tam

Carol Ying-Ying Szeto

Maureen A Sartor

Mario Medvedovic

Shuk-Mei Ho

Abstract

Introduction

Materials and Methods

Animals and Hormonal Treatment

Microarray Hybridization

Microarray Data Normalization and Analysis

Identification of Estrogen-Regulated Gene Expression

Pathway and Network Analysis

Reverse Transcription and Real-Time Quantitative Polymerase Chain Reaction

Results

T + E2 and T + DES Treatments Differentially Induced Dysplasia in LP and VP, Respectively

Hierarchical Clustering Identified Differential Action of E2 and DES in the Two Prostate Lobes

Figure 1.

Identification of Estrogen-Induced Differentially Expressed Genes Related to Dysplasia in Rat LP or VP

Table 1.

Table 2.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Identification of Estrogen-induced Differentially Expressed Genes That Are Not Related to Dysplasia in Rat LP or VP

Genes Insensitive to the T + E2 and T + DES Treatments

Figure 6.