Cellular Expression of Beta-Microseminoprotein(b-MSP) mRNA and Its Protein in Untreated

Prostate Cancer

Toshifumi Tsurusaki,1* Takehiko Koji,2 Hideki Sakai,1 Hiroshi Kanetake,1Paul K. Nakane,2 and Yutaka Saito1

1Department of Urology, Nagasaki University School of Medicine, Nagasaki, Japan2Department of Histology and Cell Biology, Nagasaki University School of Medicine,

Nagasaki, Japan

BACKGROUND. Previous studies have shown that beta-microseminoprotein (b-MSP) maybe used as a diagnostic marker for prostate cancer. However, the level of expression of b-MSPin prostate cancer detected by immunohistochemistry (IHC) has varied from one study toanother.METHODS. We analyzed the expression of both b-MSP mRNA and its protein in a largesample of prostate tumors from 104 patients with untreated prostate cancer, using bothnonradioactive in situ hybridization (ISH) and IHC.RESULTS. Our results showed that 72 and 96 of 104 specimens were negative for b-MSPmRNA (69.2%) and b-MSP (92.3%), respectively. Furthermore, a reduced expression of bothb-MSP mRNA and its protein was detected in all malignant epithelial tissues compared withbenign epithelia. Not all malignant tissue samples negative for b-MSP mRNA were negativefor b-MSP (6.7%), and vice versa (29.8%). Other tissue samples were either negative for both(62.5%) or positive for both (1.0%).CONCLUSIONS. Our results showed a lower level of expression of b-MSP in prostate cancertissue, compared with benign prostate tissue. This phenomenon may be mainly due to thepresence of reduced levels of b-MSP mRNA. Prostate 35:109–116, 1998.© 1998 Wiley-Liss, Inc.

KEY WORDS: prostate cancer; beta-microseminoprotein; in situ hybridization; immu-nohistochemistry

INTRODUCTION

Human b-MSP, also known as prostatic inhibinpeptide (PIP), beta-inhibin, or prostatic secretory pro-tein of 94 amino acids (PSP94), is one of three majorproteins present in the prostate gland, i.e., b-MSP,prostate acid phosphatase (PAP), and prostate-specificantigen (PSA). b-MSP is a small (14-kDa) cystein-richprotein found in the seminal plasma and is secreted inlarge quantities by the prostatic glandular epithelium[1–3]. The b-MSP gene is located in chromosome10q11.2 [4] and its structure has been determined byboth cDNA [1,5,6] and genomic sequencing [7–9]. b-MSP is used as an immunohistochemical marker forprostate cancer and as a serum tumor marker, similar

to PSA and PAP [10]. However, the biological functionof b-MSP has not yet been defined.

The expression of b-MSP seems to be independentof androgens, and the synthesis and secretion of b-MSP persist even after androgen deprivation [11,12].In contrast, the expression of PAP and PSA is highlydependent on steroids [10,13], and the circulating lev-els of PAP and PSA are reduced following hormone

Contract grant sponsor: Ministry of Education, Science, Sports andCulture of Japan; Contract grant number: Grant-in-Aid 06404059.*Correspondence to: Toshifumi Tsurusaki, M.D., Department ofUrology, Nagasaki University School of Medicine, 1-7-1 Sakamoto,Nagasaki 852-8501, Japan. E-mail: ttsuru-ngs@umin.u-tokyo.ac.jpReceived 11 June 1997; Accepted 18 September 1997

The Prostate 35:109–116 (1998)

© 1998 Wiley-Liss, Inc.

ablation therapy [10]. Results from our laboratory aswell as those of other investigators have shown thatthe expression of b-MSP is reduced in prostate cancer,as demonstrated by immunohistochemistry (IHC) us-ing either monoclonal or polyclonal antibodies [14–16]. We also reported that when IHC was conductedusing b-MSP monoclonal antibody produced in ourlaboratory, b-MSP was not expressed in prostate can-cer cells [17]. However, the mechanism of reduced b-MSP expression in prostate cancer is still unclear andcontroversial.

In vivo administration of human b-MSP to intactadult male rats increased the rate of apoptosis of pros-tate cells [18]. In a model of prostate adenocarcinoma,i.e., the Dunning R3327G rat tumor (DT), several in-vestigators have shown that b-MSP not only inhibitedthe growth of cultured DT cells, but also suppressedtumor growth in vitro [10,19,20]. A dose-dependentinhibition of clonogenic cell growth and DNA syn-thetic activity by purified b-MSP was observed in tu-mor cell cultures. Furthermore, the inhibitory activityof b-MSP was similar in both androgen-dependentand androgen-independent DT cell lines [20]. Thesefindings suggest that b-MSP may be useful as a noveland apparently nontoxic form of therapy for hormone-independent prostate cancer [18–20].

b-MSP mRNA has been detected in a prostate can-cer cell line, LNCaP, but not in PC-3 and DU145 [6].Two forms of b-MSP cDNA have been detected inprostate tissue and LNCaP, probably resulting fromalternative splicing. The short form of b-MSP mRNA,that lacks the third exon, is designated PSP57 mRNA.Interestingly, PSP57 mRNA was localized within thenuclei, but its protein product could not be detected[21]. Thus, a reduced expression of b-MSP in prostatecancer does not necessarily mean a decreased level ofb-MSP transcription.

In the present study, we investigated the regulatorymechanism of reduced expression of b-MSP in pros-tate cancer by examining the expression of b-MSPmRNA and its protein in the same specimen. Studieswere conducted on tissue samples obtained from 109patients with benign and malignant prostate tumors,using nonradioactive in situ hybridization (ISH) andIHC. In nonradioactive ISH, thymine-thymine (T-T)dimerized oligonucleotide probes were used. Theseprobes were originally developed and evaluated byour group as a very sensitive method with excellentresolution [22]. Our results showed that in almost allcases, reduced b-MSP expression was clearly associ-ated with a loss or very low level of b-MSP mRNAexpression in prostate cancer cells. However, therewas no significant correlation between the degree ofb-MSP expression and Gleason grade.

MATERIALS AND METHODS

Tissue Preparation

Prostatic biopsy specimens were obtained from 104patients who were diagnosed with prostate cancer atthe Department of Urology, Nagasaki University Hos-pital, between June 1983–March 1996. The mean age of104 patients with untreated prostate cancer at diagno-sis was 72.0 ± 8.3 years (± SD) and ranged from 47–90years. In addition, 5 benign samples consisting of 2benign prostatic hyperplasia (BPH) and 3 prostate tis-sue samples without prostatic disease, representingnormal control specimens, were obtained from biop-sies performed due to a strong suspicion of prostatecancer based on elevated serum PSA levels and/orabnormal findings at either digital rectal examinationor transrectal ultrasonograghy. The mean age at diag-nosis for the patients involved had been 73.6 ± 7.1years (± SD). All tissue specimens were obtained using16- or 18-gauge needle biopsies prior to administra-tion of any treatment. Biopsies of the prostate glandwere obtained using the transperineal or transrectalapproach in all 109 patients. It is true that a biopsyspecimen does not always reflect the entire tumor.However, a bias in tissue sampling was minimized inthe present study by utilizing a sextant biopsy in mostof the present cases. The tissue samples were fixed in10% neutral buffered formalin and embedded in par-affin. Sections (6 mm in thickness) were mounted on3-aminopropyltriethoxysilane-coated slides. Informedconsent was obtained from every patient before bi-opsy. Biopsy specimens were stained with hematoxy-lin and eosin for histopathological examination. Gradeof malignancy was determined in each sample usingthe Gleason grading system [23], and the tissue areacontaining the primary site was selected for furtherinvestigation. These malignant tissue samples usuallycontained normal tissue or benign hyperplasia, whichwere also used as positive control.

Preparation of Oligo-DNA Probes

A 44-base sequence complementary to b-MSPmRNA (nucleotides 159–202, a part of exon 3) wasselected due to the existence of a short form of itsmRNA, which lacked the exon 3 part. A computer-assisted search (GenBank Release 95.0, 1996) of theabove antisense sequence, as well as of the sense se-quence, showed no significant homology with anyknown sequences. These antisense and sense se-quences were synthesized together with two and threeTTA repeats at the 58- and 38-ends of sequences, andwere used as probes after haptenization with T-Tdimer. The T-T dimer was introduced into oligo-

110 Tsurusaki et al.

DNAs by UV irradiation (254 nm), using a dose of12,000 J/m2. The generation of T-T dimer was verifiedimmunochemically, using mouse monoclonal anti-T-TIgG (Kyowa Medex, Tokyo, Japan).

Dot-Blot Hybridization

Unless otherwise specified, all procedures wereconducted at room temperature (RT; 20–25°C). Twomicrofilter drops of the sense oligo-DNA solutionwere pipetted onto nitrocellulose membranes that hadbeen pretreated with 20 × SSC (1 × SSC = 0.15 M NaClin 0.015 M sodium citrate buffer, pH 7.0) in a series ofspots at 1 pg–10 ng/spot. After air-drying, the filterswere baked at 80°C for 2 hr, and incubated at 37°C for2 hr with a prehybridization medium containing 10mM Tris/HCl (pH 7. 4), 0.6 M NaCl, 1 mM EDTA, 1 ×Denhardt’s solution, 500 mg/ml yeast tRNA, 250 mg/ml salmon testicular DNA (Sigma Chemical Co., St.Louis, MO), and 40% deionized formamide (NacalaiTesque, Kyoto, Japan). The membranes were hybrid-ized overnight at 37°C with 1 mg/ml T-T dimerizedoligo-DNA probe in a medium containing 10 mMTris/HCl (pH 7.4), 0.6 M NaCl, 1 mM EDTA, 1 × Den-hardt’s solution, 250 mg/ml yeast tRNA, 125 mg/mlsalmon testicular DNA, 10% dextran sulfate, and 40%deionized formamide. After successive washings with2 × SSC (10 min, twice), 1 × SSC (10 min, twice), andPBS (10 mM sodium phosphate buffer, pH 7.4, includ-ing 0.15 M NaCl) over a period of 10 min, the mem-branes were immersed for 60 min in a blocking solu-tion that contained 5% bovine serum albumin (BSA),500 mg/ml normal mouse IgG (Sigma Chemical Co.),100 mg/ml yeast tRNA, and 100 mg/ml salmon tes-ticular DNA in PBS. The reaction with HRP-linkedmouse anti T-T dimer IgG, which was diluted at 1:80with blocking solution, lasted for 3 hr. The membraneswere then washed with 0.075% Brij35 (Sigma Chemi-cal Co.) in PBS (15 min, four times), and HRP siteswere visualized using a solution containing 3,38-diaminobenzidine-4 HCl (DAB), H2O2, CoCl2, andNiSO4(NH4)2SO4 [24].

In Situ Hybridization (ISH)

ISH was performed according to the method de-scribed previously [22]. Briefly, the sections were de-paraffinized and rehydrated by standard procedures.This was followed by treatment with 0.3% H2O2 inmethanol (15 min) to inactivate endogenous peroxi-dase, with 0.2 N HCl (RT, 20 min), and with 100 mg/ml proteinase K (37°C, 15 min). After fixation with 4%paraformaldehyde in PBS for 5 min, the sections wereimmersed in 2 mg/ml glycine in PBS (30 min) andkept in 40% deionized formamide in 4 × SSC untilused for hybridization. Hybridization was carried out

overnight at 37°C with 2 mg/ml T-T dimerized anti-sense oligo-DNA for b-MSP dissolved in the hybrid-ization medium described above. In the next step, theslides were washed with 50% formamide in 2 × SSCcontaining 0.075% Brij35 followed by PBS. The sec-tions were stained immunohistochemically as de-scribed previously [22], and the sites of peroxidasewere visualized as described above.

The hybridization signal was considered positivewhen the accumulated black deposits in individualcells exceeded the background level. When stainingwas almost similar to that of negative control, thesample was regarded as ‘‘negative’’ (−). On the otherhand, ‘‘weak positive’’ (+) staining was assigned whenthe signal was present, but localized to the perinucleararea only. ‘‘Strong positive’’ (++) staining representeda strong and clear signal in the cytoplasm.

Control Experiments for ISH

To evaluate the specificity of b-MSP mRNA signals,various types of control experiments were conductedon sections adjacent to the malignant area [25]. Anumber of consecutive tissue sections were hybridizedwith T-T dimerized oligo-DNA complementary to 28SrRNA as a positive control [26], with T-T dimerizedb-MSP sense oligo-DNA as a negative control in everyrun. Another group of tissue sections was hybridizedwith b-MSP antisense probe in the presence of an ex-cess amount (50-fold) of either homologous or nonho-mologous unlabeled oligo-DNA probe, to confirm thesequence specificity of the signal. To eliminate pos-sible involvement of proteins and DNA in signal for-mation, a number of sections were digested with 100mg/ml of RNase-A at 37°C for 1 hr before the postfix-ation step. In some sections, stringent washing condi-tions were applied after hybridization with the b-MSPantisense probe.

Immunohistochemistry (IHC)

The procedure of immunohistochemistry for b-MSP has been described previously [16,17]. Briefly,tissue sections were deparaffinized and hydrated us-ing standard procedures. They were then treated with0.3% H2O2 in methanol for 20 min to inactivate endog-enous peroxidase, and incubated in a Block Ace solu-tion (Dainippon Pharmaceutical, Osaka, Japan) con-taining 2% BSA and 10% goat serum for 60 min toreduce nonspecific background staining. After theseprocedures, the specimens were incubated for 2 hrwith monoclonal b-MSP antibody in PBS at 15 mg/ml,and then incubated for 1 hr with rabbit biotinylatedanti-mouse IgG (Zymed Laboratories, San Francisco,CA) at 1:500 in PBS. After the final incubation, the

Expression of b-MSP in Prostate Cancer 111

TABLE I. Distribution of 104 Prostatic BiopsySpecimens According to Gleason Grading System

Gleasons’s primary gradea No. of patients (%)

1 1 (1.0)2 4 (3.8)3 26 (25.0)4 51 (49.0)5 22 (21.2)Total 104 (100.0)

aPrimary grade is the grade of the most predominant areawithin malignant parts of each case. Fig. 1. Colorimetric dot-blot hybridization. Various amounts (1

pg–10 ng/spot) of b-MSP sense oligo-DNA, which were fixed ontonitrocellulose membranes, were hybridized with T-T dimerizedantisense oligo-DNA (top) or T-T dimerized sense oligo-DNA(bottom). At least 1 pg DNA was detected specifically.

Fig. 2. b-MSP mRNA expression in benign prostatic tissue using nonradio-active in situ hybridization. A: 28S rRNA antisense probe, as positive control.B: b-MSP sense probe, as negative control. C: b-MSP antisense probes. b-MSP mRNA showed strong but diffuse expression in the cytoplasmic area onlyof prostatic epithelia. D: Competition control. Competition with excessamount (50-fold) of unlabeled probe greatly suppresses the signal. E: Non-competition control. Noncompetition with excess amount (50-fold) of non-homologous unlabeled oligo-DNA does not suppresses the signal. F: Excessstringency control. Washing at excessively high stringency removes the signalfrom the prostatic epithelia. G: RNase-A pretreated control. Treatment withRNase before hybridization with b-MSP antisense probe eliminates the signalin the prostatic epithelia (×400).

112 Tsurusaki et al.

sections were subjected to a chromogen solution con-sisting of 50 mg/ml DAB and 0.05% H2O2 in 0.05 MTris buffer (pH 7.2) for 5 min, followed by counter-staining with methylgreen, dehydration, and mount-ing. The intensity of b-MSP staining was semiquanti-tatively graded from negative (−) to strong positive(++), with (−) indicating no detectable staining, (+)indicating weak positive staining, and (++) indicatingstrong staining.

Statistical Analysis

The chi-square test was used to analyze the corre-lation between the degree of b-MSP mRNA expressionand primary grade, and between the degree of b-MSPmRNA expression and the degree of b-MSP. P < 0.05denoted statistical significance.

RESULTS

Gleason Grading of Prostate Cancer

The primary Gleason grade of 104 transperineal bi-opsy specimens ranged from grade 1–5 with a meanprimary grade of 3.86 ± 0.83 (median, 4.0), as shown inTable I. Of the 104 specimens, one was categorized asgrade 1 (1%), 4 as grade 2 (4%), 26 as grade 3 (25%), 51as grade 4 (49%), and 22 as grade 5 (21%).

Dot-Blot Hybridization With T-T DimerizedSynthetic Oligo-DNA

As shown in Figure 1, when b-MSP sense oligo-DNA on the membrane was hybridized with T-Tdimerized b-MSP antisense oligo-DNA or T-T dimer-ized unrelated oligo-DNA (b-MSP sense oligo-DNA),the b-MSP antisense probe detected as little as 1 pgsense DNA, while unrelated oligo-DNA detected nosignal. These findings indicate that the antisense probesynthesized in our laboratory was specific and hadadequate sensitivity to be useful in ISH studies.

b-MSP mRNA Expression Assessed by ISH

We analyzed 5 benign prostate tissues and 104prostate malignant tumors using a b-MSP oligo-DNAantisense probe. Preliminary control experimentswere also conducted to confirm the sequence specific-ity of the b-MSP mRNA signal on benign specimens.A representative set of control experiments is shownin Figure 2. When a section of BPH was hybridizedwith a T-T dimerized 28S complementary probe, a sig-nal was detected in the cytoplasmic area as well as inthe nucleoli (Fig. 2A), whereas no staining was foundwith a T-T dimerized oligo-DNA b-MSP sense probe

(Fig. 2B) in an adjacent section. When adjacent sectionswere hybridized with b-MSP antisense probe in thepresence of an excess homologous unlabeled oligo-DNA, no staining of b-MSP mRNA was observed (Fig.2D). On the other hand, b-MSP mRNA staining wasnot altered by excess amounts of nonhomologous un-labeled oligo-DNA (Fig. 2E). When the sections werewashed at high stringency or digested with RNase-A,no staining was observed (Fig. 2F,G).

All benign specimens were evaluated as stronglypositive, i.e., the glandular and ductal epithelial cellswere positive for b-MSP mRNA, but other cells, suchas basal, stromal, endothelial and vascular smoothmuscle cells, were negative. Staining for b-MSPmRNA was localized to the cytoplasmic area of epi-thelial cells, predominantly in the perinuclear area. Atypical staining pattern is shown in Figure 2C.

In prostate cancer tissue samples, staining for b-MSP mRNA was negative in malignant epithelia com-pared with benign prostate epithelia, and the signalwas significantly less intense in cancerous tissue onthe whole than in benign epithelium (P < 0.0001).There was no correlation between degree of b-MSPmRNA signal and Gleason grade. b-MSP transcriptswere detected in only 32 of the 104 cancerous speci-mens (30.8%), 21 of which were weakly positive (+)and 11 strongly positive (++). The number of b-MSPmRNA-positive specimens in each primary grade isreported in Table II. A typical staining pattern of b-MSP mRNA-strongly positive (++) malignant tissue isshown in Figure 3C.

b-MSP Expression Assessed by IHC

IHC of b-MSP in benign prostatic tissue showed astaining pattern similar to that observed in b-MSP

TABLE II. b-MSP mRNA Expression Assessed by In SituHybridization in Benign and Malignant Specimens*

b-MSP mRNA signal

− + ++

Benign 0 0 5Primary grade of prostate cancer1 1 0 02 3 1 03 20 4 24 35 10 65 13 6 3Total 72 21 11

*The signal levels of b-MSP mRNA in cancer samples weresignificantly lower than those of benign specimens (P < 0.0001),and b-MSP mRNA expression levels in the different grades didnot differ significantly.

Expression of b-MSP in Prostate Cancer 113

mRNA. In most malignant areas, the distribution ofb-MSP signal was very heterogeneous, and stainingwas not observed in some parts. A typical example ofa malignant tissue that strongly (++) expressed bothb-MSP mRNA and its protein is shown in Figure 3E.Ninety-six cancer specimens (92.3%) were negative, 6(5.8%) were weakly stained (+), and only 2 (1.9%) werestrongly positive for b-MSP. There was no significantcorrelation between the expression level of b-MSP andGleason grade (P = 0.370), although there was a ten-dency towards a stronger staining pattern in poorly

differentiated tumors than in well-differentiated tu-mors (positive rate: grade 1, 0%; grade 2, 0%; grade 3,3.8%; grade 4, 5.9%; and grade 5, 18.2%), as shown inTable III.

Correlation Between b-MSP Protein and mRNAExpression in Prostate Cancer

Specimens negative for b-MSP were not alwaysnegative for b-MSP mRNA (31 cases, 29.8%), and viceversa (7 cases, 6.7%). Other specimens were eitherpositive for both (1 case, 1.0%) or negative for both (65

Fig. 3. b-MSP mRNA and the protein expression of a grade 5 cancer tissue, using nonradioactive in situ hybridization and immunohis-tochemistry. A: Hematoxylin and eosin staining. B: 28S rRNA antisense probe, as positive control. C: b-MSP antisense probe. b-MSPmRNA showed strong but diffuse expression in the cytoplasmic area only of malignant epithelia. D: b-MSP sense probes, as negative control.E: b-MSP monoclonal antibodies. b-MSP protein showed diffuse localization in the cytoplasm of the malignant epithelial cells (×200).

114 Tsurusaki et al.

cases, 62.5%). There was no correlation between thedegree of expression of b-MSP and that of b-MSPmRNA (P = 0.179), as shown in Table IV.

DISCUSSION

The major finding of the present study was thepresence of a significantly low level of expression ofb-MSP mRNA in malignant tissue samples (93/104cases, 89.4%, and 72 negative and 21 weakly positivespecimens) relative to that in benign specimens. More-over, the expression of b-MSP mRNA could not bedetected in a large proportion of cancer specimens(69.2%).

b-MSP is secreted mainly by prostate glandular epi-thelium. A number of investigators have previouslyreported that both the secretion and synthesis of b-MSP were reduced in prostate cancer tissues [14–16].Using IHC with a monoclonal antibody raised in ourlaboratory, we have also shown a lack of expression ofb-MSP in almost all malignant tissues from patientswith prostate cancer [17]. Thus, the present results,that more than 92% of prostate cancer specimens werenegative for b-MSP, are consistent with the previousreports.

Only a few studies have used molecular analysis ofb-MSP expression to examine prostate tumors. In onesuch study examining the expression of b-MSP mRNAin two cultured prostate cancer cell lines (PC-3 andDU145), the expression level was lower in these celllines as compared to the LNCaP cell line or BPH tis-sues, by Northern blot analysis [6]. However, to ourknowledge, no reports have examined the cellular ex-pression of b-MSP mRNA in prostate cancer tissues.Therefore, we believe that the present study is the firstdemonstration of expression of b-MSP mRNA at a cel-

lular level using nonradioactive ISH. Our resultsclearly demonstrated that b-MSP mRNA is located inthe majority of benign glandular epithelial cells and asmaller number of malignant epithelia, whereas itssignal intensity in prostate cancer was lower than thatof benign tissues. In b-MSP mRNA-negative and b-MSP-negative samples (65 cases, 62.5%), it is possiblethat the absence of b-MSP mRNA may account for theloss of b-MSP protein in prostate cancer cells. How-ever, in b-MSP mRNA-positive and b-MSP-negativesamples (31 cases, 29.8%), some translational disor-ders of b-MSP mRNA or an increase in the rate ofeither its secretion or its degradation may be respon-sible for the discrepancy. It is also possible that a de-fective or alternatively spliced mRNA, which could bedetected by our oligo-DNA probe, may have occurred,resulting in a loss of reactivity of its final product us-ing our monoclonal antibody. On the other hand, inb-MSP mRNA-negative and b-MSP-positive samples(7 cases, 6.7%), there may be a difference in the half-life of b-MSP mRNA and its protein.

In the conventional prostate cancer cell lines, LN-CaP with b-MSP expression has a more differentiatednature than both DU145 and PC-3 without b-MSP ex-pression. The expression of b-MSP seems to be corre-lated with the differentiation status of the tumor cells,based upon the results with established cell lines.However, we could not find any significant correla-tion between tumor grade and b-MSP expression invivo.

The human b-MSP gene has been assigned to10q11.2 [4]. Many chromosomal regions that may har-bor putative prostate cancer suppressor genes havebeen identified. Among these, three regions on chro-mosome 10 (10p, 10q24→qter, and 10q24→25) repre-sent loss of heterozygosity in prostate cancer cells [27–30]. The gene can be ruled out as a tumor-suppressorgene. However, since b-MSP is known as a possibleinhibitor of rat prostate tumor cell growth in vivo andin vitro [20], a complete loss or reduction of b-MSPmay contribute to the development of prostate cancer.

In conclusion, we analyzed the expression of b-MSP, at both mRNA and protein levels using ISH and

TABLE III. b-MSP Expression Assessed byImmunohistochemistry in Benign and

Malignant Specimens*

b-MSP signal

− + ++

Benign 0 0 5Primary grade of prostate cancer1 1 0 02 4 0 03 25 1 04 48 3 05 18 2 2Total 96 6 2

*The signal levels of b-MSP in cancer samples were significantlylower than those of benign specimens (P < 0.0001), and b-MSPexpression levels in the different grades did not differ signifi-cantly.

TABLE IV. Correlation Between b-MSP mRNAExpression and b-MSP Signal in Prostate Cancer*

b-MSP signal

b-MSP mRNA signal

− + ++

− 65 21 10+ 6 0 0++ 1 0 1

*There is no significant correlation between b-MSP mRNA sig-nal and b-MSP signal levels (P = 0.179).

Expression of b-MSP in Prostate Cancer 115

IHC, in 104 biopsy specimens of untreated prostatecancer. Although no significant correlation was de-tected between the expression of b-MSP mRNA orprotein and Gleason grade, we found that the levels ofb-MSP mRNA expression as well as its protein werelow in most prostate cancer tissues. We postulate thatthe reduced expression of b-MSP may play an impor-tant role in the development of prostate cancer.

ACKNOWLEDGMENTS

We are grateful to Dr. K. Abe for pathological ex-aminations, Dr. A. Yoshii and Dr. Y. Iwasaki for theirhelp in conducting the in situ hybridization study, andT. Shimogama for his technical assistance. We alsothank Dr. F.G. Issa from the Department of Medicine,University of Sydney, for the careful reading and ed-iting of the manuscript.

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