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ANTICANCER RESEARCH 26: 1253-1260 (2006)
Significance of Pituitary Tumor Transforming
Gene 1 (PTTG1) in Prostate Cancer
XUHUI ZHU1, ZEBIN MAO2, YANQUN NA1, YINGLU GUO1, XIANGHONG WANG3 and DIANQI XIN1
1Department
of Urology, Peking University First Hospital and Institute of Urology, Peking University, Beijing 100034;
of Biochemistry and Molecular Biology, Peking University Health Science Center, Beijing 100083;
3Department of Anatomy, The University of Hong Kong, SAR, China
2Department
Abstract. Recently, the pituitary tumor transforming gene 1
(PTTG1) has been suggested to be an oncogene. To investigate
whether PTTG1 plays a positive role in the pathogenesis of
prostate cancer, PTTG1 protein expression was examined in
prostate tissue samples by immunohistochemistry. PTTG1
expression was detected in a high percentage of prostate cancer
tissues (34/41, 82.9%), but to a much lesser extent in nonmalignant tissues (5/14, 35.7%). To further confirm these results,
the expression vectors containing either the PTTG1 or antisensePTTG1 gene were transfected into a prostate cancer cell line,
LNCaP, and the cell proliferation rate was studied, as well as
tumorigenicity in the LNCaP cells expressing different levels of
the PTTG1 protein. Ectopic PTTG1 gene expression promoted
prostate cancer cell proliferation and tumorigenesis both in vitro
and in nude mice. In contrast, down-regulation of PTTG1 led to
suppression of tumor cell growth. These results suggest that
PTTG1 may be a potential prognostic marker for prostate cancer
and that the down-regulation of PTTG1 may be a therapeutic
target in the suppression of prostate cancer growth.
The pituitary tumor transforming gene (PTTG) was originally
isolated from rat pituitary tumor GH4 cells by differential
display PCR (1), and the human homologous cDNA was
subsequently cloned from fetal liver. Human PTTG1 is 85%
similar to rat PTTG1 at the cDNA level and 89% similar to
rat PTTG1 at the amino acid sequence level (2). In addition,
PTTG1 is expressed in a cell cycle-dependent manner with a
peak in the G2/M-phase (3). The PTTG1 protein seems to be
involved in several of the important mechanisms of cell
proliferation and differentiation signaling pathways. Abnormal
Correspondence to: Dianqi Xin, Associate Professor, Department
of Urology, Peking University First Hospital and Institute of
Urology, Peking University, No. 8, Xishiku Street, Xicheng
District, Beijing 100034, China. Tel: 8610-66551122-2604, Fax:
8610-66551032, e-mail: [email protected]
Key Words: PTTG1, prostate cancer.
0250-7005/2006 $2.00+.40
overexpression of PTTG1 causes the inhibition of chromatid
separation, resulting in chromosomal gain or loss (4).
Overexpression of PTTG1 also increases cell proliferation,
induces cell transformation in vitro and promotes
tumorigenesis in nude mice (5, 6). PTTG1 is abundantly
expressed in human tumors including ovarian (7), esophageal
(8), pancreatic (9), kidney (10), hemopoietic system (11, 12)
and colorectal tumors (13).
PTTG1 encodes for a securin involved in the regulation of
chromatid separation during cell division. Conceivably, when
PTTG1 is abnormally high in cells, disruption of cell division
and chromosomal instability may occur and, thereby, the cells
become vulnerable to the accumulation of more mutations
during ensuing divisions. Subsequent chromosomal
aneuploidy and genetic instability may lead to the activation
of proto-oncogenes or loss of heterozygosity of tumor
suppressors, resulting in malignant transformation. Moreover,
PTTG1 also regulates the secretion of the basic fibroblast
growth factor (bFGF) (14), which induces angiogenesis, a key
determinant and rate-limiting step in tumor progression and
metastatic spread. These lines of evidence indicate that
overexpression of PTTG1 may play an important role in the
development and progression of human cancer.
Prostate cancer is one of the leading causes of cancerrelated death in men in the Western world. Although a large
percentage of prostate cancers are manageable with androgen
depletion therapy at an early stage of the disease, the majority
of them will progress to the androgen-independent stage after
2-3 years and, currently, there is no effective way to control
tumor growth at this stage(15). Therefore, it is essential to
identify novel markers that are specifically expressed in tumor
cells for the early prognosis of prostate cancer. In this study,
using clinical tissue specimens, an up-regulation of the PTTG1
protein in prostate cancer and its positive correlation with
Gleason grading were demonstrated. In addition, to study the
direct effect of PTTG1 gene overexpression on prostate
cancer growth, the PTTG1 gene was ectopically expressed into
a prostate cancer cell line, LNCaP, while it was also
inactivated by antisense technology in the same cell line. Our
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ANTICANCER RESEARCH 26: 1253-1260 (2006)
results demonstrated that PTTG1 is a key factor in the growth
of prostate cancer cells and that inactivation of the PTTG1
gene may be a therapeutic target for suppression of prostate
cancer cell growth.
Materials and Methods
Prostate samples and cell culture. Fifty-five prostate specimens were
obtained from a tissue bank in the Institute of Urology, Peking
University, China. Forty-one specimens were diagnosed as prostate
carcinoma and 14 as benign prostate hyperplasia. Of the 41 prostate
carcinoma samples, normal prostatic tissues adjacent to the tumors
were available from 18 cases. The samples were fixed in 10%
formaldehyde and paraffin-embedded. Frozen prostate cancer tissues
from 3 surgical samples and normal prostate tissue samples from 3
postmortem examinations were also obtained for Western blot
analysis. LNCaP, an androgen-dependent human prostate cancer cell
line, was grown in RPMI 1640 medium supplemented with 10% fetal
calf serum and 2 mM L-glutamine, in a 5% CO2 humidified
atmosphere at 37ÆC.
Immunohistochemistry. Paraffin sections of 5 Ìm thickness were used
for immunohistochemistry. The slides were rehydrated in xylene and
heated for antigen retrieval. Rabbit anti-human PTTG1 polyclonal
antibody and secondary were incubated with the sections,
respectively (Santa Cruz Biotechnology Inc., CA, USA). Signals were
developed by horseradish peroxidase-conjugated streptavidin and
diaminobenzidine. Cytoplasmic staining was semi-quantitated by
assessment in at least 5 randomly-selected light microscopic fields
(x400). The cytoplasmic staining was graded into negative staining
(–), weak (+), moderate (++) and strong staining (+++) by two
independent observers, and the results were expressed as the average
staining intensity.
Western blot analysis. Prostate tissues or cells were homogenized and
lysed in 2% SDS containing 1 mM phenylmethyl-sulfonylfluoride,
2 Ìg/ml aprotinin and 200 Ìg/ml leupeptin. The protein concentration
was determined by the Bradford assay using BSA as a standard.
Sixty Ìg protein were separated in 12% SDS-PAGE and transferred
to a nitrocellulose membrane. After blocking, the membrane was
blotted by the primary antibody 1:500 rabbit anti-human PTTG1
polyclonal antibody or 1:1000 goat anti-human actin polyclonal
antibody (Santa Cruz Biotechnology Inc.) for 24 h at 4ÆC. The
primary antibody was recognized by a secondary antibody (anti-rabbit
IgG) linked to horseradish peroxidase. The enhanced
chemiluminescence (ECL) method was used to detect the conjugated
horseradish peroxidase. Blotted signals were visualized by positive
bands on Hyperfilm ECL and were quantitated using a scanning
densitometer. Immunoblotting with actin antibody was used for
comparison of the sample loading in each lane.
Construction of recombinant plasmid expressing human PTTG1 and
stable transfection of LNCaP cells. Total RNA was isolated and PCR
was performed using primers as follows: forward primer: 5’-AGA
ATG GCT ACT CTG ATC TATG and reverse primer: 5’-CAC
AAA CTC TGA AGC ACT AAG to amplify the PTTG1 gene. The
PCR conditions involved an initial denaturing step of 95ÆC for 3 min,
32 cycles of 94ÆC for 30 sec, 50ÆC for 45 sec and 72ÆC for 60 sec. At
the end of the amplification, there was an elongation step at 72ÆC for
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10 min. The PCR product was purified and cloned into the pGEM-TEasy vector (Promega Corp., Madison, WI, USA), and the coding
region of human PTTG1 cDNA was confirmed by sequencing. The
recombinant pGEM plasmid was digested with EcoRI and the insert
was cloned into the EcoRI site of the eukaryotic expression vector
pIRES2-EGFP (Clontech, Palo Alto, CA, USA) in a sense or antisense direction. The two recombinant pIRES2-EGFP plasmids were
confirmed again by Sal I digestion and sequencing. LNCaP cells were
transfected with the two plasmids containing either sense or antisense
of the PTTG1 gene using Lipofectamine 2000 (Invitrogen, Carlsbad,
CA, USA), following the manufacturer’s protocol. After transfection
for 24 h, the cells were serially-diluted and grown in selection medium
containing 500 Ìg/ml G418 for 2 weeks. Finally, 3 cell lines were
generated: LNCaP cells stably transfected with sense PTTG1 cDNA
(LNCaP/PTTG1), with anti-sense PTTG1 cDNA (LNCaP/ASPTTG1) and with the vector (LNCaP/vector). PTTG1 protein
expression in the 3 cell lines was examined by Western blot.
Flow cytometry. Cells stably expressing sense, anti-sense PTTG1
cDNA or empty vector were trypsinized, washed with PBS and fixed
in 2 ml 70% cold ethanol at 4ÆC overnight, then treated with
propidium iodide and ribonuclease A for 30 min. The cell cycle
analysis was performed using a fluorescence-activated cell sorter.
3-(4,5-Dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
assay. To study the effect of PTTG1 overexpression on proliferation,
the MTT assay was performed using CellTiter 96 (Promega),
following the manufacturer’s protocol. Stably transfected LNCaP cells
were cultured in RPMI 1640 medium supplemented with 10% fetal
calf serum and 2 mM L-glutamine at 2x103 cells/well in a 96-well plate
and incubated at 37ÆC for 1-7 days. To measure cell viability,
dimethyl sulphoxide (DMSO) was added to each well and the plate
was further incubated at 37ÆC for 1 h. Absorbance at 570 nm was
measured using an ELISA reader. The absorbance is directly
proportional to the number of living cells in a culture. For each timepoint, the experiment was carried out in triplicate and was repeated
at least twice. The data were analyzed using GraphPad Prism
(GraphPad Software, Inc., San Diego, CA, USA).
Colony formation assay. Cells (5x103) were plated into a 100-mm
tissue culture dish containing G418 with RPMI 1640 medium
supplemented with 10% fetal calf serum and incubated for 2 weeks to
allow colonies to develop. The medium was replaced every week. To
count the colony number, the medium was removed and the colonies
were washed with PBS, stained with 0.5% crystal violet for 5 min,
then washed with de-ionized water to remove the excess stain.
Stained colonies larger than 1 mm in diameter were counted. Each
colony formation assay was carried out in triplicate and repeated at
least 3 times. All the data obtained from the colony formation assays
were analyzed using GraphPad Prism.
In vivo tumorigenicity. LNCaP/PTTG1, LNCaP/AS-PTTG1 or
LNCaP/vector transfected LNCaP cells (1x106 in 250 Ìl of regular
RPMI 1640 medium mixed with an equal volume of Matrigel) were
subcutaneously inoculated into the armpits of nude mice (male, aged
4 to 6 weeks). The length and width of the tumor were measured
weekly. The tumor volume was calculated using the formula: volume
= (length x width2)/2. Tumor formation was defined as a size of >40
mm3. After 2 months, the animals were sacrificed and the tumors
were weighed.
Figure 1. PTTG1 expression in prostate tissues. A. PTTG1 expression in prostate cancer, adjacent normal tissues and benign prostate hyperplasia (BPH) examined by immunostaining. Note that the
PTTG1 protein is undetectable in non-malignant tissues while it is up-regulated in cancer tissues. B. Western blotting analysis of PTTG1 expression in the non-malignant and cancer tissues (indicated
as T). Note that PTTG1 protein expression levels are much lower in the BPH and normal tissues compared to the cancer tissues.
Zhu et al: PTTG1 Gene in Prostate Cancer
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ANTICANCER RESEARCH 26: 1253-1260 (2006)
Table I. PTTG1 expression in prostate cancer, benign prostate hyperplasia
and normal prostate tissue adjacent to the cancer.
Table II. PTTG1 expression in different clinical stage and differentiation
of prostate cancer.
Immunostaining for PTTG1
Sample
Clinical course
(ABCD stage)
No. of cases
Positive
(+ to +++)
Negative
(–)
Prostate cancer
Benign prostate hyperplasia
Normal prostate tissue
adjacent to the cancers
41
14
18
34 (82.9%)*
5 (35.7%)*
0 (0%)
7
9
18
Total
73
39 (53.4%)
34
A
B
C
Differentiation of cancer cells
(Gleason score)
Well Moderate
Poor
No. of cases
5
22
14
5
27
6
Immunostaining
4
20
10
1
23
5
PTTG1
(+ – +++)
(80%) (90.9%) (71.4%) (20%) (85.2%)* (83.3%)*
Immunostaining
PTTG1 (–)
1
2
4
4
4
1
* p<0.05, Student's t-test, as compared to that of normal prostate
tissues adjacent to the cancers.
* p<0.05 as compared to that of the well-differentiated group.
Statistical analysis. The results are expressed as mean±SEM.
Statistical analysis was performed using analysis of variance
(ANOVA) and Student's t-test by the SPSS11.0 statistical package,
taking p values less than 0.05 as significant.
no statistical significance was found between moderately- and
poorly-differentiated cancers (p<0.05). These results suggest
that high PTTG1 expression may indicate unfavorable
prognosis in prostate cancer patients.
Results
Differential PTTG1 expression in malignant and non-malignant
prostate tissues. Immunohistochemistry was performed to
estimate the PTTG1 expression in 41 prostate cancer, 14
benign prostate hyperplasia and 18 normal prostate tissues
adjacent to the cancers (Figure 1A). In contrast to the
negative results observed in the non-malignant tissues, PTTG1
was detected in 34 out of 41 (82.9%) of the cancer and 5 out
of 14 (35.7%) of the benign prostate hyperplasia (Table I). To
further confirm these results, Western blotting was performed
on 3 pairs of malignant and non-malignant prostate tissues.
As shown in Figure 1B, the expression of PTTG1 was much
lower in the non-malignant tissues compared to the cancerous
tissues. These results suggest an up-regulation of PTTG1
protein expression in prostate cancer.
Correlation of PTTG1 expression with clinical stage and Gleason
score of prostate cancer tissues. To further study if there was a
correlation between PTTG1 expression and prostate cancer
staging, we analyzed the PTTG1 expression among different
stages of prostate cancer specimens. It was found that positive
PTTG1 immunostaining in clinical stages A, B and C were 4/5
(80%), 20/22 (90.9%) and 10/14 (71.4%), respectively. No
statistical difference (p>0.05) was found among the 3 stages.
For the differentiation degree of the cancer cells, Gleason
scoring was commonly used. Positive PTTG1 immunostaining
in well-, moderately- and poorly-differentiated cancers were
1/5 (20%), 23/27 (85.2%) and 5/6 (83.3%), respectively (Table
II). The positive PTTG1 rate in immunostaining in welldifferentiated cancers was significantly lower than that in
moderately- and poorly-differentiated cancers (p<0.05), but
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Effect of PTTG1 overexpression on cell proliferation. In order
to study the direct effect of PTTG1 on prostate cancer cell
growth, expression vectors containing the PTTG1 cDNA
(PTTG1), antisense PTTG1 (AS-PTTG1) as well as the
control vector were transfected into LNCaP cells and the
following stable transfectants generated: LNCaP/PTTG1,
LNCaP/AS-PTTG1 and LNCaP/vector. Western blotting
analysis showed (Figure 2A) that, while LNCaP/PTTG1 had
much higher levels of PTTG1, the LNCaP/AS-PTTG1 cells
showed the lowest PTTG1 expression compared to the vector
control (LNCaP/vector).
Cell cycle analysis showed (Figure 2B, arrows) that the
percentage of S+G2- phase cells was much higher in the
LNCaP/PTTG1 cells (S+G2=60.8%) but much lower in the
LNCaP/AS-PTTG1 cells (S+G2=26.7%) compared to the
vector control (S+G2=45.8%). In contrast, the percentage of
cells in the G1-phase was lower in the PTTG1 transfectants
(G1=39.2%) but higher in the AS-PTTG1 transfectants
(G1=73.3%) compared to the vector control (G1=54.2%)
(Figure 2B). These results suggest that overexpression of
PTTG1 promotes cell cycle progress and the inactivation of
PTTG1 results in cell cycle G1 arrest.
To further confirm the flow cytometry results and study
whether PTTG1 had any effect on the cell proliferation rate,
the MTT assay was performed. As shown in Figure 2C,
LNCaP/PTTG1 cells exhibited a much higher proliferation rate
(upper dotted line) than the vector control (solid line), while
LNCaP/AS-PTTG1 cells (lower dotted line) showed a lower
proliferation rate, especially at later time-points (i.e. days 4-5)
compared to the vector control. These results indicated that
PTTG1 plays a positive role in the proliferation of prostate
cancer cells.
Zhu et al: PTTG1 Gene in Prostate Cancer
Figure 2. Effect of PTTG1 expression on prostate cancer cell proliferation.
Vectors containing the PTTG1, AS-PTTG1 were transfected into LNCaP
cells and stable transfectants were generated. A. PTTG1 expression in
LNCaP/ PTTG1, LNCaP/AS-PTTG1 and LNCaP/vector examined by
Western blot. B. Cell cycle analysis of the vector control, PTTG1 and
AS-PTTG1 transfectants. C. MTT assay of cell proliferation rate. **
p<0.01 as compared to the LNCaP/vector.
Effect of PTTG1 on tumorigenicity of LNCaP cells. The colony
forming assay showed that the LNCaP/PTTG1 cells formed
more and larger colonies of 150±6 colonies/dish as compared
to the control groups (p<0.01). In contrast, wild-type LNCaP,
LNCaP/vector and LNCaP/AS-PTTG1 exhibited 40±1, 30±6
and 6±1 colonies/dish, respectively (Figure 3A). To further
confirm these results, 3 transfectant cell lines were injected into
nude mice and tumorigenesis was studied. As shown in Figure
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ANTICANCER RESEARCH 26: 1253-1260 (2006)
Figure 3. Effect of PTTG1 on in vitro and in vivo tumorigenicity of
LNCaP cells. A. Colony formation assay showing decreased colony
forming ability of AS-PTTG1 cells (panel D) compared to the cell lines
with high levels of PTTG1 (panels B-C). B. Tumor formation of LNCaP/
PTTG1 (middle), LNCaP/AS-PTTG1 (right) and LNCaP/vector (left) in
nude mice (circled area). Each mouse was injected subcutaneously with
1x106 cells and sacrificed after 2 months.
3B and Table III, after 2 months large tumors were formed in
the mice injected with LNCaP/PTTG1 (circled area). In
contrast, no tumor formation was found when the mice were
inoculated with LNCaP/AS-PTTG1, and much smaller tumors
were formed in mice inoculated with the LNCaP/vector. These
results suggest that overexpression of PTTG1 in LNCaP cells
promoted tumorigenesis, while inactivation of the PTTG1 gene
suppressed the tumorigenicity of LNCaP cells in nude mice.
Discussion
In this study, using clinical prostate tissues, PTTG1 was found
to be highly expressed in prostate cancer tissues, but at low
levels in normal prostate tissues (Figure 1). In addition, higher
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PTTG1 expression was found to be closely correlated to the
Gleason score but not to the clinical stage of the disease
(Table I). Since the Gleason score shows a better correlation
with the aggressiveness of prostate cancer, including cell
proliferation, aneuploidy, activation of oncogenes and
mutations of tumor suppressor genes (16-20), our results
suggest that increased PTTG1 protein expression may be an
indicator of poor clinical outcome in prostate cancer patients.
These results also agree with previous reports on pituitary and
colorectal cancers (7-9), supporting the hypothesis that
PTTG1 may be a potential oncogene.
The results generated from the cell culture and animal
experiments in this study suggest that the ability of LNCaP cells
to proliferate and form tumors was greatly affected by the
Zhu et al: PTTG1 Gene in Prostate Cancer
Table III. In Vivo tumorigenesis of LNCaP/PTTG1 in nude mice.
LNCaP
cell line
inoculated
LNCaP/vector
LNCaP/PTTG1
LNCaP/AS-PTTG1
No. of
inoculated
animals
Tumor
formation
after 2 months
Tumor
volume
(cm3)
6
6
6
3/6
5/6
0/6
0.22±3
0.51±1
0
7
8
9
expression levels of PTTG1. When LNCaP cells was ectopically
expressed the PTTG1 gene, their replication processes and
tumorigenesis were significantly enhanced (Figures 2 and 3). As
a consequence, PTTG1 may be a marker for invasive prostate
cancer as indicated by the immunostaining results. The fact that
the down-regulation of PTTG1 in prostate cancers suppressed
tumor cell proliferation and tumor formation in nude mice
strongly suggests a novel therapeutic target for the suppression
of prostate cancer growth. Our results are also supported by
previous studies on NIH 3T3 cells, where overexpression of
PTTG1 led to cellular transformation in vitro and promoted
tumor formation in vivo (4).
In conclusion, our observations suggest that PTTG1
expression was substantially enhanced in prostate cancer tissues
compared with normal prostate tissue. PTTG1 expression in
tumors had a significant positive correlation to their Gleason
score. It was also demonstrated that the up- or down-expression
of PTTG1 can significantly change the cell cycle progression,
the in vitro proliferation rate and the in vivo tumorigenesis of
human LNCaP cells. PTTG1 may, therefore, play an important
role in the early molecular events leading to the generation,
progression and prognosis of prostate carcinoma. It may have
the potential to serve as a therapeutic target for prostate cancer.
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Received September 23, 2005
Revised January 13, 2006
Accepted January 26, 2006
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