NANOGP8 is a retrogene expressed in cancers Jingyu Zhang 1,2, *, Xia Wang 1,2, *, Meixiang Li 1 , Jin Han 1 , Bing Chen 1 , Bin Wang 1 and Jianwu Dai 1 1 Laboratory of Molecular and Developmental Biology, Institute of Genetics and Developmental Biology , Chinese Academy of Sciences, Beijing, China 2 The Graduate School, Chinese Academy of Sciences, Beijing, China Nanog is a recently identified transcription factor that plays key roles in self-renewal and maintenance of plu- ripotency in inner cell mass and embryonic stem (ES) cells [1–3]. It is generally believed that the Nanog gene is specifically expressed in human ES cells and germ lineage cells and its expression may be controlled by an interaction between OCT4 and other proteins through an adjacent pair of highly conserved Octamer- and Sox-binding sites of 5¢-flanking region of Nanog [4,5]. Nanog expression is rapidly down-regulated dur- ing ES cell differentiation, and constitutive expression of Nanog gene inhibits ES cell differentiation [3]. The regulation of Nanog gene expression and the nature of its target genes emerged as central issues in pluripotent stem cell biology [2]. Pseudogenes are common in mammalian genomes such as human and mouse and are defined as inactive versions of functional genes, i.e. they do not produce functional, full-length protein [6]. There are about 20 000 pseudogenes in human genomes [7]. It has been hypothesized that pseudogenes, especially those that are transcribed, have regulatory roles because of their high level of sequence similarities and conservation [8–10]. Recently, pseudogenes have been speculated to be involved in the epigenetic regulation of gene activities [11]. It is interesting that human Nanog has unusually high number of pseudogenes, 11 in total [12,13]. Among them, NANOGP8 is theoretically unique since it has a complete open reading frame and an Alu element in the 3¢-UTR which is homologous to that of Nanog. The possibility that NANOGP8 may be a retrogene rather than a pseudogene was entertained, but was considered unlikely as NANOGP8 has not been identified in any expressed sequence tags (ESTs) [12]. In the current study, we found NANOGP8 expressed in several cancer cell lines and all the cancer Keywords Nanog; NANOGP8; pseudogene; retrogene; tumorigenesis Correspondence J. Dai, Laboratory of Molecular and Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China. Tel ⁄ Fax: +86 010 82614426 E-mail: jwdai@genetics.ac.cn *The authors contributed equally to this work. (Received 11 November 2005, revised 26 January 2006, accepted 20 February 2006) doi:10.1111/j.1742-4658.2006.05186.x Nanog is a transcription factor that plays key roles in the self-renewal and maintenance of pluripotency in human embryonic stem (ES) cells. Among Nanog’s 11 pseudogenes, NANOGP8 theoretically could be a retrogene, but was considered unlikely as it has not been identified in any expressed sequence tags (ESTs). In this study, we found that NANOGP8 was expressed in several cancer cell lines and in all cancer tissues tested. The complete coding sequence was cloned and the sequence is highly homolog- ous to that of Nanog. We were also able to detect its protein expression using anti-Nanog antibody in recombinant Escherichia coli and some can- cer cell lines tested. In addition, expression of NANOGP8 in NIH3T3 cells can promote cell proliferation. The expression of NANOGP8 in cancer cell lines and cancer tissues suggests that NANOGP8 may play important roles in tumorigenesis. This work not only has potential significance in stem cell and cancer research, but it also raises the possibility that some of the human pseudogenes may have regulatory functions. Abbreviations ES, embryonic stem; ESTs, expressed sequence tags; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide. FEBS Journal 273 (2006) 1723–1730 ª 2006 The Authors Journal compilation ª 2006 FEBS 1723 tissues tested. No expression was detected in primarily cultured fibroblasts. At the same time, NANOGP8 was shown to prompt cell proliferation. These data indicated that NANOGP8 might play important roles in tumorigenesis. Results NANOGP8 is transcribed in cancer cell lines and tumor tissues Human Nanog pseudogenes comprised 10 processed pseudogenes and one tandem duplicate [12]. Certain regions of these pseudogenes were highly conserved. The coding regions of 70% of Nanog pseudogenes share about 90% sequence identity [13]. Sequence com- parison of the mRNA revealed the presence of the 5¢-UTR sequence in Nanog, which is absent from other Nanog pseudogenes. We used this feature to dis- criminate Nanog and its pseudogenes. NANOGP8 lied in chromosome 15 and shares the greatest similarity to Nanog. It contains a complete open reading frame, which might be transcribed in physiological conditions. To determine whether NANOGP8 could be transcribed, we designed human Nanog specific primers based on its 5¢-UTR, and the expected PCR product is 444 basepairs. We also designed a pair of universal primers that would amplify NANOGP8, Nanog, and Nanog’s other pseudogenes due to their homology, and the expected PCR product is 403 basepairs. Figure 1 showed the lack of expres- sion of Nanog and its pseudogenes in normal human primarily cultured fibroblasts; Nanog was expressed in ovary teratocarcinoma cell line PA-1 and testicular embryonic carcinoma NTERA-2, and sequencing the 444 basepairs PCR product confirmed the findings (Table 1). Furthermore, when the PCR products were cloned and sequenced, the results indicated that NANOGP8 was expressed in human osteosarcoma cell line OS732, human hepatoma cell line HepG2 and human breast adenocarcinoma cell line MCF-7 (Table 1). At the same time, other pseudogenes were found in OS732, HepG2 and MCF-7, while Nanog gene was also expressed in HepG2 and MCF-7 but not in OS732 (Table 1 and Fig. 1). In this study, we found that NANOGP8 was transcribed in all of the human cancer tissues tested (Table 1 and Fig. 2) as well as in several cancer cell lines. In contrast, Nanog and NANOGP8 were not expressed in normal primarily cultured fibroblast cell line or fetal liver epithelium cells (data not shown). It is interesting that only NANOGP8 was expressed in OS732 cell line, uterine cervix and breast tumor tissues, while Nanog gene was undetectable (Table 1). Thus, it is true that NANOGP8 was transcribed in cancer cell lines and tumor tissues. Fig. 1. Detection of Nanog, NANOGP8 and other pseudogenes in tumor cell lines. RT-PCR analysis the expression of Nanog, NANOGP8 and other pseudogenes in human fibroblasts and tumor cell lines OS732, HepG2, MCF-7, THP-1, HeLa, PA-1 and NTERA-2. Sequencing analysis of PCR products (Table 1) confirmed that NANOGP8 was expressed in cell lines OS732, HepG2, and MCF-7 while Nanog was expressed in HepG2, MCF-7, PA-1 and NTERA-2. No-RT data were also shown. Table 1. The sequencing results of RT-PCR product (403 basepairs) clones with the universal primers. Cells or tissues tested Total sequenced clones Sequenced clones of Nanog Sequenced clones of NANOGP8 Others Normal cells Human fibroblasts 0000 Tumor cell lines OS732 13 0 5 6(NANOGP4) 2(NANOGP5) HepG2 12 2 3 6(NANOGP4) 1(NANOGP5) MCF-7 15 2 2 3(NANOGP4) 8(NANOGP5) THP-1 0 0 0 0 HeLa 0 0 0 0 Teratocarcinoma cell lines PA-1 7 7 0 0 NTERA-2 6 6 0 0 Tumor tissues Uterine cervix 5 0 1 1(NANOGP2) 2(NANOGP4) 1(NANOGP7) Breast 13 0 8 1(NANOGP4) 1(NANOGP5) 3(NANOGP7) Urinary bladder 8 1 4 1(NANOGP2) 2(NANOGP7) NANOGP8 is a retrogene expressed in cancers J. Zhang et al. 1724 FEBS Journal 273 (2006) 1723–1730 ª 2006 The Authors Journal compilation ª 2006 FEBS NANOGP8 complete coding sequence was obtained from urinary bladder cancer Using RT-PCR, the complete coding sequence of NANOGP8 was cloned from urinary bladder cancer tissues and its sequence was found to be highly homol- ogous to Nanog. There are six alternations over 918 sites (Fig. 3 and Supplementary material Fig. 1) and only one change in the inferred amino acid sequence from 253 Gln in Nanog to His in NANOGP8. Thus, it is likely that NANOGP8 and Nanog genes have sim- ilar functions. NANOGP8 and ⁄ or Nanog protein was detected in cell lines The expression levels of NANOGP8 appear relatively low in the cancer cell lines and neoplastic tissues compared to Nanog in EC cells (Figs 1 and 2). Given the very high degree of homology between Nanog and NANOGP8 on the basis of nucleotide sequences, it is likely that the commercially available Nanog antibod- ies could recognize NANOGP8-translated protein. To confirm this hypothesis, we first constructed GST- NANOP8 fusion protein expressed in recombinant Escherichia coli and then detected NANOP8 using anti-Nanog antibody. Nanog antibody could recognize the fusion protein (60 kDa) (Fig. 4B). To directly examine NANOGP8 protein expression, nuclear pro- tein extracts were used for western blot assay. Accord- ing to Table 1 and Fig. 1, both NANOGP8 and Nanog were transcribed in HepG2, while Nanog was not transcribed in OS732. Thus, it is likely the protein of NANOGP8 and ⁄ or Nanog was detected in cancer line HepG2 and the protein of NANOGP8 was found in cancer line OS732 using anti-Nanog antibody and the observed molecular weight (34 kDa) of the pro- tein was consistent with its predicted full-length sequence (Fig. 4A). Therefore, the above findings Fig. 2. Detection of Nanog, NANOGP8 and other pseudogenes in tumor tissues. RT-PCR analysis of Nanog, NANOGP8 and other pseudogenes expression in human fibroblasts and human carci- noma tissues of the uterine cervix, breast and urinary bladder is shown. NANOGP8 was expressed in all of the human cancer tis- sues that were tested. NANOGP8 was not expressed in human fibroblasts. Nanog was expressed in urinary bladder tumor tissues and PA-1 cell lines. No-RT data were also shown. Fig. 3. Sequence alignment of human Nanog and NANOGP8 genes. The differences in nucleotides and their positions are shown. The translational start site is defined as +1. There is only one change in the inferred amino acid sequence from Gln (CAG759) in Nanog to His (CAC759) in NANOGP8. A B Fig. 4. Western blot detection of NANOG and ⁄ or NANOGP8 pro- tein. Detection of NANOGP8 protein in (B) E. coli (1) and E. coli with NANOGP8 (2) and NANOGP8 in (A) OS732 and Nanog or NANOGP8 in MCF-7 and HepG2 using anti-Nanog antibody. The lack of expression of both Nanog and NANOGP8 in human fibro- blasts is also shown. Actin (43 kDa) was used as loading control. J. Zhang et al. NANOGP8 is a retrogene expressed in cancers FEBS Journal 273 (2006) 1723–1730 ª 2006 The Authors Journal compilation ª 2006 FEBS 1725 showed that NANOGP8 was translated and it sugges- ted NANOGP8 is a retrogene but not a pseudogene. NANOGP8 was localized in the nuclei of transfected cells Human Nanog is a transcriptional regulator and is localized in the nucleus [2,3]. Due to the high homol- ogy between Nanog and NANOGP8, NANOGP8 is likely a nuclear protein as well. To confirm this, we constructed a NANOGP8 and GFP fusion protein. As shown in Fig. 5, the fusion protein was localized in the nuclei of transfected NIH3T3 (Fig. 5 A and 5B), while GFP in the control group was present diffused in the cytoplasm (Fig. 5C,D). Therefore, NANOGP8 is also a nuclear protein. NANOGP8 promotes cells to enter into S phase Cell cycle analysis was performed in NANOGP8 trans- fected NIH3T3 (pQCXIN-NANOGP8 vector) and mock control (pQCXIN vector) by flow cytometry (Fig. 6A). The percentage of S phase in NANOP8- transfected cells was 53.3%, which was higher in comparison with the mock control (46.5%). The difference was statistically significant (P<0.05). The results were obtained from three independent clones. These results indicate that expression of exogenous NANOGP8 gene promotes cells to enter into S phase of the cell cycle. NANOGP8 promotes cell proliferation The increase in the percentage of cells in S phase sug- gests that NANOGP8 may promote cell proliferation [14]. To confirm this, we examined the effect of NANOGP8 overexpression on NIH3T3 cell growth. The results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (Fig. 6B) indicated that the proliferation rate of NANOGP8-transfected cells was significantly increased. Discussion Pseudogenes are thought to be ‘molecular fossils’, which can be used as a model to study the rate of nuc- leotide substitution, insertion and deletion in genome [15]. Recent experimental data have indicated that Fig. 5. Nuclear location of NANOGP8-GFP. The NANOGP8-GFP (A,B) and GFP (C,D) vector were introduced into NIH3T3, respectively, and photographed in bright fields and fluorescent field (B,D) and merged photos (A,C). NANOGP8 is a retrogene expressed in cancers J. Zhang et al. 1726 FEBS Journal 273 (2006) 1723–1730 ª 2006 The Authors Journal compilation ª 2006 FEBS some pseudogenes were transcribed and might be func- tionally active [9,10]. On the other hand, great care must be taken in validation of some assays for inter- vention of pseudogenes [16]. Nanog is a recently identified transcriptional factor that plays important role in regulating pluripotency and self-renewal of ES cells [2,3]. Recently, Nanog’s 11 pseudogens were identified, including 10 processed pseudogenes and one tandem duplicate [12]. They share sequence homology to the Nanog coding region, but lack the potential to produce a functional protein except the NANOGP8 because of critical mutations [12]. NANOGP8, bearing close similarity to Nanog, theoretically could be a retrogene, but this was consid- ered unlikely as it has not been identified in any ESTs. The EST database provides tremendous gene expres- sion information from many types of tissues or cells. Theoretically any gene transcriptions could be found. We carried out an EST search, where we found it is difficult to distinguish Nanog from NANOGP8 in some ESTs (Supplementary material Fig. 2). This maybe because of the limitation of each clone’s sequence information in EST database and the high similarity between NANOGP8 and Nanog, with only six nucleotides alternations and one amino acid muta- tion being found between them. In addition, NANOGP8 expression is extremely low compared with that of Nanog and this might contribute at least parti- ally to why NANOGP8 ESTs have not been detected from human cancers and cancer cell lines. To investigate whether NANOGP8 could be tran- scribed, we designed human Nanog-specific primers and also designed a pair of primers that would amplify NANOGP8, Nanog, and Nanog’s other pseudogenes due to their homology. By using these primers for PCR analysis and DNA sequencing, we would be able to identify the transcribed pseudogenes. Our results showed the lack of expression of NANOGP8 and Nanog in normal human primarily cultured fibroblasts, fetal liver epithelium cells (data not shown). Further- more, when all the PCR products were sequenced, the results confirmed that NANOGP8 was expressed in HepG2, MCF-7, OS732 as well as all the human can- cer tissues tested. In addition, we have obtained the complete NANOGP8 coding region from cancer tissues by RT- PCR excluding the genomic DNA contamination. Analysis of the nucleotide sequences in Nanog and NANOGP8 demonstrated that there are six alterna- tions, which resulted in only one amino acid change in the predicted protein sequence from (Gln, 757CAG) in Nanog and (His, 757CAC) in NANOGP8. Since NANOGP8 has an intact open-reading fragment, so it has the potential to encode a full protein. We then expressed NANOGP8 in E. coli, which could be detec- ted by western blot after GST affinity purification. Fur- ther, anti-Nanog antibody can recognize translated NANOGP8 protein in OS732. These data suggested that NANOP8 is a retrogene rather than a pseudogene. Because cancer cells and stem cells share many com- mon characters, such as unlimited proliferation, it has been suggested that similar mechanisms might be involved in regulating cancer cells and stem cells [17]. Our preliminary results showed that forced expression of Nanog gene in 3T3 fibroblasts could greatly increase cell proliferation rate [18]. When NANOGP8 was sta- bly transfected into NIH3T3 cells, the cells were pro- moted to enter into the S stage and, at the same time, MTT growth assay showed increased cell proliferation. Our findings that NANOGP8 is expressed in cancer cell lines and cancer tissues suggest that NANOGP8 60 50 40 30 20 10 0 Mock Mock NANOGP8 NANOGP8 1.8 A B 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 day 1 day 2 day 3 day 4 % Absorbance S stage percentage Fig. 6. FACS analysis results. FACS analysis of cells transfected with NANOGP8 and the mock ones (A) and the MTT assay (B). The percentage of NANOGP8 transfected cells at S phase is 53.3 ± 2.4 and of the mock cells is 46.5 ± 1.3. We examined the effect of NANOGP8 gene expression on NIH3T3 cells growth by MTT assay. NIH3T3 transfected with NANOGP8 gene versus the mock ones, P < 0.05 (except day 1); there are no significant differences between the different clones (data not shown). Data are presented as mean ± SD. The experiments were repeated for three times. Data were analyzed using the Student’s t-test. J. Zhang et al. NANOGP8 is a retrogene expressed in cancers FEBS Journal 273 (2006) 1723–1730 ª 2006 The Authors Journal compilation ª 2006 FEBS 1727 may play important roles in tumorigenesis. Oct4, another stem cell self-renewal gene, was found to be up-regulated in breast, pancreatic and colon cancers and expressed in several cancer cell lines such as HeLa and MCF-7 [19–21]. These findings suggested that this type of gene might be involved in tumorigenesis. Nanog was reported recently to be expressed only in some germ cell tumors and breast carcinoma [22–24]. Our results showed the transcription of NANOGP8, but not Nanog, in the human osteosarcoma cell line OS732, so it is likely that only NANOGP8 was trans- lated in this cell line detected by the western blot experiment. Our future goal is to analyze how the NANOGP8 gene is regulated in various cancers. We hypothesize that NANOGP8 may function in tumor cell self-renewal due to the high homology between Nanog and NANOGP8 genes. This work not only has potential significance in stem cell and cancer research, it also raises the possibility that some of the human pseudogenes may in fact be retrogenes and may have important functions in gene regulation. Experimental procedures Total RNA extract and RT-PCR Total RNA was extracted from cell lines and tissues using Trizol (Invitrogen, Carlsbad, CA, USA) reagent following the manufacturer’s instuctions. Total RNA was digested with RNAase-free DNase I (TaKaRa Carlsbad, CA, USA) at 37°C for 30 min and inactivated at 60°C for 10 min. With total RNA (2 lg) as the template and oligo(dT) as the primer, the first cDNA was synthesized in 25 lL reac- tion system with Moloney murine leukemia virus (MMLV) reverse transcriptase (Promega, Madison, WI, USA). First- strand cDNA and RNA without reverse transcription were amplified with b-actin primers to confirm the success of RT reaction and no genomic DNA contamination. At the same time, no-RT control (RT reaction without reverse transcrip- tase) was carried out to further exclude the DNA contamin- ation. cDNA template (3 lL) was used in a 25 lL reaction volume with rTaq DNA polymerase or LA Taq TM DNA polymerase with GC buffer (TaKaRa). Human NANOGP8 mRNA was amplified by RT-PCR using total RNAs extracted from urinary bladder cancer tissue. For NANOGP8, the sense primer 5¢-ATGAGTGTGGATC CAGCTTGTCC-3¢ and antisense primer 5¢-CACGTCTT CAGGTTGCATGTTCA-3¢, for Nanog-403 (and ⁄ or its pseudogenes), the sense primer 5¢-ATGCCTGTGATTTG TGGGCC-3¢ and antisense primer 5¢-GCCAGTTGTTTT TCTGCCAC-3¢, for Nanog-444, the sense primer 5¢-ATTA TAAATCTAGAGACTCC-3¢ and antisense 5¢-TTGTTT GCCTTTGGGACTGGT-3¢, for b-actin, the sense primer 5¢-TCACCACCACGGCCGAGCG-3¢ and antisense 5¢-TCTCCTTCTGCATCCTGTCG-3¢ were used. DNA was amplified with an initial enzyme activation step at 94°C for 5 min, followed by 30 cycles (b-actin) or 34 cycles (Nanog- 403; Nanog-444; NANOGP8) of 94°C for 40 s, 55°C for 40 s and 72°C for 40 s (b-actin; Nanog-403; Nanog-444) or 90 s (NANOGP8). The PCR products were analyzed by 1.2% ag- arose gel electrophoresis and the bands were extracted using gel extraction kit (Omega, Bio-Tek, Doraville, GA, USA). DNA fragments extracted were ligated into T vector (Pro- mega) and sequencing analysis was carried out on positive clones identified. Cell culture, cancer and normal tissues All cancer cell lines including OS732, HepG2, MCF-7, THP-1, HeLa, PA-1, NTERA-2 were cultured in Dul- becco’s modified Eagle’s medium (DMEM) (Hyclone, Logan, UT, USA) supplemented with 2 mml-glutamine (Gibco-BRL, Gathersburg, MD, USA), 100 · nonessential amino acid solution (Hyclone), 100 mm sodium pyruvate (Hyclone), penicillin-streptomycin solution (Hyclone), and 10% fetal bovine serum (Hyclone). For primarily cultured fibroblast and fetal liver epithelium cells, 20% fetal bovine serum was added. All cancer tissues used were obtained from the tissue bank at ZhaoYang Hospital (Beijing, China) and patients gave written consent for the use of these tissues for research purposes. Nuclear protein extraction Nuclear protein extraction was performed as described [25]. In brief, cells were subsequently rinsed with ice-cold NaCl ⁄ P i (Hyclone), NaCl ⁄ P i containing 1 mm Na 3 VO 4 and 5mm NaF, and hypotonic buffer (NaCl ⁄ P i including 20 mm Hepes, 20 mm NaF, 1 mm Na 3 VO 4 ,1mm Na 4 P 2 O 7 , 0.4 lm microcystin, 1 mm EDTA, 1 mm EGTA, 1 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, 1 lgÆmL )1 each leupeptin, aprotinin and pepstatin). They were lysed with ice-cold hypotonic buffer with 0.2% NP-40. The nuclear pellets were collected by centrifuge at 16 000 g for 20 s and then resuspended in 150 lL high salt buffer (hypotonic buffer containing 420 mm NaCl and 20% gly- cerol). The pellets were rocked gently on ice for 30 min and centrifuged at 16 000 g for 20 min to separate the nuclear proteins. Protein concentration was determined by Brad- ford method [26]. Fusion protein expression and GST purification GST fusion proteins were expressed and prepared according to the manufacture’s instructions from Amersham Bioscien- ces (Piscataway, NJ, USA). In brief, E. coli BL21 cells carry- ing NANOGP8 were grown in LB medium and induced by NANOGP8 is a retrogene expressed in cancers J. Zhang et al. 1728 FEBS Journal 273 (2006) 1723–1730 ª 2006 The Authors Journal compilation ª 2006 FEBS the addition of 0.1 mm (isopropyl thio-b-d-galactoside). The pellet obtained from 500 mL of solution was suspended and sonicated for 30 s in 70 mL of 10 mm Tris ⁄ HCl. The resulting supernatant was subjected to affinity chromato- graphy on a glutathione-Sepharose column (Amersham Biosciences) and then eluted with 50 mm Tris and 10 m m glutathione (pH ¼ 8.0). Western blot For western blot analysis, equal protein (30 lg) was examined by 10% (w ⁄ v) SDS ⁄ PAGE. Proteins on the gel were transferred onto a nitrocellulose membrane in 1.44% glycine, 0.3% Tris (pH ¼ 8.4), 20% methanol at 80 V for 1 h, and the membrane was then blocked with NaCl ⁄ P i , 5% milk, 0.3% Tween-20. The membrane was probed with polyclonal goat anti-human Nanog (1 : 1000, AF1997, R&D Systems, Minneapolis, MN, USA) or monoclonal mouse anti-human Actin (1 : 500, SC-8432, Santa Cruz, Santa Cruz, CA, USA). Results were detec- ted using the WesternBreeze Ò kit (Invitrogen). X-ray films were scanned with a GDS8000 Gel Image Analysis Sys- tem (Ultra-Violet Products, Cambridge, UK). Expression constructs and cell transfection The GFP cDNA was cloned from pEGFP-N1 vector and inserted into pQCXIN between the BamHI and EcoRI sites. The NANOGP8 was amplified by RT-PCR and inser- ted into pQCXIN between AgeI and PacI sites. The GFP and(or) NANOGP8 were(was) ligated into the pQCXIN vector to produce the pQCXIN-GFP, pQCXIN- NANOGP8, and pQCXIN-NANOGP8-GFP. NIH3T3 cells were transfected with the expression vector pQCXIN, pQCXIN-NANOGP8 and pQCXIN-NANOGP8-GFP using Lipofectamine TM 2000 according to the manufac- turer’s instructions. Stable clones were selected and isolated in media containing 500 lgÆmL )1 G418 (Invitrogen) for cell cycle analysis or MTT assays. For nuclear localization, NIH3T3 cells were performed using Lipofectamine TM 2000 and then were taken photos with Zeiss 200 inverted fluores- cent microscopy (Carl Zeiss). Cell cycle analysis and MTT assay Cell cycle was measured by the propidium iodide (PI) stain- ing method. In brief, cells (1 · 10 6 ) were washed twice with cold NaCl ⁄ P i , fixed in 5 mL 70% ethanol at 4°C overnight. Cells were rinsed twice with NaCl ⁄ P i and resuspended in 500 lL NaCl ⁄ P i with 50 lgÆmL )1 RNaseA solution at 37°C for 30 min 50 mgÆmL )1 PI was added to the incubated solu- tion. Percentages of 15–20thousand cells in G 0 ⁄ G 1 , S and G 2 ⁄ M phase of the cell cycle were analyzed on a FACScali- bur and by Modifit software. For the MTT assay, cells were plated at 1 · 10 4 per 24-well plates and were cultured for 1–4 days. Viable cells were evaluated by adding 100 lL of 2.5 mgÆmL )1 MTT cultured in 37°C for 4 h. After the removal of MTT solution, 100 lL dimethyl sulfoxide were added to each well and gently shaken for 10 min. The absorbance was determined at 492 nm with plate reader (Sunrise, Tecan, Gr ¨ odig, Austria). Data analysis Data were analyzed by Student’s t-test. A value of P < 0.05 was considered statistical significance. Acknowledgements This work was supported by grants from Chinese Academy of Sciences (KSCW2-SW-205; KSCW2-SW- 218), from NSFC (30428017) and from The Chinese 973 Program (2004CB117404; 2005CB522603). References 1 Zaehres H, Lensch MW, Daheron L, Stewart SA, Itsko- vitz-Eldor J & Daley GQ (2005) High-efficiency RNA interference in human embryonic stem cells. Stem Cells 23, 299–305. 2 Chambers I, Colby D, Robertson M, Nichols J, Lee S, Tweedie S & Smith A (2003) Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 113, 643–655. 3 Mitsui K, Tokuzawa Y, Itoh H, Segawa K, Murakami M, Takahashi K, Maruyama M, Maeda M & Yama- naka S (2003) The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 113, 631–642. 4 Kuroda T, Tada M, Kubota H, Kimura H, Hatano SY, Suemori H, Nakatsuji N & Tada T (2005) Octamer and Sox elements are required for transcriptional cis regula- tion of Nanog gene expression. Mol Cell Biol 25, 2475– 2485. 5 Rodda DJ, Chew JL, Lim LH, Loh YH, Wang B, Ng HH & Robson P (2005) Transcriptional regulation of Nanog by OCT4 and SOX2. J Biol Chem 280, 24731– 24737. 6 Vanin EF (1985) Processed pseudogenes: characteristics and evolution. Annu Rev Genet 19, 253–272. 7 Harrison PM, Hegyi H, Balasubramanian S, Luscombe NM, Bertone P, Echols N, Johnson T & Gerstein M (2002) Molecular fossils in the human genome: identifi- cation and analysis of the pseudogenes in chromosomes 21 and 22. Genome Res 12, 272–280. 8 McCarrey JR & Riggs AD (1986) Determinator-inhibi- tor pairs as a mechanism for threshold setting in J. Zhang et al. NANOGP8 is a retrogene expressed in cancers FEBS Journal 273 (2006) 1723–1730 ª 2006 The Authors Journal compilation ª 2006 FEBS 1729 development: a possible function for pseudogenes. Proc Natl Acad Sci USA 83 , 679–683. 9 Korneev SA, Park JH & O’Shea M (1999) Neuronal expression of neural nitric oxide synthase (nNOS) pro- tein is suppressed by an antisense RNA transcribed from an NOS pseudogene. J Neurosci 19, 7711–7720. 10 Hirotsune S, Yoshida N, Chen A, Garrett L, Sugiyama F, Takahashi S, Yagami K, Wynshaw-Boris A & Yoshiki A (2003) An expressed pseudogene regulates the messenger-RNA stability of its homologous coding gene. Nature 423, 91–96. 11 Yano Y, Saito R, Yoshida N, Yoshiki A, Wynshaw- Boris A, Tomita M & Hirotsune S (2004) A new role for expressed pseudogenes as ncRNA: regulation of mRNA stability of its homologous coding gene. J Mol Med 82, 414–422. 12 Booth HA & Holland PW (2004) Eleven daughters of NANOG. Genomics 84, 229–238. 13 Pain D, Chirn GW, Strassel C & Kemp DM (2005) Multiple retropseudogenes from pluripotent cell-specific gene expression indicates a potential signature for novel gene identification. J Biol Chem 280, 6265–6268. 14 Takagi S, McFadden ML, Humphreys RE, Woda BA & Sairenji T (1993) Detection of 5-bromo-2-deoxyuri- dine (BrdUrd) incorporation with monoclonal anti- BrdUrd antibody after deoxyribonuclease treatment. Cytometry 14, 640–648. 15 Mighell AJ, Smith NR, Robinson PA & Markham AF (2000) Vertebrate pseudogenes. FEBS Lett 468, 109– 114. 16 Ruud P, Fodstad O & Hovig E (1999) Identification of a novel cytokeratin 19 pseudogene that may interfere with reverse transcriptase-polymerase chain reaction assays used to detect micrometastatic tumor cells. Int J Cancer 80, 119–125. 17 Normile D (2002) Cell proliferation. Common control for cancer, stem cells. Science 298, 1869. 18 Zhang JY, Wang X, Chen B, Suo GL, Zhao YH, Duan ZY & JW.Dai. (2005) Expression of Nanog gene promotes NIH3T3 cell proliferation. Biochem Biophys Res Commun 338, 1098–1102. 19 Monk M & Holding C (2001) Human embryonic genes re-expressed in cancer cells. Oncogene 20, 8085– 8091. 20 Looijenga LH, Stoop H, de Leeuw HP, de Gouveia Brazao CA, Gillis AJ, van Roozendaal KE, van Zoelen EJ, Weber RF, Wolffenbuttel KP, van Dekken H et al. (2003) POU5F1 (OCT3 ⁄ 4) identifies cells with pluripo- tent potential in human germ cell tumors. Cancer Res 63, 2244–2250. 21 Tai MH, Chang CC, Kiupel M, Webster JD, Olson LK & Trosko JE (2005) Oct4 expression in adult human stem cells: evidence in support of the stem cell theory of carcinogenesis. Carcinogenesis 26, 495–502. 22 Ezeh UI, Turek PJ, Reijo RA & Clark AT (2005) Human embryonic stem cell genes OCT4, NANOG, STELLAR, and GDF3 are expressed in both seminoma and breast carcinoma. Cancer 104, 2255–2265. 23 Hoei-Hansen CE, Almstrup K, Nielsen JE, Brask SS, Graem N, Skakkebaek NE, Leffers H & Rajpert-De Meyts E (2005) Stem cell pluripotency factor NANOG is expressed in human fetal gonocytes, testicular carci- noma in situ and germ cell tumours. Histopathology 47, 48–56. 24 Clark AT, Rodriguez RT, Bodnar MS, Abeyta MJ, Cedars MI, Turek PJ, Firpo MT & Reijo Pera RA (2005) Human STELLAR, NANOG, and GDF3 genes are expressed in pluripotent cells and map to chromo- some 12p13, a hotspot for teratocarcinoma. Stem Cells 22, 169–179. 25 Sadowski HB & Gilman MZ (1993) Cell-free activation of a DNA-binding protein by epidermal growth factor. Nature 362, 79–83. 26 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle protein-dye binding. Anal Biochem 72, 248–254. Supplementary material The following supplementary material is available online: Fig. S1. Sequence alignment of human Nanog and NANOGP8 (NgP8) genes. The differences in nucleo- tides (gray shaded with red characters) are shown. There is only one change in the inferred amino acid sequence from Gln (CAG 759, Q) in Nanog to His (CAC 759, H) in NANOGP8. Fig. S2. Sequence alignment of human Nanog, NANOGP8 and ESTs gb|CX163754.1| or gb|CX786835.1| or gb|CD642640.1| or gb|BF893620.1| obtained from NCBI EST database. The nucleotides of ESTs consistent to NANOGP8 are marked by D, The nucleotides of ESTs consistent to Nanog are marked by *; the nucleotides of ESTs different from Nanog and NANOGP8 are marked s. These ESTs are difficult to be identified as Nanog or NANOGP8. This material is available as part of the online article from http://www.blackwell-synergy.com NANOGP8 is a retrogene expressed in cancers J. Zhang et al. 1730 FEBS Journal 273 (2006) 1723–1730 ª 2006 The Authors Journal compilation ª 2006 FEBS . resulted in only one amino acid change in the predicted protein sequence from (Gln, 757CAG) in Nanog and (His, 757CAC) in NANOGP8. Since NANOGP8 has an intact. digested with RNAase-free DNase I (TaKaRa Carlsbad, CA, USA) at 37°C for 30 min and inactivated at 60°C for 10 min. With total RNA (2 lg) as the template and oligo(dT)