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Identification of Androgen-Responsive Genes in the Rat Ventral Prostate by Complementary Deoxyribonucleic Acid Subtraction and Microarray

Department of Urology (F.J., Z.W.), Department of Molecular Pharmacology and Biological Chemistry (Z.W.), Robert H. Lurie Comprehensive Cancer Center (Z.W.), The Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611

Address all correspondence and requests for reprints to: Zhou Wang, Department of Urology, Tarry 11-715, The Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611. E-mail: wangz{at}northwestern.edu.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
We have reported the identification of approximately two dozen androgen-responsive genes, on the basis of their induction by androgens in the castrated rat ventral prostate, using PCR-based subtractive hybridization. The same prostatic cDNA samples were subjected to a modified subtractive hybridization, resulting in the identification of 21 new androgen-responsive genes, of which 14 were known and 7 were novel genes. To complement our subtraction study, we have used an Incyte rat cDNA microarray, consisting of 8951 known genes and expressed sequence tags, and found that 162 genes were up-regulated and 143 genes were down-regulated 2.3-fold or more by androgens. As expected, the genes isolated from our subtraction overlap with genes found with microarray. Northern blot was carried out on all of the genes identified by subtraction and a few selected genes from microarray. All of the assayed genes are regulated by androgens, validating our subtraction and microarray studies. The identified genes can be classified into several functional groups, including metabolism, protein chaperoning and trafficking, protein synthesis, secretions, cell cycle and apoptosis, structural and extracellular matrix proteins, and novel proteins. Identification of these androgen-responsive genes will contribute to the understanding of androgen action in the normal and cancerous prostate.

	Introduction

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ANDROGENS ARE REQUIRED for the structural and functional integrity of the prostate. The involvement of androgens in the pathogenesis of prostate cancer has been well documented. However, the exact role of androgens in prostate cancer progression remains elusive. The androgen ablation therapy developed by Huggins (1) has been the standard treatment for metastatic prostate cancer. Though the initial clinical response of prostate cancer patients to androgen ablation is usually favorable, the progression to androgen-independent disease is almost inevitable. Understanding of androgen action in the prostate may lead to novel approaches for prevention and treatment of prostate cancer.

Androgen action is mediated by the androgen receptor, a ligand-dependent transcription factor that regulates gene expression. The identification of androgen-responsive genes is essential for the molecular characterization of androgen action. We have isolated about two dozen androgen-responsive genes by subtractions in the rat ventral prostate model (2). Considering the evolutionary conservation of androgen action in the prostate, the rat prostate is an excellent in vivo model for studying androgen action. Some of the genes identified in our previous subtractions are regulated by androgens in LNCaP (lymph node carcinoma of the prostate) human prostate cancer cells, which further validates the use of rat prostate for identification of androgen-responsive genes. Other groups also have used LNCaP and CWR-22, another human prostate cancer cell line, to identify androgen-regulated genes (3, 4, 5). Various methods, such as differential display (6, 7, 8), serial analysis of gene expression (4, 9), expressed sequence tag (EST) quantitation (10), subtraction (2), and microarray (5), have been used in identification of androgen-responsive genes. The identified androgen-responsive genes include prostate-specific antigen (11), NKX3.1 (6), probasin (12), hKLK2 (human prostate-specific kallikrein) (13), and prostatein C3 (14).

The identification and characterization of androgen-responsive genes have advanced prostate cancer research in a variety of ways. One of the best-characterized androgen-responsive genes, prostate-specific antigen, is widely used as a diagnostic marker for prostate cancer. Other androgen-responsive-genes, such as hKLK2, have been proposed as potential biomarkers for prostate cancer (15). NKX3.1 is a potential tumor suppressor gene for prostate cancer (16, 17, 18). Loss of NKX3.1 expression in human prostate cancers correlates with tumor progression (19). The promoter of probasin has been successfully used to target transgenes in the mouse prostate to generate mouse transgenic models for prostate cancer (20).

In this paper, we report a comprehensive gene expression analysis of androgen action in the rat ventral prostate, using both subtractive hybridization and microarray approaches. This study has resulted in the identification of more than 100 androgen-responsive genes, which will lead to a better understanding of androgen regulation of proliferation, cell survival, and differentiation in the prostate. We showed that some of the genes isolated from the rat prostate were also regulated by androgens in LNCaP cells, providing further evidence for the evolutionary conservation of androgen action in the prostate.

	Materials and Methods

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Materials
Dimethyl sulfoxide, EDTA, NaCl, SDS (sodium dodecyl sulfate), Tris, guanidinium, and CsCl were purchased from Fisher Biotech. [³²P]Deoxy-CTP (3000 Ci/mM) was obtained from NEN Life Science Products (Boston, MA). The membrane for Northern blot was purchased from Micron Separations, Inc. (Westboro, MA). X-ray films were from Eastman Kodak Co. (Rochester, NY).

Animals
Young adult male Sprague Dawley rats (250–300 g) were purchased from Harlan Sprague Dawley, Inc. (Indianapolis, IN) and were maintained at the Northwestern University animal facility. Care and maintenance of rats followed a protocol approved by the Institutional Animal Care and Use Committee. Castration of the rats involved excision of the testes, fat pads, and epididymis. Surgical procedures were performed in a room dedicated to animal surgery. For each experimental condition, at least three rats were used, to minimize individual variations. After castration, rats were left untreated for 7 d before androgen replacement. Treatment of castrated rats with exogenous androgen was carried out by daily sc injections of testosterone propionate, at 2 mg/rat, for 14 h, 2 d, 7 d, and 14 d. Normal rats were treated with the same amount of androgen for 2 d. At the end of animal manipulations, the ventral, lateral, and dorsal lobes of the prostate were removed and immediately frozen in liquid nitrogen for subsequent RNA isolation.

Subtractive hybridization
A PCR-based subtractive hybridization was performed essentially the same as previously described (2), with minor modifications. The driver library was constructed from the mRNA of 7-d castrated adult rat ventral prostate. The tracer library was constructed from mRNA of 7-d castrated adult rat ventral prostate after 48-h androgen replacement. The PCR products of androgen-up-regulated genes (U1-U25) isolated from our previous study (2) were added to the driver library to suppress their enrichment so that unidentified androgen-up-regulated genes could be enriched favorably during subtractive hybridizations.

Microarray
Total RNAs of the ventral prostate from 7-d castrated rats and 7-d castrated rats after 2-d androgen treatment were sent to Incyte, Inc. for microarray analysis. Selection of poly(A)+ RNA, generation of cDNA, fluorescent labeling, and hybridization to the gene chip were performed by IncyteGenomics (Palo Alto, CA). Briefly, mRNA was reverse-transcribed in the presence of 5' Cy3- or Cy5-labeled random 9-mer (Operon Technologies, Alameda, CA). The paired reactions were combined and purified with a TE-30 column (CLONTECH Laboratories, Inc., Palo Alto, CA). Fluorescently labeled probes were then applied to the array for hybridization. After hybridization, the array was washed with decreasing ionic strength, and Cy3 and Cy5 fluorescence signals were detected. We examined 8951 rat genes and ESTs on the rat toxicity array (IncyteGenomics) and analyzed the results with the software GEMtool 2.4. Background-subtracted element signals were used to calculate Cy3/Cy5 ratios.

Total RNA isolation and Northern blot
The guanidinium/CsCl gradient method was used to isolate total RNA from the rat prostate, and the Rneasy mini kit (QIAGEN, Valencia, CA) was used in the isolation of total RNA from LNCaP cells, following manufacturer’s instructions. Total RNA (10 µg) was electrophoresed through a 1% agarose-formaldehyde gel, then transferred to a nylon membrane by capillary blotting and cross-linked to the membrane by UV irradiation. The transferred membrane was then hybridized at 42 C overnight with cDNA probes labeled by random priming with [³²P]deoxy-CTP in a solution containing 5x SSPE (sodium chloride sodium phosphate EDTA), 2x Denhardt’s solution, 0.1% SDS, 100 µg/ml denatured salmon sperm DNA, and 50% formamide. After hybridization, the membrane was washed at room temperature with 1x saline sodium citrate (SSC) and 0.1% SDS for 20 min, followed by three 20-min washes at 65 C with 0.2x SSC and 0.1% SDS. When heterologous probes were used, the membrane was washed at low stringency with 2x SSC and 0.1% SDS for 20 min, twice, at room temperature, followed by one 20-min wash at 52 C with 0.5x SSC and 0.25% SDS. After washing, the membranes were exposed to x-ray film with an intensifying screen at -80 C. The inserts of positive clones from the subtractive hybridization were used as templates for probe synthesis. For genes identified by microarray, we obtained cDNA clones from ATCC (Manassas, VA) or other investigators. We have purchased the following IMAGE clones: 3493618, 833335, 4071435, and 3946771 [for mouse GADD45 (growth arrest and DNA-damage-inducible, ), mouse geminin, human DAD1 (defender against cell death 1), and human GADD45, respectively]. The full-length cDNA of human tissue inhibitor of metalloprotease 3 (TIMP3) was a gift from Dr. Nancy Colburn, and the full-length cDNA of human 51-kDa FK506-binding protein (FKBP51) was a gift from Dr. Gail Baughman.

Cell culture
LNCaP cell line was purchased from ATCC and routinely maintained in RPMI 1640 with 10% FBS, 1% glutamine, and 1% penicillin-streptomycin at 37 C in a humidified atmosphere containing 5% CO₂. LNCaP cells were cultured in phenol red-free RPMI 1640 with 10% charcoal-stripped FBS for 2 d before they were treated with various concentrations of synthetic androgen analog mibolerone (Mib; NEN Life Science Products).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Identification of genes up-regulated by androgens, in the rat ventral prostate, by a modified cDNA subtraction
We have previously identified about two dozen genes up-regulated by androgens in the rat ventral prostate using a PCR-based subtractive hybridization (2). It was predicted that some genes were not identified in this screening (2). To identify more androgen-up-regulated genes, a modified subtraction was performed. In the same manner previously described (2), the driver library was constructed from the mRNA of 7-d castrated rat ventral prostate, and the tracer library was constructed from the mRNA of 7-d castrated rat ventral prostate treated with androgens for 48 h. There should be little or no change in cell number and composition in the prostate within 48 h of androgen replacement. The only modification was to add the previously identified androgen-up-regulated genes (2) into the driver library to suppress their enrichment and prevent them from competing with the enrichment of other unidentified genes (see Refs. 2 and 21 for experimental details). Southern blot analysis confirmed the elimination of those already-isolated genes and enrichment of other genes during three rounds of subtractive hybridizations (data not shown).

Screening of the enriched library resulted in the identification of 22 distinct cDNA fragments. Each independent cDNA fragment was sequenced. Only one gene (prostate-binding protein C2A) from the already identified genes that were added to the driver library was still enriched and isolated, further indicating the success of this subtraction. Our analysis revealed 21 new androgen-up-regulated genes, of which 14 genes were known and the other 7 genes were novel. The identities of the isolated genes were listed in Table 1. Northern blot analysis was used to confirm their regulation by androgens. Because all three lobes of the rat prostate are responsive to androgen manipulation, the potential regulation of these genes by androgens in the dorsal and lateral lobes of the rat prostate was also studied. The genes regulated in all three lobes might play more essential roles in androgen-regulated cell growth, differentiation, and apoptosis. Interestingly, most of the known genes identified were regulated in all three lobes of the rat prostate, whereas most of the novel genes were regulated in the ventral prostate only (Fig. 1). Four of these known genes, rat 22-kDa prostatic glycoprotein (22), Cyclin D1 (23), FKBP51 (5), and SCAP [SREBP (sterol regulatory element-binding protein) cleavage-activating protein] (24), have been previously shown to be up-regulated by androgens. The other 10 genes were not previously known to respond to androgens.

View larger version (88K): [in this window] [in a new window]   Figure 1. Northern blot analysis of the androgen-regulated genes isolated from the modified subtraction. Ventral, dorsal, and lateral prostates were isolated from 7-d castrated adult rats (-) and 7-d castrated adult rats with androgen replacement for 48 h (+). A, Known genes. 22-kDa, 22-kDa prostatic glycoprotein; AP, alkaline phosphatase; SAS, sialic acid synthase; Aqp5, aquaporin 5; SCD2, stearoyl-CoA desaturase 2; SCGF, stem cell growth factor; COL1A2, procollagen type 1 2; VL30, VL30 element. B, Novel genes (A-1 through A-7). ß-Actin was used as a control for sample loading and transfer.

An Incyte rat toxicology array was used in this study. There were 8951 sequence-verified elements on this array, including 5341 known genes. Two samples were used for comparison of gene expression, one of which was RNA from the castrated rat ventral prostate, and the other was from the castrated rat ventral prostate treated with androgens for 48 h. Two fluorescent cDNAs, one tagged with Cy3 and another with Cy5, were hybridized simultaneously to one cDNA microarray chip, and the fluorescence signals generated with each dye were determined by confocal microscopy. The differential expression of each element on the array was then calculated from the relative intensity of the Cy3-vs.-Cy5 fluorescent signal. The plot of the differential expression of the 8951 genes is shown in Fig. 2. Most genes were not altered by androgens. Approximately 1.8% (i.e. 162) of genes were up-regulated by androgens 2.3-fold or more, with 35 genes activated at least 3-fold. In the same analysis, 1.6% (i.e. 143) of genes were down-regulated by androgens 2.3-fold or more, with 17 genes repressed at least 3-fold.

View larger version (53K): [in this window] [in a new window]   Figure 2. cDNA microarray analysis of gene regulation by androgens in the rat ventral prostate. Each dot corresponds to the Cy3 (x-axis) and Cy5 (y-axis) fluorescent intensity of one single element on the microarray. Two-fold, 5-fold, and 10-fold changes in expression are indicated as parallel lines. Cy3 and Cy5 dyes were tagged to cDNA from 7-d castrated adult rat ventral prostate and 7-d castrated adult rat ventral prostate after 48-h androgen replacement, respectively.

View larger version (85K): [in this window] [in a new window]   Figure 3. Northern blot analysis of androgen-regulated genes isolated from the microarray study. Ventral, dorsal, and lateral prostates were isolated from 7-d castrated adult rats (-) and 7-d castrated adult rats with androgen replacement for 48 h (+). The expression of GADD45, DAD1, geminin, and TIMP3 was determined. ß-Actin was used as a control for sample loading and transfer.

View larger version (71K): [in this window] [in a new window]   Figure 4. Time course of androgen induction of androgen-responsive genes in the rat ventral prostate. C7: 7-d Castrated adult rat; 14h, 2d, 7d, 14d: 7-d castrated adult rat treated with testosterone for 14 h, 2 d, 7 d, or 14 d, respectively; N, normal rat; N2d, normal rat treated with testosterone for 2 d. The expression of DAD1, GADD45, and TIMP3 was determined by Northern blot analysis. The amount and quality of total RNA loaded in the gels were examined by staining the transferred membrane with methylene blue.

Classification of androgen-responsive genes
We combined all the known androgen-up-regulated genes isolated by our previous subtractions (2) and the present modified subtraction. These genes can be sorted into different categories according to their functions (see Table 4). These categories are metabolism, chaperone and trafficking proteins, secreted proteins, cell cycle, structural and extracellular matrix proteins, and novel proteins. Therefore, these important cellular pathways are targets of androgens during prostate regrowth (see Table 4).

We were unable to classify genes down-regulated by androgens (Table 3). Proteins encoded by down-regulated genes seem to perform diverse functions. Two genes, TRPM-2 (32) and IGF binding protein-3 (27), have been shown to be down-regulated by androgens previously.

View larger version (47K): [in this window] [in a new window]   Figure 5. Northern blot analysis of androgen-regulated gene expression in LNCaP cells. LNCaP cells were cultured in phenol red-free RPMI 1640 with 10% charcoal-stripped FBS for 2 d before treatment with various concentrations of Mib (0.01, 0.1, 1, 10, 100 nM) for 48 h. The expression of three genes (FKBP51, DAD1, and GADD45) was determined. ß-Actin was used as a control for sample loading and transfer.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The microarray analysis identified many more androgen-regulated genes than our two subtraction studies combined. Moreover, half of the known genes isolated from subtractions were also identified in the microarray analysis. On the other hand, one advantage offered by subtractions is the identification of novel genes not present on microarray (Table 1). We found significant overlap between the genes identified by these two different approaches, which indicates the success and complementation of both methods in our studies.

The mechanism of androgen action should be conserved evolutionarily. Some of the genes isolated from the rat ventral prostate are also regulated by androgen in LNCaP cells, for example: FKBP51, DAD1, and GADD45 (Fig. 5). Therefore, the androgen-responsive genes isolated from the rat prostate can be readily studied in human prostate cancer models. The rat ventral prostate is an excellent in vivo model for isolating androgen-responsive genes.

Our results show that androgens activate and suppress a wide range of genes in the rat prostate in vivo, demonstrating the complexity of the androgen-responsive gene-expression program. This is not surprising, considering the multiple roles played by androgens in the prostate. A major functional category activated by androgens is genes involved in metabolism. A great number of genes from this category are lipogenic genes associated with the cholesterol and lipid synthesis pathway. Our result supports previous findings that androgens coordinately regulate lipogenic gene expression (24, 30). The lipogenic genes isolated from subtractions are farnesyl diphosphate synthase, stearoyl-CoA desaturase 2, SCAP, and LDL receptor. The lipogenic genes isolated from the microarray analysis are farnesyl diphosphate synthase, cytosolic 3-hydroxy 3-methylglutaryl CoA synthase, squalene epoxidase, lanosterol 14--demethylase, PI esterase, acyl-CoA synthase, liver carboxylesterase, and 2,3-oxidosqualene:lanosterol cyclase. SCAP has been shown to play a pivotal role in the lipogenic effects of androgens in LNCaP cells (24). Here, we showed that SCAP was up-regulated by androgens in the rat prostate in vivo. SCAP, together with other lipogenic genes, mediates the lipogenic effect of androgens in rat prostate in vivo.

Another major category of genes is involved in protein chaperoning and trafficking pathways (Tables 2 and 4). These genes might be required for the production of androgen-induced protein secretions in the prostate (Tables 2 and 4). Four genes in this category (calreticulin, CaBP1, endoplasmin, and calumenin) encode calcium-binding proteins. Calcium influx has been implicated in castration-induced apoptosis in the prostate (33). These androgen-responsive calcium-binding proteins might be involved in intracellular calcium homeostasis. To support this hypothesis, we showed previously that androgen-stimulated calreticulin expression protected LNCaP cells from cytotoxic intracellular Ca²⁺ overload induced by Ca²⁺ ionophore A23187 (34). An additional four genes in this category (FKBP51, FKBP13, cyclophilin, and cyclophilin B) belong to the same peptidylprolyl isomerase family. One of these genes, FKBP51, has been shown to be up-regulated during androgen-independent prostate cancer progression (5). Another interesting gene in this category is ORP150, which suppresses hypoxia-induced cell death (35). Its up-regulation by androgens might be relevant to prostate cell survival under hypoxic condition.

One major physiological function of the prostate is to produce secreted proteins. Five secreted proteins up-regulated by androgens were identified in the microarray study. These genes are glandular kallikrein, IGF-I, enkephalinase (neutral endopeptidase), alkaline phosphatase, and bone morphogenetic protein-6 (BMP-6). All of them, except for BMP-6, were reported previously to respond to androgens. Interestingly, except neutral endopeptidase, all of these genes have also been shown to be up-regulated during prostate cancer progression (36, 37, 38, 39). On the other hand, neutral endopeptidase seems to be down-regulated in prostate cancer (40, 41), and it is inhibitory to prostate cancer cells (42). Both kallikrein and alkaline phosphatase have been proposed as potential prostate cancer markers (43, 44). BMP-6 was shown to be strongly expressed in prostate cancer bone metastasis (45). Responsiveness of BMP-6 expression to androgen manipulation suggests that the high BMP-6 expression in prostate cancer bone metastasis may be androgen-dependent. It will be interesting to investigate whether BMP-6 is involved in the metastasis of prostate cancer cells to the bone.

Genes involved in cell cycle progression and apoptosis were also up-regulated by androgens in the prostate. Genes involved in both positive and negative cell cycle progression were identified. Cyclin D1, PCNA, and cdc2 are required for cell cycle progression, whereas geminin and GADD45 are inhibitory to cell cycle progression. Androgen regulation of both stimulatory and inhibitory cell cycle regulatory genes argues that androgens control the balance between cell cycle progression and cell cycle inhibition, thereby maintaining the homeostasis of the prostate. The cell cycle inhibitory genes activated by androgens might also be involved in androgen-mediated prostate cell differentiation. DAD1 is an antiapoptotic gene, and its disruption induced massive apoptosis in both Drosophila and mice (46, 47). DAD1 might be involved in androgen-mediated prostate cell survival. Abnormal expression of the genes associated with cell cycle progression or apoptosis might be involved in prostate cancer progression.

No obvious pattern exists for genes down-regulated by androgens from the microarray analysis. One possible explanation is that androgens may not directly target down-regulated genes. However, androgen-down-regulated genes could still be important in androgen action. One interesting gene confirmed to be down-regulated by androgens is TIMP3. TIMP3 possesses antitumor activity, by virtue of metastasis inhibition and induction of apoptosis (48). It is possible that TIMP3 may play an important role in prostate cancer progression.

In conclusion, a significant number of androgen-responsive genes were identified in our study, providing a strong foundation for elucidating the molecular mechanism of androgen action in the prostate. Some novel targets of androgens, especially genes associated with cell cycle progression, apoptosis, and differentiation, could be involved in prostate cancer development and progression. Further research is required to determine the roles of those genes in prostate cancer.

	Acknowledgments

 
We thank Nancy Colburn for human TIMP3 cDNA, Gail Baughman for human FKBP51 cDNA, Xiaoyan Cai and Jomol Cyriac for technical assistance, and members of Wang lab for critical reading.

	Footnotes

 
This work was supported, in part, by NIH Grant R01-DK-51193 and NIH P50-CA-90386 Prostate Cancer SPORE (Special Program of Research Excellence).

Abbreviations: BMP-6, Bone morphogenetic protein-6; CoA, coenzyme A; DAD1, defender against cell death 1; EST, expressed sequence tag; FPPS, farnesyl pyrophosphate synthase; FKBP51, 51-kDa FK506-binding protein; GADD45, growth arrest and DNA-damage-inducible, ; hKLK, human prostate-specific kallikrein; LDL, low-density lipoprotein; LNCaP, lymph node carcinoma of the prostate; Mib, mibolerone; ORP150, oxygen-regulated protein 150 kDa; PCNA, proliferating cell nuclear antigen; SCAP, SREBP cleavage activating protein; SDS, sodium dodecyl sulfate; SREBP, sterol regulatory element-binding protein; SSC, saline sodium citrate; TIMP3, tissue inhibitor of metalloprotease 3.

Received July 15, 2002.

Accepted for publication December 18, 2002.

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