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Endocrinology Vol. 142, No. 5 2138-2146
Copyright © 2001 by The Endocrine Society

ARTICLES

Rodent PSP94 Gene Expression Is More Specific to the Dorsolateral Prostate and Less Sensitive to Androgen Ablation than Probasin1

Yushi Imasato2, Toru Onita2, Madeleine Moussa, Hideki Sakai, Franky L. Chan, Jim Koropatnick, Joseph L. Chin and Jim W. Xuan

Department of Surgery (Y.I., T.O., J.L.C., J.W.X.), Microbiology and Immunology (J. K.) and Pathology (M.M., J.K.), University of Western Ontario, London, Ontario, Canada N6A 4G5; Department of Anatomy, Hong Kong Chinese University (F.L.C.), Hong Kong, China; and Department of Urology, Nagasaki University School of Medicine (Y.I., T.O., H.S.), Nagasaki 852-8501, Japan

Address all correspondence and requests for reprints to: Jim W. Xuan, Urology Research Laboratory, London Health Sciences Center, 375 South Street, London, Ontario, Canada N6A 4G5. E-mail: jim.xuan{at}lhsc.on.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To date, the rodent ventral prostate (VP) has been the focus of many studies on androgen action, less attention has been directed to the lateral prostate (LP) and the dorsal prostate (DP). The rodent VP has no clear homologous counterpart in the human prostate. The rodent LP and DP is the only prostate lobe comparable to the peripheral zone of the human prostate, where hormone-induced prostate cancer mainly occurs. To explore its utility for prostate targeting, we have studied the gene expression of PSP94 with rat probasin (rPB), a gene commonly used for prostate targeting in prostate cancer research and a gene typically responsive to androgen regulation. Firstly, we demonstrated PSP94 gene transcription being more specific to the LP and DP lobes than rPB, where rPB RNA was detected in the LP and DP and other lobes at different levels. Secondly, we found that PSP94 gene transcription decreased relatively slowly in response to androgen deprivation but recovered rapidly in response to testosterone replacement after complete ablation of PSP94 transcription. In the VP, gene transcripts of rPB were specifically responsive to androgen deprivation; however, they responded relatively slowly in the LP and DP. RNase protection experiments indicated that the slow response was not due to abnormal persistence of PSP94 messenger RNA specifically in the DP and LP lobes in comparison with rPB. Thirdly, Western blot analysis revealed that both PSP94 and rPB expression is specific to the LP and DP at the protein level, exhibiting slow responses to testosterone replacement after castration. We conclude that PSP94 gene expression at the transcriptional level is more specific to the LP and DP than rPB and thus less sensitive to androgen ablation. This may have clinical implications for strategies to target the prostate in cancer therapy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE RODENT MALE sex accessory organ is notable for its complex structure, being composed of the lateral prostate (LP), dorsal prostate (DP), ventral prostate (VP), coagulation gland (CG, or anterior prostate) and seminal vesicles (SV) (1). To date, the ventral prostate has been the focus of many studies on androgen action (2, 3 ; for review, see Ref. 4), because androgen deprivation by castration induces rapid cell death in the ventral prostate via apoptosis. However, less attention has been directed to the LP and DP. The VP has no clear homologous counterpart in the prostate of higher animals, whereas the LP and DP are considered to be similar to the prostate structure seen in higher animals and human, for review see (5, 6, 7).

The Noble rat prostate cancer (CaP) can be induced exclusively in the LP and DP with simultaneous administration of specific doses of androgens (testosterone, T) and estrogens (estradiol, E2) (8). The TRAMP (transgenic adenocarcinoma of mouse prostate) model was constructed by integrating regulatory sequences of a rat probasin gene (rPB) to direct the expression of SV40 T antigen (Tag) in the mouse germline (9, 10). TRAMP represents a major advance in CaP research for targeting heterologous gene expression to the prostate because tumors induced were targeted mostly to the LP and DP. However, higher levels of expression of SV40 Tag (10) and CAT (11) were also observed in the VP in rPB driven transgenic mice. As with other previously developed prostate targeting vectors, TRAMP is androgen responsive with fewer tumors developing if animals are castrated at an early age (10).

PSP94 (prostate secretory protein of 94 amino acids), also known as ß-microseminoprotein (12, 13), is one of the three most abundant secretory proteins (PSP94, PAP: prostatic acid phosphatase and PSA: prostatic-specific antigen) of the prostate gland (14); for review see Ref. 12). Studies of PSP94 in experimental animals, including primates (15, 16), pig (17), rat (18), and mice PSP94 (19) have been undertaken. Research interest on PSP94 has been based on the protein as a potential CaP biomarker and tumor suppressor. PSP94 expression in CaP tissue demonstrated by immunohistochemistry (IHC) and in situ hybridization (ISH) showed a decreased level in tumors (13, 20, 21), whereas serum PSP94 determined by enzyme linked immunosorbent assay (ELISA) was elevated in CaP patients (22). Our studies suggested that PSP94 may be used as an androgen independent CaP marker (23).

The LP and DP specific expression of PSP94 has been described, although no detailed data were given (18). Recently, we identified PSP94 is exclusively expressed in the LP and DP (19, 24, 25). Because a few genes are used for prostate targeting, and thus far no human homologous counterpart of rat probasin has been identified, this study will explore the potential utility of PSP94 in a clinical setting for prostate targeting and gene therapy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Castration and androgen replacement, and anatomy of experimental rat
Castrated Sprague Dawley (300–500 g) rats were purchased commercially (Charles River, Québec, Canada) and were monitored histologically for infection during the course of experiments. Androgen replacement injection was carried out as standard procedure (26). In brief, castrated rats were injected im with testosterone (Taro Inc., Bramalea, Ontario, Canada) dissolved in sesame oil at a dosage of 2 mg/rat·day for 2 weeks, and control group were injected only with sesame oil. The rat prostate gland complexes were dissected into individual lobes according to the description and definition as reported (1).

Total cellular RNA isolation and RT-PCR
Trizol solution (Life Technologies, Inc., Burlington, Ontario, Canada) was used to isolate total RNA according to manufacturer’s instructions. RT-PCR was performed as previously reported (16, 27). First strand complementary DNA (cDNA) was synthesized using oligo dT primer, total RNA, and MMLV reverse transcriptase (Life Technologies, Inc.) according to manufacturer’s protocol.

Quantitative RT-PCR of PSP94 cDNA with internal references
GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as an internal messenger RNA (mRNA) reference. Primer pairs used are: GADPH primer 1 5' CCA CCT TCT TGA TGT CAT CA 3', GADPH primer 2 5' TAT TGG GCG CCT GGT CAC CA 3', which were used in PCR, generated a 752-bp fragment located at 5' end of the mouse GADPH cDNA (GenBank Accession No. M32599). Primers used for rodent PSP94 amplification were used as previously reported (19). Primers used for rat probasin were: Primer 1 5' TAC CTA CAG AGC TCA CAC ACG ATG 3'; Primer 2 5' CTA ATT CTG ATC TTG TTT GGA CAG G 3', which are located at the 3' and 5' ends of the rPB coding region (9). A number of PCR cycles were first tested for logarithmic amplification by comparing intensity of ethidium bromide staining of amplified cDNA fragments separated in 6% PAGE. PCR samples (10 µl) for amplification of GAPDH (752 bp) were taken every 5 cycles up to 20 cycles of amplification of the cDNA. PCR products (10 µl) up to 40 cycles of amplification of PSP94 and rPB were taken after the initial 20 cycles.

Competitive and quantitative RT-PCR of PSP94 cDNA with external references
The two primers used for RT-PCR and cloning of rat PSP94 cDNA can be used to amplify mouse PSP94 as well (19), so that mouse or rat cDNA can be used as an external competitor against each other in the competitive and quantitative RT-PCR test. The two cDNAs amplified competitively will have very similar molecular weight, but can be separated by PAGE after HindIII digestion, which cleaves only mouse PSP94 cDNA at the beginning of exon 2 (19). Figure 4AGo shows the result using mouse PSP94 cDNA as a competitor in PCR amplification of the first strand cDNA of rat PSP94. Two fragments (~390 bp and ~50 bp) resulting from HindIII digestion of mouse PSP94 cDNA PCR products were revealed (lanes 3–5). Except the weak and short fragment from mouse, the intensity of all fragments (see Fig. 4AGo) was scanned by a densitometer (PhosphorImager Model S1, Molecular Dynamics, Inc., Sunnyvale, CA). As shown in the plot (see Fig. 4BGo), mouse long fragments were found to be inversely correlated with that of rat PSP94 PCR bands. Because the amount of mouse cDNA as an external competitor can be precisely determined, a comparable amount of rat cDNA/mRNA to be tested can also be rather precisely determined according to densitometric scanning on the gel. For example, the rat mRNA as determined in Fig. 4BGo is comparable to approximately 0.65 ng of the competitor mouse PSP94 cDNA, which implied that rat mRNA was present at a concentration of approximately 5 x 1010 molecule/mg wet weight LP tissue.



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  		Figure 4. Quantitative RT-PCR analysis of PSP94 gene expression in castrated prostate tissue samples. A, 1.5% agarose gel electrophoresis analysis of competitive RT-PCR products. Mouse cDNA plasmid was used as an internal competitor and the amounts (ng) added to PCR mixture for amplification of rat PSP94 were as indicated. PCR products were digested by HindIII and two HindIII fragments were seen and the intensity of the small fragment (~50 bp) correlated with the large mouse cDNA bands (~400 bp). Control (lane 2) contains the product of a PCR with no cDNA (either rat or mouse) added. DNA molecular weight standards used were from bottom: 154, 201, 220, 298, 344, 396, 506 bp. B, Analysis of gel A by densitometric scanning. Graph compares densitometric (volume) changes of PCR products with different amounts of external competitor mouse PSP94 cDNA (only large fragment scanned) and rat PSP94 in the competitive PCR. C, PSP94 mRNA in castration and testosterone replacement experiments. PSP94 levels (109–10 molecules/mg wet tissue) were determined by the same methods used in (A). Results from mean values of duplicate samples were used to calculate plotted values.

 
Ribonuclease (RNase) protection analysis
DIG (digoxigenin)-labeled PSP94 and rPB RNA probes were generated by in vitro transcription with DIG-labeled UTP, according to the manufacturer’s protocol (Roche Molecular Biochemicals, Laval, Québec, Canada). Briefly, approximately 1 µg of rat PSP94 cDNA (in pBluescript) (19) was linearized and transcribed using T7 RNA polymerase (Promega Corp., Madison, WI) to synthesize the antisense probe. The sense probe was synthesized at a reverse direction using T3 RNA polymerase (Promega Corp.). For preparation of rPB single-strand probes, plasmid DNA was similarly linearized by two enzymes and transcribed using T3 RNA polymerase or T7 RNA polymerase, as required. The labeled RNA probes were first analyzed by formaldehyde agarose gel electrophoresis and then quantified by dot-blot analysis.

RNase protection assay. Approximately 7 µg of purified rat prostate RNA were mixed with 2 ng of DIG-labeled PSP94 or rPB RNA probe. The mixtures were placed in hybridization buffer (80% formamide, 100 mM sodium citrate, 300 mM sodium acetate, 1 mM EDTA, pH 6.4), and incubated at 95 C for 3 min, then at 43 C for overnight (16~18 h). The RNA hybrids were digested with RNase (at a final concentration of 0.05 µg/µl RNase A, 12.5 µg/µl RNase T1) at 37 C for 30 min. To eliminate nonspecific digestion to double-stranded, hybridized RNA, different dilution times and combinations of RNase A and RNase T1 were tested, and DIG probes digested with RNase was used as a control. RNase digestion was loaded on 5% polyacrylamide 8 M urea gels. The DIG-chemiluminescent reaction was conducted following the manufacturer’s instructions (Roche Molecular Biochemicals). As a negative control, a sense probe was used to hybridize RNA. Lack of RNase protection was taken as evidence of specific hybridization of antisense probes.

In situ hybridization
Freshly dissected rat prostate and male accessory gland samples were fixed in 4% paraformaldehyde in PBS for 48 to 72 h, followed by processing and embedding in paraffin. Nonradioactive in situ hybridization was performed according to a previously reported protocol (25) with some modifications. In brief, deparaffinized and rehydrated sections were treated with 0.2 N HCl followed by 0.3% Triton X-100 in PBS, 20 µg/ml proteinase K (Life Technologies, Inc.) at 37 C for 30 min. Sections were prehybridized in a buffer containing 4 x SSC, 2 x Denhardt’s solution, 10% dextran sulfate, 50% deionized formamide, 0.2 mg/ml salmon sperm DNA, and 0.25 mg/ml yeast transfer RNA at 37 C for 1 h, and hybridized with DIG-labeled PSP94 or rPB RNA probes at a concentration of 0.8 µg/ml at 55 C overnight (16~18 h). After hybridization, sections were treated by RNase digestion and subsequent posthybridization washing. The DIG-color reactions were conducted following the manufacturer’s instructions (Roche Molecular Biochemicals). As a negative control, a sense probe was used.

Western blotting analysis
Fresh dissected prostate lobes (~ 0.2 g) were homogenized in 1 ml ice-cold 0.01 M PBS and then centrifuged at 10,000 x g. The clear cell extract was designated as the lysate and was analyzed by 15% SDS-PAGE electrophoresis and western blotting. The chemiluminescent method was used to detect specific protein (ECL Western Blotting kit, Amersham Pharmacia Biotech, Oakville, Ontario, Canada) according to the protocol provided by the manufacturer. The first antibody used was a polyclonal antibody to recombinant rat GST-PSP94 (24) and R23, a polyclonal Ab to rat probasin (a gift from Dr. R. Matusik). Horseradish peroxidase (HRP) conjugated swine antirabbit IgG (Dimension Laboratories, Mississauga, Ontario, Canada) was used as a secondary antibody.

Recombinant DNA techniques and DNA sequence analysis
RT-PCR product of rPB mRNA was confirmed by DNA sequencing directly on PCR DNA. Automatic DNA sequencing was performed on double-strand DNA in a DNA sequencer (ABI Model 377, Perkin-Elmer Corp., Norwalk, CT). Probasin cDNA (CM-40, from Dr. R. Matusik) was subcloned by insertion of a PstI fragment of original rPB clone into pBluescript vector.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PSP94 gene expression is less responsive to androgen ablation in castrated rats
Our clinical studies (21, 23) indicate that PSP94 expression in human tumor cells persists while the patient is under antiandrogen hormone therapy. To confirm this observation in a rodent model, RT-PCR analysis of prostate samples from castrated rats (n = 4) was first performed. Figure 1Go shows the results of RT-PCR detection of PSP94 mRNA (A), using a house-keeping gene GAPDH as an internal reference (B). As shown by 6% PAGE analysis of RT-PCR samples after 40 cycles of amplification, PSP94 transcripts are present only in DP and LP among five male sex accessory gland samples from castrated rats. PSP94 transcripts persisted for 3 weeks after castration, indicating a slow response to androgen ablation. Figure 1DGo shows the relative density of PSP94 normalized against the density of the control GAPDH. Because rat probasin (rPB) has been reported to be an androgen responsive gene (7, 9, 26), PCR with rPB specific primers was performed on the same first strand cDNA (RNA) samples as shown in Fig. 1Go, A and B. Figure 1CGo shows that rPB mRNA existed in all five male accessory gland samples in normal rats, in contrast to PSP94, an LP- and DP-specific gene (19, 25). Furthermore, rPB gene transcription was significantly suppressed only in the VP one week after castration (shown by result of densitometric scanning in Fig. 1EGo). In the coagulation gland, it was suppressed by weeks 1.5–2. In contrast, no significant suppression of rPB transcripts in the LP and DP was observed by RT-PCR in the 2 weeks after castration.



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  		Figure 1. A, RT-PCR analysis of total RNA extracts of tissue samples of prostate and male accessory gland dissected from normal rat and from rats 1–4 weeks (W1–4) after castration. PCR amplification was first tested for logarithmic amplification as described in Materials and Methods. PCR samples of PSP94 were taken after 40 cycles of amplification of first strand cDNA synthesized from 1 µg total RNA separately for each lobe (rat n = 4). B, RT-PCR of GAPDH as an internal reference mRNA. The same amount of first strand cDNA (as shown in A) was used for PCR amplification of GAPDH, and samples were analyzed after 20 cycles of PCR amplification. C, RT-PCR of rat probasin mRNA. The same amount of first strand cDNA (as shown in A) was used for PCR amplification of rPB, and samples were analyzed after 40 cycles of PCR amplification. The molecular weight standards (STD) used are (from bottom); 201, 220, 298, 344, 396, 506, 517, 1018 bp. D and E, Densitometric scanning of RT-PCR products in gels A (PSP94), B (GAPDH), and C (rPB). Graphs show relative density (volume) of PSP94 (D) and rPB (E) signals in the prostate lobes and male sex accessory glands normalized against GAPDH density. VP was highlighted for rPB (E) showing the quick response after W1.

 
In situ hybridization confirm the differential suppression of PSP94 and rPB by androgen
To confirm the results of the RT-PCR analysis of castrated prostate samples, in situ hybridization experiments were performed (rats n > 4). As shown in Fig. 2Go, PSP94 showed strong signals (A–E) only in the LP (A) and DP (B), and no positive signals in the VP, CG, and SV (Fig. 2Go, A–E). These positive signals were localized to the cytoplasm of the glandular epithelial cells, but were not seen in the stroma. Although rPB (Fig. 2Go, F–J) showed varying degrees of staining in all five male sex accessory gland samples at different levels, the VP was weaker than in the LP and DP but was still positive. Three weeks After castration, all glands were atrophied due to androgen deprivation. Positive signals for PSP94 mRNA from samples were seen only in the LP and DP, but these signals were weak compared with signals from normal rat tissue (Fig. 2Go, K–O). Also, positive signals for rPB were weaker in all lobes compared with those from normal rat tissue. In particular, rPB signals in the VP in castrated rats were almost completely absent (Fig. 2Go, P–T).



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  		Figure 2. Nonradioactive in situ hybridization (ISH) of PSP94 or rPB mRNA in rat prostate and male sex accessory gland (rats n > 4). A–E, ISH of PSP94 mRNA in normal rat prostate (A, LP; B, DP; C, VP; D, CG; E, SV). Positive signals were seen only in the cytoplasm of glandular epithelial cells in LP and DP. x100. F–J, ISH of rPB mRNA in normal rat prostate and male sex accessory gland (F, LP; G, DP; H, VP; I, CG; J, SV). Positive signals were seen in all lobes. x100. K–O, ISH of PSP94 mRNA in castrated (3 weeks after castration) rat prostate (K, LP; L, DP; M, VP; N, CG; O, SV). Positive signals were weaker than those of the normal rat prostate. x100. P–T, ISH of rPB mRNA in castrated (3 weeks) rat prostate. Positive signals were weaker than those of normal rat prostate (especially in VP). x100.

 
RNase protection experiments indicated that the slow response specifically in the DP and LP lobes to androgen ablation was not due to abnormal persistence of PSP94 mRNA as compared with rPB
To determine whether the relatively muted responsiveness of PSP94 to androgen ablation is due to abnormal lobe-specific stability of PSP94 mRNA in the LP and DP, a RNase protection experiment was performed to analyze RNA samples from normal prostate and male sex accessory gland, and rPB was used as a control. Hybridization reaction took place between RNA samples and DIG labeled single-strand probes, including sense and antisense PSP94 and rPB probes. As shown in Fig. 3Go, RNase (A and T1) treatment of hybrids showed positive signals only in the LP and DP for PSP94 mRNA, whereas positive signals were found in the LP, DP, and VP for rPB mRNA. This analysis of PSP94 and rPB mRNA confirmed results previously obtained by RT-PCR and ISH. Next, the stability of PSP94 and rPB mRNA was compared by RNase protection experiments using prostate lobe samples. Only normal LP was tested, because as we have reported (24) that the majority (85%) of rat PSP94 protein are presented in LP. Freshly dissected rat prostate LP samples were incubated at 37 C for 0.5 h, 1, and 2 h. Total RNA samples were extracted immediately and subjected to RNase protection analysis. As indicated in Fig. 3BGo, RNase (A and T1) protected hybrids of PSP94 mRNA (~500 nucleotides) were detectable after 30 min of incubation of LP tissue, which diminished and then disappeared after 1 or 2 h incubation at 37 C. Similarly, degradation of rPB mRNA (~650 nucleotides) was evident within 1–2 h incubation. A control experiment was performed using the same amount (1 µg) of total cellular RNA used for RNase protection reactions, and by RT-PCR amplification of transcripts from the house-keeping gene GAPDH (Fig. 3CGo), demonstrating that the amounts of RNA used for RNase protection in each test were approximately comparable. Figure 3DGo shows the result of normalization of both PSP94 and rPB densities in the gel against control of GAPDH. Figure 3EGo demonstrates the similarity of both PSP94 and rPB mRNAs in terms of decreasing percentages of relative densities in the course of in vitro incubation of LP samples.



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  		Figure 3. Ribonuclease (RNase) protection experiment of hybrids between DIG labeled single-strand probes and cDNAs of PSP94 and rPB. A, RNase protection tests of total RNA (1 µg) extracted from normal prostate lobes (rats, n = 4). Controls of either sense strand or antisense probes were not hybridized with RNA. A control (antisense probe + RNase) showed a lack of hybridization with RNA. A control (sense probe + RNase) was first hybridized with total RNA (1 µg) from LP then treated with the same amount of RNase as test groups. B, Stability test of mRNA of PSP94 and rPB in LP. To assess the degradation of mRNAs in LP, fresh LP tissues (0 h) were incubated for the time periods indicated. One microgram total RNA for each test was used for RNase protection. C, Control experiment: RT-PCR amplification of 1 µg total RNA for each lobe and each test was performed as in Fig. 1Go for semiquantitative measurement the content of GAPDH mRNA. The molecular weight standards shown (from bottom): 344, 396, 506, 517 bp. D, Densitometric scanning of film B and gel D. Graphs show relative density (GAPDH -1, ratio of volumes of sample density against GAPDH) of PSP94 and rPB signals in lateral prostate. E, Graph shows decreases of density (shown percentages of the normal LP signals) during incubation hours of LP samples.

 
Quantitative RT-PCR analysis of PSP94 gene expression in the LP and DP after androgen replacement to castrated rats
A competitive RT-PCR for quantitation of PSP94 mRNA molecules in samples has been developed (Fig. 4Go, A and B, see Materials and Methods) and was used to determine responsiveness of PSP94 after androgen replacement for castrated rats. Figure 4CGo is a graph showing a quantitative RT-PCR determination of PSP94 molecules presented in two of the castrated rats tested in Figs. 2Go and 3Go, and in castrated rats after androgen replacement injection. Testosterone replacement injection was started after 2 weeks, when PSP94 transcripts had almost completely dissipated. Slower decrease of PSP94 levels in the LP and DP was observed as compared with rPB, which was reported to be significantly decreased after 3 days of castration (26). Two weeks after castration, significant depression of PSP94 transcripts was observed (from 5–6 x 1010 mRNA/mg wet tissue to 2–5 x 109 mRNA/mg wet tissue). Testosterone injection into castrated rats at this stage resulted in a quick response to androgen up-regulation. As shown in Fig. 4Go, three consecutive daily supplemental high dosage testosterone injections (2 mg/day·rat) resulted in complete recovery of PSP94 transcription to a normal level. Exceptionally high levels of PSP94 transcripts observed after 3 days of injection may be due to excessive androgen administration.

Results of Western blotting experiments indicate that both PSP94 and rPB are the LP and DP-specific and show slow response to androgen ablation
Because gene transcripts may not provide a precise reflection of protein levels in tissues, both PSP94 and rPB protein were tested in prostate samples from castrated and castration/androgen replacement rats. Firstly, a 15% SDS-PAGE was loaded with about 10 µg of total cellular protein for all five male sex accessory gland samples from rats after 1 to 4 weeks of castration. According to Coomassie blue staining (data not shown), Western blots were prepared using re-adjusted amounts of protein lysate samples to test approximately equal amounts of protein for each sample. Association with PSP94 antibody showed that PSP94 protein (at an apparent molecular mass of ~16 kDa) in the LP and DP decreased one week after castration and was almost completely eliminated by two weeks (Fig. 5AGo). Subsequently, the tissue protein level of PSP94 was measured by the same Western blotting procedures as in Fig. 5AGo on rats subjected to testosterone replacement injection (+T) 2 weeks after castration (-T). As shown in Fig. 5BGo, PSP94 protein levels recovered rapidly within 1 day (15 days after castration) of testosterone injection. After 3–7 days’ treatment with testosterone, PSP94 in the LP returned to precastration levels.



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  		Figure 5. Western blot analysis of tissue cell lysates from normal and castrated rats (rat, n = 4) associated with polyclonal antibodies against both rat PSP94 and rPB. First, fresh tissue lysate samples (10 µg protein/lane) from W1 toW4 after castration were loaded onto a 15% SDS-PAGE and stained with Coomassie Blue (data not shown). For preparation of Western blots, approximately an equal and comparable amount of cellular protein was re-adjusted and loaded. Standard prestained protein markers (low range, from Life Technologies, Inc.) used to measure molecular weight of PSP94 and rPB were loaded and coblotted. A, Result of Western blot analysis of PSP94 in normal and castrated rats. Blots (A, B) were reacted with PSP94 antiserum (1:10,000 dilution). B, Western blotting analysis of PSP94 protein level in DP and LP samples from castrated rats. Castrated rats were injected with testosterone replacement (+T) at day1, 3, 7, and 14 following two weeks after castration. Control (-T) is a castrated rat group with out testosterone injection. C, Western blot analysis of rPB protein level in tissue protein samples (10 µg/lane) from prostate complexes of two normal rats. D, Western blot analysis of rPB protein level in castrated rats. E, Western blot analysis of rPB protein level. Castrated rats were injected with testosterone replacement at Day 1, 3, 7, and 14 following 2 weeks after castration. Only injection samples were shown and control (-T, not shown) was similar to that of B. Polyclonal antibody against rPB (R23) was diluted 1:5000 (C–E). Duplicated bands of proteins tested were seen in western blots (D–F), which may indicate the possible isoforms of native PSP94 as reported previously (17 18 ), or isoforms formed due to disulfide bond reassociation during preparation of PAGE samples boiling with ß-mercaptoethanol.

 
A parallel study was performed using a specific anti-rPB antibody in the same prostate samples used for PSP94 measurement. Firstly, lysates from normal rats were tested (Fig. 5CGo). With the exception of the CG, rPB protein was specific to the DP and LP at an apparent molecular mass of approximately 20 kDa, even in the presence of rPB mRNA in the VP (see Figs. 1Go and 3Go). Western blot analysis was using the same total tissue lysate samples tested for PSP94 from castrated rats (see Fig. 5BGo). Similar to PSP94, rPB protein decreased significantly after 1 week of castration and was almost completely eliminated by 2 weeks (Fig. 5DGo). With testosterone replacement injection 2 weeks following castration, the depression of rPB in the LP and DP was partially reversed after 3 days of testosterone administration (Fig. 5EGo). Complete recovery of rPB protein occurred after 7 days of androgen replacement (Fig. 5EGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In terms of morphology and anatomy, human and rodent prostate are very different. The rodent ventral prostate represents the most significant difference. The LP and DP are considered to be closely related to regions of human prostate in which cancer develops (for review see Refs. 5, 7, 28). To date, less research has been directed to these two similar lobes, also called the dorsolateral prostate (DLP), in contrast to the VP. The rodent VP and DLP are known to exhibit marked cytological, biochemical and functional differences including differential susceptibility to carcinogens. Rat DLP is embryologically similar to the peripheral zone of the human prostate, whereas rat CG is similar to the human central zone (for review see Refs. 5, 7, 28). Consequently, the high zinc (6) and citrate (28) content of rat and human prostates, and the mechanism of PRL regulation of DLP function (6), are likely associated with a common lineage of epithelial cells (for review see Refs. 5, 6, 28). Because the prostate is an androgen-dependent gland and the incidence of carcinoma rises markedly in aged men, it has been suggested that protracted stimulation of this tissue by sex hormones likely plays an important role in the carcinogenic process. Simultaneous exposure of intact rats to certain dosages of testosterone (T) and 17ß-estradiol (E2) consistently induces intraductal dysplasia exclusively in the DLP (8), which closely resembles the prostatic intraepithelial neoplasia (PIN) in the human gland.

In this report, we have demonstrated that PSP94 gene expression through a comparative study, is more specific to the LP and DP lobes than rPB, a gene commonly used for prostate targeting in prostate cancer research and a gene typically responsive to androgen regulation (7, 9, 26). This conclusion is demonstrated by a very sensitive RT-PCR technique, and by ISH and RNase protection experiments in which rPB RNA transcripts were found to be present in the VP, SV, and CG at different levels. Secondly, we have found that PSP94 gene transcription responds relatively slowly to androgen deprivation, as demonstrated by RT-PCR, quantitative RT-PCR (Figs. 1Go and 4Go) and in situ hybridization (Fig. 2Go). RNase protection experiments also ruled out the possibility that the slow response is due to exceptional persistence of PSP94 mRNA in the LP and DP. In contrast to PSP94, we found that rPB gene transcription is specifically responsive to androgen deprivation in the VP. In the LP and DP it is relatively slowly responsive, the same pattern of response as PSP94 in the LP and DP.

The above observations may be explained by the differential expression of PSP94 and rPB in the DLP, along with differential responses in DLP to androgen ablation. Various studies have shown the rapid response of the VP to androgen action (2, 29), whereas regressive changes in the DLP, and especially the lateral prostate (LP) have been shown to be much slower. Even at the morphological level, (i.e. by lobe wet weight), regressive changes in the DLP are almost indistinct as compared with striking regressive changes of CG and VP (29). Consequently, RNA transcripts of both PSP94 and rPB in DLP are slowly responsive to androgen ablation. There have been several reports of androgen-independent effects of PRL on the different lobes of the rat prostate, including mitochondrial zinc and citric acid production and regulation (for review, see Ref. (6).

PSP94 transcripts quickly responds to androgen replenishment after its expression has been completely abrogated by castration. This may be explained by the fact that androgen is the terminal differentiation product necessary for maintaining the basic prostate function. All prostate genes are ultimately susceptible to suppression by androgen withdrawal and all will resume normal function if androgen replacement is instituted. Most prostate researchers accept the definition of "androgen independence" as persistence of gene transcription hours or a maximum of a few days after castration (2, 3, 26). Normally all prostate genes respond to androgen replacement after castration.

We have found that rat probasin gene expression, in contrast to PSP94, is not exclusively restricted to the DLP, as there is, although less abundant, transcription in the VP. This result is consistent with the previous report that rPB driven CAT or SV40 Tag expression is targeting not only to the DLP but also other lobes such as the VP (10, 11, 30, 31 ; for review see Ref. 32). This may be the reason that the rat probasin gene responds more rapidly to androgen deprivation.

It is of interest that both rPB and PSP94 proteins detected by antibodies against respectively rat probasin and PSP94 showed both to be DLP specific. Compared with the rapid response of rPB gene transcription to castration (3 days to complete suppression), both rPB and PSP94 protein levels in the DLP showed slow response to androgen. Testosterone replacement had a pronounced effect on rats, which had been castrated two weeks previously, showing strong recovery of both proteins after only 3 days testosterone injection. One may postulate that protein production with both PSP94 and rPB is under the control of a generalized DLP-specific translational factor, which may override the non-DLP-specific gene transcripts/mRNA by rPB in the VP and other male sex accessory glands (the SV and CG). Matusik and colleagues found that the VP is relatively resistant to SV40Tag expression and only hyperplasia, or occasionally low-grade dysplasia, was identified as opposed to cancer in the DLP lobe, even in the presence of levels of transgene expression in this lobe (11, 30, 31) However, there may still be a DLP specific element in the PSP94 gene structure, which controls PSP94 gene expression at the protein level in the DLP, primarily in the LP.

Our goal is to explore the clinical utility of a DLP tissue specific element in the PSP94 gene for use in targeting prostate cells for ablation by gene therapy techniques. Clearly, identification of a rodent DLP-specific element will not be directly useful in humans. However, because the majority of human PSP94 are secreted from the prostate (14, 33) with only trace amounts appearing in other tissues, identification of a mouse genetic element important in DLP-specific PSP94 expression is information essential in finding a similar prostate tissue specific element in the human PSP94 gene. In this regard, the PSP94 gene may be of more clinical utility than that of rPB, for which no human homologue has been identified to date. Because there are several pathways for regulation of DLP gene expression independent of androgen control, DLP-specific expression of PSP94 may be more valuable than rPB in evaluating the effectiveness of androgen deprivation therapy in treatment of CaP.


   Acknowledgments
 
The authors thank Dr. R. Matusik for the kind permission to use rat probasin cDNA clone and polyclonal antibody for this comparative study.


   Footnotes
 
1 This work was supported by grants from the Medical Research Council of Canada (MT-15390), The Kidney Foundation of Canada, The Prostate Cancer Research Foundation of Canada, Internal Research Funds of London Health Sciences Center, an RGC Earmarked Research Grant (CUHK 4131/00M) (to F.L.C.), a Grant-in-Aid (No. 11671562) from the Ministry of Education, Science, Sports and Culture of Japan, and also from Procyon Biopharma Inc. (Montréal, Québec, Canada). Back

2 These two authors contributed equally to this work. Back

Received November 13, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Lee C 1987 Gross dissection of three lobes of the rat prostate. In: Coffey DS, Bruchovsky N, Gardner Jr WA, Resnick M, Karr JP (eds) Current Concepts and Approaches to the Study of Prostate Cancer. Alan R. Liss, Inc., New York, pp 577–582
  2. Wang Z, Tufts R, Haleem R, Cai X 1997 Genes regulated by androgen in the rat ventral prostate. Proc Natl Acad Sci USA 94:12999–13004[Abstract/Free Full Text]
  3. Blok LJ, Kumar MV, Tindall DJ 1995 Isolation of cDNAs that are differentially expressed between androgen-dependent and androgen-independent prostate carcinoma cells using differential display PCR. The Prostate 26:213–224[Medline]
  4. Tenniswood M 1997 Apoptosis, tumour invasion and prostate cancer. Br J Urol [Suppl 2] 79:27–34
  5. Price D 1963 Comparative aspects of development and structure in the prostate. In: Andervont HB (ed) Biology of the Prostate and Related Tissues. U.S. Department of Health, Education, and Welfare, Public Health Serves, National Cancer Institute, Bethesda, MD, 1–27
  6. Costello LC, Franklin RB 1994 Effect of prolactin on the prostate. Prostate 24:162–166[Medline]
  7. Matusik RJ, Kreis C, McNicol P, Sweetland R, Mullin C, Fleming WH, Dodd JG 1986 Regulation of prostatic genes: role of androgens and zinc in gene expression. Biochem Cell Biol 64:601–607[Medline]
  8. Leav I, Ho SM, Ofner P, Merk FB, Kwan PW, Damassa D 1988 Biochemical alterations in sex hormone-induced hyperplasia and dysplasia of the dorsolateral prostates of Noble rats. J Natl Cancer Inst 80:1045–1053[Abstract/Free Full Text]
  9. Rennie PS, Bruchovsky N, Leco KJ, Sheppard PC, McQueen SA, Cheng H, Snoeck R, Hamel A, Bock ME, MacDonald BS, Nickel BE, Chang C, Liao S, Cattini PA, Matusik RJ 1993 Characterization of two cis-acting DNA elements involved in the androgen regulation of the probasin gene. Mol Endocrinol 7:23–36[Abstract/Free Full Text]
  10. Greenberg NM, Demayo F, Finegold MJ, Medina D, Tilley WD, Aspinall JO, Cunha GR, Donjacour AA, Matusik RJ, Rosen JM 1995 Prostate cancer in a transgenic mouse. Proc Natl Acad Sci USA 92:3439–3443[Abstract/Free Full Text]
  11. Yan Y, Sheppard PC, Kasper S, Lin L, Hoare S, Kapoor A, Dodd JG, Duckworth ML, Matusik RJ 1997 Large fragment of the probasin promoter targets high levels of transgene expression to the prostate of transgenic mice. Prostate 32:129–139[CrossRef][Medline]
  12. Hara M, Kimura H 1989 Two prostate-specific antigens, {gamma}-seminoprotein and ß-microseminoprotein. J Lab Clin Med 113:541–548[Medline]
  13. Hyakutake H, Sakai H, Yogi Y, Tsuda R, Minami Y, Yushita Y, Kanetake H, Nakazono I, Saito Y 1993 ß-microseminoprotein immunoreactivity as a new prognostic indicator of prostatic carcinoma. The Prostate 22:347–355[Medline]
  14. Tremblay J, Frenette G, Tremblay RR, Dupont A, Thabet M, Dube JY 1987 Excretion of three major prostatic secretory proteins in the urine of normal men and patients with benign prostatic hypotrophy or prostate cancer. The Prostate 10:235–243[Medline]
  15. Nolet S, St-louis D, Mbikay M, Chretien M 1991 Rapid evolution of prostatic protein PSP94 suggested by sequence divergence between Rhesus monkey and human cDNA. Genomics 9:775–777[CrossRef][Medline]
  16. Xuan JW, Wu D, Guo Y, Garde S, Shum D, Mbikay M, Zhong R, Chin JL 1997 Molecular cloning and gene expression analysis of PSP94 (prostate secretory protein of 94 amino acids) in primates. DNA Cell Biol 16:627–638[Medline]
  17. Fernlund P, Granberg L, Roepstorf P 1994 Amino acid sequence of ß-microseminoprotein from porcine seminal plasma. Arch Biochem Biophy 309:70–76[CrossRef][Medline]
  18. Fernlund P, Granberg L, Larson I 1996 Cloning of ß-microseminoprotein of the rat: a rapidly evolving mucosal surface protein. Arch Biochem Biophy 334:73–82[CrossRef][Medline]
  19. Xuan JW, Kwong J, Chan FL, Ricci M, Imasato Y, Sakai H, Fong GH, Panchal C, Chin JL 1999 cDNA, genomic cloning and gene expression analysis of mouse PSP94 (prostate secretory protein of 94 amino acids). DNA Cell Biol 18:11–26[CrossRef][Medline]
  20. Tsurusaki T, Koji T, Sakai H, Kanetake H, Nakane PK, Saito Y 1998 Cellular expression of ß-microseminoprotein (ß-MSP) mRNA and its protein in untreated prostate cancer. The Prostate 35:109–116[CrossRef][Medline]
  21. Sakai H, Tsurusaki T, Kanda S, Koji T, Xuan JW, Saito Y 1999 Prognostic significance of ß-microseminoprotein mRNA expression in prostate cancer. The Prostate 38:278–284[CrossRef][Medline]
  22. Huang C, Brassil D, Rozzel M, Schellhammer PF, Wright Jr GL 1993 Comparison of prostate secretory protein with prostate specific antigen and prostatic acid phosphatase as a serum biomarker for diagnosis and monitoring patients with prostate carcinoma. The Prostate 23:201–212[Medline]
  23. Imasato Y, Xuan JW, Sakai H, Izawa JI, Saito Y, Chin JL, Moussa M 2000 PSP94 expression after androgen deprivation therapy: a comparative study with prostate specific antigen in benign prostate and prostate cancer. J Urol 164:1819–1824[CrossRef][Medline]
  24. Kwong J, Chan FL, Jiang S, Guo YZ, Imasato Y, Sakai H, Koropatnik J, Chin JL, Xuan JW 1999 Differential expression of PSP94 in rat prostate lobes as demonstrated by an antibody against recombinant GST-PSP94. J Cell Biochem 74:406–417[CrossRef][Medline]
  25. Kwong J, Xuan JW, Choi HL, Chan PSF, Chan FL 2000 PSP94 (or ß-microseminoprotein) is a secretory protein specifically expressed and synthesized in the lateral lobe of the rat prostate. Prostate 42:219–229[CrossRef][Medline]
  26. Dodd JG, Sheppard PC, Matusik RJ 1983 Characterization and cloning of rat dorsal prostate mRNAs. J Biol Chem 258:10731–10737[Abstract/Free Full Text]
  27. Xuan JW, Chin JL, Guo.Y, Chambers AF, Finkelman MA, Clarke MW 1995 Alternative splicing of PSP94 (prostate secretory protein of 94 amino acids) mRNA in prostate tissue. Oncogene 11:1041–1047[Medline]
  28. Costello LC, Franklin RB 1998 Novel role of zinc in the regulation of prostate citrate metabolism and its implications in prostate cancer. Prostate 35:285–296[CrossRef][Medline]
  29. Yamashita A, Hayashi N, Sugimura Y, Cunha GR, Kawamura J 1996 Influence of diethylstilbestrol, Leuprolelin (a luteinizing hormone-releasing hormone analog), Finasteride (a 5 {alpha}-reductase inhibitor), and castration on the lobar subdivisions of the rat prostate. Prostate 29:1–14[Medline]
  30. Greenberg NM, DeMayo FJ, Scheppard PC, Barrios R, Lebovitz R, Finegold M, Angeopoulou R, Dodd JG, Duckworth ML, Rosen JM, Matusik RJ 1994 The rat probasin gene promoter directs hormonally and developmentally regulated expression of a heterologous gene specifically to the prostate in transgenic mice. Mol Endocrinol 8:230–239[Abstract/Free Full Text]
  31. Kasper S, Sheppard PC, Yan Y, Pettigrew N, Borowsky AD, Prins GS, Dodd JG, Duckworth ML, Matusik RJ 1998 Development, progression and androgen-dependence of prostate tumors in probasin-large T antigen transgenic mice: a model for prostate cancer. Lab Invest 78:319–333[Medline]
  32. Green JE, Greenberg NM, Ashendel CL, Barrett JC, Boone C, Getzenberg RH, Henkin J, Matusik R, Janus TJ, Scher HI 1998 Workgroup 3: transgenic and reconstitution models of prostate cancer. Prostate 36:59–63[CrossRef][Medline]
  33. Dube JY, Frenette G, Paquin R, Chapdelaine P, Tremblay J, Tremblay RR, Lazure C, Seidah N, Chretien M 1987 Isolation from human seminal plasma of an abundant 16-kDa protein originating from the prostate, its identification with a 94-residue peptide originally described as ß-inhibin. J Androl 8:182–189[Abstract/Free Full Text]



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