TIPUH1 encodes a novel KRAB zinc-finger protein highly expressed in human hepatocellular carcinomas

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Abstract

To achieve a better understanding of mechanisms that underlie hepatocarcinogenesis and to identify novel target molecules for diagnosis and therapy of hepatocellular carcinoma (HCC), we previously analysed gene-expression profiles of 20 HCC tissues on a cDNA microarray. Among the genes upregulated in the tumor tissues compared with their nontumor counterparts, we focused on a novel gene termed transcription-involved protein upregulated in HCC (TIPUH1) that putatively encoded a 500-amino-acid protein containing 12 zinc-finger domains and a Kruppel-associated box domain. Multiple-tissue northern blot analysis revealed it's testis- and placenta-specific expression in normal tissues. Colony-formation assay in soft agar showed that TIPUH1 conferred anchorage-independent growth to NIH3T3 cells, suggesting its oncogenic activity. Conversely, specific siRNA for TIPUH1 knocked down its expression in HCC cells, which resulted in their growth inhibition. We identified four TIPUH1-interacting proteins including TIF1β, a transcription-intermediary protein, and three involved in pre-mRNA processing (hnRNPU, hnRNPF, and Nucleolin), suggesting that overexpressed TIPUH1 may play a role in hepatocarcinogenesis by regulating transcription and/or RNA processing of growth control genes. These data may contribute to a better understanding of liver neoplasia, and to the development of novel strategy for treatment of HCCs.

Introduction

Hepatocellular carcinoma (HCC) is one of the most prevalent and intractable of human cancers, causing as many as half a million deaths per year worldwide (Parkin et al., 2001). Chronic inflammation due to infection to hepatitis B or C viruses (HBV or HCV) is considered to be a major causative factor for development of HCC. Although recent medical advances have made great progress in diagnosis, many patients with HCCs are not diagnosed until the disease has reached an advanced stage. In addition, most HCC patients cannot be cured by surgical resection because they suffer from severe liver dysfunction and/or their tumors are widespread. Therefore prognosis of patients with HCC remains poor. It is a pressing concern to identify sensitive tumor markers, develop novel effective chemotherapeutic drugs, and discover preventive strategies.

Although the underlying etiology of HCC has been rather well characterized, the molecular mechanisms of hepatocarcinogenesis are still not well understood. Accumulating evidence suggests that multiple processes involving genetic and epigenetic alterations in a number of genes underlie hepatocarcinogenesis. Several growth factors including transforming growth factor-α (TGF-α) (Grisham, 2001) and insulin-like growth factor-2 (IGF-2) have been implicated in development of HCC. Altered expression of E-cadherin, MMP-1 and -7, MT1-MMP, gankyrin, SMYD3, WDRPUH, and PEG10 also appears to be associated with the development and/or progression of the disease (Feitelson et al., 2002; Hamamoto et al., 2004; Silva et al., 2005). Tumor-suppressor genes such as p53 and AXIN1 can also play crucial roles in hepatocarcinogenesis (Tanaka et al., 1993; Satoh et al., 2000). However, in spite of this growing body of genetic knowledge, no effective drugs are available at present for treatment of HCCs.

In order to gain further insight into the mechanisms underlying hepatocarcinogenesis and to develop novel therapeutic approaches to liver cancer, we earlier analysed gene-expression profiles on cDNA microarrays representing 23 040 genes, and identified 165 transcripts that were upregulated in HCC (Okabe et al., 2001). In the present study we focused on one of them, a gene (later termed transcription-involved protein upregulated in HCC (TIPUH1)) that would encode a novel Kruppel-type zinc-finger protein. We found that TIPUH1 exerts oncogenic activity, and that its overexpression confers anchorage-independent growth of NIH3T3 cells. Furthermore, suppression of TIPUH1 retarded the growth of HCC cells in culture. Taken together, overexpressed TIPUH1 is involved in the proliferation and/or survival of cancer cells. Analysis of the molecular entities that immunoprecipitated with TIPUH1 identified four TIPUH1-interacting proteins, TIF1β, hnRNPU, hnRNPF, and Nucleolin. Our findings should contribute to a better understanding of HCC and to development of novel anti-HCC drugs that would specifically target TIPUH1.

Results

Identification of a novel human gene upregulated in hepatocellular carcinoma

Using a genomewide cDNA microarray containing 23 040 genes, we previously analysed expression profiles of 20 HCCs, and identified a number of genes showing altered expression between cancer tissues and corresponding noncancerous tissues (Okabe et al., 2001). Among 165 genes that were frequently upregulated in the HCCs, we focused on a novel gene with an in-house accession name of C6242, as it was overexpressed in the majority of those tumors. The gene corresponded to an EST archived as Hs.454685, and was annotated as LOC115509 in the UniGene database of the National Center for Biotechnology Information. Semiquantitative reverse transcription-polymerase chain reaction (RT–PCR) corroborated its elevated expression in seven of 10 additional HCCs compared to corresponding noncancerous liver tissues (Figure 1a). Moreover, its expression was augmented in all seven hepatoma cell lines examined (Figure 1b). Our microarray data had shown that this gene was not upregulated in breast, colon, bladder or prostate cancers (data not shown).

Figure 1
figure1

Expression and putative protein structure of transcription-involved protein upregulated in HCC (TIPUH1). (a) Semiquantitative RT–PCR analysis of TIPUH1 in 10 HCCs (T) and their corresponding noncancerous liver tissues (N). Expression of GAPDH served as a control. (b) Expression of TIPUH1 in hepatoma cells and noncancerous liver tissue. (c) Northern blot analysis of TIPUH1 in a panel of 16 normal adult human tissues. β-Actin served as a control. (d) Schematic presentation of putative structure of TIPUH1 protein.

Expression, isolation, and characterization of the novel gene

Multiple-tissue Northern blotting using the C6242 cDNA as a probe revealed abundant expression of a 2.7-kb transcript specifically in the testis and placenta, but this transcript was not detected in any of the 14 other tissues examined (Figure 1c). As no EST clone in public databases contained the most 5′ part of this gene, we searched for candidate exons from the genomic sequence encompassing the gene (GenBank accession number: NM_138447.1) using GENSCAN and the Gene Recognition and Assembly Internet Link (GRAIL) programs. Subsequent exon connection and 5′ rapid amplification of cDNA ends (5′ RACE) experiments confirmed the sequence of the 5′ part of the transcript. The assembled cDNA sequence consisted of 2739 nucleotides containing an open reading frame encoding a 500-amino-acid protein. The gene consisted of three exons spanning an approximately 7-kb genomic region on chromosomal band 16p11.2. The Simple Modular Architecture Research Tool (SMART) program suggested that the predicted protein included a Kruppel-associated box (KRAB) and 12 Kruppel (Cys2-His2)-type zinc-finger domains (Figure 1d). This structure is typical of members of the single largest class of transcription factors within the human genome. Therefore, we termed this gene as TIPUH1. A search for amino-acid homologies in public databases identified a mouse protein (accession number: BC052084.1) and a rat protein (accession number: NM_173330.1), each of which was 83% identical to TIPUH1. These mammalian homologues were unnamed or hypothetical proteins with unknown functions.

Subcellular localization of TIPUH1

We prepared plasmids expressing FLAG-tagged TIPUH1 and transiently transfected them into SNU475 and HepG2 hepatoma cells. Immunoblot analysis using extracts from HEK293 cells transfected with the plasmid showed a 56-kDa band corresponding to the tagged TIPUH1 protein (Figure 2a). Immunocytochemical staining of the SNU475 and HepG2 cells and fluorescent microscopy revealed that the tagged TIPUH1 protein was present in the nuclei (Figure 2b). The PSORT II program consistently predicted subcellular localization of TIPUH1 protein in the nucleus.

Figure 2
figure2

Subcellular localization of transcription-involved protein upregulated in HCC (TIPUH1). (a) Western blot analysis of exogenously expressed FLAG-tagged TIPUH1 protein in HEK293 cells. Empty p3xFLAG-CMV10 (mock) vector served as a negative control. (b) FLAG-tagged TIPUH1 protein was expressed exogenously into SNU475 (HCC) and HepG2 hepatoma cells, and stained with anti-FLAG monoclonal antibody. The protein was visualized by rhodamine-conjugated secondary anti-mouse IgG antibody or FITC-conjugated secondary anti-mouse IgG antibody (left panel). Nuclei were counterstained with 4′,6′-diamidine-2′-phenylindole dihydrochloride (DAPI) (middle panel); merged images (Rhodamine or FITC and DAPI) are shown in the right panel. Magnifications × 400 (SNU475); × 600 (HepG2).

Function of TIPUH1 on cell growth

To investigate the effect of TIPUH1 on cell growth, we established NIH3T3 cells expressing TIPUH1 stably (NIH3T3-TIPUH1 cells), and performed a colony-formation assay in soft agar (Figure 3a). The growth of NIH3T3-TIPUH1 cells was similar to that of controls (NIH3T3-mock cells) when they were maintained in normal culture media. Notably, NIH3T3-TIPUH1 cells formed colonies in soft agar, whereas NIH3T3-mock cells produced no colonies (Figure 3a). This observation was confirmed by three independent experiments. These data suggest that TIPUH1 transforms NIH3T3 cells and confers anchorage-independent cell growth.

Figure 3
figure3

Growth-promoting effect of transcription-involved protein upregulated in HCC (TIPUH1). (a) Anchorage-independent growth of NIH3T3-TIPUH1 cells in soft agar. The representative picture of NIH3T3-TIPUH1 cells expressing stably exogenous TIPUH1 (upper panel), and that of NIH3T3-mock cells (lower panel) were shown. The number of colonies was counted after 3 weeks of plating and shown in the histogram (right panel). *Significant difference (P<0.05) determined by a Student's t-test. (b) Gene-knockdown effect and growth-suppressive effect of plasmids expressing four different TIPUH1-siRNAs or EGFP-siRNA, and mock plasmid. Semiquantitative RT–PCR was carried out to examine expression of TIPUH1 48 h after transfection of the plasmids into HepG2 cells (lower panel). Viability of HepG2 cells transfected with the plasmids was analysed in triplicate by MTT assays on day 7 after transfection and subsequent maintenance of cells with an appropriate concentration of G418 (upper panel). (ce) Growth-suppressive effect of TIPUH1-siRNA-02 in Huh7 (c), Alexander (d), and SNU449 cells (e). *Significant difference (P<0.05) determined by a Student's t-test.

To test whether elevated expression of TIPUH1 may play a crucial role in the proliferation of HCC cells, we prepared plasmids expressing four kinds of siRNA to TIPUH1 and transfected them, together with a neomycin-resistance gene, into HepG2 cells that expressed TIPUH1 abundantly. As shown in Figure 3b, plasmids expressing siRNA-02 most effectively suppressed TIPUH1 expression compared with mock (psiU6BX) or those expressing siRNA to EGFP (psiU6BX-EGFP), whereas others showed a mild gene-silencing effect, or none. We subsequently tested whether suppression of TIPUH1 affected cell growth and found that transfection of siRNA-02 into HepG2 cells reduced their viability when compared with cells transfected with mock or EGFP (Figure 3b). On the other hand, siRNAs-01, -03, and -04 brought about mild decreases in the numbers of surviving cells, compared with siRNA-02. We also investigated the effect of siRNA-02 on three additional HCC cell lines (Huh7, Alexander, and SNU449) and obtained similar results (Figure 3c–e), indicating that suppressed expression of TIPUH1 was associated with a decrease in cell viability, either as a consequence of reduced cell growth or induction of apoptosis. These data suggested that TIPUH1 might play an essential role in proliferation and/or survival of HCC cells.

Identification of TIPUH1-interacting proteins

To investigate the biological function of TIPUH1 we searched for proteins that would interact with it by means of immunoprecipitation (IP) and mass spectrometric (MS) assays. We transfected HEK293 cells with plasmids expressing FLAG-tagged TIPUH1, empty vector (mock), or unrelated FLAG-tagged protein of 70 kDa (WDRPUH). After incubating the cells, we immunoprecipitated proteins from lysates using an anti-FLAG antibody conjugated with agarose beads. Comparison of the bands of the immunoprecipitants on sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) identified at least four bands, corresponding to proteins of 120, 100, 75, and 53 kDa, which were immunoprecipitated specifically from lysates of cells transfected with TIPUH1 (data not shown). These bands were excised and subsequently analysed by matrix-assisted laser desorption/ionization mass spectrometry. The 120-kDa protein corresponded to hnRNPU; the 100-kDa protein to a transcriptional corepressor, TIF1β (also termed Kap-1), the 75-kDa protein to nucleolin, and the 53-kDa protein to hnRNPF.

Using anti-FLAG antibody we immunoprecipitated extracts from cells transfected with p3xFLAG-TIPUH1 or mock vector, and confirmed that hnRNPU was co-immunoprecipitated from cells with TIPUH1, but not from cells containing mock vector (Figure 4a). Conversely, IP with anti-hnRNPU antibody co-immunoprecipitated FLAG-tagged TIPUH1 from cells transfected with p3xFLAG-TIPUH1 but not those with mock (Figure 4b). We also examined interaction between exogenous TIPUH1 and endogenous nucleolin in HEK293 cells. After IP of FLAG-tagged TIPUH1 with anti-FLAG antibody, we performed immunoblotting with anti-nucleolin antibody and detected a band of approximately 75 kDa (Figure 4c). Conversely, IP with anti-nucleolin antibody co-immunoprecipitated FLAG-tagged TIPUH1 from cells transfected with p3xFLAG-TIPUH1 but not those with mock vector (Figure 4d). We also confirmed associations between TIPUH1 and TIF1β (Figure 4e) or hnRNPF (Figure 4f) by IP of extracts from cells expressing FLAG-tagged TIPUH1 with anti-FLAG antibody.

Figure 4
figure4

Interaction of transcription-involved protein upregulated in HCC (TIPUH1) with hnRNPU, TIF1β, nucleolin or hnRNPF. HEK293 cells were transiently transfected with p3xFLAG-TIPUH1, or p3xFLAG empty vector (mock). Lysates were immunoprecipitated with murine anti-FLAG M2 antibody conjugated with agarose beads and specific antibodies to interacting proteins. (a and b) Co-immunoprecipitation of FLAG-TIPUH1 and endogenous hnRNPU. Whole-cell extracts from cells exogenously expressing FLAG-TIPUH1 were used as positive controls. (c and d) Co-immunoprecipitation of FLAG-TIPUH1 and endogenous nucleolin. (e) Co-immunoprecipitation of FLAG-TIPUH1 and endogenous TIF1β. (f) Co-immunoprecipitation of FLAG-TIPUH1 and endogenous hnRNPF.

Although further studies are needed to prove the interaction between endogenous TIPUH1 and these proteins, the data provided here should help for the clarification of the biological function of TIPUH1, and the identification of novel therapeutic strategies to HCC.

Discussion

Although we have shown that TIPUH1 expression is elevated in the majority of hepatocarcinomas, the mechanism(s) of its augmentation remains unresolved. Several studies have reported gain or amplification of chromosome 16p, where TIPUH1 is located (Wilkens et al., 2001; Kim et al., 2003; Raidl et al., 2004). Therefore genetic amplification of TIPUH1 may be involved in the enhancement. Alternatively, other mechanism(s), such as transcriptional activation or increased mRNA stability, may play a role for the upregulation. TIPUH1 encodes a putative protein of 500 amino acids (aa) that contains a KRAB domain at the N-terminus and Cys2-His2-type zinc-fingers in tandem. Kruppel-associated box/Cys2-His2 zinc-finger proteins (KZFP) constitute the single largest class of potential regulators of gene expression (Mark et al., 1999). The KRAB domain is a conserved motif at the N-terminus of a large number of zinc-finger proteins, comprising a 75-amino-acid stretch that can be divided into the KRAB-A domain, a 45-aa-long minimal repression module, and the KRAB-B domain (Witzgall et al., 1994). TIPUH1 protein contains only the KRAB-A domain that, as in other KZFPs, is located in its N-terminal region. The basic structural unit of the Kruppel-type zinc-finger is a conserved peptide of 28 aa, which is often repeated (Looman et al., 2002). As TIPUH1 consists of only one KRAB domain and 12 Kruppel-type zinc-finger domains, it appears to be a novel member of the KZFP family.

Kruppel-associated box-containing proteins function as transcriptional repressors when tethered to template DNAs through their DNA-binding domains (Witzgall et al., 1994; Collins et al., 2001; Looman et al., 2002). This transcriptional silencing is thought to be mediate via interaction of the KRAB box with the ubiquitous corepressor TIF1β (Nielsen et al., 1999; Collins et al., 2001). TIF1β, also named KAP-1 or KRIP-1, can interact with numerous KRAB domains to enhance KRAB-mediated repression and to silence transcription when it is directly bound to DNA (Collins et al., 2001). Experimental evidence suggests that this silencing activity results from recruitment of a histone deacetylase complex (Underhill et al., 2000) and/or HP-1 proteins that represent chromatin constituents associated with silencing of euchromatic genes (Nielsen et al., 1999; Collins et al., 2001). TIF1β can use its plant homeo-domain and bromo-domains to link KZFPs to Mi-2α and other components of the nucleosome-remodeling and deacetylase complex (Schultz et al., 2001). Our identification of TIF1β as one of the TIPUH1-binding proteins fits well with that notion. In addition, KRAB-containing proteins could bind to promoters RNA polymerase I, II, and III to repress their transcriptional activity, and might affect splicing of RNA as a post-transcriptional mechanism (Urrutia, 2003). Therefore, by forming a complex with TIF1β, TIPUH1 might repress transcription by way of chromatin remodeling through histone modification, by inhibiting RNA polymerase activities, and/or by reducing the stability of RNA. Along with elevated TIPUH1 expression, our microarray data showed that TIF1β was also significantly upregulated in the majority of the HCC samples analysed (data not shown); this observation implied that enhancement of both TIPUH1 and TIF1β in HCC cells may repress expression of genes associated with apoptosis, cell cycle arrest, or growth suppression.

We also documented interaction of TIPUH1 with hnRNPU, hnRNPF, and nucleolin, all of which are involved in mRNA or rRNA processing. hnRNP proteins package nascent pre-mRNA as it is transcribed in vivo, and a discrete hnRNP particle is constituted from more than 20 hnRNP proteins (Reed and Magni, 2001). Those proteins are responsible for appropriate packaging of RNAs that must be retained in the nucleus, such as RNAs with introns, or mutant RNAs that are unable to enter the spliceosome assembly pathway (Reed and Magni, 2001). They are involved not only in pre-mRNA processing, transport, localization, stability, and translation of mRNA, but also in alternative splicing (Caceres and Kornblihtt, 2002) and regulation of telomerase (Ford et al., 2002). In addition, hnRNP-U associates with a transcription factor, Yes-associated protein (YAP), to modulate its transactivation of Bax (Howell et al., 2000). Future studies may help to clarify whether elevated expression of TIPUH1 reflects augmented generation and subsequent processing of mRNA and/or rRNA in cancer cells, or a deregulation of RNA-engineering that results in production of aberrant transcripts.

Recent studies have disclosed that KZFPs play important roles during cell differentiation and development, cell proliferation, apoptosis, and neoplastic transformation (Urrutia, 2003). Some KZFP proteins are also implicated in cancer; for example, human Kruppel-related gene 1 (HKR1) is highly expressed in lung cancers, particularly tumors treated with cisplatin (Oguri et al., 1998). TIPUH1 could be involved in hepatocarcinogenesis either through a transcriptional-repressive mechanism or through modification of mRNA splicing, by associating with its binding proteins.

As it was expressed exclusively in the testis and placenta among 16 normal tissues and its expression was enhanced in the great majority of HCC cases we examined, TIPUH1 appears to be a novel cancer-testis antigen. Therefore, immunotherapies targeting TIPUH1 could be explored as a good therapeutic option. In fact, the suppression of TIPUH1 expression by a gene-specific siRNA, resulting in growth inhibition, indicated that inhibitors of TIPUH1 should be effective anti-HCC drugs; moreover, they should be relatively safe because expression of TIPUH1 is weak or absent in normal adult human tissues except for the testis and placenta. We identified four proteins that interact with the gene product: transcriptional corepressor TIF1β and three proteins involved in RNA engineering, hnRNPU, hnRNPF, and nucleolin. The data suggest that TIPUH1 is a key factor for proliferation of HCC cells, through regulation of transcription and/or mRNA processing. The four TIPUH-interacting proteins we identified may facilitate screening for inhibitors of TIPUH1.

Materials and methods

Cell lines

Human embryonic kidney 293 (HEK293) cells were obtained from IWAKI; human hepatoma cell lines Alexander and HepG2, and mouse a fibroblast cell line NIH3T3, were from the American Type Culture Collection (ATCC). Another human hepatoma cell line, Huh7, was obtained from the Japanese Collection of Research Bioresources (JCRB), whereas SNU398, SNU423, SNU449, and SNU475 were obtained from the Korean cell-line bank. All cell lines were grown in monolayers in appropriate media: Dulbecco's modified Eagle's medium (DMEM) for Alexander, Huh7, HepG2, and HEK293, and RPMI1640 for SNU398, SNU423, SNU449, and SNU475. All media were supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic solution (Sigma, St. Louis, MO, USA), and cells were maintained at 37°C in humidified air containing 5% CO2.

Semiquantitative reverse transcription-polymerase chain reaction

Semiquantitative RT–PCR was carried out as described elsewhere (Silva et al., 2005). Amplification proceeded for 4 min at 94°C for denaturing, followed by 35 cycles for TIPUH1 and 20 for GAPDH, at 94°C for 30 s, 56°C for 30 s, and 72°C for 45 s, in the GeneAmp PCR system 9700 (Perkin-Elmer, Foster City, CA, USA). Primer sequences for GAPDH were: forward, 5′-IndexTermACAACAGCCTCAAGATCATCAG-3′ and reverse, 5′-IndexTermGGTCCACCACTGACACGTTG-3′; and for TIPUH1, forward, 5′-IndexTermGTGGCACTGTGGTGTTACCTTAT-3′ and reverse 5′-IndexTermCCTCTAAACCTTTGCCTACGACT-3′.

Northern blot analysis

Human multiple-tissue blots (Clontech, Palo Alto, CA, USA) were hybridized with a 32P-labeled cDNA fragment of TIPUH1). Prehybridization, hybridization, and washing were performed according to the supplier's recommendations. The blots were autoradiographed with intensifying screens at −80°C for 120 h.

5′ rapid amplification of cDNA ends

5′ rapid amplification of cDNA ends experiments were carried out using a Marathon cDNA amplification kit (Clontech) according to the manufacturer's instructions. For the amplification of the 5′ part of TIPUH1 cDNA, we used a gene-specific reverse primer (5′-IndexTermTAGATTCTGGGCGCACTTGTGGCTCTCC-3′) and the AP-1 primer supplied in the kit. The cDNA template was synthesized from human testis mRNA (Clontech).

Construction of plasmids expressing TIPUH1

The entire coding region of TIPUH1 was amplified by RT–PCR using gene-specific primers 5′-IndexTermGGGGTACCACCATGGCGCCACCTTCG-3′ and 5′-IndexTermCGGAATTCATGGGCGTTGCCCCTCTGACTGG-3′. The PCR products were cloned into an appropriate cloning site of pcDNA3.1 (Invitrogen, Carlsbad, CA, USA), p3xFLAG-CMV-10 (Sigma), or pcDNA3.1myc/His (Invitrogen) vector.

Immunoprecipitation and Western blot analysis

HEK293 cells, which do not express TIPUH1, were transfected with p3xFLAG-TIPUH1 or mock vector. Extraction of protein, IP, and Western blot analysis were performed as described in our previous report (Silva et al., 2005). Anti-FLAG M2 antibody conjugated with agarose (Sigma) was used for IP, and mouse anti-FLAG M2 (Sigma) antibody, anti-TIF1β, anti-hnRNPF, anti-hnRNPU, and anti-nucleolin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used for Western blot analyses. Horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (IgG) (Amersham Pharmacia Biotechnologies, Little Chalfont, UK) or donkey anti-goat IgG (Santa Cruz) served as the secondary antibody for the ECL Detection System (Amersham).

Immunocytochemical staining

SNU475 and HepG2 cells transfected with p3xFLAG-TIPUH1 or p3xFLAG-mock (empty) plasmids were fixed with PBS containing 4% paraformaldehyde for 15 min. Immunocytochemical staining using mouse anti-FLAG antibody (Sigma) was carried out as described elsewhere (Satoh et al., 2000). Nuclei were counter-stained with 4′,6′-diamidine-2′-phenylindole dihydrochloride (DAPI).

Colony-formation assay

We established NIH3T3 cells expressing TIPUH1 stably (NIH3T3-TIPUH1), and performed colony-formation assay in soft agar. Briefly, NIH3T3-TIPUH1 cells and control NIH3T3-mock cells were suspended in DMEM containing 10% FCS and 0.4% low melting temperature agarose (Cambrex Bio Science, Baltimore, USA), and plated onto solidified 0.53% agarose containing DMEM with 10% FCS in a 10 cm dish at a density of 1 × 104 cells/dish. Number of colonies was counted 3 weeks after the plating.

Gene-silencing effect of TIPUH1 siRNAs

Plasmids expressing TIPUH1 siRNAs were prepared by cloning double-stranded oligonucleotides into the psiU6BX vector as described elsewhere (Shimokawa et al., 2003). The oligonucleotides used for TIPUH1 siRNAs were as follows: for si-01, 5′-IndexTermCACCAACGAAACACCGATGACTGGGTTCAAGAGACCCAGTCATCGGTGTTTCGTT3′ and 5′-IndexTermAAAAAACGAAACACCGATGACTGGGTCTCTTGAACCCAGTCATCGGTGTTTCGTT-3′; for si-02, 5′-IndexTermCACCAATCACCGGACCACACACACATTCAAGAGATGTGTGTGTGGTCCGGTGATT-3′ and 5′-IndexTermAAAAAATCACCGGACCACACACACATCTCTTGAATGTGTGTGTGGTCCGGTGATT-3′; for si-03, 5′-IndexTermCACCAAACCTTGCCTACGACATGTTTTCAAGAGAAACATGTCGTAGGCAAGGTTT-3′ and 5′-IndexTermAAAAAAACCTTGCCTACGACATGTTTCTCTTGAAAACATGTCGTAGGCAAGGTTT-3; and for si-04, 5′-IndexTermCACCAAAAGGTTTCCGTTAGCCCCGTTCAAGAGACGGGGCTAACGGAAACCTTTT-3′ and 5′-IndexTermAAAAAAAAGGTTTCCGTTAGCCCCGTCTCTTGAACGGGGCTAACGGAAACCTTTT-3′. psiU6BX-TIPUH1, psiU6BX-EGFP (Shimokawa et al., 2003) and psiU6BX-mock (empty) plasmids were transfected into HepG2, Huh7, SNU449, and Alexander cells, using FuGENE6 reagent or Nucleofector according to the suppliers' recommendations (Roche Diagnostics, Mannheim, Germany and Amaxa, Cologne, Germany). Total RNA was extracted from the cells 48 h after transfection. Silencing of the TIPUH1 gene by the specific constructs was confirmed by RT–PCR. Cell viability was measured using Cell-counting kit-8 (DOJINDO, Kumamoto, Japan) 7 days after transfection according to the supplier's recommendations. The data were subjected to analysis of variance and the Student's t-test.

Matrix-assisted laser desorption/ionization mass spectrometry

Proteins immunoprecipitated with anti-FLAG antibody from cells transfected with p3xFLAG-TIPUH1, mock, or with p3xFLAG-WDRPUH expressing an unrelated protein (Silva et al., 2005) were separated by SDS–PAGE. Detection of specific bands and subsequent analysis of the protein by matrix-assisted laser desorption/ionization mass spectrometry was performed as described previously (Silva et al., 2005).

Accession codes

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Acknowledgements

We are grateful to Ms Yuka Yamane for excellent technical assistance. This work was supported in part by Research for the Future Program Grant (00L01402) from the Japan Society for the Promotion of Science.

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Correspondence to Y Nakamura.

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Silva, F., Hamamoto, R., Furukawa, Y. et al. TIPUH1 encodes a novel KRAB zinc-finger protein highly expressed in human hepatocellular carcinomas. Oncogene 25, 5063–5070 (2006) doi:10.1038/sj.onc.1209517

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Keywords

  • TIPUH1
  • hepatocellular carcinoma
  • TIF1β; hnRNPU
  • nucleolin

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