Through a genome-wide cDNA microarray, we identified that the paternally expressed gene 10 (PEG10) was highly expressed in a great majority of hepatocellular carcinomas, although its expression was absent in normal liver cells. Exogenous expression of PEG10 conferred oncogenic activity and transfection of hepatoma cells with antisense S-oligonucleotides suppressing PEG10 resulted in their growth inhibition. Additional experiments revealed that PEG10 protein associated with SIAH1, a mediator of apoptosis, and that overexpression of PEG10 decreased the cell death mediated by SIAH1. These findings suggested that development of drug(s) inhibiting PEG10 activity could be a novel approach for the treatment of hepatocellular carcinomas.

HCC3 is one of the most common malignancies worldwide. Although several novel therapeutic modalities have been developed in recent years, prognosis of advanced HCC remains poor. Molecular investigations have disclosed involvement of alterations of TP53, CTNNB1, and AXIN1 in hepatocarcinogenesis (1, 2, 3) but only in a limited fraction of HCCs. Thus, discovery of new target molecules that are critically involved in a majority of cases and expressed specifically in tumors will be essential for improving therapeutic intervention and prognosis of hepatic cancers.

Microarray technologies have enabled researchers to obtain comprehensive data about gene expression, not only in experimental models but also in human cancers (4, 5). In a previous report (6), we compared expression profiles of 20 HCCs with their corresponding noncancerous liver tissues using a cDNA microarray consisting of 23,040 genes. Those experiments disclosed a number of genes that appeared to be involved in hepatocarcinogenesis and revealed moreover that expression profiles were different between hepatitis B virus-positive and hepatitis C virus-positive HCCs.

To identify ideal therapeutic targets, we chose to investigate genes that were commonly and exclusively up-regulated in HCCs, using data obtained from the microarray. In the work reported here, we isolated the entire transcript of a gene that was selectively expressed in cancerous tissues. This gene was eventually found to be identical to PEG10(7). Exogenous expression of PEG10 promoted growth of certain HCC cell lines that did not manifest endogenous expression of this gene. In addition, we demonstrated interaction of PEG10 protein with SIAH proteins, which play important roles in apoptosis. Our data raise novel insights into mechanisms of hepatocarcinogenesis and suggest that PEG10 might serve as a novel molecular target for treatment of HCCs.

Cell Lines and Tissue Specimens.

HEK293 cells and human hepatoma cell lines HepG2, Huh7 and Alexander were obtained from the American Type Culture Collection (Manassas, VA). SNU423, SNU449, and SNU475 were obtained from the Korea cell line bank. All cell lines were grown in monolayers in appropriate media supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic solution (Sigma, St. Louis, MO) and maintained at 37°C in air containing 5% CO2. All HCCs and corresponding noncancerous liver tissues were obtained with informed consent from patients who underwent hepatectomy.

RT-PCR.

RT-PCR experiments were carried out in 20-μl volumes of PCR buffer (TaKaRa, Tokyo, Japan), with 4 min at 94°C for denaturing followed by 20 (for GAPDH) or 30 (for PEG10 and SIAH1) cycles of 94°C for 30 s, 56°C for 30 s, and 72°C for 30 s in the GeneAmp PCR system 9700 (Perkin-Elmer, Foster City, CA). Primer sequences were as follows: for GAPDH, forward 5′-ACAACAGCCTCAAGATCATCAG-3′ and reverse 5′-GGTCCACCACTGACACGTTG-3′; for PEG10, forward 5′-AACAACAACAACAACTCCAAGC-3′ and reverse 5′-TCTGCACCTGGCTCTGCAG-3′; and for SIAH1, forward 5′-TCCAACAATGACTTGGCGAGT-3′ and reverse 5′-CTTTTTCTGTGTGTGGCAGAG-3′.

Northern Blot Analysis.

Human multiple tissue blots (Clontech, Palo Alto, CA) were hybridized with a 32P-labeled PEG10 cDNA. Prehybridization, hybridization, and washing were performed according to the supplier’s recommendations. The blots were autoradiographed with intensifying screens at −80°C for 24 h.

Immunoblotting.

The polyclonal antibody to PEG10 was purified from sera of immunized rabbits with recombinant GST-PEG10 protein produced in Escherichia coli. Cell extracts were prepared using lysis buffer [150 mm NaCl, 1% Triton X-100, 50 mm Tris-HCl (pH 7.4), and 1 mm DTT, with complete Protease Inhibitor Cocktail (Boehringer Mannheim, Mannheim, Germany). Proteins were separated by 10% SDS-PAGE and immunoblotted with the rabbit anti-PEG10 antibody. Horseradish peroxidase-conjugated goat antirabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA) served as the secondary antibody for the ECL Detection System (Amersham Pharmacia Biotech, Piscataway, NJ).

Immunohistochemical Staining.

Cultured cells on chamber slides were fixed with PBS containing 4% paraformaldehyde for 15 min, then rendered permeable with PBS containing 0.1% Triton X-100 for 2.5 min at room temperature. Frozen sections from primary HCCs and noncancerous liver tissue were fixed with acetone for 15 min. The cells were incubated with 2% BSA in PBS for 24 h at 4°C and hybridized with the anti-PEG10 antibody. Antibodies were stained with fluorescent substrate-conjugated antirabbit secondary antibody (ICN Pharmaceuticals, Costa Mesa, CA). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole. Fluorescent images were obtained with an Eclipse E800 microscope (Nikon, Tokyo, Japan).

Colony Formation Assay and Growth Suppression Assay.

Cells transfected with plasmid vector expressing the entire coding region of PEG10 using FuGENE6 reagent according to the supplier’s protocol (Boehringer Mannheim) were cultured with an appropriate concentration of geneticin for 2 weeks, fixed with 100% methanol, and stained by Giemsa solution. Colonies > 1 mm were counted 2 weeks after transfection of pcDNA 3.1(+), pcDNA 3.1(−)/PEG10, or pcDNA 3.1(+)/PEG10. Cells transfected with sense (5′-CCTCGCGTGGTGAGTA-3′) or antisense (5′-TACTCACCACGCGAGG-3′) S-oligonucleotides of PEG10 were stained in the same manner.

Flow Cytometry.

A total of 1 × 105 cells was collected by trypsinization at the given time points and fixed in 70% cold ethanol. Cells treated with RNase and propidium iodide (50 μg/ml) in PBS were analyzed by a FACScan (Becton Dickinson, San Jose, CA).

Yeast Two-Hybrid Experiment.

A yeast two-hybrid assay was performed with the Matchmaker GAL4 Two-Hybrid System 3 according to the manufacturer’s protocols (Clontech). We cloned the entire coding sequence of PEG10 into the EcoRI-SalI site of pAS2-1 vector as bait and screened a human testis cDNA library (Clontech).

In Vitro Protein-binding Assay.

The entire coding regions of SIAH1 and SIAH2 were amplified using primers 5′-CGCGAATTCCGCCCACAGAAATGAGCC-3′ and 5′-CATCTCGAGACATGGAAATAGTTACATTGATGC-3′ or 5′-TGCGAATTCCATGGTTGGTTCGGAGC-3′ and 5′-GTGCTCGAGGACAACATGTAGAAATAGTAAC-3′, respectively, and cloned into appropriate cloning sites of pET21b vector (Novagen, Madison, WI) or pCMV-Flag5 (Sigma). Recombinant His-tagged SIAH-1 protein was prepared using the Xpress system (Invitrogen, Carlsbad, CA) according to the manufacturer’s recommendations. ProBond histidine affinity resin (Invitrogen) incubated with or without 10 μg of His-tagged SIAH-1 protein at 4°C for 1 h, followed by extensive washing with binding buffer [20 mm NaH2PO4, 500 mm NaCl (pH 7.8)], was then incubated with 50 μg of cell lysates from SNU423-PEG10 cells exogenously overexpressing PEG10 in NP40 lysis buffer [150 mm NaCl, 50 mm Tris (pH 8.0), and 1% NP40]. After the resin was washed with wash buffer (20 mm NaH2PO4 and 500 mm NaCl) twice each at pH 7.8, pH 6.0, and pH 5.5, protein was eluted with elution buffer (300 mm imidazole in wash buffer). The eluted proteins were analyzed by immunoblotting using anti-His probe antibody (Santa Cruz Biotechnology) or anti-PEG10 antibody. Similarly, GST or GST-PEG10 fusion protein, immobilized on Glutathione Sepharose 4B beads (Amersham Pharmacia Biotech, Uppsala, Sweden), was incubated with lysates from HEK293-SIAH2 cells overexpressing Flag-tagged SIAH2. Bound proteins were eluted with elution buffer [120 mm NaCl, 50 mm Tris-HCl (pH 8.0), and 20 mm glutathione (Sigma)] and analyzed by immunoblotting using anti-Flag (Sigma) and anti-PEG10 antibody.

Construction of Adenovirus Expressing SIAH1.

Generation and preparation of adenovirus-expressing SIAH1 was achieved using the Adenovirus Expression Vector Kit (TaKaRa) according to the supplier’s protocol. First, the entire coding region of SIAH1 was amplified and cloned into an appropriate site of the pcDNA3.1/myc-C vector (Invitrogen). Subsequently, the fragment of myc-tagged SIAH1 was cloned into the cosmid vector pAxCAwt supplied in the kit. (TaKaRa).

Identification of a Novel Gene Frequently Up-Regulated in HCCs.

Using a genome-wide cDNA microarray consisting of 23,040 genes, we identified a gene that was commonly up-regulated among a total of 20 HCCs (6). Elevated expression of PEG10 in the tumors was confirmed by TaqMan PCR (data not shown). A multitissue Northern blot using the cDNA as a probe showed a 6.4-kb transcript predominantly expressed in placenta, testis, and ovary (Fig. 1 A). Using the 5′ rapid amplification of cDNA ends method, we obtained cDNA sequences that covered almost the entire transcript of this gene (GenBank accession no. AB049150). Simple Modular Architecture Research Tool (version 3)4 suggested that the predicted protein contained a coiled-coil motif (codons 1–50) as well as a zinc-finger motif (codons 294–310).

Expression of PEG10 in HCC Cell Lines and Primary HCCs.

To investigate the role of PEG10 in HCCs, we generated rabbit polyclonal antibody to the gene product. Using this antibody, we evaluated expression of this protein in six hepatoma cell lines. HepG2, Huh7, and Alexander cells constitutively expressed Mr 40,000 PEG10 protein (Fig. 1,B), and immunohistochemical staining disclosed that PEG10 was located in both nucleus and cytoplasm in those cells (Fig. 1,C). Among 16 primary HCCs that were different from the 20 HCCs used for the cDNA microarray analysis, we detected strong nuclear and cytoplasmic staining of PEG10 in the tumor tissue from 15 cases but not in their corresponding normal tissues (Fig. 1 D).

Promotion of Growth of Human Hepatoma Cells by PEG10.

To test the effects of PEG10 gene transfer on growth of hepatoma cells, we transfected an expression plasmid containing PEG10 to two cell lines (SNU423 and SNU475), which had shown no endogenous expression of PEG10 protein (Fig. 1,B). Compared with mock or antisense plasmid clones, the PEG10 sense plasmid vector promoted colony formation in both cell lines (Fig. 2,A). To additionally investigate the growth-promoting effects of PEG10, we generated stable transfectants using SNU423 cells in which endogenous PEG10 expression was absent (Fig. 2,B). The PEG10 stable transfectant cells revealed significant growth promotion compared with the parental or mock cells (Fig. 2,C). Under conditions of serum starvation (0.1% FBS), the mock cells rapidly underwent growth arrest, but stable PEG10-expressing cells continued to proliferate (Fig. 2 D).

Suppression of Growth of Hepatoma Cells by Antisense Oligonucleotides of PEG10.

To examine whether suppression of PEG10 would retard growth and/or induce death of HCC cells, we designed various antisense S-oligonucleotides. Among them, antisense S-oligonucleotides encompassing the first exon-intron boundary, but not other antisense or control S-oligonucleotides, significantly decreased endogenous expression of PEG10 in Alexander and Huh7 cells that constitutively express abundant PEG10 (Fig. 2,E for Huh7 and data not shown). Transfection of the antisense S-oligonucleotides significantly reduced number of viable cells in these two cell lines (Fig. 2 F for Huh7, and data not shown), but no growth-suppressive effect was observed when we introduced the antisense S-oligonucleotides into SNU423 cells, which do not express endogenous PEG10 (data not shown).

Interaction of PEG10 with SIAH-1 and SIAH-2.

To examine the oncogenic mechanism of PEG10, we searched for PEG10-interacting proteins using a yeast two-hybrid screening system because the NH2-terminal region of PEG10 also contains a coiled-coil motif that generally allows for protein-protein interactions. Among the clones identified, those homologous to Drosophila seven in absentia (SIAH1 and SIAH2) interacted with PEG10 by simultaneous transformation with pAS2.1-PEG10 and pACT2-SIAH1 or SIAH2 (Fig. 3,A). To confirm the interaction of PEG10 with SIAH1, we prepared recombinant His-tagged SIAH1 protein and detected this association when PEG10 protein was expressed in mammalian cells (Fig. 3,B). In addition, we demonstrated association of GST-PEG10 fusion protein with flag-tagged SIAH2 protein expressed in HEK293 cells (Fig. 3 C).

Effect of PEG10 on the Cell Death Induced by Gene Transfer of SIAH1 in HCC Cells.

Others had reported that expression of human SIAH1 was increased during p53-dependent arrest of the cell cycle and that it induced apoptosis in several cell lines (8, 9). Hence, we hypothesized that PEG10 may affect the apoptosis mediated by SIAH1. To examine this hypothesis, we generated recombinant adenoviruses expressing myc-tagged SIAH1 protein (Ad-SIAH1) and LacZ (Ad-LacZ). Semiquantitative RT-PCR demonstrated that expression of SIAH1 was decreased in all six hepatoma cell lines examined, compared with normal liver tissue (Fig. 4,A). We infected five hepatoma cell lines (HepG2, Huh7, Alexander, SNU423, and SNU475), each of which revealed transfection efficiencies of 67.3–100% at an MOI of 100, with Ad-LacZ; immunoblot analysis using anti-myc antibody confirmed exogenous expression of myc-tagged SIAH1 protein after 24 h of infection (Fig. 4,B). The increase in cell death was marked in cultures infected with Ad-SIAH1 in all five cell-lines. Consistently, an MTT assay after 72 h of infection demonstrated a dose-dependent decrease of viability when cells were infected with Ad-SIAH1 (Fig. 4,C). Flow cytometry demonstrated that induction of SIAH1 expression significantly increased the numbers of cells in G2-M and sub-G1 populations, and a terminal deoxynucleotidyl transferase-mediated nick end labeling assay corroborated that the number of apoptotic cells infected with Ad-SIAH1 was significantly greater than cells with Ad-LacZ (Fig. 4,D). To determine whether overexpression of PEG10 protein could protect hepatocytes from cell death induced by SIAH1, we constructed SNU423-PEG10 cells, which stably express exogenous PEG10, and infected them with Ad-SIAH1. Compared with the parental SNU423 or control SNU423-mock cells, SNU423-PEG10 cells showed significantly greater viability 48 h after being infected with Ad-SIAH1 (Fig. 4 E), indicating the protective role of PEG10 from SIAH1-mediated cell death.

Chronic hepatitis because of hepatitis B or hepatitis C virus is considered a major risk factor for HCC, but each virus may contribute to hepatocarcinogenesis via a different pathway (10, 11). For example, our studies of gene expression profiles of HCCs using a genome-wide cDNA microarray have revealed different expression patterns between hepatitis B virus-positive and hepatitis C virus-positive tumors, implying different character, although many genes are commonly up-regulated in both types of tumor. Because PEG10, a gene that was up-regulated in a great majority of HCCs (35 of 36 HCCs analyzed by the cDNA microarray, RT-PCR, and/or immunostaining), was expressed at an undetectable level in matched noncancerous liver tissues, PEG10 could potentially serve as a diagnostic marker and might be an ideal molecular target for development of drugs to treat patients with primary HCC.

Transfer of PEG10 into hepatoma cells that expressed no detectable endogenous PEG10 protein elicited significant growth promotion activity. Serum starvation did not suppress the growth of cells that expressed PEG10 at high levels. Because transfer of PEG10 into HEK293, Cos7, and NIH3T3 cells did not promote growth (data not shown), our data indicate that the oncogenic activity of PEG10 is likely to be specific to hepatocytes.

PEG10 shows 61.4% homology to murine myelin expression factor 3, the product of which is thought to function as a transcriptional factor and to control expression of myelin basic protein during brain development (12). Myelin expression factor 3 protein consists of 235 amino acids with a zinc finger domain in the COOH-terminal region. Conservation of this domain may indicate that PEG10 itself functions as a transcriptional factor.

We found that PEG10 protein was able to interact with SIAH1 and SIAH2 proteins, which are homologues of Drosophila seven in absentia (sina); the latter is involved in the fate of R7 photoreceptor cells of Drosophila during eye development (13). SIAH1 protein is involved in ubiquitin-mediated proteolysis of several proteins, including kinesin-like DNA binding protein, BAG-1, and DCC (deleted in colorectal cancer) via its RING finger domain (14, 15, 16). This gene is located on chromosomal band 16q12-q13, a region frequently deleted in tumors arising from various tissues, including HCCs (17, 18), suggesting that SIAH1 might function as a tumor suppressor. Furthermore, SIAH1 was shown to interact with the tumor suppressor adenomatous polyposis coli and facilitates degradation of β-catenin through formation of a degradation complex that is independent of glycogen synthase kinase-3β (19, 20). Notably, we observed a marked decrease of PEG10 protein by exogenous expression of SIAH1 in Huh7 and HepG2 cells (data not shown), suggesting that SIAH1 may exert its tumor-suppressive function by degrading oncogeneic proteins, including BAG-1, β-catenin, and PEG10. Although we carried out immunoprecipitation assays using overexpression of PEG10 together with SIAH1, we were unable to detect their association, indicating that SIAH1-associated PEG10 might be quickly degraded through ubiquitin-proteosome pathway. The reduced expression of SIAH1 in HCC cell lines and induction of apoptosis after transfer of exogenous SIAH1 into HCC cells suggest that SIAH1 plays an important role in suppressing hepatocarcinogenesis.

In our experiments, cells that stably expressed PEG10 revealed a significant decrease in cell death in response to Ad-SIAH1, suggesting that imbalance between expression of PEG10 and SIAH1 may be involved in hepatocarcinogenesis through inhibition of apoptosis. PEG10 was isolated as a paternally expressed gene from a newly defined imprinted region at 7q21 (7). Loss of imprinting might be involved in the elevated expression of this gene in HCCs, although we found no evidence to support it. Hence, the actual mechanisms by which the expression of PEG10 is deregulated in HCCs remain to be investigated.

Finally, we have demonstrated that reduction of PEG10 expression by treatment with antisense S-oligonucleotides decreases growth of HCC cells significantly. Interestingly, the antisense sequences suppressed growth only of HCC cells that endogenously expressed PEG10, not in cell lines that did not. Because expression of this gene was enhanced in the majority of HCC tissues and very low or absent among all normal adult human tissues, except gonadal glands, suppression of PEG10 might be an ideal therapeutic strategy for treating primary HCCs. Although additional functional analysis of PEG10 is required, the data provided here should contribute to a more profound understanding of hepatocarcinogenesis and to development of novel therapeutic approaches.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by “Research for the Future” Program Grant 00L01402 from the Japan Society for the Promotion of Science.

3

The abbreviations used are: HCC, hepatocellular carcinoma; HEK293, human embryonic kidney 293; PEG10, the paternally expressed gene 10; GST, glutathione S-transferase; RT-PCR, reverse transcription-PCR; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MOI, multiplicity of infection; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

4

Internet address: http://smart.embl-heidelberg.de.

Fig. 1.

Expression of PEG10 in adult human tissues and primary HCCs. A, multiple tissue Northern blot analysis of PEG10 in adult human tissues. B, gene and protein expression of PEG10 in six hepatoma cell lines. RT-PCR was carried out using a PEG10-specific PCR primer set (top panel). GAPDH served as an internal control. Immunoblotting was performed using anti-PEG10 antibody. The amount of protein applied in the SDS-PAGE was evaluated by the Coomassie Brilliant Blue (CBB) staining. C, subcellular localization of PEG10 protein in HCC cell lines (magnification, ×600). D, immunohistochemical staining of PEG10 in a primary HCC and the corresponding noncancerous liver tissue. (magnification, ×600).

Fig. 1.

Expression of PEG10 in adult human tissues and primary HCCs. A, multiple tissue Northern blot analysis of PEG10 in adult human tissues. B, gene and protein expression of PEG10 in six hepatoma cell lines. RT-PCR was carried out using a PEG10-specific PCR primer set (top panel). GAPDH served as an internal control. Immunoblotting was performed using anti-PEG10 antibody. The amount of protein applied in the SDS-PAGE was evaluated by the Coomassie Brilliant Blue (CBB) staining. C, subcellular localization of PEG10 protein in HCC cell lines (magnification, ×600). D, immunohistochemical staining of PEG10 in a primary HCC and the corresponding noncancerous liver tissue. (magnification, ×600).

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Fig. 2.

Effect of PEG10 on cell growth in human hepatoma cell lines. A, colony formation by SNU423 and SNU475 cells after PEG10 gene transfer. Relative number of colonies transfected with plasmids expressing PEG10 to mock vector was calculated (mean ± SD). B, expression of PEG10 protein in stable transfectant (SNU423-PEG10) cells. The amount of protein applied in the SDS-PAGE was evaluated by Coomassie Brilliant Blue (CBB) staining. C, Growth curve of SNU423-PEG10 cells cultured in RPMI 1640 with 10% FBS. D, growth curve of SNU423-PEG10 cells under serum-starved conditions (cultured in RPMI 1640 with 0.1% FBS). E, reduced expression of PEG10 in Huh7 cells transfected with either sense or antisense S-oligonucleotides. F, relative number of viable Huh7 cells after transfection with the sense or antisense S-oligonucleotides. Relative number of viable cells was examined by MTT assay (mean ± SD).

Fig. 2.

Effect of PEG10 on cell growth in human hepatoma cell lines. A, colony formation by SNU423 and SNU475 cells after PEG10 gene transfer. Relative number of colonies transfected with plasmids expressing PEG10 to mock vector was calculated (mean ± SD). B, expression of PEG10 protein in stable transfectant (SNU423-PEG10) cells. The amount of protein applied in the SDS-PAGE was evaluated by Coomassie Brilliant Blue (CBB) staining. C, Growth curve of SNU423-PEG10 cells cultured in RPMI 1640 with 10% FBS. D, growth curve of SNU423-PEG10 cells under serum-starved conditions (cultured in RPMI 1640 with 0.1% FBS). E, reduced expression of PEG10 in Huh7 cells transfected with either sense or antisense S-oligonucleotides. F, relative number of viable Huh7 cells after transfection with the sense or antisense S-oligonucleotides. Relative number of viable cells was examined by MTT assay (mean ± SD).

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Fig. 3.

Interaction between PEG10 and SIAH1/2 proteins. A, yeast two-hybrid experiment. pAS2-1 or pAS2-1 containing PEG10 were cotransfected into yeast strain AH109 with library vectors containing SIAH1 or SIAH2. B, in vitro protein-binding assay of PEG10 and SIAH1. Protein from cells expressing PEG10 was incubated with resin conjugated with or without SIAH1. Eluted protein was analyzed by immunoblotting using anti-His and anti-PEG10 antibodies. In the last two lanes, his-tagged SIAH1 and cell lysate containing PEG10 were loaded directly on the gel as controls. C, in vitro protein-binding assay of PEG10 and SIAH2. Protein from cells expressing flag-tagged SIAH2 was incubated with GST-PEG10 or GST and purified with Glutathione Sepharose beads. In the right three lanes, GST-PEG10 fusion protein, GST, and cell lysate containing flag-tagged SIAH2 protein were loaded as controls.

Fig. 3.

Interaction between PEG10 and SIAH1/2 proteins. A, yeast two-hybrid experiment. pAS2-1 or pAS2-1 containing PEG10 were cotransfected into yeast strain AH109 with library vectors containing SIAH1 or SIAH2. B, in vitro protein-binding assay of PEG10 and SIAH1. Protein from cells expressing PEG10 was incubated with resin conjugated with or without SIAH1. Eluted protein was analyzed by immunoblotting using anti-His and anti-PEG10 antibodies. In the last two lanes, his-tagged SIAH1 and cell lysate containing PEG10 were loaded directly on the gel as controls. C, in vitro protein-binding assay of PEG10 and SIAH2. Protein from cells expressing flag-tagged SIAH2 was incubated with GST-PEG10 or GST and purified with Glutathione Sepharose beads. In the right three lanes, GST-PEG10 fusion protein, GST, and cell lysate containing flag-tagged SIAH2 protein were loaded as controls.

Close modal
Fig. 4.

Effects of SIAH1 gene transfer on viability of hepatoma cells. A, expression of SIAH1 in hepatoma cell lines examined by RT-PCR. mRNA extracted from normal liver tissue was used for comparison. B, expression of exogenous myc-tagged SIAH1 protein in SNU423 cells by adenovirus-mediated SIAH1 gene transfer (100 MOI). C, assessment of cell viability by MTT assay after infection with Ad-SIAH1 at MOIs of 20, 50, and 100. D, apoptosis induced by Ad-SIAH1 (100 MOI) in Huh7 cells; sub-G1 population of cells infected with Ad-SIAH1 or Ad-LacZ was examined 48 h after infection by fluorescence-activated cell sorting (left panels). Apoptotic cells were detected in green or yellow by terminal deoxynucleotidyl transferase-mediated nick end labeling assay (right panels). E, Effect of PEG10 expression on cell death induced by SIAH1. Cell viability was examined by MTT assay 48 h after infection with Ad-SIAH1 in PEG10-expressing cells (SNU423-PEG10), the parental (SNU423), or control (SNU423-Mock) cells. Data (mean ± SD) represent MTT activity of cells with Ad-SIAH1 relative to those with Ad-LacZ.

Fig. 4.

Effects of SIAH1 gene transfer on viability of hepatoma cells. A, expression of SIAH1 in hepatoma cell lines examined by RT-PCR. mRNA extracted from normal liver tissue was used for comparison. B, expression of exogenous myc-tagged SIAH1 protein in SNU423 cells by adenovirus-mediated SIAH1 gene transfer (100 MOI). C, assessment of cell viability by MTT assay after infection with Ad-SIAH1 at MOIs of 20, 50, and 100. D, apoptosis induced by Ad-SIAH1 (100 MOI) in Huh7 cells; sub-G1 population of cells infected with Ad-SIAH1 or Ad-LacZ was examined 48 h after infection by fluorescence-activated cell sorting (left panels). Apoptotic cells were detected in green or yellow by terminal deoxynucleotidyl transferase-mediated nick end labeling assay (right panels). E, Effect of PEG10 expression on cell death induced by SIAH1. Cell viability was examined by MTT assay 48 h after infection with Ad-SIAH1 in PEG10-expressing cells (SNU423-PEG10), the parental (SNU423), or control (SNU423-Mock) cells. Data (mean ± SD) represent MTT activity of cells with Ad-SIAH1 relative to those with Ad-LacZ.

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