Human Molecular Genetics Advance Access originally published online on December 23, 2005
Human Molecular Genetics 2006 15(2):201-211; doi:10.1093/hmg/ddi430
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Stem-cell protein Piwil2 is widely expressed in tumors and inhibits apoptosis through activation of Stat3/Bcl-XL pathway
1Institute of Human Genetics, 2Department of Hematology and Oncology, 3Department of Gastroentropathology and 4Department of Pathology, University of Göttingen, 37073 Göttingen, Germany
* To whom correspondence should be addressed at: Institute of Human Genetics, University of Göttingen, Heinrich Düker Weg 12, 37073 Göttingen, Germany. Tel: +49 5513919669; Fax: +49 551399303; Email: knayern{at}gwdg.de
Received July 1, 2005; Accepted November 17, 2005
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The genes of the piwi family are defined by conserved PAZ and Piwi domains and play important roles in stem-cell self-renewal, RNA silencing and translational regulation in various organisms. Both, mouse and human Piwil2 genes, members of the piwi gene family, are specifically expressed in testis. We report here enhanced expression of the human Piwil2 gene in testicular seminomas, but not in testicular non-seminomatous tumors. Expression of the Piwil2 gene was also found in different tumors examined, including prostate, breast, gastrointestinal, ovarian and endometrial cancer of human and in breast tumors, rhabdomyosarcoma and medulloblastoma of mouse. Therefore, Piwil2 can be categorized as a novel member of cancer/testis antigens. To identify genes activated by Piwil2, RNA isolated from NIH-3T3 cells expressing constitutively Piwil2 were compared with RNA samples from control NIH-3T3 cells using a cancer gene array. Induction of high-level expression of the antiapoptotic gene Bcl-XL was observed in cells expressing Piwil2. Furthermore, increased Bcl-XL expression correlated with increase of signal transducer and activator of transcription 3 (Stat3) expression. Gene silencing of Piwil2 with its small interference RNA suppressed Stat3 and Bcl-XL expression and induced apoptosis. A causal link between Piwil2 expression and inhibition of apoptosis and enhanced proliferation was demonstrated in cells expressing Piwil2. Furthermore, results of soft agar assay indicated that Piwil2 overexpression induced transformation of fibroblast cells. In summary, our results demonstrate that Piwil2 is widely expressed in tumors and acts as an oncogene by inhibition of apoptosis and promotion of proliferation via Stat3/Bcl-XL signaling pathway. Expression of Piwil2 in a wide variety of tumors could be a useful prognostic factor that could have also diagnostic and therapeutic implications.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In connection with tumors, the issue of stem cells has been raised for several decades. As tumor cells also exhibit self-renewal capacity, it seems plausible that their regulation is similar to that of the stem cells. Dysregulation of stem-cell self-renewal is a likely requirement for the development of cancer. In addition, most cancers comprise a heterogenous population of cells with marked differences in their proliferative potential as well as the ability to reconstitute the tumor upon transplantation. Cancer stem cells are a minor population of tumor cells that possess the stem-cell property of self-renewal. This new model for cancer will have significant ramifications for the way we study and treat cancer. In addition, through targeting the cancer stem cell and its dysregulated self-renewal, our therapies for treating cancer are likely to improve (1
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The Piwi genes represent the first class of genes known to be required for stem-cell self-renewal in diverse organisms (2). These genes are highly conserved during evolution and play essential roles in stem-cell self-renewal, gametogenesis and RNA interference (RNAi) in diverse organisms ranging from Arabidopsis to human. In the Drosophila ovary, piwi is required in somatic signaling cells to maintain germline stem cells. Piwi has a role as a cell-autonomous promoter of germline stem-cell division. Removing Piwi protein from single germline stem cells significantly decreases the rate of their division (3). Overexpression of piwi in somatic cells causes an increase both in the number of germline stem cells and the rate of their division. Thus, in Drosophila, piwi is a key regulator of stem-cell division—its somatic expression modulates the number of germline stem cells and the rate of their division, while its germline expression also contributes to promoting stem-cell division in a cell-autonomous manner (3). In mammals, piwi genes are expressed specifically in testis and play a key role in spermatogenesis (4). In the mouse genome, two piwi homologs have been identified (Miwi and Mili or Piwil2). Miwi-null mice do not complete spermatogenesis, but arrest occurs at the beginning of the round spermatid stage (5), significantly downstream of the germline stem-cell division stage (5). The Mili-null mice showed arrest of spermatogenesis at the spermatocyte stage, which is reminiscent of the phenotype of Mvh (mouse vasa homolog)-null mice (6). In human, eight members of argonaute family are identified. Argonaute family were classified into two subfamilies: the Piwi subfamily, Piwil1 (hiwi), Piwil2 (hili), Piwil3 and Piwil4 (hiwi2) and the eIF2C/AGO subfamily. All four members of Piwi subfamily are expressed mainly in testis (7). Human Piwil1 (hiwi) is specifically expressed in both normal and malignant male germ cells in a maturation stage-dependent pattern, in which it might function in germ cell proliferation (8). It was also demonstrated that Piwil1 (hiwi) is expressed in a variety of primitive hematopoietic cells and may play a role in determining or regulating hematopoietic stem-cell development (9). Altogether, the data from different organisms suggest a key role of piwi genes in stem-cell division. The evidence that the piwi genes play essential roles in stem-cell division is the basis of our hypothesis that overexpression of these genes leads to disturbance of cell division, causes tumors and, therefore, plays a role as dose-dependent oncogenic fate determinants.
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RESULTS |
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Piwil2 is expressed specifically in testis and in a wide variety of tumors
Expression of mouse and human Piwil2 genes was analyzed using RT–PCR and primers specific for mouse and human Piwil2 gene. Testis-specific expression was obtained in 20 normal adult human organs and nine normal mouse organs (Fig. 1A and B). Using immunostaining analysis with anti-Piwil2-antibody, a staining could be only detected in spermatogonia and spermatocytes in mouse (Fig. 1C) and human (Fig. 1D). These results confirm the observation obtained by other groups (4
,7). In RNAs isolated from different human and mouse cancer cell lines, a specific RT–PCR product was observed in most examined cell lines, seven of 10 murine cell lines (Fig. 2A) and nine of 10 human cell lines (Fig. 2B). No expression was detected in NIH-3T3 cells (Fig. 2A). To examine expression of Piwil2 in tumor tissues, RNAs were isolated from different mouse and human solid tumors and were subjected to RT–PCR analysis. In mouse, three types of tumors with their corresponding normal tissues were examined. Whereas, in breast tumor, rhabdomyosarcoma and medulloblastoma expression of Piwil2 was detected, no expression was observed in normal breast, muscle and cerebellum (Fig. 2C). In human, in different tumor types, expression of Piwil2 was detectable using RT–PCR (Fig. 2D). In three of four different ovarian tumors, four of four different prostate carcinoma, four of four patients with tumors in lymphatic gland and in seven of seven breast tumors, expression of Piwil2 was detected by RT–PCR analysis (Fig. 2D). To examine expression of Piwil2 on protein level, immunostaining was performed using antibody against Piwil2 protein. In mouse, expression of Piwil2 was detected in transformed testicular germ cell line GC-1 in proliferating cells (Fig. 3A). Although expression of Piwil2 was not detected in normal skeletal muscle and cerebellar tissues (Fig. 3B), Piwil2 was detectable in corresponding tumor tissues, rhabdomyosarcoma and medulloblastoma (Fig. 3B). In human, Piwil2 was found in cytoplasm of breast tumor cell line MDA-MB-231 (Fig. 4A). In all four breast tumor tissues from different patients, expression of Piwil2 was observed (Fig. 4B–E). No expression was detectable in normal breast tissue (Fig. 4F). Furthermore, immunohistochemical analysis resulted in other tumors either in dispersed (Fig. 4K, L, P, Q, R and S) or in clonal form (Fig. 4G, I, J, M, N, O and T). These results indicate that Piwil2 is specifically expressed in spermatogonia of testis and ectopically in most tumor cell lines and tumor tissues.
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Piwil2 is overexpressed in testicular germ cell tumors
To examine expression of Piwil2 in human testicular germ cell tumors, a human testicular cancer profiling array with RNAs from seminomas, non-seminomatous tumors and surrounding normal testicular tissue was hybridized with a Piwil2-specific probe. As shown in Figure 5A and B, an overexpression of Piwil2 was observed in seminomas (nine of 10). No enhanced expression of Piwil2 was detected in testicular non-seminomatous tumors (Fig. 5B).
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Identification of Piwil2 downstream targets
To search for potential molecular downstream targets of Piwil2, we established NIH-3T3 cell lines stably expressing Piwil2 using a fusion gene harboring coding region of Piwil2 under control of CMV promoter (Fig. 6A). Expression of Piwil2 in the stable cell line NIH3T3-pcDNA-Piwil2 was examined on RNA (Fig. 6B) by RT–PCR and on protein level by western blot analysis (Fig. 6C) and immunostaining (Fig. 6D) using mouse anti-Piwil2 antibody. It was demonstrated that Piwil2 is transcribed and translated properly in NIH3T3-pcDNA-Piwil2 cell line. Expression profiles of NIH-3T3 cells expressing stably full length Piwil2 was compared with control cell line NIH3T3-pcDNA (Fig. 7A) using a Cancer Pathway Finder Array. This analysis revealed activation of genes related to cell growth, adhesion and apoptosis (Table 1 and Fig. 7A). Clearly, an activation of Bcl-XL was observed in cells expressing Piwil2 (Fig. 7A). To examine whether expression of Bcl-XL is regulated directly by Piwil2 or indirectly by upstream regulatory factors of Bcl-X, expression of the signal transducers and activators of transcription 3 (Stat3) and serine/threonine kinase Akt, two upstream regulatory factors of Bcl-X (10
,11) were examined. Whereas a slight increase (about 2-fold) was observed in expression level of Akt, a clear activation of Stat3 was obtained in cells stably expressing Piwil2 (Fig. 7B and C). A slight overexpression of Stat2 and reduction in expression of RelA and NF-Kappa B were observed (Fig. 7B and C). No change was detected in expression of Ets2. To analyze further the association between Piwil2 expression and expression of Stat3, Akt, Bcl-XL and cyclin D1, expression of these genes were examined in human testicular germ cell tumor cell line Tera-1 and mouse breast tumor tissues. In human and mouse, association between Piwil2 overexpression and overexpression of Stat3, Akt and cyclin D1 were demonstrated (Fig. 7D).
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On protein level, coimmunostaining with anti-Piwil2 and anti-Stat3 antibodies showed presence of cytoplasmic (Fig. 8A) and nuclear (Fig. 8B) forms of Stat3 protein in Piwil2 expressing cells. Furthermore, activation of Stat3 expression was demonstrated by Western blot analysis using anti-Stat3 antibody in Piwil2 expressing NIH3T3 cells (Fig. 8C).
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Finally, to analyze whether Piwil2 expression regulates expression of Stat3, Akt, Bcl-XL and cyclin D1, we examine the effect of direct down-regulation of Piwil2 using its small interference RNA small interference RNA (siRNA) on expression level of Stat3, Akt, Bcl-XL and cyclin D1 in GC-1 cells. RT–PCR was used to demonstrate no change in the Piwil2 level in GC-1 cells transfected with control siRNA (Fig. 8D), whereas there was an obvious decrease in expression of Piwil2 following transfection with the Piwil2 siRNA after 24 h (Fig. 8D). Suppression in Piwil2 expression resulted in down-regulation of Stat3, Bcl-XL and cyclin D1 (Fig. 8D). These results suggest the role of Piwil2 as a regulatory factor of Stat3/Bcl-XL/cyclin D1 pathway.
Effect of Piwil2 overexpression on cellular phenotypes
To study the causal effect of Piwil2 expression on cellular phenotypes, NIH3T3 cells stably expressing Piwil2 (NIH3T3-pcDNA-Piwil2) were compared with control cells NIH3T3-pcDNA concerning apoptosis, cell proliferation and transformation. We used BD ApoAlert DNA fragmentation Kit for measurement of apoptosis. The BD ApoAlert DNA Fragmentation Assay Kit detects apoptosis-induced nuclear DNA fragmentation via a fluorescence assay. Using two different techniques, microscopically and cytometrically, a significant decrease of apoptosis was observed in NIH-3T3 cells expressing Piwil2 (Fig. 9A and B) in multiple experiments. Using a combined apoptosis assay and Piwil2 immunostaining, it was demonstrated that apoptotic cells do not express Piwil2 (Fig. 9C) and cells expressing Piwil2 are not apoptotic (Fig. 9C). Transfection of GC-1 cells which express Piwil2 endogenously resulted in apoptosis of these cells (Fig. 9D). Here, it was also demonstrated that GC-1 cells with a suppression of Piwil2 expression undergo apoptosis (Fig. 9D). Quantification of apoptosis rate of GC-1 cell transfected with Piwil2 siRNA indicated an increase of apoptotic cells compared with untransfected GC-1 cells (Fig. 9E).
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In proliferation assay, an increased proliferative activity of NIH3T3 cells expressing Piwil2 was obtained (Fig. 10A) in multiple experiments. Increased proliferation was observed if the number of starting cells was higher than 9000 (P<0.01) and not detectable if the starting cell number was low (for example 2000 and 6500 starting cells in Fig. 10A).
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To examine the correlation between Piwil2 expression and cell cycle, 2 kb of human Piwil2 5' flanking region was linked to coding region of EGFP (Fig. 10B). This fusion construct was transfected into the HeLa cells and expression of EGFP was observed after 48 h. Expression of EGFP was always observed in proliferative active cells with condensed metaphase chromosomes (Fig. 10B). In addition, it was demonstrated that NIH3T3-pcDNA-Piwil2 cells with constitutive expression of Piwil2 are more rounded than NIH3T3-pcDNA cells (data not shown) and formed significantly more colonies in soft agar (Fig. 10C).
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DISCUSSION |
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We showed that Piwil2 in mouse and human is expressed specifically in testis and in most tumors. Therefore, Piwil2 appears to fall in the category of cancer–testis antigens (CTAs). CTAs were the first human tumor-associated antigens to be characterized at the molecular level (12
). Specific genes are expressed in the testis and in tumors of varying histological origin. In testis, CTAs are exclusively present in cells of the germ cell lineage, though there is a lot of variation in the expression profile during different stages of germ cell development (13). The tissue expression pattern supports the notion that these antigens could be targets for active specific immunotherapy. Therefore, the wide range of tumors in which Piwil2 have been detected urges further efforts to develop effective specific immunotherapeutic procedures.
In human, an elevated expression of Piwil2 was observed in testicular germ cell tumors. This expression pattern mimics expression pattern of Piwil1 in testicular germ cell tumors. In normal human testes, Piwil1 (hiwi) is specifically expressed in germ cells, with its expression detectable in spermatocytes and round spermatids during spermatogenesis (8). Enhanced expression of Piwil1 was found in 12 out of 19 sampled testicular seminomas originating from embryonic germ cells with retention of germ cell phenotype. In contrast, no enhanced expression was detected in 10 non-seminomatous testicular tumors that originate from the same precursor cells as seminomas, yet have lost their germ cell characteristics (8). These results indicate a possible synergistic effect of piwi genes on initiation and progression of testicular germ cell tumors.
We demonstrated that Piwil2 activated the expression of Bcl-XL. Bcl-X belongs to bcl-2 gene family. Members of the bcl-2 family of genes serve as regulators of cell death, either promoting (Bax, Bak, Bok, Bik, Blk, Hrk, Bad, Bid, Diva and EGL-1) or inhibiting it (Bcl-2, Bcl-X, Bcl-w, Mcl-1 and CED-9) (10,14,15). Furthermore, we showed that Piwil2 is able to induce the expression of Stat3 and to enhance slightly expression of Akt, two upstream regulators of Bcl-XL (11,16). The signal transducers and activators of transcription (STAT) factors function as downstream effectors of cytokine and growth factor receptor signaling. In mouse, the Stat3 activation is required and sufficient to maintain the undifferentiated state of ES cells (17,18). When compared with normal cells and tissues, constitutively activated STATs have been detected in a wide variety of human cancer cell lines and primary tumors (19). STATs are activated by tyrosine phosphorylation, which is normally a transient and tightly regulated process. In tumor cells, constitutive activation of STATs is linked to persistent activity of tyrosine kinases, including Src, epidermal growth factor receptor, Janus kinases, Bcr-Abl and many others. Such oncogenic tyrosine kinases are often activated as a consequence of permanent ligand/receptor engagement in autocrine or paracrine cytokine and growth factor signaling or represent autonomous constitutively active enzymes as a result of genetic alterations found in tumor, but not in normal cells. Persistent signaling of specific STATs, in particular Stat3 and Stat5, has been demonstrated to directly contribute to oncogenesis by stimulating cell proliferation and preventing apoptosis. STATs participate in oncogenesis through up-regulation of genes encoding apoptosis inhibitors and cell-cycle regulators such as Bcl-XL, Mcl-1, cyclins D1/D2 and c-Myc (19). These results demonstrate that Piwil2 independently targets two important cellular signaling pathways, Stat3/Bcl-X and Stat3/cyclin D1 and, therefore, can act as an oncogenic factor in tumorigenesis of different tissues. Block of constitutive Stat3 signaling results in growth inhibition and apoptosis of Stat3-positive tumor cells in vitro and in vivo.
Association of Piwil2 expression and expression of Stat3, Bcl-XL, cyclin D1 and Akt was demonstrated in testicular germ cell tumor cell lines, Tera-1 (Fig. 7D) and GC-1 (Fig. 8D), and mouse breast tumor tissue (Fig. 7D). In Tera-1 cells, an association was found between elevated Piwil2 expression and increased expression of Stat3, Bcl-XL, cyclin D1 and Akt, whereas in mouse breast tumor, this association was found only with Stat3 and cyclin D1, but not with Bcl-XL. This indicates that Piwil2 acts through different signaling pathways in tumors with epithelial and stromal origins. Interestingly, although there are differences between Piwil2 expression and expression of Bcl-XL, cyclin D1 and Akt in stromal cell line NIH3T3 expressing Piwil2 (Fig. 7B) and epithelial tumors (Figs 7D and 8D), no differences were observed between elevated Piwil2 expression and increased Stat3 expression in stromal and epithelial derived tumors and cancer cell lines.
The question that remains to be discussed is how Piwil2 regulates expression of the genes which are involved in apoptosis and proliferation and specially Piwil2/Bcl-XL pathway. Piwil2 belongs to the PPD (PAZ Piwi domain) proteins. These proteins have well-established roles in RNAi. RNAi is a process utilized by eukaryotes to modulate gene expression at pre- and post-transcriptional levels (20). The Piwi domain, comprising approximately 300 amino acids has been shown to mediate the interaction of PPD proteins with Dicer (21). Recent structural and bioinformatic analyses suggest that Piwi domains share similarity with endonucleases, an observation that led to the discovery that PPD proteins are directly involved in cleavage of targeted mRNAs during the effector stage of RNAi (22). The mechanisms by which PPD proteins mediate translational suppression and chromatin silencing are not known at this time. Bioinformatic analyses have revealed that many of the RNAi targets in plants are transcription factors (23,24). Accordingly, it is possible that overexpression of Piwil2 activates endogenous RNAi machinery which induce indirectly expression of Stat3/Bcl-XL.
Other interesting observation is the increase of Piwil2 expression in the proliferating cells. (Figs 3A and 10B). Of course, it is not hard to imagine how Piwil2 may indirectly affect cell-cycle progression through their involvement in gene-silencing pathways that regulate expression of transcription factors like Stat3. It has recently become evident that PPD proteins and Dicer also function in siRNA-independent pathways that regulate cell-cycle events (25). Accordingly, it is important to consider the aberrant expression of PPD proteins and Dicer in human cancers. We observed a difference in cell-proliferation rate when we started from 9500 cells per well. This supposed the role of cell–cell interaction in Piwil2 mediated cell proliferation, because the possibility for cells to form aggregate is greater with this starting cell number.
Of particular significance is the observation that seminoma tumors are often associated with increased levels of Hiwi mRNA (8). Coincidentally, Hiwi was first reported as a gene that is expressed in undifferentiated hematopoietic stem cells, but not in differentiated cells (9). Likewise, expression of the D. melanogaster orthologue of Hiwi, Piwi, had previously been reported to promote mitosis in stem cells (3). In addition, the chromosomal locus 12q24.33 that includes the Hiwi gene has been linked to testicular germ cell tumors (26). Together, these observations are consistent with a scenario in which overexpression of certain PPD proteins like Piwil2 is associated with increased mitosis in undifferentiated cells. The role of other PPD protein family members in this process is less clear.
In order to examine whether other signaling pathways are also involved in Piwil2-mediated increase of Bcl-XL and cyclin D1, we investigated changes in the expression level of Stat2, RelA, Ets2 and NFkB2 in NIH3T3 cells expressing constitutively Piwil2. All these proteins are known as regulatory factors of Bcl-XL and cyclin D1 (27–29). A slight increase in Stat2 expression and a slight reduction in expression of RelA and NFkB2 were observed. No change in the expression level of Ets2 was obtained. These results indicated that Stat2, RelA, Ets2 and NFkB2 are not mainly involved in Piwil2-mediated regulation of Bcl-XL and cyclin D1 in NIH3T3-pcDNA-Piwil2 cells.
Our results demonstrate that Piwil2 inhibits apoptosis and stimulates proliferation through activation of Stat3/Bcl-XL and enhancement of Stat3/cyclin D1 signaling pathways. Inhibition of constitutively signaling pathways by repression of Piwil2 expression can inhibit tumor cell growth in vitro and in vivo and provides a novel means for therapeutic intervention in human cancer. Furthermore, specific expression of Piwil2 in the testis and its aberrant expression in various tumors make this molecule an attractive candidate as a cancer prognostic and diagnostic marker.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Plasmid construction
Plasmid pcDNA-Piwil2 was constructed using complete coding region (cDNA) of mouse Piwil2 gene obtained by RT–PCR from a testicular RNA. The PCR product was cloned into pcDNA plasmid (BCCM/LMBP Plasmid collection, Gent, Belgium) containing promoter of cytomegalovirus (CMV). For hpiwil2-EGFP construct, 2 kb of flanking region of human Piwil2 gene was amplified and cloned into a plasmid vector containing coding region of EGFP gene and SV40 polyadenylation signals. Both plasmids contain neomycine resistance gene under control of SV40 early enhancer and promoter elements for positive selection. The final constructs were sequenced completely.
Cell culture
Mouse NIH3T3 fibroblasts were maintained in Dulbecco's modified Eagle's medium (DMEM) (PAN, Aidenbach, Germany) supplemented with 10% fetal bovine serum (FBS), 1% glutamine (200 mM) and 1% penicillin/streptomycin. GC-1 was purchased from America Tissue Cell Collection (ATCC, Rockville, MD, USA) and maintained in DMEM containing 15 mM HEFES and supplemented with 0.45% glucose (w/w), 10% FBS and 2% penicillin (50 U/ml)/streptomycin (50 µg/ml). The human breast cancer cell line MDA-MB-231 was obtained from the ATCC and maintained in DMEM supplemented with sodium pyruvate (1 mM), 10% FBS, 2% penicillin/streptomycin. HeLa cells were cultured in DMEM containing 10% heat-inactivated FBS, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were cultured at 37°C in a humidified atmosphere of 5% CO2. Cells were plated at a density of 106 cells/ml in 25 cm2 culture flask and the monolayer was transfected with a freshly prepared liposome solution (55°C) containing HEPES–NaCl and 1 µg/µl Clonfectin (BD Clontech, Palo Alto, CA, USA). For the transfection, 4 µg of each construct DNA was mixed with 100 µl serum-free medium and 8 µg of Clonfectin and incubated for 30 min at RT. Cells were incubated in this solution for 4 h in a 5% CO2 incubator. After washing in PBS (37°C), expansion medium was added and cells were further cultivated for 3 days. Hela cells were observed under a fluorescence microscope (Olympus BX60) after 48 h. Stable NIH3T3 transfectants were selected by cultivating in medium containing 1 mg/ml of G-418 (PAN, Aidenbach, Germany) for 4 weeks. This cell line was designated as NIH3T3-pcDNA-Piwil2. As control, NIH3T3 cells were transfected with pcDNA plasmid and the stable cell line was designated as NIH3T3-pcDNA.
Western blot analysis
Sample proteins and controls were resolved via polyacrylamide gel electrophoresis. Proteins were then transferred to nitrocellulose membranes using standard methods (Invitrogen, Carlsbad, CA, USA). Blots were blocked with 5% non-fat dried milk (skim milk) freshly made in 1' PBS-T (0.1% Tween-20 in phosphate-buffered saline), rocked on a rotating shaker for 1 h at room temperature, rinsed three times in 1x PBS-T, probed with the indicated primary antibody, 1:500 diluted in PBS-T: piwil2 (rabbit, polyclonal peptide Ab; EP34147- dpvrplfrgptpvhp), 1:1000 diluted in PBS-T: Stat3 (rabbit, monoclonal Ab; Epitomics, CA, USA) for overnight at 4°C and rinsed three times in PBS-T. The blot was probed with an enzyme-linked secondary antibody (horseradish peroxidase) in 1x PBS-T (1:10 000) for 2 h at room temperature; excess secondary antibody was rinsed off with three rinses in PBS-T for 20 min each and then chemiluminescent detection reagents were used to reveal results. Immunoblotting for 
-tubulin was performed to verify equivalent protein loading.
Northern blot hybridization
Total RNA was isolated from NIH3T3-pcDNA cells and NIH3T3-pcDNA-Piwil2 cells using the RNeasy Mini kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. Equal amounts of RNA were fractionated by formaldehyde agarose gel electrophoresis. Filters were hybridized with probes for Bcl-XL, Akt, cyclin D1, RelA, NFkB2, Ets2, Stat2 and Stat3, and after stripping, reprobed with human elongation factor-2 to ensure RNA concentrations and integrity.
RNA preparation and RT–PCR analysis
Total RNA was extracted from mouse and human tissues (normal and tumor) using TRIzol reagent (GIBCO-BRL) and from mouse and human cell lines (normal and tumor) using RNeasy Mini kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. Single strand cDNAs were prepared from 5 µg RNA using a reverse transcriptase reaction (Invitrogen, Karlsruhe, Germany) and each PCR amplification was performed using Piwil2-specific primers (mouse: 5'-GCACAGTCCACGTGGTGGAAA-3', 5'-TCCATAGTCAGG ACCGGAGGG-3', human: 5'-CAGGCAGAGGCCATGTATTT-3', 5'-AACATGCCGACC TCATGCT-3'), mouse cyclin D1(5'-TGACACCAATCTCCTCAACG-3', 5'-AGCTTGTTC ACCAGAAGCAG-3'), mouse Stat3 (5'-TAGCCGATTCCTGCAAGAGT-3', 5'-AGCCAGC TCTTATCAGTCA-3'), mouse Akt (5'-TATTGGCTACAAGGAACGGC-3', 5'-TCTTCAT GGCATAGTAGCAA-3'), mouse Bcl-XL (5'-TCGAAGAGAATAGGACTGAG-3', 5'-TCA AAGCTCTGATACGCGGT-3'), human Bcl-XL (5'-ATCAATGGCAACCCA TCCTG-3', 5'-GTAAGTGGCCATCCAAGCTG-3'), human cyclin D1 (5'-TGCATGTTCGTGGCCTCT AA-3', 5'-CAGTCTGGGTCACACTTGAT-3'), human Akt (5'-ACGCCATGAAGATCCT CAAG-3', 5'-TTAATGTGCCCGTCCTTGTC-3'), human Stat3 (5'-ATTGACCAGCAGTA TAGCCG-3', 5'-TTCCAGCTGCTGCATCTTCT-3') and GAPDH primers (mouse: 5'-CA CCACCAACTGCTTAGCC-3', 5'-CGGATACA TTGGGGGGTAGG-3', human: 5'-CC AGCAAGAGCACAAGAGGAAGAG-3', 5'-AGCACGGGATACTTTATTAGATG-3') used as a standard. The cycling conditions were as follows: 5 min at 94°C, five cycles of 30 s at 94°C, 30 s at 57–61°C, 1 min at 72°C, 30 cycles of 30 s at 94°C, 45 s at 56°C, 1 min at 72°C and 72°C for 10 min. In siRNA experiments, the cycling conditions were as follows: 5 min at 94°C, 30 s at 94°C, 24 cycles (Piwil2), 23 cycles (Stat3), 30 cycles (Akt), 25 cycles (Bcl-XL, cyclin D1), 27 cycles (GAPDH) of 30 s at 60°C, 50 s at 72°C, 5 min at 72°C. In tera1 and mouse breast tumor RT–PCR experiments, the cycling conditions were as follows: 5 min at 94°C, 30 s at 94°C, 28 cycles (Piwil2, Stat3, Akt, Bcl-XL, cyclin D1), 30 cycles (GAPDH) of 45 s at 60°C, 1 min at 72°C, 5 min at 72°C.
Human total RNAs from colon, bone marrow, brain, small intestine, fetal brain, fetal liver, heart, kidney, spinal cord, lung, placenta, prostate, salivary gland, skeletal muscle, spleen, testis, stomach, thyroid, trachea and uterus were purchased from BD Biosciences (BD Clontech). Mouse total RNA was isolated from spleen, lung, heart, brain, kidney, skeletal muscle, ovary, liver, testis, breast and cerebellum. Furthermore, from the following human and mouse tumor tissues, RNA was isolated for RT–PCR analysis: mouse: rhabdomyosarcoma (n=3), medulloblastoma (n=3) and breast tumors (n=2); human: colon cancer (n=2), ovarian dysgerminoma (n=2), malignant mixed mullerian tumor (MMMT) of the endometrium (n=2), clear cell renal cell carcinoma (n=2), gastrointestinal stromal tumor (n=4), stromal sarcoma of endometrium (n=2), adenocarcinoma of endometrium (n=2), squamous cell carcinoma of pancreas (n=1), normal woman blood (n=4), adenocarcinoma of pancreas (n=1), ovarian cancer (n=4), prostate carcinoma (n=4), mamacarcinoma (n=7) and lymphatic gland tumors (n=5).
Following cell lines were used for isolation of RNA and RT–PCR analysis: mouse cell lines: transformed germ cells (GC-1 and GC-2), transformed Leydig cell line (MA-10), teratocarcinoma cell line F9, non-transformed spermatocyte (GC-4) and Sertoli cell (15P-1) line and non-testicular tumor cell lines PCT (pituarity gland), BT (breast tumor) and NS 20Y (neuroblastoma); human cell lines: PC3 (prostate cancer), MDA-MB-231 (breast cancer), LNCAP (prostate cancer), HeLa (cervical cancer), Jurkat (T-cell leukemia), MCF-7 (breast adenocarcinoma), Daudi (Burkitt's lymphoma), 2102EP (embryonal carcinoma), DU-145 (prostate cancer), H12.1 (embryonal carcinoma).
Apoptosis assay
Apoptosis was determined by two methods using ApoAlert DNA fragmentation assay. The BD ApoAlertTM DNA Fragmentation Assay Kit detects apoptosis-induced nuclear DNA fragmentation via a fluorescence assay. The established cell lines (NIH3T3-pcDNA, NIH3T3-pcDNA-Piwil2 and GC-1 transfected siRNA of Piwil2) were fixed in 4% formaldehyde, processed for an apoptosis assay using the ApoAlert DNA fragmentation kit (BD Clontech) as described by the manufacturer, and apoptotic cells were observed and counted under a fluorescence microscope. Apoptotic rates were also analyzed by flow cytometry using FITC/propidium iodide (PI) staining. Staining was performed according to the manufacturer's instruction (BD Clontech). Fluorescence intensity measurements were done after excitation at 488 nm and detection at 520–530 nm for green fluorescence for apoptotic cells and at 665–685 nm for PI for viability of cells.
Proliferation assay
NIH3T3-pcDNA and NIH3T3-pcDNA-Piwil2 cells were seeded at 2000, 6500 and 9500 cells/well in 96-well plates, respectively. Cells were cultivated in microtiter plates for 2 and 10 h. Quantification of cell proliferation was performed using Quantos cell proliferation assay kit (Stratagene, La Jolla, CA, USA). Fluorescence of a DNA-dye complex from lysed cells was determined using a microtiter plate-reading fluorometer with filters appropriate for 355 nm excitation and 460 nm emission.
Soft agar colony assay
NIH3T3-pcDNA, NIH3T3-pcDNA-piwil2 and GC-1 cells were mixed with cell culture medium containing 0.8% agar. This cell suspension of 2000 cells/well was immediately plated in six-well plates coated with 0.3% agar in cell culture medium (2 ml per well) and cultured at 37°C with 5% CO2. After 2 weeks, the top layer of the culture was stained with 0.001% Crystal violet for 1 h (Sigma-Aldrich). The culture was analyzed in triplicate, and colonies larger than 100 µm in diameter were counted.
Cancer array hybridization
Human Cancer Profiling Array II (Clontech) was hybridized with a human Piwil2 cDNA probe overnight and washed according to manufacturer's instruction. Quantitative evaluation of the array was performed by phosphor imager analysis using a molecular imager FX (BioRad, Hercules, CA, USA). For identification of differentially expressed genes, radioactive Cancer PathwayFinder Gene Array (GEArray Q series, SuperArray, Frederick, USA) was used. Hybridization procedures were performed using a GEArray kit (SuperArray, Frederick, USA) as described by the manufacturer.
Histology and immunohistochemistry
NIH3T3-pcDNA, NIH3T3-pcDNA-Piwil2, MDA-MB-231, GC-1 and GC-1 transfected with Piwil2 siRNA were seeded on culture slides (FALCON, Le Pont De Claix, France) to 50–60% confluence. Cells were fixed in 4% paraformaldehyde solution, washed three times in PBS and then incubated with an anti-Piwil2 antibody arised against peptide (dpvrplfrgptpvhp) and/or anti-Stat3 antibody as primary antibodies in a humidifying chamber at 4°C overnight. After washing, culture slides were incubated with a 1:500 FITC-conjugated in goat anti-mouse IgG and/or 1:1000 Cy3-conjugated in goat anti-rabbit IgG (Sigma-Aldrich) secondary antibody. Slides were washed three times in PBS and mounted in DAPI mounting solution (Vector Laboratories, Burlingame, CA, USA). The cells were observed under a fluorescence microscope (Olympus BX60).
Mouse (skeletal muscle, cerebellum, medulloblastoma, rhabdomyosarcoma) and human tissue samples (testis, testicular germ cell tumors, coccyx teratoma, ovarian dysgerminoma, ovarian teratoma, adenocarcinoma of colon, gastrointestinal stromal tumors, clear cell renal cell carcinoma, MMMT of endometrium, stromal sarcoma of endometrium and adenocarcinoma of endometrium) were fixed in 4% paraformaldehyde in PBS, paraffin embedded and sectioned (3 µm). To remove paraffin from the slide, the paraffin-coated slide was washed three times with xylol for 3 min each, ethanol(100, 96, 90, 80, 70, 50%) for 2 min each, dH2O for 5 min and finally three times in PBS for 3 min and then incubated with anti-Piwil2 antibody in a humidifying chamber at 4°C overnight. After washing with PBS, primary antibody treated slides were incubated with 1:500 FITC- or 1:100 alkaline phosphatase-conjugated anti mouse IgG (Sigma-Aldrich) for 2 h, washed with PBS (three times) for 3 min, stained with DAPI (Vector Laboratories, Burlingame, CA, USA) and then observed under a fluorescence microscope (Olympus BX60).
siRNA experiments
GC-1cells were used for siRNA transfection. Cells were plated at 2.0x105cells per well in a six-well tissue culture plate. Following 24 h in culture, cells were transfected with 80 nM piwil2 siRNA (Invitrogen siRNA). The piwil2 siRNA used was forward (5'-ACACAGCAUUC CGGCCUCCUUCAAA-3') and reverse (5'-UUUGAAGGAGGCCGGA AUGCUGUGU-3'). Luciferase double-stranded RNA (Eurogentec, Belgium) was used as a control RNAi treatment. Using 3 µl per well Lipofectamine 2000 (Invitrogen, Karlsruhe, Germany) according to the manufacturer's protocol. Growth medium was replaced after 8 h without loss of transfection activity. In 0, 24, 48, 72 h after transfection, the cells were harvested for total RNA. For apoptosis assay, in 24 h after transfection, the cells were performed as described earlier. All experiments were carried out in triplicate.
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ACKNOWLEDGEMENTS |
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We thank Ch. Müller, D. Meyer, I. Schwandt and B. Sadowski for excellent technical assistance. We thank Dr I.M. Adham and Professor Dr H. Hahn for providing of mouse breast tumor tissues, rhabdomyosarcoma and medulobasltoma and Dr P Burfeind for providing of cell lines. This work was supported by the grant (Forschungsförderungsprogramm Stammzelle to K.N.) of the University of Göttingen.
Conflict of Interest statement. KN and WE are inventors on a patent describing the use of Piwil2 for diagnosis and treatment of tumors.
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