Absence of PIWIL2 (HILI) expression in human bladder cancer cell lines and tissues
Parvaneh Nikpoura, Mehdi Forouzandeh-Moghaddamb, Seyed Amir-Mohsen Ziaeec, Olusola Y. Dokund,
Wolfgang Arthur Schulzd, Seyed Javad Mowlaa,*
aDepartment of Genetics, Faculty of Basic Sciences, Tarbiat Modares University, P.O. Box 14115-175, Tehran, Iran
bDepartment of Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, P.O. Box 14115-115, Tehran, Iran
cUrology and Nephrology Research Center and Shahid Labbafinejad Medical Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
dDepartment of Urology, Heinrich Heine University, Moorenstr. 5, 40225 Du ¨sseldorf, Germany
Recent studies have shown many similarities between basic
mechanisms mediating stem and cancer cell self-renewal [1,2].
The stem cell model for cancer hypothesizes that a key event in
tumorigenesis is the disruption of genes involved in the regulation
of stem cell self-renewal [1,2]. Therefore, the study of stem cell
self-renewal genes has a great potential to expand our under-
standing of carcinogenesis and to develop novel strategies for
preventing and treating cancers .
Argonaute proteins,alsoknown asPAZ (Piwi/Argonaute/Zwille)
Piwi (P-element induced wimpy testis) domain (PPD) proteins,
are members of a well-conserved family that is expressed in a
variety of organisms, from fission yeasts to humans. The family
can be divided into two subfamilies, Piwi and Ago, based on their
primary sequence homology and expression pattern. Piwi
subfamily members are expressed only in germ lineage cells,
whereas members of the Ago subfamily are expressed ubiqui-
The Piwi subfamily represents the first class of genes known to
be essential for stem cell self-renewal in various organisms .
Members of Piwi family play important roles in RNA silencing ,
translational regulation , gametogenesis  as well as stem cell
self-renewal . In human, four members of the family have been
identified: PIWIL1 (HIWI), PIWIL2 (HILI), PIWIL3 and PIWIL4
(HIWI2). All four members of Piwi family are primarily expressed
in testis . Expression of human PIWIL2 (HILI), located on 8p21.3,
has been reported in a variety of tumor cells originating from not
only germ but also somatic cells, including prostate, breast,
gastrointestinal, ovarian and endometrial cancers . Ectopic
expression of the gene has also been reported in tumors of breast,
rhabdomyosarcoma, and medulloblastoma of mouse, where it
apparently inhibits apoptosis through activation of Stat3/Bcl-XL
To investigate a potential involvement of PIWIL2 in human
bladder cancer, we examined the expression of the gene in bladder
specimens with or without malignant lesions as well as a series of
Cancer Epidemiology 33 (2009) 271–275
A R T I C L EI N F O
Accepted 29 June 2009
A B S T R A C T
Background: PIWIL2, a member of Argonaute family of proteins, is exclusively expressed in testis and
functions in development and maintenance of germline stem cells. Recently, ectopic expression of
PIWIL2 has been reported in a variety of human and mouse tumors. To investigate a potential
involvement of PIWIL2 in human bladder cancer, we examined its expression in several human bladder
cancer cell lines, normal uroepithelial cell cultures, and some bladder tissues. Methods: Relative
expression of PIWIL2 was determined by real-time quantitative RT-PCR in fifteen bladder carcinoma cell
lines, six normal uroepithelial cell cultures and seventy tissue specimens of tumor, tumor margins and
morphologically normal tissues of bladder. Specific primers for PIWIL2, TBP and GAPDH (as two internal
controls) were used for reverse transcription polymerase chain reaction technique. Results: Real-time
qRT-PCR demonstrated high PIWIL2 expression in testis tissue, but at least 240-fold lower expression in
all examined cell lines. The highest expression outside testis was observed in one of six primary cultures
of normal uroepithelial cells, but even lower expression of PIWIL2 was detected in any of the examined
tumor and non-tumor tissues. Conclusion: Lack of PIWIL2 expression in most tissues along with its
aberrant expression in some tumors candidate the gene as an attractive tumor marker for some
neoplasms. However, our study indicates that PIWIL2 does not play a role in carcinogenesis of human
? 2009 Elsevier Ltd. All rights reserved.
* Corresponding author. Tel.: +98 21 82883464; fax: +98 21 82883463.
E-mail address: email@example.com (S.J. Mowla).
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cell lines from invasive bladder carcinomas and normal uroe-
pithelial cell cultures.
2. Methods and materials
2.1. Cell lines and cell culture
Collection of Microorganisms and Cell Cultures (DSMZ, Braunsch-
weig), and were cultured in DMEM (Gibco Life Technologies,
Karlsruhe, Germany), supplemented with 10% fetal calf serum and
100 mg/ml penicillin/streptomycin, as described elsewhere .
Normal uroepithelialcells (NUECs) were prepared from the ureters
of nephrectomy patients by a standard method  with minor
modifications . Normal cells were routinely maintained in
keratinocyte serum-free medium (KSFM, Gibco Life Technologies)
supplemented with 50 mg/ml bovine pituitary extract (BPE), 5 ng/
the first passage, at which nonepithelial cells were removed, they
were used for experiments.
2.2. Tissue samples
The bladder tumor and non-tumor tissue samples were
obtained from Urology and Nephrology Research Center (UNRC)
at Tehran, as described in our previous studies [12,13]. A total of
seventytissue samples wereexaminedfor PIWIL2expression. They
consisted of 32 non-tumor and 38 tumor samples of bladder.
Table 1 summarizes some characteristics of the patients with
bladder cancer. Testis tissues from two healthy men undergoing
TESE (testicular sperm extraction) operation was obtained from
Shayan-Mehr Medical Center and used in all assays, as a positive
control. The experimental design was approved by the Ethics
Committees of Tarbiat Modares University and the UNRC. The
patients’ written informed consents were obtained prior to
2.3. RNA extraction
Total RNA was isolatedfrom sub-confluent cellculturesand cell
lines using Qiazol reagent (Qiagen, Hilden, Germany) and purified
via RNeasy columns (Qiagen, Hilden, Germany). RNX plus solution
(Cinnagen, Tehran, Iran) was used for extraction of total RNA from
powdered tissues according to the manufacturer’s instructions.
cDNA synthesis was performed using SuperScriptII reverse
transcriptase (Promega, Mannheim, Germany) or RevertAid
MMuLV Reverse Transcriptase (Fermentas, Vilnius, Lithuania)
with oligo-dT primers as described previously [12–14].
2.4. Real-time qRT-PCR
Real-time qRT-PCR assays were performed using the Light-
Cycler II (Roche, Germany), in the case of all cell lines and primary
cell cultures or Rotor-gene 3000 (Corbett, Australia) for tissue
samples. Real-time qRT-PCR for PIWIL2, GAPDH and TBP mRNAs
was performed using specific primers (Table 2) with the
QuantiTect SYBR Green PCR kit (Qiagen, Hilden, Germany) or
the LightCycler-FastStart DNA Master PLUS SYBR Green I kit
(Roche, Mannheim, Germany). The PCR for the genes included an
initial denaturation step at 95 8C for 10 min, followed by 40
amplification cycles consisting of denaturation at 95 8C for 10 s,
55 8C (59 8C for GAPDH) for 20 s and an extension at 72 8C for 20 s.
All measurements were performed in at least triplicates.
The identity of PCR products were further confirmed on a 2%
agarose gel, stained with ethidium bromide, and visualized under
the ultraviolet light. Relative gene expression was calculated using
the standard curve method.
3.1. Optimization of RT-PCR reaction
carried out for both ordinary and real-time RT-PCR reactions on
5637 cell line, using specific primers for human PIWIL2 gene. Since
PIWIL2 has been reported to be expressed at the mRNA level in
human normal testis tissue, we used a testis tissue sample as a
positive control in all RT-PCR reactions. Electrophoresis of the PCR
products on agarose gels demonstrated single bands with the
expected sizes for the amplified PIWIL2 (303 bp) and TBP (128 bp)
segments. The results also revealed the absence of PIWIL2
expression in 5637 cell line (Fig. 1A). Analysis of gene expression
using real-time PCR showed a unique melting curve without
primer dimers (Fig. 1B), which was further confirmed by agarose
gel separation and staining. To optimize our quantitation
technique, a cDNA synthesized from a testis tissue sample was
used in serial dilution and a PCR efficiency near to 100% was
obtained (Fig. 1C).
3.2. No expression of PIWIL2 gene in bladder normal uroepithelial
cells and carcinoma cell lines
After optimization of the real-time PCR, expression of PIWIL2
gene was analysed using real-time qRT-PCR in the bladder
carcinoma cell lines 253J, 5637, 639V, 647V, BFTC905, BFTC909,
HT1376, J82, RT4, RT112, SD, SW1710, UMUC3, VMCub I and
VMCub II, almost all derived from invasive bladder cancers. The
results of real-timeqRT-PCR experiments demonstrated that while
expression of PIWIL2 in testis tissue was easily detectable, its
expression was very low or undetectable in all examined cell lines.
A brief description of patients with bladder cancera.
Mean age, years
Age range, years
60.94 ? 13.46
aValues in parentheses are percents.
Sequences of the designed PCR primers and the expected sizes of the amplicons.
DesignationSequenceProduct size (bps)
P. Nikpour et al./Cancer Epidemiology 33 (2009) 271–275
The highest level in any urothelial cell was observed in one of six
primary cultures of normal uroepithelial cells (NUEC111) (Fig. 2).
The expression level in this cell culture was 240-fold lower than
that in the testis tissue.
3.3. PIWIL2 is not expressed in bladder tumor and non-tumor tissues
Next,we investigatedtherelativeexpression ofthe PIWIL2gene
in human bladder tissues. No expression of PIWIL2 was detected in
any of the examined tumor and non-tumor tissues of bladder. To
make sure that the lack of PIWIL2 expression was not due to a
technical problem, a positive control of a testis tissue sample
accompanied each reaction (Fig. 3). Moreover, analysis of the
control gene, TBP, proved that RNA extraction and cDNA synthesis
had proceeded correctly. From the PIWIL2 value in testis and the
PIWIL2 in benign or tumorous bladder tissue cannot exceed 0.5% of
the level in testicular tissue.
The genes of the Piwi subfamily have so far been described in
various organisms from jelly fish to human [4,15–21]. They belong
to the Argonaute family of proteins, which function primarily in
development and maintenance of germline stem cells .
Although the Piwi genes were initially isolated from a human
testis cDNA library , further analysis of the expression pattern of
PIWIL1 (HIWI) and PIWIL2 (HILI) revealed that they are also
expressed in a wide range of tissues including prostate, ovary,
small intestine, heart, brain, liver, skeletal muscle and kidney
[9,22,23]. Lee et al.  found enhanced expression of PIWIL2 in
testicular seminomas, but not in testicular nonseminomatous
tumors. PIWIL2 was also expressed in human and mouse tumors of
various tissues. Over-expression of mouse Piwil2 in a fibroblast cell
line increased Bcl-XLexpression, which correlated with increased
silencing via small interfering RNA suppressed Stat3 and Bcl-XL
expression and induced apoptosis. Lee et al.  concluded that
PIWIL2 acts as an oncogene by inhibiting apoptosis and promoting
proliferation via a Stat3/Bcl-XLsignaling pathway. The finding that
the expression of PIWIL family members may contribute to the
development of various epithelial solid tumors prompted us to
investigate the potential expression of PIWIL2 in human bladder
Since there was no report on PIWIL2 expression in bladder
cancer, we first started our investigation by screening fifteen
different bladder carcinoma cell lines and six primary cultures of
the normal uroepithelial cells for PIWIL2 gene expression. Except
uroepithelial cells, we failed to observe any expression of the gene
in other normal uroepithelial cells. This finding is consistent with a
previous study on the PIWIL2 gene , which indicated that the
gene is not expressed in normal somatic cells. Interestingly, we did
not observe expression of PIWIL2 in any of the examined bladder
carcinoma cell lines as well. In human, expression of PIWIL2 has
been reported in prostate cancer cell lines (PC-3, LNCAP, DU-145),
Fig. 1. Optimization of PIWIL2 RT-PCR. (A) RT-PCR analysis of PIWIL2 and TBP (as an
internal control) expression in the 5637 carcinoma cell line and a testis tissue
sample (as a positive control). (B) A unique melting curve without primer dimers
showing specific amplification of PIWIL2 on real-time PCR. (C) Amplification curves
of a testicular cDNA dilution series for determination of amplification efficiency.
Fig. 2. Relative expression of PIWIL2 in fifteen bladder cancer cell lines, six normal
urothelial cells and a testis tissue sample. Histograms comparing the relative gene
expression of PIWIL2 to GAPDH as determined by quantitative RT-PCR. Values
shown represent the mean ? STD.
Fig. 3. Relative expression of PIWIL2 in seventy tumor and non-tumor tissue
samples of bladder. No expression of PIWIL2 (normalized to TBP) was observed in
bladder tumor and non-tumor tissues as determined by real-time qRT-PCR. A testis
tissue sample was used as the positive control (the right dashed bar). Values shown
represent the mean ? STD.
P. Nikpour et al./Cancer Epidemiology 33 (2009) 271–275
breast cancer cell lines (MDA-MB-231, MCF-7), cervical cancer
(HeLa), T-cell leukemia (Jurkat), Burkitt’s lymphoma (Daudi) and
embryonal carcinoma (2102EP) . However, it is difficult to
estimate the level of expression in those cancers because the paper
by Lee et al. did not use quantitative RT-PCR. Absence of PIWIL2
expression in all of the examined bladder carcinoma cell lines may
indicate that this gene does not play a role in carcinogenesis of
human bladder carcinoma.
We then extended our study to assess PIWIL2 gene expression
in humanbladder tissue specimens.Again, therewas nodetectable
expression of the gene in tissues obtained from tumors or the non-
tumor specimensobtained from the margin of the same tumors. To
make sure that the lack of PIWIL2 expression inbladder tissues was
not due to a technical problem, we examined the expression of the
gene in a testis tissue specimen. As expected, a high expression of
PIWIL2 was found in testis sample, further confirming the absence
of the PIWIL2 expression in bladder cancers. Our result confirms
the findings of Lee et al.  in so far as they found no expression of
PIWIL2 in any of the examined human normal somatic tissues.
However, we also observed no expression of PIWIL2 in any of the
examined bladder tumor tissues.
This finding may indicate that ectopic expression of PIWIL2 is
not essential for the pathogenesis of human bladder carcinoma.
Accordingly, since over-expression of the Bcl-XL anti-apoptotic
gene has been reported in bladder carcinoma [24,25], there must
be other regulators for targeting Stat3/Bcl-XLsignaling pathway in
So far, the expression profile of other PIWIL family members has
been investigated in different tumor entities. Sharma et al. 
analyzed PIWIL1 (HIWI) gene expression in eight immortalized
leukemia cell lines but found no detectable expression by RT-PCR.
Similarly, Qiao et al.  reported that PIWIL1 is only over-
expressed in seminomas, but not in nonseminomas or in somatic
tumors of the adult testis. On the other hand, PIWIL1 over-
expression has been reported in different gastric cancer cell lines
and tissues . In patients with soft-tissue sarcoma, elevated
PIWIL1 mRNA transcript levels were associated with an increased
risk of tumor-related death . Furthermore, Grochola et al. 
reported elevated levels of PIWIL1 mRNA transcripts in 40 out of 56
microdissected PDAC (ductal adenocarcinoma of the pancreas)
tissues. In anattemptto prepare antibodies against humanPIWIL3,
Wang et al.  reported the detection of PIWIL3 protein in the
cytoplasm of human astrocytic glioma and meningioma. In
addition, transient transfection of PIWIL4, the only member of
the PIWIL family that is ubiquitously expressed in human tissues,
induces histone H3 lysine methylation at the p16Ink4a(CDKN2A)
locus, resulting in the downregulation of this tumor suppressor
gene . To the best of our knowledge, besides the above listed
tumor types, no other reports on the role of PIWIL1, 3 and 4
expressions in tumor development have been published. There-
fore, further investigating the potential expression of these genes,
at both mRNA and protein levels, in various tumor types would
expand our knowledge on the potential role of PIWIL family on
carcinogenesis. Interestingly, certain other genes restricted to
testis, i.e. cancer-testis antigens, have been observed to be
ectopically expressed in bladder, e.g. several MAGE-A genes
Finding new molecular markers with differential expression in
tumor and normal cells has a pivotal role in diagnosis and
treatment of cancer. The PIWIL family of proteins, as regulators of
stem cell self-renewaland cancer proliferation,has the potential to
be utilized in future as new tumor markers in a variety of cancer
types. The present data report for the first time that PIWIL2 is not
expressed in either human normal uroepithelial cells or bladder
carcinoma cell lines and tissues; suggesting that the gene is not an
essential player in the tumorigenesis of bladder cancer.
Conflict of interest
The authors have no conflict of interest.
We are grateful to Ms. Nasim Hatefi and Nasim Ghorbanmehr
for their assistance in collecting bladder and testis tissue samples.
P.N. is supported partially by Iranian nanotechnology initiative. All
the samples were provided by Shahid Labbafinejad Medical Center
and the experiments were performed in Tarbiat Modares and
Heinrich-HeineUniversities. This work was supported, inpart, by a
research grant from the Urology and Nephrology Research Centre
 Reya T, Morrison SJ, Clarke MF, Weissman IL. Stem cells, cancer, and cancer
stem cells. Nature 2001;414(6859):105–11.
 Al-Hajj M, Clarke MF. Self-renewal and solid tumor stem cells. Oncogene
 Bui M, Reiter RE. Stem cell genes in androgen-independent prostate cancer.
Cancer Metast Rev 1998;17(4):391–9.
of the Argonaute family in the human genome small star, filled. Genomics
 Cox DN, Chao A, Baker J, Chang L, Qiao D, Lin H. A novel class of evolutionarily
conservedgenes defined bypiwi areessential forstem cellself-renewal. Genes
 Kuramochi-Miyagawa S, Watanabe T, Gotoh K, Totoki Y, Toyoda A, Ikawa M,
et al. DNA methylation of retrotransposon genes is regulated by Piwi family
members MILI and MIWI2 in murine fetal testes. Genes Dev 2008;22(7):908–
 Carmell MA, Xuan Z, Zhang MQ, Hannon GJ. The Argonaute family: tentacles
that reach into RNAi, developmental control, stem cell maintenance, and
tumorigenesis. Genes Dev 2002;16(21):2733–42.
 Kuramochi-Miyagawa S, Kimura T, Ijiri TW, Isobe T, Asada N, Fujita Y, et al.
Mili, a mammalian member of piwi family gene, is essential for spermatogen-
esis. Development (Cambridge England) 2004;131(4):839–49.
 Lee JH, Schutte D, Wulf G, Fuzesi L, Radzun HJ, Schweyer S, et al. Stem-cell
protein Piwil2 is widely expressed in tumors and inhibits apoptosis through
activation of Stat3/Bcl-XL pathway. Human Mol Genet 2006;15(2):201–11.
 Swiatkowski S, Seifert HH, Steinhoff C, Prior A, Thievessen I, Schliess F, et al.
Activities of MAP-kinase pathways in normal uroepithelial cells and urothelial
carcinoma cell lines. Exp Cell Res 2003;282(1):48–57.
 Southgate J, Hutton KA, Thomas DF, Trejdosiewicz LK. Normal human urothe-
lial cells in vitro: proliferation andinduction ofstratification. LabInvest; JTech
Methods Pathol 1994;71(4):583–94.
 Atlasi Y, Mowla SJ, Ziaee SA, Bahrami AR. OCT-4, an embryonic stem cell
marker, is highly expressed in bladder cancer. Int J Cancer 2007;120(7):1598–
 Shafaroudi AM, Mowla SJ, Ziaee SA, Bahrami AR, Atlasi Y, Malakootian M.
Overexpression of BMI1, a polycomb group repressor protein, in bladder
tumors: a preliminary report. Urol J 2008;5(2):99–105.
 Hoffmann MJ, Muller M, Engers R, Schulz WA. Epigenetic control of CTCFL/
BORIS and OCT4 expression in urogenital malignancies. Biochem Pharmacol
 Bohmert K, Camus I, Bellini C, Bouchez D, Caboche M, Benning C. AGO1 defines
a novel locus of Arabidopsis controlling leaf development. EMBO J 1998;17(1):
 Moussian B, Schoof H, Haecker A, Jurgens G, Laux T. Role of the ZWILLE gene in
the regulation of central shoot meristem cell fate during Arabidopsis embry-
ogenesis. EMBO J 1998;17(6):1799–809.
 Reinke V, Smith HE, Nance J, Wang J, Van Doren C, Begley R, et al. A global
profile of germline gene expression in C. elegans. Mol Cell 2000;6(3):
 Seipel K, Yanze N, Schmid V. The germ lineand somaticstem cell gene Cniwi in
the jellyfish Podocoryne carnea. Int J Dev Biol 2004;48(1):1–7.
 Cox DN, Chao A, Lin H. Piwi encodes a nucleoplasmic factor whose activity
modulates the number and division rate of germline stem cells. Development
(Cambridge England) 2000;127(3):503–14.
 Tan CH, Lee TC, Weeraratne SD, Korzh V, Lim TM, Gong Z. Ziwi, the zebrafish
homologue of the Drosophila piwi: co-localization with vasa at the embryonic
genital ridge and gonad-specific expression in the adults. Mech Dev
 Kuramochi-Miyagawa S, Kimura T, Yomogida K, Kuroiwa A, Tadokoro Y,
Fujita Y, et al. Two mouse piwi-related genes: miwi and mili. Mech Dev
 Liu X, Sun Y, Guo J, Ma H, Li J, Dong B, et al. Expression of hiwi gene in human
gastric cancer was associated with proliferation of cancer cells. Int J Cancer
P. Nikpour et al./Cancer Epidemiology 33 (2009) 271–275
 Sharma AK, Nelson MC, Brandt JE, Wessman M, Mahmud N, Weller KP, et al. Download full-text
Human CD34(+) stem cells express the hiwi gene, a human homologue of the
Drosophila gene piwi. Blood 2001;97(2):426–34.
 Gazzaniga P, Gradilone A, Silvestri I, Gandini O, Giuliani L, Vincenzoni A, et al.
Variable levels of bcl-2, bcl-x and bax mRNA in bladder cancer progression.
Oncol Reports 1998;5(4):901–4.
 Kirsh EJ, Baunoch DA, Stadler WM. Expression of bcl-2 and bcl-X in bladder
cancer. J Urol 1998;159(4):1348–53.
 Qiao D, Zeeman AM, Deng W, Looijenga LH, Lin H. Molecular characterization
of hiwi, a human member of the piwi gene family whose overexpression is
correlated to seminomas. Oncogene 2002;21(25):3988–99.
 Taubert H, Greither T, Kaushal D, Wurl P, Bache M, Bartel F, et al. Expression of
the stem cell self-renewal gene Hiwi and risk of tumour-related death in
patients with soft-tissue sarcoma. Oncogene 2007;26(7):1098–100.
 Grochola LF, Greither T, Taubert H, Moller P, Knippschild U, Udelnow A, et al.
The stem cell-associated Hiwi gene in human adenocarcinoma of the pan-
creas: expression and risk of tumour-related death. Br J Cancer 2008;99(7):
 Wang XL, Chen XM, Gao HJ, Huang ZG. Preparation and distribution of
polyclonal antibodies against human PIWIL3 protein in tumor tissues.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi 2008;24(7):714–6.
 SugimotoK,Kage H,Aki N,Sano A,Kitagawa H,Nagase T,et al.Theinductionof
H3K9 methylation by PIWIL4 at the p16Ink4a locus. Biochem Biophys Res
 Fradet Y, Picard V, Bergeron A, LaRue H. Cancer-testis antigen expression in
bladder cancer. Prog Urol 2006;16(4):421–8.
 Picard V, Bergeron A, Larue H, Fradet Y. MAGE-A9 mRNA and protein expres-
sion in bladder cancer. Int J Cancer 2007;120(10):2170–7.
P. Nikpour et al./Cancer Epidemiology 33 (2009) 271–275