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Ninjurin1 increases p21 expression and induces cellular senescence in human hepatoma cells
Journal of Hepatology 41 (2004) 637–643 www.elsevier.com/locate/jhep
Ninjurin1 increases p21 expression and induces cellular senescence in human hepatoma cells Takashi Toyama1,*, Yutaka Sasaki2, Masayoshi Horimoto1, Kenya Iyoda2, Takayuki Yakushijin2, Kazuyoshi Ohkawa1, Tetsuo Takehara2, Akinori Kasahara3, Toshiyuki Araki4, Masatsugu Hori1, Norio Hayashi2 1
Department of Internal Medicine and Therapeutics, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan 2 Department of Molecular Therapeutics, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan 3 Department of General Medicine, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan 4 Department of Pathology and Immunology and Internal Medicine, Washington University Medical School, St. Louis, MO 63110, USA
Background/Aims: Ninjurin1 is a novel adhesion molecule that has a role in promoting nerve regeneration. Although ninjurin1 is ubiquitously expressed in various human tissues, including the liver, the biologic functions of ninjurin1 in tissues other than the nervous system remain unknown. The aim of this study was to investigate the function of ninjurin1 in hepatocytes. Methods: The effect of ninjurin1 overexpression was examined in Huh-7 hepatoma cells. Ninjurin1 expression was examined by Western blot in human hepatocellular carcinoma tissues as well as their adjacent liver tissues. Results: Ninjurin1-overexpressing clones exhibited strong growth inhibition due to G1 cell cycle arrest, which is associated with a posttranscriptional increase in p21WAF1/Cip1, a decrease of cyclin-dependent kinase 2 activity and the hypophosphorylation of Rb. The ninjurin1-overexpressing clones had increased senescence-associated b-galactosidase activity and autofluorescent pigment, characteristic features of cellular senescence. The levels of ninjurin1 expression were higher in hepatocellular carcinoma tissues than those in adjacent liver tissues. Conclusions: The present study provides the first evidence that ninjurin1 is able to induce the senescence program. Ninjurin1 may be involved in the regulation of cellular senescence in the liver during carcinogenesis. q 2004 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Keywords: Ninjurin1; Cellular senescence; p21; G1 Arrest; Carcinogenesis; Hepatocellular carcinoma
1. Introduction Ninjurin1 (nerve injury-induced protein 1) is a novel adhesion molecule first isolated as a gene induced in Schwann cells after nerve injury [1]. Ninjurin1 is located on the cell surface of Schwann cells and neurons, and has an important role in neurite regeneration via homophilic interactions between neuronal axons and Schwann cells Received 15 January 2004; received in revised form 9 June 2004; accepted 25 June 2004; available online 20 July 2004 * Corresponding author. Address: Department of Molecular Therapeutics, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. Tel.: C81-6-6879-3636; fax: C81-6-6879-3639. E-mail address: [email protected] (T. Toyama).
[2]. In addition, ninjurin1 is ubiquitously expressed in various human tissues originating from epithelial cells (e.g. liver, kidney, thymus, adrenal gland) [3]. However, the biological relevance of ninjurin1 on tissues other than the nervous system remains unknown. In the present study, we explored the biologic and biochemical consequences of ninjurin1 overexpression in a human hepatoma cell line. Ninjurin1 expression in Huh-7 hepatoma cells induced p21 elevation and G1 cell cycle arrest. Furthermore, ninjurin1-overexpressing cells exhibited the characteristic features of cellular senescence: increased senescence-associated (SA) b-galactosidase activity and the presence of autofluorescent pigment. In addition, the levels of ninjurin1 expression were higher in human hepatocellular carcinoma (HCC) tissues than in their
0168-8278/$30.00 q 2004 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jhep.2004.06.027
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adjacent liver tissues. The present study suggests a possible role of ninjurin1 in regulating cellular senescence pathologically arising in the liver. 2. Materials and methods 2.1. Antibodies Anti-human ninjurin1 mouse polyclonal antibody and the anti-pRb antibody (14001A) were obtained from Pharmingen/Transduction Laboratories (San Diego, CA). Polyclonal antibodies for p21 (C-19), p27 (F-8), cyclinD1 (M-20), cyclinE (M-20), cyclinA (H-432), cyclin-dependentkinase2 (CDK2) (H-298), CDK4 (C-22), and CDK6 (C-21) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).
2.2. Establishment of ninjurin1-transfected Huh-7 cells Huh-7 cells, a human hepatoma cell line, were obtained from the Japanese Cancer Research Resource Bank Cell Bank Center (Tokyo, Japan). Cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% decomplemented fetal calf serum (FCS) and antibiotics. Human ninjurin1 cDNA was excised from a pT7T3 plasmid [2] and inserted into a cloning site of pcDNA3.1 plasmid (Invitrogen, Groningen, the Netherlands). The construction was verified by sequencing (ABI Prism310; Perkin–Elmer, Foster City, CA). The ninjurin1 expression plasmid was transfected into Huh-7 cells using a Lipofectin reagent (Gibco BRL/Life Technologies, Grand Island, NY). Selection was performed via the addition of 100 mg/ml Zeosin (Invitrogen, Co. Ltd., Japan). Using this method, the ninjurin1-transfected cells generated much fewer Zeosinresistant colonies than cells transfected with vector alone (mock clones). In addition, because Zeosin-resistant clones grew slowly, it took over a month to pick the slowest growing colony. After the Zeosin-resistant clones were screened for ninjurin1 expression by immunoblotting, four positive single clones were selected. Two positive clones (Nin1 and Nin2) and mock clones were used for the experiments, but similar results were obtained using the other two clones, and the magnitude of the effects observed were proportional to the amount of ninjurin1 expressed by each clone.
2.3. Growth curves The control cells and ninjurin1-overexpressing clones were seeded at a density of 1!104 cells/35 mm-diameter dish. At 1, 2, 3, and 4 days after plating, these cells were removed from the culture plate by short exposure to trypsin and EDTA (GIBCO-BRL/Life Technologies). The total cell number was determined by trypan blue staining via microscopy.
2.4. Cell-cycle analysis Exponentially growing cultures of the control cells and ninjurin1overexpressing clones were trypsinized and collected. Cell pellets were fixed in 70% ethanol and stored at K20 8C. On the day of the assay, the fixed cells were collected by centrifugation, and the pellets were resuspended in PBS containing 0.2 mg/ml propidium iodide, and 1 mg/ml RNase. After 30 min incubation, the cell suspension was filtered through a 60 mM Spectra mesh filter and analyzed with a FACS Caliber flow cytometer using CellQuest software (Becton Dickinson Co. Chino Hills, CA). The percentages of cells in different phases of the cell cycle were determined with the ModFit 2.0 computer program (Verity Software House, Inc., Topsham, ME).
2.5. Immunocytochemical detection of ninjurin1 Huh-7 cells were fixed with paraformaldehyde and permeabilized with 0.05% Triton X-100. After blocking with Block Ace (Snow Brand Milk Product Co., Sapporo, Japan) containing 10% normal goat serum, cells were incubated for 1 h with anti-ninjurin1 polyclonal antibody. The cells were further incubated with Alexaw 488 goat anti-mouse IgG (Molecular Probes Inc., Eugene, OR). Images were acquired by confocal laser microscopy using a Zeiss LSM510 (Carl Zeiss Co., Ltd., Germany).
2.6. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) Cells were fixed with neutral buffered formaldehyde on day 3 after plating. Apoptotic cells were detected using an In situ Apoptosis Detection Kit (WAKO, Tokyo, Japan) according to the manufacturer’s instructions. The percentage of TUNEL-positive cells relative to total cells was determined by counting 100–300 cells in 10 randomly chosen fields per cover slide using a Nikon Microphot-FXA (Nikon, Tokyo, Japan).
2.7. Western blot analysis Whole cell lysates were prepared in Triton lysis buffer. Protein concentration was determined using a BCA protein assay kit (Pierce Chemical, Rockford, IL). Identical amounts of protein were separated on an acrylamide gel, and transferred to Hybond-P membranes (Amersham Pharmacia Biotech Co. Ltd., Buckinghamshire, UK). The membrane was blocked and then incubated with each antibody. The membrane was further incubated with the horseradish peroxidase-conjugated immunoglobulin. Detection was performed using the enhanced chemiluminescence (ECL) assay protocol (Pierce Chemical). The signal intensities were analyzed using an NIH image program (National Institutes of Health, Bethesda, MD).
2.8. RNA isolation and northern blot analysis Total cellular RNA was prepared using Trizol reagent (GIBCOBRL/Life Technologies) in accordance with the manufacturer’s instructions. For Northern blot analysis, 20 mg of RNA was separated on 1.0% agarose formaldehyde gels and transferred onto Hybond-N membranes (Amersham Pharmacia Biotech Co. Ltd.), and crosslinked to the membrane with a GS GENE Linker (BioRad Laboratories, Hercules, CA). mRNAs were detected by hybridization with random-primer-labeled probes (Amersham Pharmacia Biotech Co. Ltd.).
2.9. Kinase assay In vitro phosphorylation of Rb by CDKs was detected by the phosphoFinde (Boston Biologicals, Inc, Bedford, MA) system following the manufacturer’s instructions. Whole cell lysates were prepared in Triton lysis buffer, with 20 mg of total cell protein per sample being precleared with 15 ml of protein G-Sepharose beads (Amersham Pharmacia Biotech Co. Ltd.). Immunoprecipitation of CDKs was performed with 15 ml of each anti-CDK polyclonal antibody. For the kinase reaction, immunocomplexes were incubated in kinase buffer supplemented with ATP-g-S and Rb kinase substrate (Boston Biologicals). Samples were then separated on an acrylamide gel and transferred to Hybond-P membranes. The membrane was blocked and then incubated with PhosphoFind antibody (Boston Biologicals). The membrane was further incubated with horseradish peroxidase-conjugated antibody. Detection of the phosphorylated Rb was performed using the ECL assay protocol (Pierce Chemical).
2.10. Senescence-associated b-galactosidase SA-b-galactosidase activity was detected using a SA-b-galactosidase staining kit (Cell Signaling Technology, Inc., Beverly, MA) according to the manufacturer’s instructions. The control cells and ninjurin1-overexpressing clones were fixed and stained at pH 6.0 with X-gal. Clear blue cytoplasmic staining was regarded as positive. The percentage of SA-b-galactosidase activity-positive cells relative to total cells was determined by counting 100–300 cells in 10 randomly chosen fields per dish using phase-contrast microscopy (ECLIPSE TS 100, Nikon).
2.11. Autofluorescence The control cells and ninjurin1-overexpressing clones cultured on LabTekw four-chamber slides (Nalge Nunc International Corp, Naperville, IL) were fixed with neutral buffered formaldehyde. Images (!200) were then obtained using a green filter (FT510, Carl Zeiss Co., Ltd.), with 60-s exposure using a fluorescence microscope (Axioplan2, Carl Zeiss Co., Ltd.).
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2.12. Human hepatocellular carcinoma tissues Twenty pairs of HCCs and adjacent nontumor counterparts were obtained at the time of surgical resection. All adjacent nontumor tissues were diagnosed as chronic hepatitis (nZ9) or cirrhosis (nZ11). Samples of normal liver tissue adjacent to hepatic metastatic tumors were obtained at the time of surgery and served as controls.
2.13. Statistical analysis The data were analyzed with Statview software (SAS Institute Inc., Cary, NC) and are expressed as meansGstandard deviation. The differences were compared using Bonferroni’s t-test among group if oneway ANOVA showed P!0.01. The difference was considered statistically significant if P!0.01.
3. Results 3.1. Overexpression of ninjurin1 in human hepatoma cells We introduced the ninjurin1 gene into Huh-7 cells as described in the Section 2 and established stable transfectants that constitutively expressed human ninjurin1. Among the four clones isolated, two, designated as Nin1 and Nin2, expressed relatively large amounts of human ninjurin1, judging from Western blot analyses. Nin1 and Nin2 showed approximately 2.3- and 2.5-fold increases in ninjurin1 protein, respectively, when compared to the mock clones, which had been transduced with an empty plasmid (Fig. 1(a)). In the immunocytochemical experiments, ninjurin1transduced cells showed stronger expression of ninjurin1 than the mock clones (Fig. 1(c)). Of note is the finding that ninjurin1 overexpression caused dramatic alterations in cell morphology, as shown in Fig. 2. Nin1 and Nin2 cells exhibited increased size and a flattened morphology. No morphological change was observed in the mock clones. Analysis of cell number after trypan blue staining revealed strong inhibition of cell growth in ninjurin1overexpressing clones (Fig. 3). The parental and mock clones were maintained with a doubling time of approximately 36 h, whereas Nin1 and Nin2 had a doubling time of approximately 4 days.
Fig. 1. Expression of ninjurin1 in ninjurin1-transfected Huh-7 cells. (a) (Top) Western blot analysis of ninjurin1 expression in control cells and ninjurin1-overexpressing clones (Nin1 and Nin2). (Bottom) The proteins visualized by Coomassie blue staining indicate that equal amounts of protein were loaded in each lane. One representative experiment of three is shown. (b) (Top) Northern blot analysis of ninjurin1 mRNA in the control cells and ninjurin1-overexpressing clones. (Bottom) Expressions of 18S and 28S rRNA indicate that equal amounts of RNA were loaded in each lane. (c) Immunocytochemical analysis of ninjurin1 expression in mock and Nin1 cells. Images were acquired at !200 magnification by confocal laser microscopy using a Zeiss LSM510.
the growth-inhibitory effect of ninjurin1 was primarily due to cell cycle arrest rather than cell death. 3.3. Cell-cycle regulatory genes To further characterize the nature of the cell-cycle arrest caused by ninjurin1 overexpression, the expression of several cell-cycle regulatory genes was examined by Western blot analysis. As shown in Fig. 6, the level of
3.2. Ninjurin1 provokes cell arrest To determine the mechanisms of the reduced rate of cell growth, cell cycle analysis was conducted using flow cytometry. As shown in Fig. 4, ninjurin1 overexpression increased the percentage of the total cell population in the G0–G1 phase, decreased the percentage of cells in the S phase, but had no consistent effect on the percentage of cells in the G2-M phase, compared to the controls. Thus, ninjurin1-overexpressing cells were arrested at the G1 phase. We performed a TUNEL assay to examine the possible effects of enforced ninjurin1 expression on apoptosis. As shown in Fig. 5, the percentage of apoptotic cells in the ninjurin1-overexpressing clones was significantly lower than that in the control cells. Taken together,
Fig. 2. Effect of ninjurin1 overexpression on cell morphology in Huh-7 cells. Phase contrast micrographs at !200 magnification show the parental, mock-transfected, and ninjurin1-overexpressing Huh-7 cells (Nin1 and Nin2). Nin1 and Nin2 cells exhibited increased size and a flattened morphology. No changes in morphology were detected in the mock clones. (BarZ50 mm).
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Fig. 3. Effect of ninjurin1 overexpression on cell growth. The total cell number was determined by trypan blue staining via microscopy at the times indicated. Data represent meansGSD of three experiments performed in triplicate. (*P!0.01).
expression of cyclinA declined, but those of cyclinD and cyclinE were not altered in ninjurin1-overexpressing clones, when compared to the control clones. We next examined the expression of the catalytic partners of these cyclins, CDK2, CDK4, and CDK6. The levels of expression of all CDKs were consistently suppressed by ninjurin1 overexpression.
Fig. 4. Cell cycle profile of ninjurin1-overexpressing Huh-7 cells. (a) Cell cycle distribution of ninjurin1-overexpressing clones was analyzed by flow cytometry. Exponentially growing cultures of Huh-7 cells or their derivatives were trypsinized and collected. Propidium iodidestained cells were analyzed by flow cytometry as described in Materials and Methods. (b) Values in the table indicate the percentage of the total cell population at the indicated phase of the cell cycle; data represent the meansGSD of three experiments performed in triplicate. *P!0.01.
Fig. 5. Apoptotic effect of ninjurin1 overexpression in Huh-7 cells. (a) Apoptotic cells were detected by TUNEL assay. Apoptotic cells are indicated with arrows (BarZ20 mm). (b) Apoptotic cells with nuclei stained brown were counted under a light microscope, and their percentages are shown. *P!0.01.
We also examined the levels of expression of CDK inhibitors p21 and p27. There was no change in the level of p27, but the level of expression of p21 was greatly upregulated. On average, the expression level of p21 increased approximately 5-fold in both Nin1 and Nin2, compared to the mock clones. Northern blot analysis revealed that there was no apparent difference between the levels of p21 mRNA, whereas the mRNA levels of CDK2, CDK4, and CDK6 were repressed (Fig. 7(c)). These results indicated that CDK2, CDK4, and CDK6 expressions in Nin1 and Nin2 were transcriptionally downregulated, but p21 expression was not. In summary, enforced ninjurin1 expression resulted in elevated p21 and in repressed cyclinA, CDK2, CDK4, and CDK6 expression. The decline in the level of cyclinA expression is likely to be a consequence of the G1 arrest rather than its cause, since the products of this gene are believed to be one of the downstream events stimulated by E2F and to participate in the second half of the cell cycle [4]. The activity of the cyclin–CDK kinase complexes is tightly controlled by several mechanisms, including synthesis of the cyclin and CDK proteins, phosphorylation of CDK, and binding of CDK-inhibitory proteins [5]. We further examined CDK-associated kinase activity in the control cells and ninjurin1-overexpressing cells. As shown in Fig. 7(a), Rb phosphorylation by CDK2 immunoprecipitates, but not by CDK4 or CDK6 immunoprecipitates, was significantly decreased in the ninjurin1-overexpressing clones.
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Fig. 7. Effect of ninjurin1 overexpression on CDKs and p21 in Huh-7 cells. (a) Analysis of CDK-associated Rb kinase activity in Huh-7 cells. CDKs were immunoprecipitated from whole cell lysates extracted from the control cells and the ninjurin1-overexpressing clones. The kinase activity of the immune complexes were measured using Rb protein as substrate. (b) Western blot analysis of the Rb protein in the control cells and the ninjurin1-overexpressing clones. ppRb indicates the hyperphosphorylated form of pRb. (c) Northern blot analysis of the CDKs and p21 mRNA in the control cells and the ninjurin1overexpressing clones. Expression of 18S and 28S rRNA indicate that equal amounts of RNA were loaded in each lane.
In addition, declines in the levels of hyperphosphorylated Rb (Fig. 7(b)) were also observed in ninjurin1-overexpressing clones. The data indicate that CDK2 activity, but not CDK4 or CDK6 activity, is downregulated in ninjurin1-overexpressing cells, although the levels of expression of all CDKs are consistently suppressed. Therefore, the levels of expression of CDKs do not seem to be important determinants for the CDK activities. It is more likely that inhibition of CDK2 activity is a consequence of ninjurin1-induced upregulation of p21, since p21 can efficiently inhibit cyclinE-CDK2 activity, but not cyclinD-dependent CDK4/6 activity [6]. Taken together, cell cycle arrest by enforced ninjurin1 expression seems to be associated with posttranscriptional elevation of p21 and a significant decrease in the CDK2 activity, but not CDK4 and CDK6 activities.
proliferative capacity and become senescent [7–9]. Therefore, to investigate whether ninjurin1 induces cellular senescence, we examined two additional senescenceassociated markers: detection of SA-b-galactosidase activity at pH 6.0 [10] and autofluorescence intensity [10,11]. Fig. 8 shows representative results of the SA-bgalactosidase activity. The percentage of SA-b-galactosidase-positive cells in ninjurin1-overexpressing populations was 26% in Nin1 and 21% in Nin2. In contrast, less than 1% of the parental and mock clones were SA-b-galactosidasepositive. The lysosomes of senescent cells accumulate lipofuscin, an autofluorescent pigment that is thought to be a heterogeneous mixture of lipid and protein products of peroxidation [11]. To determine the presence of autofluorescent pigments, we performed fluorescence microscopy on the Huh7 cells. As shown in Fig. 9(a), Nin1 and Nin2 cells displayed intense cytoplasmic, granular fluorescence. Flow cytometric analysis revealed that both Nin1 and Nin2 cells showed a discernable increase in fluorescence in comparison to the mock clones (Fig. 9(b)). Taken together, the data indicated that enforced expression of ninjurin1 induces not only cell-cycle arrest, but, more importantly, cellular senescence in hepatoma cells.
3.4. Cellular senescence
3.5. Expression of ninjurin1 in human liver tissues
The observed characteristics, such as a flat enlarged morphology, arrest with a G1 DNA content, increased expression of p21 and resistance to spontaneous apoptosis, are similar to those of cells that have suppressed their
We examined the expression of ninjurin1 by Western blot analysis in human liver tissues (Fig. 10). The levels of ninjurin1 expression in chronic diseased livers adjacent to HCC tissues increased in comparison with normal liver
Fig. 6. Effect of ninjurin1 overexpression on cell cycle regulators in Huh7 cells. Whole cell lysates (20 mg) extracted from the control cells and the ninjurin1-overexpressing clones were subjected to Western blot analysis with antibodies against cell-cycle regulators. The proteins visualized by Coomassie blue staining indicate that equal amounts of protein were loaded in each lane. One representative experiment of three is shown.
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4. Discussion
Fig. 8. Microscopic analysis of senescence-associated b-galactosidase staining in ninjurin1-overexpressing Huh-7 cells. Control cells and the ninjurin1-overexpressing clones cultured in 6-well plates were fixed and stained with senescence-associated b-gal staining kit as described in “Materials and Methods.” Photomicrographs (!200) were obtained using a phase-contrast microscope (ECLIPSE TS 100, Nikon, Tokyo, Japan) (BarZ50 mm).
tissues. Furthermore, the levels of ninjurin1 expression in HCC were significantly higher than those in the adjacent nontumor liver tissues. In other words, ninjurin1 expression was upregulated in chronic hepatitis or cirrhosis, and further increased in HCC. A recent report has shown that cellular senescence cells increase in the same order: normal liver, chronic hepatitis, and HCC [12]. This suggests that ninjurin1 is a mediator for activating the senescence program during the course of chronic liver disease.
Fig. 9. Effect of ninjurin1 overexpression on autofluorescence in Huh-7 cells. (a) Fluorescent photomicrographs (!200) of the control cells and the ninjurin 1-overexpressing clones. (b) Flow cytometric analysis of ninjurin1-overexpressing Huh-7 cells on autofluorescence. The assay was performed in triplicate and the results are representative of three independent experiments.
The present study clearly demonstrated that enforced expression of ninjurin1 upregulates p21 expression and triggers a cellular response leading to growth arrest and senescence in human hepatoma cells. Furthermore, increase of ninjurin1 expression was associated in vivo with both the development of HCC and the growth-inhibitory phase of liver regeneration (Fig. 10 and unpublished data). Ninjurin1-overexpressing clones displayed the following characteristics: (1) a flattened, enlarged cell morphology, G1 cell cycle arrest, and resistance to spontaneous apoptosis, which are commonly observed with senescent fibroblasts [13–15]; (2) accumulation of lipofuscin granules, which is a specific marker for senescent cells, evidenced by increased autofluorescence [11]; and (3) SA-b-galactosidase staining (pH 6.0), which is another specific biochemical marker of senescence [10]. Thus far, it has been established that ninjurin1 induces cell–cell adhesion by homophilic binding. The present study provides the first evidence that ninjurin1 provokes the cellular senescence program. Human fibroblasts undergo a limited number of cell divisions before entering the irreversible arrest of senescence [16]. With regard to the molecular mechanisms in the senescence process, various kinds of genes, for example, p53 [17], Rb [18], and cell cycle regulator genes p21 [13,14] and p16 [15], are involved in establishing and maintaining
Fig. 10. Expression of ninjurin1 in human and animal liver. (a) Expression of ninjurin1 was assessed by Western blot analysis in 20 pairs of HCC (T), adjacent nontumor liver counterparts (NT), all of which were chronic diseased livers (NT), and two normal livers (normal). Shown is a representative blot. The optical density of each band was analyzed from the Scion image. Ninjurin1 expression in adjacent nontumor liver tissues was higher than in normal liver tissues, and that in HCC was higher than in adjacent nontumor liver tissues.
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cellular senescence. These molecules commonly suffer lossof-function mutations in various human cancers, and are recognized as tumor suppressors. The Huh-7 hepatoma cells used in the present study carry a mutated form of the p53 gene at codon 220 [19]. This mutant p53 lacks a DNA binding motif and is unable to induce p21 expression [20,21]. Therefore, the observed increase in p21 expression of the ninjurin1-expressing clones is independent of p53. In addition, Northern blot analysis indicated that the large increase in p21 protein expression was not due to an increase in transcription, but to posttranscriptional regulation. This mechanism is consistent with a recent report suggesting that the posttranscriptional induction of p21 is important for the establishment of cellular senescence [22]. Although the underlying mechanism is not clear, ninjurin1 is capable of increasing the expression of p21. Since p21 was originally identified via the phenomenon of senescence [13] and is thought to be one of the key regulators of senescence [14], p21 expression induced by ninjurin1 may be an important mediator in activating the senescence program. Cellular senescence is not only observed in cultured cells such as fibroblasts [16] and mammary epithelial cells [23], but also occurs in vivo. In fact, senescent cells accumulate in chronic hepatitis, probably due to repeated destruction and regeneration of hepatocytes, and further increase in HCC tissue [12,24]. We found that ninjurin1 expression was upregulated in chronic hepatitis and further increased in HCC tissues, suggesting a role of ninjurin1 in mediating cellular senescence in chronic diseased livers as well as HCC. Cellular senescence is considered to function as a powerful tumor suppressive mechanism, limiting the proliferative capacity of cells in vivo [7,8]. Therefore, growth past senescent barriers may be a pivotal event in the earliest stages of carcinogenesis [12,23]. If so, hepatoma cells must acquire multiple alterations by which they can overcome ninjurin1-mediated senescence to grow in vivo. To better understand the role of ninjurin1 in the liver, further study is needed to examine whether inhibition of ninjurin1 expression affects senescence in hepatocytes as well as hepatocarcinogenesis. Acknowledgements This work was supported by a grant-in-aid (No. 1670499) from the Ministry of Education, Science, Sports, and Culture of Japan.
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Ninjurin1 increases p21 expression and induces cellular senescence in human hepatoma cells Takashi Toyama1,*, Yutaka Sasaki2, Masayoshi Horimoto1, Kenya Iyoda2, Takayuki Yakushijin2, Kazuyoshi Ohkawa1, Tetsuo Takehara2, Akinori Kasahara3, Toshiyuki Araki4, Masatsugu Hori1, Norio Hayashi2 1
Department of Internal Medicine and Therapeutics, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan 2 Department of Molecular Therapeutics, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan 3 Department of General Medicine, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan 4 Department of Pathology and Immunology and Internal Medicine, Washington University Medical School, St. Louis, MO 63110, USA
Background/Aims: Ninjurin1 is a novel adhesion molecule that has a role in promoting nerve regeneration. Although ninjurin1 is ubiquitously expressed in various human tissues, including the liver, the biologic functions of ninjurin1 in tissues other than the nervous system remain unknown. The aim of this study was to investigate the function of ninjurin1 in hepatocytes. Methods: The effect of ninjurin1 overexpression was examined in Huh-7 hepatoma cells. Ninjurin1 expression was examined by Western blot in human hepatocellular carcinoma tissues as well as their adjacent liver tissues. Results: Ninjurin1-overexpressing clones exhibited strong growth inhibition due to G1 cell cycle arrest, which is associated with a posttranscriptional increase in p21WAF1/Cip1, a decrease of cyclin-dependent kinase 2 activity and the hypophosphorylation of Rb. The ninjurin1-overexpressing clones had increased senescence-associated b-galactosidase activity and autofluorescent pigment, characteristic features of cellular senescence. The levels of ninjurin1 expression were higher in hepatocellular carcinoma tissues than those in adjacent liver tissues. Conclusions: The present study provides the first evidence that ninjurin1 is able to induce the senescence program. Ninjurin1 may be involved in the regulation of cellular senescence in the liver during carcinogenesis. q 2004 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Keywords: Ninjurin1; Cellular senescence; p21; G1 Arrest; Carcinogenesis; Hepatocellular carcinoma
1. Introduction Ninjurin1 (nerve injury-induced protein 1) is a novel adhesion molecule first isolated as a gene induced in Schwann cells after nerve injury [1]. Ninjurin1 is located on the cell surface of Schwann cells and neurons, and has an important role in neurite regeneration via homophilic interactions between neuronal axons and Schwann cells Received 15 January 2004; received in revised form 9 June 2004; accepted 25 June 2004; available online 20 July 2004 * Corresponding author. Address: Department of Molecular Therapeutics, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. Tel.: C81-6-6879-3636; fax: C81-6-6879-3639. E-mail address: [email protected] (T. Toyama).
[2]. In addition, ninjurin1 is ubiquitously expressed in various human tissues originating from epithelial cells (e.g. liver, kidney, thymus, adrenal gland) [3]. However, the biological relevance of ninjurin1 on tissues other than the nervous system remains unknown. In the present study, we explored the biologic and biochemical consequences of ninjurin1 overexpression in a human hepatoma cell line. Ninjurin1 expression in Huh-7 hepatoma cells induced p21 elevation and G1 cell cycle arrest. Furthermore, ninjurin1-overexpressing cells exhibited the characteristic features of cellular senescence: increased senescence-associated (SA) b-galactosidase activity and the presence of autofluorescent pigment. In addition, the levels of ninjurin1 expression were higher in human hepatocellular carcinoma (HCC) tissues than in their
0168-8278/$30.00 q 2004 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jhep.2004.06.027
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adjacent liver tissues. The present study suggests a possible role of ninjurin1 in regulating cellular senescence pathologically arising in the liver. 2. Materials and methods 2.1. Antibodies Anti-human ninjurin1 mouse polyclonal antibody and the anti-pRb antibody (14001A) were obtained from Pharmingen/Transduction Laboratories (San Diego, CA). Polyclonal antibodies for p21 (C-19), p27 (F-8), cyclinD1 (M-20), cyclinE (M-20), cyclinA (H-432), cyclin-dependentkinase2 (CDK2) (H-298), CDK4 (C-22), and CDK6 (C-21) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).
2.2. Establishment of ninjurin1-transfected Huh-7 cells Huh-7 cells, a human hepatoma cell line, were obtained from the Japanese Cancer Research Resource Bank Cell Bank Center (Tokyo, Japan). Cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% decomplemented fetal calf serum (FCS) and antibiotics. Human ninjurin1 cDNA was excised from a pT7T3 plasmid [2] and inserted into a cloning site of pcDNA3.1 plasmid (Invitrogen, Groningen, the Netherlands). The construction was verified by sequencing (ABI Prism310; Perkin–Elmer, Foster City, CA). The ninjurin1 expression plasmid was transfected into Huh-7 cells using a Lipofectin reagent (Gibco BRL/Life Technologies, Grand Island, NY). Selection was performed via the addition of 100 mg/ml Zeosin (Invitrogen, Co. Ltd., Japan). Using this method, the ninjurin1-transfected cells generated much fewer Zeosinresistant colonies than cells transfected with vector alone (mock clones). In addition, because Zeosin-resistant clones grew slowly, it took over a month to pick the slowest growing colony. After the Zeosin-resistant clones were screened for ninjurin1 expression by immunoblotting, four positive single clones were selected. Two positive clones (Nin1 and Nin2) and mock clones were used for the experiments, but similar results were obtained using the other two clones, and the magnitude of the effects observed were proportional to the amount of ninjurin1 expressed by each clone.
2.3. Growth curves The control cells and ninjurin1-overexpressing clones were seeded at a density of 1!104 cells/35 mm-diameter dish. At 1, 2, 3, and 4 days after plating, these cells were removed from the culture plate by short exposure to trypsin and EDTA (GIBCO-BRL/Life Technologies). The total cell number was determined by trypan blue staining via microscopy.
2.4. Cell-cycle analysis Exponentially growing cultures of the control cells and ninjurin1overexpressing clones were trypsinized and collected. Cell pellets were fixed in 70% ethanol and stored at K20 8C. On the day of the assay, the fixed cells were collected by centrifugation, and the pellets were resuspended in PBS containing 0.2 mg/ml propidium iodide, and 1 mg/ml RNase. After 30 min incubation, the cell suspension was filtered through a 60 mM Spectra mesh filter and analyzed with a FACS Caliber flow cytometer using CellQuest software (Becton Dickinson Co. Chino Hills, CA). The percentages of cells in different phases of the cell cycle were determined with the ModFit 2.0 computer program (Verity Software House, Inc., Topsham, ME).
2.5. Immunocytochemical detection of ninjurin1 Huh-7 cells were fixed with paraformaldehyde and permeabilized with 0.05% Triton X-100. After blocking with Block Ace (Snow Brand Milk Product Co., Sapporo, Japan) containing 10% normal goat serum, cells were incubated for 1 h with anti-ninjurin1 polyclonal antibody. The cells were further incubated with Alexaw 488 goat anti-mouse IgG (Molecular Probes Inc., Eugene, OR). Images were acquired by confocal laser microscopy using a Zeiss LSM510 (Carl Zeiss Co., Ltd., Germany).
2.6. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) Cells were fixed with neutral buffered formaldehyde on day 3 after plating. Apoptotic cells were detected using an In situ Apoptosis Detection Kit (WAKO, Tokyo, Japan) according to the manufacturer’s instructions. The percentage of TUNEL-positive cells relative to total cells was determined by counting 100–300 cells in 10 randomly chosen fields per cover slide using a Nikon Microphot-FXA (Nikon, Tokyo, Japan).
2.7. Western blot analysis Whole cell lysates were prepared in Triton lysis buffer. Protein concentration was determined using a BCA protein assay kit (Pierce Chemical, Rockford, IL). Identical amounts of protein were separated on an acrylamide gel, and transferred to Hybond-P membranes (Amersham Pharmacia Biotech Co. Ltd., Buckinghamshire, UK). The membrane was blocked and then incubated with each antibody. The membrane was further incubated with the horseradish peroxidase-conjugated immunoglobulin. Detection was performed using the enhanced chemiluminescence (ECL) assay protocol (Pierce Chemical). The signal intensities were analyzed using an NIH image program (National Institutes of Health, Bethesda, MD).
2.8. RNA isolation and northern blot analysis Total cellular RNA was prepared using Trizol reagent (GIBCOBRL/Life Technologies) in accordance with the manufacturer’s instructions. For Northern blot analysis, 20 mg of RNA was separated on 1.0% agarose formaldehyde gels and transferred onto Hybond-N membranes (Amersham Pharmacia Biotech Co. Ltd.), and crosslinked to the membrane with a GS GENE Linker (BioRad Laboratories, Hercules, CA). mRNAs were detected by hybridization with random-primer-labeled probes (Amersham Pharmacia Biotech Co. Ltd.).
2.9. Kinase assay In vitro phosphorylation of Rb by CDKs was detected by the phosphoFinde (Boston Biologicals, Inc, Bedford, MA) system following the manufacturer’s instructions. Whole cell lysates were prepared in Triton lysis buffer, with 20 mg of total cell protein per sample being precleared with 15 ml of protein G-Sepharose beads (Amersham Pharmacia Biotech Co. Ltd.). Immunoprecipitation of CDKs was performed with 15 ml of each anti-CDK polyclonal antibody. For the kinase reaction, immunocomplexes were incubated in kinase buffer supplemented with ATP-g-S and Rb kinase substrate (Boston Biologicals). Samples were then separated on an acrylamide gel and transferred to Hybond-P membranes. The membrane was blocked and then incubated with PhosphoFind antibody (Boston Biologicals). The membrane was further incubated with horseradish peroxidase-conjugated antibody. Detection of the phosphorylated Rb was performed using the ECL assay protocol (Pierce Chemical).
2.10. Senescence-associated b-galactosidase SA-b-galactosidase activity was detected using a SA-b-galactosidase staining kit (Cell Signaling Technology, Inc., Beverly, MA) according to the manufacturer’s instructions. The control cells and ninjurin1-overexpressing clones were fixed and stained at pH 6.0 with X-gal. Clear blue cytoplasmic staining was regarded as positive. The percentage of SA-b-galactosidase activity-positive cells relative to total cells was determined by counting 100–300 cells in 10 randomly chosen fields per dish using phase-contrast microscopy (ECLIPSE TS 100, Nikon).
2.11. Autofluorescence The control cells and ninjurin1-overexpressing clones cultured on LabTekw four-chamber slides (Nalge Nunc International Corp, Naperville, IL) were fixed with neutral buffered formaldehyde. Images (!200) were then obtained using a green filter (FT510, Carl Zeiss Co., Ltd.), with 60-s exposure using a fluorescence microscope (Axioplan2, Carl Zeiss Co., Ltd.).
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2.12. Human hepatocellular carcinoma tissues Twenty pairs of HCCs and adjacent nontumor counterparts were obtained at the time of surgical resection. All adjacent nontumor tissues were diagnosed as chronic hepatitis (nZ9) or cirrhosis (nZ11). Samples of normal liver tissue adjacent to hepatic metastatic tumors were obtained at the time of surgery and served as controls.
2.13. Statistical analysis The data were analyzed with Statview software (SAS Institute Inc., Cary, NC) and are expressed as meansGstandard deviation. The differences were compared using Bonferroni’s t-test among group if oneway ANOVA showed P!0.01. The difference was considered statistically significant if P!0.01.
3. Results 3.1. Overexpression of ninjurin1 in human hepatoma cells We introduced the ninjurin1 gene into Huh-7 cells as described in the Section 2 and established stable transfectants that constitutively expressed human ninjurin1. Among the four clones isolated, two, designated as Nin1 and Nin2, expressed relatively large amounts of human ninjurin1, judging from Western blot analyses. Nin1 and Nin2 showed approximately 2.3- and 2.5-fold increases in ninjurin1 protein, respectively, when compared to the mock clones, which had been transduced with an empty plasmid (Fig. 1(a)). In the immunocytochemical experiments, ninjurin1transduced cells showed stronger expression of ninjurin1 than the mock clones (Fig. 1(c)). Of note is the finding that ninjurin1 overexpression caused dramatic alterations in cell morphology, as shown in Fig. 2. Nin1 and Nin2 cells exhibited increased size and a flattened morphology. No morphological change was observed in the mock clones. Analysis of cell number after trypan blue staining revealed strong inhibition of cell growth in ninjurin1overexpressing clones (Fig. 3). The parental and mock clones were maintained with a doubling time of approximately 36 h, whereas Nin1 and Nin2 had a doubling time of approximately 4 days.
Fig. 1. Expression of ninjurin1 in ninjurin1-transfected Huh-7 cells. (a) (Top) Western blot analysis of ninjurin1 expression in control cells and ninjurin1-overexpressing clones (Nin1 and Nin2). (Bottom) The proteins visualized by Coomassie blue staining indicate that equal amounts of protein were loaded in each lane. One representative experiment of three is shown. (b) (Top) Northern blot analysis of ninjurin1 mRNA in the control cells and ninjurin1-overexpressing clones. (Bottom) Expressions of 18S and 28S rRNA indicate that equal amounts of RNA were loaded in each lane. (c) Immunocytochemical analysis of ninjurin1 expression in mock and Nin1 cells. Images were acquired at !200 magnification by confocal laser microscopy using a Zeiss LSM510.
the growth-inhibitory effect of ninjurin1 was primarily due to cell cycle arrest rather than cell death. 3.3. Cell-cycle regulatory genes To further characterize the nature of the cell-cycle arrest caused by ninjurin1 overexpression, the expression of several cell-cycle regulatory genes was examined by Western blot analysis. As shown in Fig. 6, the level of
3.2. Ninjurin1 provokes cell arrest To determine the mechanisms of the reduced rate of cell growth, cell cycle analysis was conducted using flow cytometry. As shown in Fig. 4, ninjurin1 overexpression increased the percentage of the total cell population in the G0–G1 phase, decreased the percentage of cells in the S phase, but had no consistent effect on the percentage of cells in the G2-M phase, compared to the controls. Thus, ninjurin1-overexpressing cells were arrested at the G1 phase. We performed a TUNEL assay to examine the possible effects of enforced ninjurin1 expression on apoptosis. As shown in Fig. 5, the percentage of apoptotic cells in the ninjurin1-overexpressing clones was significantly lower than that in the control cells. Taken together,
Fig. 2. Effect of ninjurin1 overexpression on cell morphology in Huh-7 cells. Phase contrast micrographs at !200 magnification show the parental, mock-transfected, and ninjurin1-overexpressing Huh-7 cells (Nin1 and Nin2). Nin1 and Nin2 cells exhibited increased size and a flattened morphology. No changes in morphology were detected in the mock clones. (BarZ50 mm).
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Fig. 3. Effect of ninjurin1 overexpression on cell growth. The total cell number was determined by trypan blue staining via microscopy at the times indicated. Data represent meansGSD of three experiments performed in triplicate. (*P!0.01).
expression of cyclinA declined, but those of cyclinD and cyclinE were not altered in ninjurin1-overexpressing clones, when compared to the control clones. We next examined the expression of the catalytic partners of these cyclins, CDK2, CDK4, and CDK6. The levels of expression of all CDKs were consistently suppressed by ninjurin1 overexpression.
Fig. 4. Cell cycle profile of ninjurin1-overexpressing Huh-7 cells. (a) Cell cycle distribution of ninjurin1-overexpressing clones was analyzed by flow cytometry. Exponentially growing cultures of Huh-7 cells or their derivatives were trypsinized and collected. Propidium iodidestained cells were analyzed by flow cytometry as described in Materials and Methods. (b) Values in the table indicate the percentage of the total cell population at the indicated phase of the cell cycle; data represent the meansGSD of three experiments performed in triplicate. *P!0.01.
Fig. 5. Apoptotic effect of ninjurin1 overexpression in Huh-7 cells. (a) Apoptotic cells were detected by TUNEL assay. Apoptotic cells are indicated with arrows (BarZ20 mm). (b) Apoptotic cells with nuclei stained brown were counted under a light microscope, and their percentages are shown. *P!0.01.
We also examined the levels of expression of CDK inhibitors p21 and p27. There was no change in the level of p27, but the level of expression of p21 was greatly upregulated. On average, the expression level of p21 increased approximately 5-fold in both Nin1 and Nin2, compared to the mock clones. Northern blot analysis revealed that there was no apparent difference between the levels of p21 mRNA, whereas the mRNA levels of CDK2, CDK4, and CDK6 were repressed (Fig. 7(c)). These results indicated that CDK2, CDK4, and CDK6 expressions in Nin1 and Nin2 were transcriptionally downregulated, but p21 expression was not. In summary, enforced ninjurin1 expression resulted in elevated p21 and in repressed cyclinA, CDK2, CDK4, and CDK6 expression. The decline in the level of cyclinA expression is likely to be a consequence of the G1 arrest rather than its cause, since the products of this gene are believed to be one of the downstream events stimulated by E2F and to participate in the second half of the cell cycle [4]. The activity of the cyclin–CDK kinase complexes is tightly controlled by several mechanisms, including synthesis of the cyclin and CDK proteins, phosphorylation of CDK, and binding of CDK-inhibitory proteins [5]. We further examined CDK-associated kinase activity in the control cells and ninjurin1-overexpressing cells. As shown in Fig. 7(a), Rb phosphorylation by CDK2 immunoprecipitates, but not by CDK4 or CDK6 immunoprecipitates, was significantly decreased in the ninjurin1-overexpressing clones.
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Fig. 7. Effect of ninjurin1 overexpression on CDKs and p21 in Huh-7 cells. (a) Analysis of CDK-associated Rb kinase activity in Huh-7 cells. CDKs were immunoprecipitated from whole cell lysates extracted from the control cells and the ninjurin1-overexpressing clones. The kinase activity of the immune complexes were measured using Rb protein as substrate. (b) Western blot analysis of the Rb protein in the control cells and the ninjurin1-overexpressing clones. ppRb indicates the hyperphosphorylated form of pRb. (c) Northern blot analysis of the CDKs and p21 mRNA in the control cells and the ninjurin1overexpressing clones. Expression of 18S and 28S rRNA indicate that equal amounts of RNA were loaded in each lane.
In addition, declines in the levels of hyperphosphorylated Rb (Fig. 7(b)) were also observed in ninjurin1-overexpressing clones. The data indicate that CDK2 activity, but not CDK4 or CDK6 activity, is downregulated in ninjurin1-overexpressing cells, although the levels of expression of all CDKs are consistently suppressed. Therefore, the levels of expression of CDKs do not seem to be important determinants for the CDK activities. It is more likely that inhibition of CDK2 activity is a consequence of ninjurin1-induced upregulation of p21, since p21 can efficiently inhibit cyclinE-CDK2 activity, but not cyclinD-dependent CDK4/6 activity [6]. Taken together, cell cycle arrest by enforced ninjurin1 expression seems to be associated with posttranscriptional elevation of p21 and a significant decrease in the CDK2 activity, but not CDK4 and CDK6 activities.
proliferative capacity and become senescent [7–9]. Therefore, to investigate whether ninjurin1 induces cellular senescence, we examined two additional senescenceassociated markers: detection of SA-b-galactosidase activity at pH 6.0 [10] and autofluorescence intensity [10,11]. Fig. 8 shows representative results of the SA-bgalactosidase activity. The percentage of SA-b-galactosidase-positive cells in ninjurin1-overexpressing populations was 26% in Nin1 and 21% in Nin2. In contrast, less than 1% of the parental and mock clones were SA-b-galactosidasepositive. The lysosomes of senescent cells accumulate lipofuscin, an autofluorescent pigment that is thought to be a heterogeneous mixture of lipid and protein products of peroxidation [11]. To determine the presence of autofluorescent pigments, we performed fluorescence microscopy on the Huh7 cells. As shown in Fig. 9(a), Nin1 and Nin2 cells displayed intense cytoplasmic, granular fluorescence. Flow cytometric analysis revealed that both Nin1 and Nin2 cells showed a discernable increase in fluorescence in comparison to the mock clones (Fig. 9(b)). Taken together, the data indicated that enforced expression of ninjurin1 induces not only cell-cycle arrest, but, more importantly, cellular senescence in hepatoma cells.
3.4. Cellular senescence
3.5. Expression of ninjurin1 in human liver tissues
The observed characteristics, such as a flat enlarged morphology, arrest with a G1 DNA content, increased expression of p21 and resistance to spontaneous apoptosis, are similar to those of cells that have suppressed their
We examined the expression of ninjurin1 by Western blot analysis in human liver tissues (Fig. 10). The levels of ninjurin1 expression in chronic diseased livers adjacent to HCC tissues increased in comparison with normal liver
Fig. 6. Effect of ninjurin1 overexpression on cell cycle regulators in Huh7 cells. Whole cell lysates (20 mg) extracted from the control cells and the ninjurin1-overexpressing clones were subjected to Western blot analysis with antibodies against cell-cycle regulators. The proteins visualized by Coomassie blue staining indicate that equal amounts of protein were loaded in each lane. One representative experiment of three is shown.
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4. Discussion
Fig. 8. Microscopic analysis of senescence-associated b-galactosidase staining in ninjurin1-overexpressing Huh-7 cells. Control cells and the ninjurin1-overexpressing clones cultured in 6-well plates were fixed and stained with senescence-associated b-gal staining kit as described in “Materials and Methods.” Photomicrographs (!200) were obtained using a phase-contrast microscope (ECLIPSE TS 100, Nikon, Tokyo, Japan) (BarZ50 mm).
tissues. Furthermore, the levels of ninjurin1 expression in HCC were significantly higher than those in the adjacent nontumor liver tissues. In other words, ninjurin1 expression was upregulated in chronic hepatitis or cirrhosis, and further increased in HCC. A recent report has shown that cellular senescence cells increase in the same order: normal liver, chronic hepatitis, and HCC [12]. This suggests that ninjurin1 is a mediator for activating the senescence program during the course of chronic liver disease.
Fig. 9. Effect of ninjurin1 overexpression on autofluorescence in Huh-7 cells. (a) Fluorescent photomicrographs (!200) of the control cells and the ninjurin 1-overexpressing clones. (b) Flow cytometric analysis of ninjurin1-overexpressing Huh-7 cells on autofluorescence. The assay was performed in triplicate and the results are representative of three independent experiments.
The present study clearly demonstrated that enforced expression of ninjurin1 upregulates p21 expression and triggers a cellular response leading to growth arrest and senescence in human hepatoma cells. Furthermore, increase of ninjurin1 expression was associated in vivo with both the development of HCC and the growth-inhibitory phase of liver regeneration (Fig. 10 and unpublished data). Ninjurin1-overexpressing clones displayed the following characteristics: (1) a flattened, enlarged cell morphology, G1 cell cycle arrest, and resistance to spontaneous apoptosis, which are commonly observed with senescent fibroblasts [13–15]; (2) accumulation of lipofuscin granules, which is a specific marker for senescent cells, evidenced by increased autofluorescence [11]; and (3) SA-b-galactosidase staining (pH 6.0), which is another specific biochemical marker of senescence [10]. Thus far, it has been established that ninjurin1 induces cell–cell adhesion by homophilic binding. The present study provides the first evidence that ninjurin1 provokes the cellular senescence program. Human fibroblasts undergo a limited number of cell divisions before entering the irreversible arrest of senescence [16]. With regard to the molecular mechanisms in the senescence process, various kinds of genes, for example, p53 [17], Rb [18], and cell cycle regulator genes p21 [13,14] and p16 [15], are involved in establishing and maintaining
Fig. 10. Expression of ninjurin1 in human and animal liver. (a) Expression of ninjurin1 was assessed by Western blot analysis in 20 pairs of HCC (T), adjacent nontumor liver counterparts (NT), all of which were chronic diseased livers (NT), and two normal livers (normal). Shown is a representative blot. The optical density of each band was analyzed from the Scion image. Ninjurin1 expression in adjacent nontumor liver tissues was higher than in normal liver tissues, and that in HCC was higher than in adjacent nontumor liver tissues.
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cellular senescence. These molecules commonly suffer lossof-function mutations in various human cancers, and are recognized as tumor suppressors. The Huh-7 hepatoma cells used in the present study carry a mutated form of the p53 gene at codon 220 [19]. This mutant p53 lacks a DNA binding motif and is unable to induce p21 expression [20,21]. Therefore, the observed increase in p21 expression of the ninjurin1-expressing clones is independent of p53. In addition, Northern blot analysis indicated that the large increase in p21 protein expression was not due to an increase in transcription, but to posttranscriptional regulation. This mechanism is consistent with a recent report suggesting that the posttranscriptional induction of p21 is important for the establishment of cellular senescence [22]. Although the underlying mechanism is not clear, ninjurin1 is capable of increasing the expression of p21. Since p21 was originally identified via the phenomenon of senescence [13] and is thought to be one of the key regulators of senescence [14], p21 expression induced by ninjurin1 may be an important mediator in activating the senescence program. Cellular senescence is not only observed in cultured cells such as fibroblasts [16] and mammary epithelial cells [23], but also occurs in vivo. In fact, senescent cells accumulate in chronic hepatitis, probably due to repeated destruction and regeneration of hepatocytes, and further increase in HCC tissue [12,24]. We found that ninjurin1 expression was upregulated in chronic hepatitis and further increased in HCC tissues, suggesting a role of ninjurin1 in mediating cellular senescence in chronic diseased livers as well as HCC. Cellular senescence is considered to function as a powerful tumor suppressive mechanism, limiting the proliferative capacity of cells in vivo [7,8]. Therefore, growth past senescent barriers may be a pivotal event in the earliest stages of carcinogenesis [12,23]. If so, hepatoma cells must acquire multiple alterations by which they can overcome ninjurin1-mediated senescence to grow in vivo. To better understand the role of ninjurin1 in the liver, further study is needed to examine whether inhibition of ninjurin1 expression affects senescence in hepatocytes as well as hepatocarcinogenesis. Acknowledgements This work was supported by a grant-in-aid (No. 1670499) from the Ministry of Education, Science, Sports, and Culture of Japan.
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