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Expression of Nanog gene promotes NIH3T3 cell proliferation
BBRC Biochemical and Biophysical Research Communications 338 (2005) 1098–1102 www.elsevier.com/locate/ybbrc
Expression of Nanog gene promotes NIH3T3 cell proliferation Jingyu Zhang a,b, Xia Wang a, Bing Chen a, Guangli Suo a, Yanhong Zhao a, Ziyuan Duan a, Jianwu Dai a,* a
Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100080, China b Graduate School, Chinese Academy of Sciences, Beijing 100080, China Received 28 September 2005 Available online 21 October 2005
Abstract Cells are the functional elements in tissue engineering and regenerative medicine. A large number of cells are usually needed for these purposes. However, there are numbers of limitations for in vitro cell proliferation. Nanog is an important self-renewal determinant in embryonic stem cells. However, it remains unknown whether Nanog will influence the cell cycle and cell proliferation of mature cells. In this study, we expressed Nanog in NIH3T3 cells and showed that expression of Nanog in NIH3T3 promoted cells to enter into S phase and enhanced cell proliferation. This suggests that Nanog gene might function in a similar fashion in mature cells as in ES cells. In addition, it may provide an approach for in vitro cell expansion. 2005 Elsevier Inc. All rights reserved. Keywords: Nanog; Cell cycle; Cell proliferation; NIH3T3
Tissue engineering is the regeneration and remodeling of tissue in vivo for the purpose of repairing, replacing, maintaining, or enhancing organ function, as well as the engineering and growth of functional tissue substitutes in vitro for implantation to replace the damaged or diseased tissues and organs [1]. Cells are the functional elements in tissue engineering, but the use of cells in tissueengineered constructs has been hampered largely due to the limitation of in vitro cell expansion. Great efforts have been made to understand the mechanisms that control cell proliferation [2–6]. The center of cellular proliferation is the cell division cycle, which is controlled by cyclin-dependent kinases (CDKs) [7]. The activities of CDKs in turn depend on their association with cyclins [8]. The precise regulation of proliferation in response to internal and external cues is critical for organ development and tissue renewal.
*
Corresponding author. Fax: +86 010 82614426. E-mail address: [email protected] (J. Dai).
0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.10.071
Nanog is a newly identified homeodomain-bearing protein that defines embryonic stem (ES) cell identity. It is transcribed specifically in pluripotent ES and embryonic germ (EG) cells in both mouse and human [9,10]. It plays a crucial role in the maintenance of both undifferentiated state and pluripotency independently of LIF signal pathway. This has been suggested by the loss of pluripotency in Nanog-deficient ES and in Nanog-null embryos after implantation [9,10]. Nanog expression may be controlled by an interaction between OCT4 and other proteins in ES cells through an adjacent pair of highly conserved Octamer- and Sox-binding sites of 5 0 -flanking region in Nanog [11]. Nanog is an important self-renewal determinant of ES cells. However, it remains unknown whether Nanog will influence cell cycle and cell proliferation of mature cells. NIH3T3 cell line was established from mouse embryo and is useful for gene transfection [12]. It is a highly contact-inhibited cell line. To explore the function of Nanog in mature cells, we used liposome-mediated gene transfection to express exogenous Nanog gene in NIH3T3 cells. After obtaining several Nanog stably transfected clones
J. Zhang et al. / Biochemical and Biophysical Research Communications 338 (2005) 1098–1102
by selection and isolation, we examined their growth characters including cell cycle and cell proliferation. Materials and methods Cell culture and transfection. The ES cell line MESPU13 derived from mouse 129/ter strain [13] was plated on mitomycin C-treated mouse embryonic fibroblasts in high glucose DulbeccoÕs modified EagleÕs medium (DMEM) (Hyclone) supplemented with 20% fetal calf serum (characterized FBS, Hyclone), 1000 U/ml LIF, 0.1 mM b-mercaptoethanol, 2 mM L-glutamine (Gibco), and 100· non-essential amino acid solution (Hyclone), 100 mM sodium pyruvate (Hyclone), 100 U/ml penicillin (Gibco), 100 lg/ml streptomycin (Gibco). NIH3T3 cells were cultured in DMEM with 10% FBS. NIH3T3 cells were transfected with the expression vector pQCXIN (BD Clontech), pQCXIN-Nanog using Lipofectamine 2000 according to the manufacturerÕs instructions. Stable clones were selected and isolated in media containing 500 lg/ml G418 (Invitrogen). Antibiotic selection gave rise to seven stable Nanog-transfected clones and ten transfected clones with pQCXIN vector only (mock clones), some of which have been further analyzed. For nuclear location, NIH3T3 cells were transfected with pQCXIN-Nanog-GFP using Lipofectamine 2000 and then photographs were taken with Zeiss 200 inverted fluorescent microscope (Carl Zeiss). Gene cloning and expression constructs. The pQCXIN is a bicistronic expression vector designed to express a target gene along with the neomycin selection marker. The GFP cDNA was cloned from pEGFP-N1 vector and inserted into pQCXIN between the BamHI and EcoRI sites. The Nanog gene was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) using total RNA extracted from mouse ES cells and inserted into pQCXIN between NotI and BamHI sites. The GFP and (or) Nanog were (was) ligated into the pQCXIN vector to produce the pQCXIN-GFP, pQCXIN-Nanog, and pQCXIN-Nanog-GFP. Total RNA extract and semi-quantitative RT-PCR. Total RNAs were extracted from mES, NIH3T3, and NIH3T3 transfected cells using Trizol (invitrogen) reagent following the manufacturerÕs recommendations. Semi-quantitative RT-PCR was performed as described [14] with a minor modification. Prior to the first cDNA strand synthesis, total RNAs were digested with RNAase-free DNase I (TaKaRa) at 37 C for 20 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 strand was synthesized in a 25 ll reaction system with M-MLV reverse transcriptase (Promega). Firststrand cDNA and RNA without reverse transcriptase (RT) were amplified to confirm the success of RT reaction and no genomic DNA contamination. cDNA template (2 ll) was used in a 50 ll reaction volume with rTaq DNA polymerase (TaKaRa). Preliminary experiments were conducted to ensure that the measurements were performed in the exponential phase of the amplification process. For Nanog, the sense primer 5 0 ATTTGCGGCCGCATGAGTGTGGGTCTTC-3 0 and antisense primer 5 0 -CGGGATCCTCATATTTCACCTGGTGGAG-3 0 , for cyclinA, the sense primer 5 0 -CCTCGAGGCATTCGGGTCGC-3 0 and antisense primer 5 0 -TTCTTTTAAGCTCAGCTGGCC-3 0 , for m-cdk2, the sense primer 5 0 -TAGAGACTCCAGGATTTTAACG-3 0 and antisense primer 5 0 -GTGGGTTGTTTGCCTTTGGGAC-3 0 , and for b-actin, the sense primer 5 0 -AGAAGATCTGGCACCACACC-3 0 and antisense primer 5 0 TACGACCAGAGGCATACAGG-3 0 were used. Amplification following hot start (95 C for 5 min) was carried out 28 cycles for b-actin (30 cycles for Nanog, cyclinA, and cdk2) consisting of 1 min at 95 C, 40 s at 55 C, and 90 s for Nanog (1 min for b-actin, cyclinA, and cdk2) at 72 C; an additional extension time 7 min at 72 C was added at the end of the 28 or 30 cycles. PCR products were analyzed by 1.5% agarose gel electrophoresis and band intensity was measured directly on GDS8000 Gel Image Analysis System. The amount of each mRNA was expressed as a ratio between Nanog and b-actin. Nuclear protein extraction and Western blot. Nuclear protein extraction was performed as described [15]. In brief, cells were subsequently rinsed with ice-cold PBS (Hyclone), PBS containing 1 mM Na3VO4 and 5 mM NaF, and hypotonic buffer (PBS including 20 mM Hepes, 20 mM NaF,
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1 mM Na3VO4, 1 mM Na4P2O7, 0.4 lM microcystin, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, and 1 lg/ml 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 16,000g for 20 s and then resuspended in 150 ll high salt buffer (hypotonic buffer containing 420 mM NaCl and 20% glycerol). The pellets were rocked gently on ice for 30 min and centrifuged at 16,000g for 20 min to separate the nuclear proteins. Protein concentration was determined by the Bradford method. For Western blot analysis, equal nuclear proteins (30 lg) were 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 8.4), and 20% methanol at 80 V for 1 h, and the membrane was then blocked with PBS, 5% milk, and 0.3% Tween 20. The membrane was probed with rabbit antimouse Nanog (1:400, Abcam) or monoclonal mouse anti-human Actin (1:500, Santa Cruz). Results were detected using WesternBreeze kit (Invitrogen). X-ray films were scanned with a GDS8000 Gel Image Analysis System (Ultra-Violet Products). Cell cycle analysis and growth curve. The DNA contents of cells were measured by the propidium iodide (PI) staining method. Cells (1 · 106) were washed twice with cold PBS without Ca2+ and Mg2+, and fixed in 5 ml of 70% ethanol at 4 C overnight. Cells were rinsed twice with PBS without Ca2+ and Mg2+, and resuspended in 500 ll PBS with 50 lg/ml RNaseA solution at 37 C for 30 min. Fifty milligram per milliliter PI was added to the incubated solution. Percentages of 15–20,000 cells in G0/G1, S, and G2/M phases of the cell cycle were analyzed on a FACScalibur and by Modifit software. For the analysis of cell growth, cells were plated at 1 · 104 in each of the 24-well plates. Viable cells were counted from day 1 to day 7 and compared to the control. Cell counts were performed using a hemocytomer. These results were obtained from three independent clones and each reproduced three times. Data analysis. Data were analyzed by StudentÕs t test. A value of P < 0.05 was considered statistical significance.
Results and discussion Nanog alters the morphology of NIH3T3 NIH3T3 cells do not express endogenous Nanog gene (Fig. 1A). After transfection, we have selected and isolated seven stable NIH3T3 clones in which the expression of exogenous Nanog gene was confirmed by RT-PCR and Western blot. The mRNA transcriptional levels were performed by semi-quantitative RT-PCR. Expression levels were normalized using an internal b-actin control, and the changes were determined by densitometric analysis. There was significant difference (about 1.5-fold) between mES and Nanog-transfected clones in Nanog expression, while no significant change was noted among different clones (Fig. 1A). Western blot results (mES and five clones were shown) (Fig. 1B) were consistent with this finding. Under phase contrast microscopy, NIH3T3 and NIH3T3 transfected with empty pQCXIN vector were spindle-shaped with the typical fibroblastic appearance [16]. In contrast, NIH3T3 cells transfected with Nanog gene were round-shaped in culture. Their sizes appeared smaller than the normal and mock control cells. During mitosis, NIH3T3 cells round up and the attached surface areas of the cells decrease until the two resulting daughter cells reenter the interphase. This characteristic feature has been used to purify G1 cell populations
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Fig. 1. (A) Nanog expression in seven transfected clones and mES were detected by semi-quantitative RT-PCR and normalized by the housekeeping gene b-actin. (B) Nanog protein levels were determined by Western blot. Equal nuclear protein was loaded in each lane.
using a ‘‘shake-off’’ procedure [17]. NIH3T3 transfected with Nanog gene appeared round-shaped in culture suggested that the cells transfected with Nanog gene had more dividing potential.
fusion protein was localized in the nuclei of transfected NIH3T3 (Figs. 2A and B), while GFP in the mock control was present diffusely in the cytoplasm (Figs. 2C and D). Nanog promotes cells to enter into S phase
Nanog is localized in the nuclei of transfected cells Nanog containing a homeodomain suggests that it is likely to act as a transcriptional regulator [9] and should be localized in the nucleus. To confirm this, we constructed a Nanog and GFP fusion protein expression vector. The
Cell cycle analysis was performed by flow cytometry (Figs. 2E–H). The percentage of S phase in Nanog-transfected cells was 57.3% (Fig. 2G), which was significantly higher than those of normal NIH3T3 cells (47.1%) (Fig. 2E) and the mock control (46.5%) (Fig. 2F). The
Fig. 2. Nuclear localization of Nanog-GFP and flow cytometric analysis results. The Nanog-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 photographs (A,C). Original magnification: (A–D) 200·. Flow cytometric analysis of the normal NIH3T3 cells (E), the mock control (F), transfected with Nanog gene (G), and comparison among them (H). The percentages of cells in S stage are: 47.1 ± 1.6 (E), 46.5 ± 2.2 (F), and 57.3 ± 4.1 (G). Data are presented as means ± SD. Results were obtained from three independent clones and each was repeated three times. Data were analyzed using StudentÕs t test. NIH3T3 cells, the mock cells versus NIH3T3 transfected with Nanog gene, P < 0.05; NIH3T3 cells versus the mock ones, P > 0.05.
J. Zhang et al. / Biochemical and Biophysical Research Communications 338 (2005) 1098–1102
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Fig. 3. Nanog gene expression promoted cell growth. (A) The effect of Nanog gene expression on NIH3T3 cells growth. Data are given as means ± SD. Results were obtained from three independent clones and each was repeated three times. Data were analyzed using StudentÕs t test. NIH3T3 transfected with Nanog gene versus the normal NIH3T3 and mock control, P < 0.05; the normal NIH3T3 and the mock control, P > 0.05. (B) CyclinA and cdk2 expression in NIH3T3 cells, the mock control and the Nanog transfected cells. The expression of cyclinA and cdk2 was not affected by Nanog expression.
difference was statistically significant (P < 0.05) between Nanog-transfected cells and the controls (the normal NIH3T3 cells and the mock ones), and no significant difference (P > 0.05) was noted between the normal NIH3T3 and the mock control (Fig. 2H). The results were obtained from three independent clones and each was repeated three times. It showed that expression of exogenous Nanog gene promoted cells to enter into the S phase of cell cycle. Nanog promotes cell proliferation The increase in the percentage of S phase is a good indicator of cell proliferation [18], so we examined the effect of Nanog expression on NIH3T3 cell growth. Nanog transfected cells showed an increased proliferation rate compared to those of the normal and the mock control cells, while the rate of cell growth was similar between NIH3T3 cells and the mock control (Fig. 3A). These results were obtained from three independent clones and each was repeated three times.
for in vitro expansion of mature cells. By generating human cell clones carrying Nanog gene under an inducible promoter [22,23], we might be able to control the human cell growth in vitro and obtain sufficient cells when needed. In summary, we showed that expression of Nanog gene promoted NIH3T3 cells to enter into S phase and increased the cell proliferation. This suggests that Nanog gene might function in a similar fashion in mature cells as in ES cells. In addition, it may provide an approach for the in vitro cell expansion. However, the molecular mechanism for Nanog gene function in NIH3T3 is not clear. Thus, additional studies are needed to uncover the precise mechanism of how Nanog promotes cell proliferation. Acknowledgments This work was supported by the ‘‘100 Talented Scholar Program’’ and grants from Chinese Academy of Sciences (KSCX2-SW-205; KSCW2-SW-218), from NSFC (30428017), and from The Chinese 973 Program (2004CB117404; 2005CB522603).
The study of the expression of cell cycle-dependent kinases Mammalian cell proliferation is primarily regulated at the G1- to S-phase cell cycle entry point. A large number of genes can affect cell proliferation. CyclinA associated enzymes have been established as key elements of progression through the S phase of the cell cycle [19,20]. As the G1/S-Cdk activities reach a critical threshold, it triggers the transition from late G1 into S phase. CyclinA, which binds to cdk2, is expressed in S phase, forming S-Cdk. The latter is required for DNA synthesis [21]. Fig. 3B shows that the expression levels of cyclinA and cdk2 were very similar among the control, the mock control, and the cells transfected with Nanog. This suggests that the effect of Nanog on cell proliferation may not involve cyclinA and cdk2. Cells are the functional elements of regenerative medicine and tissue engineering. There are many limitations for the in vitro cell expansion. In this study, we showed that expression of Nanog gene in NIH3T3 cells promoted cell proliferation. This may provide a possible approach
References [1] J.D. Sipe, C.A. Kelley, L.A. Mcnicol, Reparative medicine: growing tissues and organs, Ann. NY Acad. Sci. 961 (2002). [2] D.G. Johnson, J.K. Schwarz, W.D. Cress, J.R. Nevins, Expression of transcription factor E2F1 induces quiescent cells to enter S phase, Nature 365 (1993) 349–352. [3] L. Wu, C. Timmers, B. Maiti, H.I. Saavedra, L. Sang, G.T. Chong, F. Nuckolls, P. Giangrande, F.A. Wright, S.J. Field, M.E. Greenberg, S. Orkin, J.R. Nevins, M.L. Robinson, G. Leone, The E2F1-3 transcription factors are essential for cellular proliferation, Nature 414 (2001) 457–462. [4] M. Duman-Scheel, L. Weng, S. Xin, W. Du, Hedgehog regulates cell growth and proliferation by inducing Cyclin D and Cyclin E, Nature 417 (2002) 299–304. [5] U. Thorsteinsdottir, A. Mamo, E. Kroon, L. Jerome, J. Bijl, H.J. Lawrence, K. Humphries, G. Sauvageau, Overexpression of the myeloid leukemia-associated Hoxa9 gene in bone marrow cells induces stem cell expansion, Blood 99 (2002) 121–129. [6] T. Reya, A.W. Duncan, L. Ailles, J. Domen, D.C. Scherer, K. Willert, L. Hintz, R. Nusse, I.L. Weissman, A role for Wnt signaling in selfrenewal of haematopoietic stem cells, Nature 423 (2003) 409–414.
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[7] L.H. Hartwell, M.B. Kastan, Cell cycle control and cancer, Science 266 (1994) 1821–1828. [8] M. Schwab, M. Tyers, Cell cycle. Archipelago of destruction, Nature 413 (2001) 268–269. [9] I. Chambers, D. Colby, M. Robertson, J. Nichols, S. Lee, S. Tweedie, A. Smith, Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells, Cell 113 (2003) 643–655. [10] K. Mitsui, Y. Tokuzawa, H. Itoh, K. Segawa, M. Murakami, K. Takahashi, M. Maruyama, M. Maeda, S. Yamanaka, The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells, Cell 113 (2003) 631–642. [11] T. Kuroda, M. Tada, H. Kubota, H. Kimura, S.Y. Hatano, H. Suemori, N. Nakatsuji, T. Tada, Octamer and Sox elements are required for transcriptional cis regulation of Nanog gene expression, Mol. Cell Biol. 25 (2005) 2475–2485. [12] N.G. Copeland, G.M. Cooper, Transfection by exogenous and endogenous murine retrovirus DNAs, Cell 16 (1979) 347–356. [13] X.L. Hu, K.G. Shang, Establishment and characterization of six ES cell lines from mouse 129/ter strain, Acta, Scientiarum Naturalium Universitatis Pekinensis 32 (1996) 248–253. [14] Y. Zhao, E. Chen, L. Li, B. Gong, W. Xie, S. Nanji, I.D. Dube, M.R. Hough, Gene expression profiling in the inductive human hematopoietic microenvironment, Biochem. Biophys. Res. Commun. 323 (2004) 703–711. [15] H.B. Sadowski, M.Z. Gilman, Cell-free activation of a DNA-binding protein by epidermal growth factor, Nature 362 (1993) 79–83.
[16] J.L. Jainchill, S.A. Aaronson, G.J. Todaro, Murine sarcoma and leukemia viruses: assay using clonal lines of contact-inhibited mouse cells, J. Virol. 4 (1969) 549–553. [17] P. Kasinathan, J.G. Knott, Z. Wang, D.J. Jerry, J.M. Robl, Production of calves from G1 fibroblasts, Nat. Biotechnol. 19 (2001) 1176–1178. [18] S. Takagi, M.L. McFadden, R.E. Humphreys, B.A. Woda, T. Sairenji, Detection of 5-bromo-2-deoxyuridine (BrdUrd) incorporation with monoclonal anti-BrdUrd antibody after deoxyribonuclease treatment, Cytometry 14 (1993) 640–648. [19] A.R. Rosenberg, F. Zindy, F. Le Deist, H. Mouly, P. Metezeau, C. Brechot, E. Lamas, Overexpression of human cyclin A advances entry into S phase, Oncogene 10 (1995) 1501–1509. [20] S.I. Kim, C.S. Park, M.S. Lee, M.S. Kwon, E.H. Jho, W.K. Song, Cyclin-dependent kinase 2 regulates the interaction of Axin with betacatenin, Biochem. Biophys. Res. Commun. 317 (2004) 478–483. [21] K. Riabowol, G. Draetta, L. Brizuela, D. Vandre, D. Beach, The cdc2 kinase is a nuclear protein that is essential for mitosis in mammalian cells, Cell 57 (1989) 393–401. [22] F. Yao, T. Svensjo, T. Winkler, M. Lu, C. Eriksson, E. Eriksson, Tetracycline repressor, tetR, rather than the tetR-mammalian cell transcription factor fusion derivatives, regulates inducible gene expression in mammalian cells, Hum. Gene Ther. 9 (1998) 1939–1950. [23] J.R. Howe, B.V. Skryabin, S.M. Belcher, C.A. Zerillo, C. Schmauss, The responsiveness of a tetracycline-sensitive expression system differs in different cell lines, J. Biol. Chem. 270 (1995) 14168–14174.
Expression of Nanog gene promotes NIH3T3 cell proliferation Jingyu Zhang a,b, Xia Wang a, Bing Chen a, Guangli Suo a, Yanhong Zhao a, Ziyuan Duan a, Jianwu Dai a,* a
Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100080, China b Graduate School, Chinese Academy of Sciences, Beijing 100080, China Received 28 September 2005 Available online 21 October 2005
Abstract Cells are the functional elements in tissue engineering and regenerative medicine. A large number of cells are usually needed for these purposes. However, there are numbers of limitations for in vitro cell proliferation. Nanog is an important self-renewal determinant in embryonic stem cells. However, it remains unknown whether Nanog will influence the cell cycle and cell proliferation of mature cells. In this study, we expressed Nanog in NIH3T3 cells and showed that expression of Nanog in NIH3T3 promoted cells to enter into S phase and enhanced cell proliferation. This suggests that Nanog gene might function in a similar fashion in mature cells as in ES cells. In addition, it may provide an approach for in vitro cell expansion. 2005 Elsevier Inc. All rights reserved. Keywords: Nanog; Cell cycle; Cell proliferation; NIH3T3
Tissue engineering is the regeneration and remodeling of tissue in vivo for the purpose of repairing, replacing, maintaining, or enhancing organ function, as well as the engineering and growth of functional tissue substitutes in vitro for implantation to replace the damaged or diseased tissues and organs [1]. Cells are the functional elements in tissue engineering, but the use of cells in tissueengineered constructs has been hampered largely due to the limitation of in vitro cell expansion. Great efforts have been made to understand the mechanisms that control cell proliferation [2–6]. The center of cellular proliferation is the cell division cycle, which is controlled by cyclin-dependent kinases (CDKs) [7]. The activities of CDKs in turn depend on their association with cyclins [8]. The precise regulation of proliferation in response to internal and external cues is critical for organ development and tissue renewal.
*
Corresponding author. Fax: +86 010 82614426. E-mail address: [email protected] (J. Dai).
0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.10.071
Nanog is a newly identified homeodomain-bearing protein that defines embryonic stem (ES) cell identity. It is transcribed specifically in pluripotent ES and embryonic germ (EG) cells in both mouse and human [9,10]. It plays a crucial role in the maintenance of both undifferentiated state and pluripotency independently of LIF signal pathway. This has been suggested by the loss of pluripotency in Nanog-deficient ES and in Nanog-null embryos after implantation [9,10]. Nanog expression may be controlled by an interaction between OCT4 and other proteins in ES cells through an adjacent pair of highly conserved Octamer- and Sox-binding sites of 5 0 -flanking region in Nanog [11]. Nanog is an important self-renewal determinant of ES cells. However, it remains unknown whether Nanog will influence cell cycle and cell proliferation of mature cells. NIH3T3 cell line was established from mouse embryo and is useful for gene transfection [12]. It is a highly contact-inhibited cell line. To explore the function of Nanog in mature cells, we used liposome-mediated gene transfection to express exogenous Nanog gene in NIH3T3 cells. After obtaining several Nanog stably transfected clones
J. Zhang et al. / Biochemical and Biophysical Research Communications 338 (2005) 1098–1102
by selection and isolation, we examined their growth characters including cell cycle and cell proliferation. Materials and methods Cell culture and transfection. The ES cell line MESPU13 derived from mouse 129/ter strain [13] was plated on mitomycin C-treated mouse embryonic fibroblasts in high glucose DulbeccoÕs modified EagleÕs medium (DMEM) (Hyclone) supplemented with 20% fetal calf serum (characterized FBS, Hyclone), 1000 U/ml LIF, 0.1 mM b-mercaptoethanol, 2 mM L-glutamine (Gibco), and 100· non-essential amino acid solution (Hyclone), 100 mM sodium pyruvate (Hyclone), 100 U/ml penicillin (Gibco), 100 lg/ml streptomycin (Gibco). NIH3T3 cells were cultured in DMEM with 10% FBS. NIH3T3 cells were transfected with the expression vector pQCXIN (BD Clontech), pQCXIN-Nanog using Lipofectamine 2000 according to the manufacturerÕs instructions. Stable clones were selected and isolated in media containing 500 lg/ml G418 (Invitrogen). Antibiotic selection gave rise to seven stable Nanog-transfected clones and ten transfected clones with pQCXIN vector only (mock clones), some of which have been further analyzed. For nuclear location, NIH3T3 cells were transfected with pQCXIN-Nanog-GFP using Lipofectamine 2000 and then photographs were taken with Zeiss 200 inverted fluorescent microscope (Carl Zeiss). Gene cloning and expression constructs. The pQCXIN is a bicistronic expression vector designed to express a target gene along with the neomycin selection marker. The GFP cDNA was cloned from pEGFP-N1 vector and inserted into pQCXIN between the BamHI and EcoRI sites. The Nanog gene was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) using total RNA extracted from mouse ES cells and inserted into pQCXIN between NotI and BamHI sites. The GFP and (or) Nanog were (was) ligated into the pQCXIN vector to produce the pQCXIN-GFP, pQCXIN-Nanog, and pQCXIN-Nanog-GFP. Total RNA extract and semi-quantitative RT-PCR. Total RNAs were extracted from mES, NIH3T3, and NIH3T3 transfected cells using Trizol (invitrogen) reagent following the manufacturerÕs recommendations. Semi-quantitative RT-PCR was performed as described [14] with a minor modification. Prior to the first cDNA strand synthesis, total RNAs were digested with RNAase-free DNase I (TaKaRa) at 37 C for 20 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 strand was synthesized in a 25 ll reaction system with M-MLV reverse transcriptase (Promega). Firststrand cDNA and RNA without reverse transcriptase (RT) were amplified to confirm the success of RT reaction and no genomic DNA contamination. cDNA template (2 ll) was used in a 50 ll reaction volume with rTaq DNA polymerase (TaKaRa). Preliminary experiments were conducted to ensure that the measurements were performed in the exponential phase of the amplification process. For Nanog, the sense primer 5 0 ATTTGCGGCCGCATGAGTGTGGGTCTTC-3 0 and antisense primer 5 0 -CGGGATCCTCATATTTCACCTGGTGGAG-3 0 , for cyclinA, the sense primer 5 0 -CCTCGAGGCATTCGGGTCGC-3 0 and antisense primer 5 0 -TTCTTTTAAGCTCAGCTGGCC-3 0 , for m-cdk2, the sense primer 5 0 -TAGAGACTCCAGGATTTTAACG-3 0 and antisense primer 5 0 -GTGGGTTGTTTGCCTTTGGGAC-3 0 , and for b-actin, the sense primer 5 0 -AGAAGATCTGGCACCACACC-3 0 and antisense primer 5 0 TACGACCAGAGGCATACAGG-3 0 were used. Amplification following hot start (95 C for 5 min) was carried out 28 cycles for b-actin (30 cycles for Nanog, cyclinA, and cdk2) consisting of 1 min at 95 C, 40 s at 55 C, and 90 s for Nanog (1 min for b-actin, cyclinA, and cdk2) at 72 C; an additional extension time 7 min at 72 C was added at the end of the 28 or 30 cycles. PCR products were analyzed by 1.5% agarose gel electrophoresis and band intensity was measured directly on GDS8000 Gel Image Analysis System. The amount of each mRNA was expressed as a ratio between Nanog and b-actin. Nuclear protein extraction and Western blot. Nuclear protein extraction was performed as described [15]. In brief, cells were subsequently rinsed with ice-cold PBS (Hyclone), PBS containing 1 mM Na3VO4 and 5 mM NaF, and hypotonic buffer (PBS including 20 mM Hepes, 20 mM NaF,
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1 mM Na3VO4, 1 mM Na4P2O7, 0.4 lM microcystin, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, and 1 lg/ml 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 16,000g for 20 s and then resuspended in 150 ll high salt buffer (hypotonic buffer containing 420 mM NaCl and 20% glycerol). The pellets were rocked gently on ice for 30 min and centrifuged at 16,000g for 20 min to separate the nuclear proteins. Protein concentration was determined by the Bradford method. For Western blot analysis, equal nuclear proteins (30 lg) were 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 8.4), and 20% methanol at 80 V for 1 h, and the membrane was then blocked with PBS, 5% milk, and 0.3% Tween 20. The membrane was probed with rabbit antimouse Nanog (1:400, Abcam) or monoclonal mouse anti-human Actin (1:500, Santa Cruz). Results were detected using WesternBreeze kit (Invitrogen). X-ray films were scanned with a GDS8000 Gel Image Analysis System (Ultra-Violet Products). Cell cycle analysis and growth curve. The DNA contents of cells were measured by the propidium iodide (PI) staining method. Cells (1 · 106) were washed twice with cold PBS without Ca2+ and Mg2+, and fixed in 5 ml of 70% ethanol at 4 C overnight. Cells were rinsed twice with PBS without Ca2+ and Mg2+, and resuspended in 500 ll PBS with 50 lg/ml RNaseA solution at 37 C for 30 min. Fifty milligram per milliliter PI was added to the incubated solution. Percentages of 15–20,000 cells in G0/G1, S, and G2/M phases of the cell cycle were analyzed on a FACScalibur and by Modifit software. For the analysis of cell growth, cells were plated at 1 · 104 in each of the 24-well plates. Viable cells were counted from day 1 to day 7 and compared to the control. Cell counts were performed using a hemocytomer. These results were obtained from three independent clones and each reproduced three times. Data analysis. Data were analyzed by StudentÕs t test. A value of P < 0.05 was considered statistical significance.
Results and discussion Nanog alters the morphology of NIH3T3 NIH3T3 cells do not express endogenous Nanog gene (Fig. 1A). After transfection, we have selected and isolated seven stable NIH3T3 clones in which the expression of exogenous Nanog gene was confirmed by RT-PCR and Western blot. The mRNA transcriptional levels were performed by semi-quantitative RT-PCR. Expression levels were normalized using an internal b-actin control, and the changes were determined by densitometric analysis. There was significant difference (about 1.5-fold) between mES and Nanog-transfected clones in Nanog expression, while no significant change was noted among different clones (Fig. 1A). Western blot results (mES and five clones were shown) (Fig. 1B) were consistent with this finding. Under phase contrast microscopy, NIH3T3 and NIH3T3 transfected with empty pQCXIN vector were spindle-shaped with the typical fibroblastic appearance [16]. In contrast, NIH3T3 cells transfected with Nanog gene were round-shaped in culture. Their sizes appeared smaller than the normal and mock control cells. During mitosis, NIH3T3 cells round up and the attached surface areas of the cells decrease until the two resulting daughter cells reenter the interphase. This characteristic feature has been used to purify G1 cell populations
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Fig. 1. (A) Nanog expression in seven transfected clones and mES were detected by semi-quantitative RT-PCR and normalized by the housekeeping gene b-actin. (B) Nanog protein levels were determined by Western blot. Equal nuclear protein was loaded in each lane.
using a ‘‘shake-off’’ procedure [17]. NIH3T3 transfected with Nanog gene appeared round-shaped in culture suggested that the cells transfected with Nanog gene had more dividing potential.
fusion protein was localized in the nuclei of transfected NIH3T3 (Figs. 2A and B), while GFP in the mock control was present diffusely in the cytoplasm (Figs. 2C and D). Nanog promotes cells to enter into S phase
Nanog is localized in the nuclei of transfected cells Nanog containing a homeodomain suggests that it is likely to act as a transcriptional regulator [9] and should be localized in the nucleus. To confirm this, we constructed a Nanog and GFP fusion protein expression vector. The
Cell cycle analysis was performed by flow cytometry (Figs. 2E–H). The percentage of S phase in Nanog-transfected cells was 57.3% (Fig. 2G), which was significantly higher than those of normal NIH3T3 cells (47.1%) (Fig. 2E) and the mock control (46.5%) (Fig. 2F). The
Fig. 2. Nuclear localization of Nanog-GFP and flow cytometric analysis results. The Nanog-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 photographs (A,C). Original magnification: (A–D) 200·. Flow cytometric analysis of the normal NIH3T3 cells (E), the mock control (F), transfected with Nanog gene (G), and comparison among them (H). The percentages of cells in S stage are: 47.1 ± 1.6 (E), 46.5 ± 2.2 (F), and 57.3 ± 4.1 (G). Data are presented as means ± SD. Results were obtained from three independent clones and each was repeated three times. Data were analyzed using StudentÕs t test. NIH3T3 cells, the mock cells versus NIH3T3 transfected with Nanog gene, P < 0.05; NIH3T3 cells versus the mock ones, P > 0.05.
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Fig. 3. Nanog gene expression promoted cell growth. (A) The effect of Nanog gene expression on NIH3T3 cells growth. Data are given as means ± SD. Results were obtained from three independent clones and each was repeated three times. Data were analyzed using StudentÕs t test. NIH3T3 transfected with Nanog gene versus the normal NIH3T3 and mock control, P < 0.05; the normal NIH3T3 and the mock control, P > 0.05. (B) CyclinA and cdk2 expression in NIH3T3 cells, the mock control and the Nanog transfected cells. The expression of cyclinA and cdk2 was not affected by Nanog expression.
difference was statistically significant (P < 0.05) between Nanog-transfected cells and the controls (the normal NIH3T3 cells and the mock ones), and no significant difference (P > 0.05) was noted between the normal NIH3T3 and the mock control (Fig. 2H). The results were obtained from three independent clones and each was repeated three times. It showed that expression of exogenous Nanog gene promoted cells to enter into the S phase of cell cycle. Nanog promotes cell proliferation The increase in the percentage of S phase is a good indicator of cell proliferation [18], so we examined the effect of Nanog expression on NIH3T3 cell growth. Nanog transfected cells showed an increased proliferation rate compared to those of the normal and the mock control cells, while the rate of cell growth was similar between NIH3T3 cells and the mock control (Fig. 3A). These results were obtained from three independent clones and each was repeated three times.
for in vitro expansion of mature cells. By generating human cell clones carrying Nanog gene under an inducible promoter [22,23], we might be able to control the human cell growth in vitro and obtain sufficient cells when needed. In summary, we showed that expression of Nanog gene promoted NIH3T3 cells to enter into S phase and increased the cell proliferation. This suggests that Nanog gene might function in a similar fashion in mature cells as in ES cells. In addition, it may provide an approach for the in vitro cell expansion. However, the molecular mechanism for Nanog gene function in NIH3T3 is not clear. Thus, additional studies are needed to uncover the precise mechanism of how Nanog promotes cell proliferation. Acknowledgments This work was supported by the ‘‘100 Talented Scholar Program’’ and grants from Chinese Academy of Sciences (KSCX2-SW-205; KSCW2-SW-218), from NSFC (30428017), and from The Chinese 973 Program (2004CB117404; 2005CB522603).
The study of the expression of cell cycle-dependent kinases Mammalian cell proliferation is primarily regulated at the G1- to S-phase cell cycle entry point. A large number of genes can affect cell proliferation. CyclinA associated enzymes have been established as key elements of progression through the S phase of the cell cycle [19,20]. As the G1/S-Cdk activities reach a critical threshold, it triggers the transition from late G1 into S phase. CyclinA, which binds to cdk2, is expressed in S phase, forming S-Cdk. The latter is required for DNA synthesis [21]. Fig. 3B shows that the expression levels of cyclinA and cdk2 were very similar among the control, the mock control, and the cells transfected with Nanog. This suggests that the effect of Nanog on cell proliferation may not involve cyclinA and cdk2. Cells are the functional elements of regenerative medicine and tissue engineering. There are many limitations for the in vitro cell expansion. In this study, we showed that expression of Nanog gene in NIH3T3 cells promoted cell proliferation. This may provide a possible approach
References [1] J.D. Sipe, C.A. Kelley, L.A. Mcnicol, Reparative medicine: growing tissues and organs, Ann. NY Acad. Sci. 961 (2002). [2] D.G. Johnson, J.K. Schwarz, W.D. Cress, J.R. Nevins, Expression of transcription factor E2F1 induces quiescent cells to enter S phase, Nature 365 (1993) 349–352. [3] L. Wu, C. Timmers, B. Maiti, H.I. Saavedra, L. Sang, G.T. Chong, F. Nuckolls, P. Giangrande, F.A. Wright, S.J. Field, M.E. Greenberg, S. Orkin, J.R. Nevins, M.L. Robinson, G. Leone, The E2F1-3 transcription factors are essential for cellular proliferation, Nature 414 (2001) 457–462. [4] M. Duman-Scheel, L. Weng, S. Xin, W. Du, Hedgehog regulates cell growth and proliferation by inducing Cyclin D and Cyclin E, Nature 417 (2002) 299–304. [5] U. Thorsteinsdottir, A. Mamo, E. Kroon, L. Jerome, J. Bijl, H.J. Lawrence, K. Humphries, G. Sauvageau, Overexpression of the myeloid leukemia-associated Hoxa9 gene in bone marrow cells induces stem cell expansion, Blood 99 (2002) 121–129. [6] T. Reya, A.W. Duncan, L. Ailles, J. Domen, D.C. Scherer, K. Willert, L. Hintz, R. Nusse, I.L. Weissman, A role for Wnt signaling in selfrenewal of haematopoietic stem cells, Nature 423 (2003) 409–414.
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J. Zhang et al. / Biochemical and Biophysical Research Communications 338 (2005) 1098–1102
[7] L.H. Hartwell, M.B. Kastan, Cell cycle control and cancer, Science 266 (1994) 1821–1828. [8] M. Schwab, M. Tyers, Cell cycle. Archipelago of destruction, Nature 413 (2001) 268–269. [9] I. Chambers, D. Colby, M. Robertson, J. Nichols, S. Lee, S. Tweedie, A. Smith, Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells, Cell 113 (2003) 643–655. [10] K. Mitsui, Y. Tokuzawa, H. Itoh, K. Segawa, M. Murakami, K. Takahashi, M. Maruyama, M. Maeda, S. Yamanaka, The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells, Cell 113 (2003) 631–642. [11] T. Kuroda, M. Tada, H. Kubota, H. Kimura, S.Y. Hatano, H. Suemori, N. Nakatsuji, T. Tada, Octamer and Sox elements are required for transcriptional cis regulation of Nanog gene expression, Mol. Cell Biol. 25 (2005) 2475–2485. [12] N.G. Copeland, G.M. Cooper, Transfection by exogenous and endogenous murine retrovirus DNAs, Cell 16 (1979) 347–356. [13] X.L. Hu, K.G. Shang, Establishment and characterization of six ES cell lines from mouse 129/ter strain, Acta, Scientiarum Naturalium Universitatis Pekinensis 32 (1996) 248–253. [14] Y. Zhao, E. Chen, L. Li, B. Gong, W. Xie, S. Nanji, I.D. Dube, M.R. Hough, Gene expression profiling in the inductive human hematopoietic microenvironment, Biochem. Biophys. Res. Commun. 323 (2004) 703–711. [15] H.B. Sadowski, M.Z. Gilman, Cell-free activation of a DNA-binding protein by epidermal growth factor, Nature 362 (1993) 79–83.
[16] J.L. Jainchill, S.A. Aaronson, G.J. Todaro, Murine sarcoma and leukemia viruses: assay using clonal lines of contact-inhibited mouse cells, J. Virol. 4 (1969) 549–553. [17] P. Kasinathan, J.G. Knott, Z. Wang, D.J. Jerry, J.M. Robl, Production of calves from G1 fibroblasts, Nat. Biotechnol. 19 (2001) 1176–1178. [18] S. Takagi, M.L. McFadden, R.E. Humphreys, B.A. Woda, T. Sairenji, Detection of 5-bromo-2-deoxyuridine (BrdUrd) incorporation with monoclonal anti-BrdUrd antibody after deoxyribonuclease treatment, Cytometry 14 (1993) 640–648. [19] A.R. Rosenberg, F. Zindy, F. Le Deist, H. Mouly, P. Metezeau, C. Brechot, E. Lamas, Overexpression of human cyclin A advances entry into S phase, Oncogene 10 (1995) 1501–1509. [20] S.I. Kim, C.S. Park, M.S. Lee, M.S. Kwon, E.H. Jho, W.K. Song, Cyclin-dependent kinase 2 regulates the interaction of Axin with betacatenin, Biochem. Biophys. Res. Commun. 317 (2004) 478–483. [21] K. Riabowol, G. Draetta, L. Brizuela, D. Vandre, D. Beach, The cdc2 kinase is a nuclear protein that is essential for mitosis in mammalian cells, Cell 57 (1989) 393–401. [22] F. Yao, T. Svensjo, T. Winkler, M. Lu, C. Eriksson, E. Eriksson, Tetracycline repressor, tetR, rather than the tetR-mammalian cell transcription factor fusion derivatives, regulates inducible gene expression in mammalian cells, Hum. Gene Ther. 9 (1998) 1939–1950. [23] J.R. Howe, B.V. Skryabin, S.M. Belcher, C.A. Zerillo, C. Schmauss, The responsiveness of a tetracycline-sensitive expression system differs in different cell lines, J. Biol. Chem. 270 (1995) 14168–14174.