The Prostate 38:278284 (1999)

Prognostic Significance of -Microseminoprotein

mRNA Expression in Prostate Cancer
Hideki Sakai,1* Toshifumi Tsurusaki,1 Shigeru Kanda,1 Takehiko Koji,2
Jim W. Xuan,3 and Yutaka Saito1
1

Department of Urology, Nagasaki University School of Medicine, Nagasaki, Japan

Department of Histology and Cell Biology, Nagasaki University School of Medicine,
Nagasaki, Japan
3
Department of Surgery, University of Western Ontario, London, Ontario, Canada

BACKGROUND. Human -microseminoprotein (-MSP or PSP94) is a small protein secreted by prostatic epithelial cells. We recently reported the presence of low levels of -MSP
mRNA expression and protein in most prostate cancer tissues.
METHODS. -MSP and mRNA expression was examined by in situ hybridization in biopsy
specimens obtained from 92 patients with prostate cancer. All tissue specimens were obtained
by needle biopsies prior to treatment. All patients subsequently received endocrine therapy.
To estimate the influence of -MSP mRNA expression and three possible prognostic factors,
i.e., patient age, clinical stage, and Gleason score, on time to progression under endocrine
therapy, univariate and multivariate analyses were performed using Coxs proportional hazards regression model.
RESULTS. Multivariate survival analysis showed that clinical stage was the strongest prognostic factor (P =0.006) and that -MSP mRNA expression was the second strongest factor (P
= 0.038) in 92 patients with stage BD disease. Analysis of only 51 patients with stage D
disease showed that -MSP mRNA expression was the only significant prognostic indicator
for progression under endocrine therapy (P = 0.003).
CONCLUSIONS. The presence of cells that express the -MSP transcript may be a novel
indicator of potentially aggressive prostate cancer. Prostate 38:278284, 1999.
1999 Wiley-Liss, Inc.

KEY WORDS:

prostate cancer; -microseminoprotein; PSP94; mRNA; prognosis

INTRODUCTION
Prostate cancer has a high incidence and is the second leading cause of death from cancer among men in
the Western world [1]. Identification of factors that
predict clinical outcome is important but can be difficult. In general, the clinical stage and histological
grade correlate to the biological behavior of prostate
cancer, and are considered as the most useful prognostic factors [2,3]. Several studies have examined the
role of other possible prognostic indicators, such as
age [2], extent of disease on a bone scan [4], DNA
ploidy [5], and the expression of prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP) in
tumor cells [6,7]. Recently, other potential prognostic
markers have also been reported [8].
1999 Wiley-Liss, Inc.

Human -microseminoprotein (-MSP), also

known as prostatic inhibin peptide (PIP), -inhibin, or
prostatic secretory protein of 94 amino acids (PSP94),
is one of the major proteins secreted by prostatic epithelial cells [911]. -MSP is a small (14 kDa) cysteinerich protein, abundant in the seminal plasma [911],
and detectable in serum and urine [12,13]. In contrast
to PSA and PAP, regulation of -MSP expression is
independent of androgens [14]. The -MSP gene is
Grant sponsor: Ministry of Education, Science, Sports, and Culture
of Japan; Grant number: 06404059.
*Correspondence to: Hideki Sakai, M.D., Department of Urology,
Nagasaki University School of Medicine, 1-7-1 Sakamoto, Nagasaki
852-8501, Japan. E-mail: hsakai@net.nagasaki-u.ac.jp
Received 19 February 1998; Accepted 3 August 1998

-MSP mRNA and Prognosis in Prostate Cancer

located on chromosome 10q11.2 [15]. Although -MSP
was initially identified as being specific to the prostate, immunohistochemical and Northern analyses
have since detected -MSP secretion in other mucusproducing cells, e.g., in tracheal epithelium [16]. Thus,
-MSP is not currently considered a prostate-specific
protein. Its biological function, however, remains unknown. It has been demonstrated that -MSP can suppress DNA synthesis and growth of prostate cells in
vitro and in vivo [17,18]. It has also been reported that
-MSP can induce apoptosis of prostate cancer cells in
an animal model system [17]. These findings suggest
that -MSP may be useful therapeutically.
We recently analyzed the expression of -MSP at
both the mRNA and protein levels in 104 biopsy specimens of untreated prostate cancer, using in situ hybridization (ISH) and immunohistochemical staining
[19]. Although no significant correlation was detected
between the expression of -MSP mRNA or protein
and Gleason grade, we found that levels of -MSP
mRNA expression and protein were low in most prostate cancer tissues. These results suggest that reduced
expression of -MSP may play an important role in the
development of prostate cancer.
In the present study, we attempted to correlate
-MSP mRNA expression with progression-free survival of prostate cancer patients treated with endocrine therapy. Multivariate survival analysis demonstrated that in patients with advanced prostate cancer,
-MSP mRNA expression was an independent prognostic indicator for progression during endocrine
therapy.
MATERIALS AND METHODS

279

antiandrogen, chlormadinone acetate. Disease progression was estimated based on evidence of biochemical failure and/or evidence of clinical progression. Biochemical failure was defined as an increase in
PSA values on two or more consecutive follow-up visits. Clinical progression was defined as an increase in
any previously measurable lesion by more than 25% in
two perpendicular diameters, or appearance of a new
metastatic tumor on chest X-ray, CT scan, or bone
scintigraphy.
All tissue specimens were obtained by transperineal or transrectal needle biopsies using 16- or 18gauge needles prior to administration of any treatment. A systematic sextant biopsy was performed in
most cases. In addition, five control samples comprising two benign prostatic hyperplasia (BPH) and three
normal prostate tissue samples were obtained from
biopsies performed due to a strong suspicion of prostate cancer based on elevated serum PSA levels and/
or abnormal findings at either digital rectal examination or transrectal ultrasonography. Informed consent
was obtained from every patient prior to biopsy. Tissue samples were fixed in 10% neutral bufferd formalin and embedded in paraffin. Sections (6 m in thickness) were mounted on 3-aminopropyltriethoxysilane-coated slides. Biopsy specimens were stained
with hematoxylin and eosin for histopathological examination. The grade of malignancy was determined
in each sample by using the Gleason grading system
[21], and the tissue area containing the primary site
was selected for further investigation. These malignant tissue samples usually contained normal tissue or
tissue with benign hyperplasia, which were also used
as positive controls.

Patients and Tissues

Preparation of Oligo-DNA Probes

We selected 92 patients with prostate cancer from a

group treated with endocrine therapy at Nagasaki
University Hospital. The inclusion criteria were the
availability of a sufficient tumor biopsy tissue and
well-documented clinical follow-up. All patients analyzed in this study were also included in our previous
study [19]. Diagnosis of prostate cancer was made between June 1984March 1996. The mean age of our
patients with untreated prostate cancer at diagnosis
was 72.7 years (range, 4790 years). In 12 patients,
cancer was localized within the prostate (stage B, or
T2N0M0 [20]), in 29 the cancer was locally advanced
(stage C, or T34N0M0), and metastatic disease was
found in the remaining 51 (stage D, or N13 or M1).
All patients received endocrine therapy after the
initial diagnosis. Fifty patients were treated with a luteinizing hormone-releasing hormone agonist and the
remainder were treated with estrogen or a steroidal

A 44-base pair sequence complementary to -MSP

mRNA (nucleotides 159202, part of exon 3) was selected due to the presence of a short form of -MSP
mRNA, which lacked exon 3 [22]. A computer-assisted
search (GenBank Release 95.0, 1996) of the above antisense sequence and the sense sequence showed no
significant homology with any known sequences.
These antisense and sense sequences were synthesized
together with two and three TTA repeats at the 5- and
3-ends of sequences, and used as probes after haptenization with a T-T dimer. The T-T dimer was introduced into oligo-DNAs by ultraviolet irradiation (254
nm), using a dose of 12,000 J/m2. The generation of a
T-T dimer was verified immunochemically using a
mouse monoclonal anti-T-T IgG (Kyowa Medex, Tokyo, Japan). Furthermore, a dot-blot hybridization
study showed that the antisense probe synthesized in
our laboratory was specific and had adequate sensi-

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Sakai et al.

tivity to be useful in ISH studies [19]. Preliminary experiments were also conducted on benign tissue specimens to confirm the sequence specificity of the -MSP
mRNA signal.
In Situ Hybridization
ISH was performed according to the method described previously [23]. Briefly, sections were deparaffinized and rehydrated by standard procedures.
This was followed by treatment with 0.3% H2O2 in
methanol for 15 min to inactivate endogenous peroxidase, 0.2 N HCl at room temperature for 20 min, and
100 g/ml proteinase K at 37C for 15 min. After fixation with 4% paraformaldehyde in phosphatebuffered saline (PBS) for 5 min, sections were immersed in 2 mg/ml glycine in PBS for 30 min and
stored in 40% deionized formamide in 4 SSC (1
SSC: 0.015 M sodium citrate, pH 7.0, supplemented
with 0.15 M NaCl) until used for hybridization. Hybridization was carried out overnight at 37C with 2
g/ml T-T-dimerized antisense oligo-DNA for
-MSP, dissolved in a hybridization medium containing 10 mM Tris/HCl (pH 7.4), 0.6 M NaCl, 1 mM
EDTA, 1 Denhardts solution, 250 g/ml yeast
tRNA, 125 g/ml salmon testicular DNA, 10% dextran sulfate, and 40% deionized formamide. In the
next step, the slides were washed with 50% formamide in 2 SSC containing 0.075% Brij35 followed by
PBS. The sections were stained immunohistochemically as described previously [23], and the sites of peroxidase activity were visualized using a solution containing 3,3-diaminobenzidine-4 HCl (DAB), H2O2,
CoCl2, and NiSO4(NH4)2SO4 [24].
The hybridization signal was considered positive
when the accumulated black deposits in individual
cells exceeded the background level and more than
20% of carcinoma cells showed such a distinct signal.
When staining was similar to that of the negative control, the sample was regarded as negative. On the
other hand, weak positive staining was assigned
when the signal was present, but localized to the perinuclear area only. Strong positive staining represented a strong and clear signal in the cytoplasm. A
number of consecutive tissue sections were hybridized
with T-T-dimerized oligo-DNA complementary to 28S
rRNA as a positive control [25] and also with T-Tdimerized -MSP sense oligo-DNA as a negative control in every run. All specimens were independently
evaluated by two observers who were blind to the
clinical outcome.
Statistical Analysis
A computer software package, StatView 4.5 (Abacus Concepts, Inc., Berkeley, CA), was used for statis-

Fig. 1. -MSP mRNA expression of a grade 5 prostate cancer

tissue, using nonradioactive in situ hybridization. A: Hematoxylin
and eosin staining. B: 28S rRNA antisense probe, as positive control. C: -MSP antisense probe. -MSP mRNA showed strong but
diffuse expression in the cytoplasmic area of only malignant epithelia. D: -MSP sense probes, as negative control (AD, 200).
These photomicrographs also appeared in our previous report
[19].

tical analysis. Difference in mean age between groups

was analyzed by Students t-test. The 2 test was used
to analyze the correlation between -MSP mRNA expression and the clinical stage or Gleason score. Progression-free survival curves were calculated according to the Kaplan-Meier method, and statistical significance was examined using the log rank test. Coxs
proportional hazards regression model was also used
for univariate and multivariate survival analyses.
RESULTS
Expression of -MSP mRNA in prostate tissue was
examined by ISH [19]. All five control specimens were
evaluated as strongly positive, i.e., the glandular and
ductal epithelial cells were positive for -MSP mRNA,
whereas other cells such as basal, stromal, endothelial,
and vascular smooth muscle cells were negative.
Staining for -MSP mRNA was localized to the cytoplasmic area of epithelial cells, predominantly in the
perinuclear area. In contrast, prostate cancer specimens exhibited -MSP mRNA staining which was heterogenous or scattered in most of the malignant epithelia. Typical staining patterns of -MSP mRNAstrongly positive and -negative malignant tissues are
shown in Figures 1 and 2, respectively.
Of the 92 cancerous specimens, 63 were negative, 18
weakly positive, and 11 strongly positive for -MSP
mRNA. Accordingly, patients were divided into two

-MSP mRNA and Prognosis in Prostate Cancer

281

TABLE I. Correlation Between -MSP mRNA

Expression and Age, Clinical Stage, and Gleason Score*
-MSP mRNA

Fig. 2. A representative example of -MSP mRNA-negative

prostate cancer (grade 4) (200).

groups: 63 negative and 29 positive for -MSP mRNA

expression. The correlations between -MSP mRNA
expression and patient age, clinical stage, or Gleason
score were analyzed. There was no significant difference in age between the two groups (Students t-test, P
= 0.91), and no significant correlation was noted between -MSP mRNA expression and clinical stage (P
= 0.27), or between -MSP mRNA expression and
Gleason score (2 test, P = 0.45, Table I). As expected,
however, the Gleason score significantly correlated
with the clinical stage (P = 0.009).
The mean and median follow-up periods of patients treated with endocrine therapy were 34 and 29
months, respectively. Biochemical and/or clinical progression was observed in 44 patients (48%). Univariate
and multivariate survival analyses with Coxs regression model were used to evaluate the influence of
-MSP mRNA expression and three possible prognostic factors, i.e., patient age, clinical stage, and Gleason
score, on time to progression. In the univariate analysis where all 92 patients treated with endocrine
therapy were included, the clinical stage (P = 0.0006)
and Gleason score (P = 0.001) were considered important prognostic factors. Multivariate analysis of the
four variables showed that clinical stage had the
strongest effect on progression-free survival (P =
0.006), while -MSP mRNA expression (P = 0.038) was
the second most significant factor associated with progression-free survival (Table II).
Because the clinical stage correlated strongly with
time to progression in hormonally-treated prostate
cancer patients, further survival analyses were performed in only 51 patients with stage D disease. Univariate analysis including this group of patients
showed only -MSP mRNA expression as the significant variable (P = 0.007), whereas patient age (P = 0.14)
and Gleason score (P = 0.16) did not influence progression-free survival. Multivariate analysis of the
three variables showed that only -MSP mRNA ex-

Mean age (years)

Clinical stage
B (T2N0M0)
C (T34N0M0)
D (N13 or M1)
Gleason score
4
5
6
7
8
9
10
Total

Negative (%)

Positive (%)

72.8

72.6

10 (16)
17 (27)
36 (57)

2 (7)
12 (41)
15 (52)

4 (6)
2 (3)
5 (8)
16 (25)
15 (24)
17 (27)
4 (6)
63 (100)

1 (3)
0 (0)
3 (10)
5 (17)
6 (21)
8 (28)
6 (21)
29 (100)

*There was no significant difference in patient age between the

two groups by Students t-test (P = 0.91), and no significant
correlation was noted between -MSP mRNA expression and
clinical stage (P = 0.27), and between -MSP mRNA expression
and Gleason score (P = 0.45) by 2 test.

pression was a significant determinant of prognosis (P

= 0.003, Table III). The progression-free survival
curves according to -MSP mRNA expression in stage
D prostate cancer patients are shown in Figure 3. A
significantly worse progression-free survival rate was
noted in patients with positive -MSP mRNA expression (P = 0.005 by log rank test). Thus, positive -MSP
mRNA expression significantly correlated with a short
time to progression in advanced prostate cancer patients treated with endocrine therapy.
DISCUSSION
Multivariate survival analyses in the present study
showed that hormonally-treated stage D prostate cancer patients with positive -MSP mRNA expression
had a significantly poorer outcome in terms of progression-free survival than those with negative expression (P = 0.003). In contrast, when all patients with
stage BD disease were included in such analysis,
-MSP mRNA expression showed only a borderline
prognostic value (P = 0.038). The prognosis for prostate cancer is generally considered to depend on the
clinical stage and tumor grade [2,3]. In this series of
patients, clinical stage and tumor grade (Gleason
score) also had strong prognostic value in the univariate analysis. However, the Gleason score was not a
significant independent prognostic factor in the multivariate analysis, as can be explained by the strong

282

Sakai et al.

TABLE II. Univariate and Multivariate Survival Analyses for 92 Prostate Cancer Patients Treated With Endocrine
Therapy in Coxs Regression Model
Univariate
Variable
Age
Clinical stage
Gleason score
-MSP mRNA expression

Multivariate

Hazard ratio

95% confidence
interval

P value

Hazard ratio

95% confidence
interval

P value

0.964
2.762
1.519
1.711

0.9301.000
1.5484.930
1.1811.952
0.9183.192

0.049
0.0006
0.001
0.091

0.971
2.466
1.238
2.043

0.9351.007
1.3004.678
0.9391.633
1.0424.008

0.11
0.006
0.13
0.038

TABLE III. Univariate and Multivariate Survival Analyses for 51 Patients With Stage D Prostate Cancer Treated With
Endocrine Therapy in Coxs Regression Model
Univariate
Variable
Age
Gleason score
-MSP mRNA expression

Multivariate

Hazard ratio

95% confidence
interval

P value

Hazard ratio

95% confidence
interval

P value

0.971
1.248
2.883

0.9351.009
0.9161.701
1.3386.211

0.14
0.16
0.007

0.957
1.169
3.436

0.9151.000
0.8431.623
1.5027.856

0.050
0.35
0.003

Fig. 3. Kaplan-Meier progression-free survival curves according

to -microseminoprotein (-MSP) mRNA expression in 51 hormonally-treated prostate cancer patients with metastasis. The
-MSP mRNA-positive group exhibited a significantly shorter period to progression than the -MSP mRNA-negative group (P =
0.005 by log rank test).

correlation between Gleason score and clinical stage.

Thus, although the presence of metastasis had the
strongest predictive value in all patients treated with
endocrine therapy, among those with metastasis, positive -MSP mRNA expression indicated a short interval to progression. Thus, our findings suggest that the
presence of cells that express the -MSP transcript
may be an indicator of potentially more aggressive
prostate cancer.

Although a number of prognostic factors for prostate cancer have been reported, e.g., proliferating cell
nuclear antigen [26], p53, bcl-2 [27], E-cadherin,
-catenin [28], and vascular density [29], most of these
factors correlated with tumor grade assessed by various criteria. Thus, such prognostic factors have only
an incidental significance, because as mentioned
above, tumor grade generally has a strong prognostic
value for prostate cancer. On the other hand, the present study demonstrated that -MSP mRNA expression did not correlate with tumor grade as assessed by
the Gleason grading system. Furthermore, multivariate analysis showed that -MSP mRNA expression
was a significant predictor for failure of endocrine
therapy in prostate cancer patients with metastasis.
Therefore, -MSP mRNA expression is considered to
be a truly independent prognostic indicator for advanced prostate cancer patients treated with endocrine therapy.
Initially we attempted to correlate the expression of
-MSP protein and -MSP mRNA with prognosis.
However, we abandoned the idea of performing such
analysis using -MSP protein expression because of an
extremely low frequency of -MSP-positive prostate
cancer. Only 6 patients (7%) of 92 included in this
study had immunohistochemically positive -MSP expression (data not shown).
The reason for the correlation between positive expression of -MSP mRNA and poor prognosis in hormonally-treated prostate cancer patients is unknown

-MSP mRNA and Prognosis in Prostate Cancer

at present. We previously analyzed the expression of
-MSP at both the mRNA and protein levels in 104
biopsy specimens of untreated prostate cancer, and
found a lower level of expression of -MSP in prostate
cancer tissue compared with benign prostate tissue
[19]. We also suggested that this phenomenon might
be mainly due to the presence of reduced levels of
-MSP mRNA [19]. Based on these findings, we expected in the present study that patients with high
expression of -MSP mRNA would show a better
prognosis than those with low levels of expression. In
contrast to our expectations, in the present study a
worse progression-free survival rate was noted in patients with positive -MSP mRNA expression. Although most of the transformed prostatic epithelial
cells lose the ability to express -MSP mRNA and produce its protein, some carcinoma cells with a high
biological potential may continue to express -MSP
mRNA through an aberrant intracellular signal transduction. Furthermore, our finding of a low frequency
of -MSP-positive prostate cancer suggests that in
such malignant cells the mRNA may be hardly translated into the wild type -MSP. In a series of preliminary immunohistochemical studies from our laboratory, we recently analyzed the expression of -MSP in
surgical specimens obtained from patients treated
with prostatectomy after neoadjuvant hormonal
therapy. Interestingly, although none of the prostate
cancers treated with androgen deprivation therapy
showed positive PSA staining, a number of high-grade
tumors (Gleason grade 4 and 5) showed positive
-MSP expression (unpublished results). While the
physiological consequences of changes in -MSP expression in malignant cells of the prostate remain to be
elucidated, we speculate that the presence of carcinoma cells expressing -MSP mRNA may contribute
to the malignant potential or the androgenindependent growth of the tumor. In this regard, assessment of -MSP mRNA expression in prostate cancer tissue samples may be useful for selection of patients who should be treated aggressively, e.g., with
cytotoxic chemotherapy. To establish the role of
-MSP mRNA as a prognostic indicator, further studies, including a prospective cohort study, are needed.

CONCLUSIONS
Multivariate survival analysis showed that in prostate cancer patients with metastasis, -MSP mRNA
expression was an independent prognostic determinant of survival during endocrine therapy, and that
this prognostic indicator was independent of histological grade. Assessment of -MSP mRNA expression in prostate cancer tissue samples may be useful

283

for the identification of a subgroup of patients with

aggressive malignant prostate cancer.
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