Ubiquitin-dependent proteolysis by the 26S proteasome plays a pivotal role in cell cycle progression as well as in tumorigenesis. In this pathway, ubiquitin-conjugating enzyme (E2), together with ubiquitin ligase (E3), transfers ubiquitin to the specific substrate protein(s); however, little is known about the potential contribution of E2 to tumorigenesis. In this study, we examined the expression levels of 17 E2 genes in 25 different human normal tissues and 24 human cancerous cell lines by using a quantitative real-time reverse transcription-PCR. Among the E2 gene family, the expression level of UbcH10 was extremely low in many of the normal tissues but prominent in the majority of cancerous cell lines. Intriguingly, UbcH10 was expressed at high levels in primary tumors derived from the lung, stomach, uterus, and bladder as compared with their corresponding normal tissues, suggesting that UbcH10 is involved in tumorigenesis or progression of the tumor. To further investigate a possible contribution of UbcH10 to malignant transformation and tumor cell proliferation, NIH3T3 cells were transfected with the expression plasmid encoding UbcH10, and stable transfectants were subsequently established. UbcH10-overexpressing cells exhibited an increased incorporation of bromodeoxyuridine, an enhanced growth rate, an increase in saturation density, and a promotion of colony formation in soft agar medium as compared with parental NIH3T3 cells and the control transfectants. Collectively, our present results provide the first evidence that UbcH10 is highly expressed in various human primary tumors and that UbcH10 has an ability to promote cell growth and malignant transformation.

Ubiquitination-dependent proteolysis is closely related to diverse cellular processes including cell cycle progression, signal transduction, and differentiation (1, 2). In this system, substrate proteins are processed for degradation by three distinct enzyme activities including the ubiquitin-activating enzyme (E1), the ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3; Refs. 1 and 2). The E1 protein activates ubiquitin in an ATP-dependent manner and then transfers it to the E2 protein through a thiol-ester linkage. Eventually, ubiquitin is transferred from the E2 protein to a lysine residue of the specific substrate protein, which requires the E3 ligase activity. Polyubiquitinated proteins are recognized by the 26S proteasome and rapidly degraded (1, 2). Accumulating evidence suggests that E3 protein plays an important role in the regulation of the cell cycle progression as well as in tumorigenesis. MDM2, which acts as an E3 ubiquitin ligase for tumor suppressor p53, directly mediates the ubiquitination and subsequent degradation of p53 (3, 4, 5). Deubiquitination of p53 by HAUSP results in the stabilization of p53 to induce p53-dependent growth suppression and apoptosis (6). Overexpression of Efp, which is a Ring finger-dependent E3 ligase and mediates proteolysis of the negative cell cycle regulator 14-3-3ς, promotes breast tumor growth (7). In addition, the APC,3 which acts as an E3 ligase at mitosis, is required for the destruction of mitotic cyclins and thereby allows progression through mitosis and mitotic exit (8, 9, 10, 11). These findings strongly suggest that down-regulation of the growth and/or tumor suppressor by ubiquitin-dependent breakdown contributes to cell cycle progression and/or tumor cell proliferation, respectively.

All known E2 proteins are structurally related and share a conserved domain of Mr 16,000 that carries the cysteine residue required for the formation of ubiquitin-E2 thiol ester (12). E2 protein catalyzes the E3-dependent multiple ubiquitination that leads to degradation of substrate proteins, and various E2 and E3 proteins function in cognate pairs and provide specificity in substrate protein ubiquitination (12, 13). Recent work provides evidence that various E2 proteins play a cell cycle-regulatory role. It has been shown that UbcH10 or Ubc4 is required for APC-dependent ubiquitination of mitotic cyclins (9, 14, 15), and dominant-negative UbcH10 blocks the ubiquitination as well as the destruction of mitotic cyclins and causes cells to accumulate in mitosis (16). In addition, Ubc2/Rad6 and Ubc3/CDC34 are specifically involved in the ubiquitination-dependent degradation of the cyclin-dependent kinase inhibitor p27 (17). Intriguingly, the expression level of UbcH10 is up-regulated in NIH3T3 cells transformed by a EWS-FLI1 fusion gene associated with Ewing’s sarcoma (18). Recently, it has been shown that the expression levels of the ubiquitin-conjugating enzyme gene Ubc9 are increased in human lung adenocarcinomas compared with those of their corresponding normal tissues (19). Thus, certain E2 proteins could be closely linked to the cell cycle progression and/or tumorigenesis. However, little is known about the potential contribution of E2 protein to the tumorigenic response mediated by ubiquitination-dependent proteolysis.

In the present study, we examined by quantitative real-time RT-PCR the expression patterns of 17 E2 genes in 25 different human normal tissues, 24 human cancerous cell lines, and various primary tumors and their corresponding normal tissues. We found that UbcH10 was highly expressed in numerous cancerous cell lines and various primary tumors as compared with matched normal tissues. Additionally, overexpression of UbcH10 in NIH3T3 cells promoted deregulated cell growth and also induced anchorage-independent growth.

Cell Culture and Transfection.

Human osteosarcoma cell lines OST and SAOS-2; human colorectal adenocarcinoma cell lines COLO320, SW480, and LoVo; human hepatocellular carcinoma cell line HepG2; human breast cancer cell lines MB453 and MB231; human malignant melanoma cell line G-361; human amelanotic melanoma cell line C32TG; human thyroid medullary carcinoma cell line TTC2; human pancreas adenocarcinoma cell line ASPC-1; and human lung adenocarcinoma cell line A549 were obtained from American Type Culture Collection. Human esophageal squamous cell carcinoma cell line EC-GI-10 was obtained from RIKEN Cell Bank. Human gastric carcinoma cell line Kato-III was obtained from Japanese Cancer Research Resources Bank. Human rhabdomyosarcoma cell lines RMS-NK and ASPS-KY were kind gifts from Dr. K. Kushida (Kanagawa Cancer Center). Human neuroblastoma cell lines SMS-KAN, SMS-KCN, IMR32, SK-N-AS, SK-N-DZ, and TGW were kind gifts from Dr. G. M. Brodeur (The University of Pennsylvania). Human neuroblastoma cell line NB-1 was kindly provided by Dr. S. Miyake (Kyoto Prefectural University of Medicine). Cells were maintained in DMEM or RPMI 1640 supplemented with 10% heat-inactivated CS (Invitrogen, Carlsbad, CA), 2 mm l-glutamine, 50 units/ml penicillin, and 50 μg/ml streptomycin at 37°C in an atmosphere of 5% CO2 in air. For transfection, NIH3T3 cells were stably transfected with the backbone expression plasmid (pcDNA3) or the expression plasmid encoding human UbcH10 by using Lipofectin transfection reagent (Invitrogen) according to the manufacturer’s instructions. Forty-eight h after transfection, cells were grown in culture medium containing 800 μg/ml G418 for 2 weeks. The resulting drug-resistant colonies were isolated and assayed for UbcH10 expression by Western analysis.

Tumor Samples.

Surgically resected tumor tissues and their corresponding normal tissues used in this study were obtained as frozen specimens from Chiba Cancer Center Hospital between 1995 and 1996 (Chiba, Japan). These tumors included six lung adenocarcinomas, three gastric adenocarcinomas, two uterine carcinomas, and six bladder carcinomas.

Plasmids.

To obtain full-length human UbcH10 cDNA, total RNA prepared from human fetal liver was reverse transcribed using oligo(dT) and Superscript II reverse transcriptase (Invitrogen). The subsequent PCR was performed with the following oligonucleotide primers: UbcH10 sense, 5′-CGCCGAATTCACGGCTTCCCAAAAC-3′; and UbcH10 antisense, 5′-TATAGCGGCCGCACAAAAAGGACAGACCATC-3′. These sense and antisense oligonucleotide primers were synthesized based on the nucleotide sequence databases (GenBank and European Molecular Biology Laboratory) and contained an engineered EcoRI and NotI restriction site (underlined), respectively. The specific amplified fragment was gel purified, digested completely with EcoRI and NotI, and subcloned into the identical restriction sites of the pcDNA3-FLAG expression plasmid to give pcDNA3-FLAG-UbcH10. The resultant expression plasmid was sequenced to confirm in-frame fusion of the UbcH10 and FLAG tag.

Immunofluorescence.

Transfected cells were grown on coverslips. After incubation for 36 h, cells were washed with ice-cold PBS, fixed with 3.7% formaldehyde in PBS for 30 min, permeabilized with 0.2% Triton X-100 for 5 min, and then blocked in PBS supplemented with 3% BSA. The cells were sequentially treated with a monoclonal anti-FLAG antibody (M2; Sigma Chemical Co., St. Louis, MO) and a rhodamine-conjugated goat antimouse IgG antibody (Invitrogen). The stained cells were visualized under a confocal laser scanning microscope (Olympus, Tokyo, Japan).

Western Blot Analysis.

Cells were washed with ice-cold PBS, scraped in SDS sample buffer (20), and sonicated briefly. Equal amounts of protein were loaded onto 15% SDS-polyacrylamide gels based on estimations from Coomassie Blue staining for each sample. The proteins were transferred onto nitrocellulose membrane filter (Toyo Roshi, Tokyo, Japan) using a semi-dry transfer apparatus (Bio-Rad). Nonspecific binding sites were blocked with 5% nonfat dry milk powder in TBST [20 mm Tris-Cl (pH 7.6), 150 mm NaCl, and 0.1% (v/v) Tween 20]. The membrane was incubated with the monoclonal anti-FLAG antibody or the polyclonal anti-actin antibody (20-33; Sigma Chemical Co.), followed by incubation with a horseradish peroxidase-conjugated goat antimouse IgG secondary antibody (Cell Signaling Technology, Beverly, MA). Enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech, Piscataway, NJ) was used for detection.

BrdUrd Incorporation during DNA Synthesis.

Cells were seeded at a density of 1 × 105 cells/60-mm dish. After incubation for 36 h, cells were treated with 10 μm BrdUrd (Roche Molecular Biochemicals, Indianapolis, IN) for 10 min. Cells were then washed three times with ice-cold PBS and fixed with 70% ethanol for 20 min at −20°C. After washing with PBS, cells were incubated with a monoclonal anti-BrdUrd antibody diluted 1:10 in incubation buffer [66 mm Tris-Cl (pH 7.5), 0.66 mm MgCl2, and 1 mm β-mercaptoethanol] for 1 h at 37°C, washed three times with PBS, incubated with a FITC-conjugated goat antimouse IgG secondary antibody diluted 1:20 in PBS for 1 h at 37°C, and again washed three times with PBS. Cell nuclei were stained with propidium iodide. The stained cells were visualized under a confocal laser scanning microscope (Olympus).

Cell Proliferation.

To evaluate cell proliferation, cells were plated onto 24-well cell culture dishes at a density of 5 × 103 cells/well in 1 ml of culture medium containing 10% or 2% CS. Cells were allowed to adhere to the bottom of the cell culture dish for 24 h. At the indicated time periods, cells were trypsinized, and cell counting was carried out in triplicates using a Coulter Counter (Coulter Electronics Ltd., Hialeah, FL).

Soft Agar Assay.

Cells (2.5 × 103) were suspended in 0.3% low-melting point agarose (SeaPlaque; BMA, Rockland, ME) dissolved in DMEM containing 10% CS and plated in 60-mm soft agar plates consisting of DMEM containing 10% CS in a 0.53% agarose medium. Agar plates were carefully placed in the incubator. After 3 weeks, the number of colonies formed with a diameter of >100 μm was scored.

Northern Blot Analysis.

Total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. Twenty-five μg of total RNA per lane were electrophoresed in a formaldehyde-containing 1% agarose gel and transferred onto a nylon membrane filter (Hybond-N+; Amersham Pharmacia Biotech) by capillary diffusion in 20× SSC. The filter was fixed by UV irradiation and then hybridized overnight at 65°C in a solution containing 7.5% dextran sulfate, 1 m NaCl, 1% N-lauroyl sarcosine, 2.5 μg/ml heat-denatured salmon sperm DNA, and a radiolabeled probe. The filter was washed twice in 0.5× SSC/0.1% N-lauroyl sarcosine at 50°C and subjected to autoradiography.

Quantitative Real-Time RT-PCR.

Total RNA was isolated using the RNeasy mini kit (Qiagen, Valencia, CA) according to the manufacturer’s recommendations. Five μg of total RNA from each sample were incubated with random primers and Superscript II reverse transcriptase (Invitrogen) to yield cDNA. Real-time RT-PCR was carried out on cDNA using the ABI Prism 7700 Sequence Detection System (TaqMan; PE Applied Biosystems, Foster City, CA). The human GAPDH primers and fluorescence (6-carboxyfluorecein)-labeled probe were used as a control following the manufacturer’s instructions. TaqMan Universal PCR Master Mix was used in the PCR reaction mixture, and PCR was performed using the following program: 95°C for 10 min; and 40 cycles of 95°C for 15 s and 60°C for 1 min. Specific primers and probes used are as follows: (a) for UbcH1A, 5′-GCAAGAAGGAGAAAGTTGAAGG-3′ (forward primer), 5′-GCAAAAACCCAGAAGACCAA-3′ (reverse primer), and 5′-CAGCAATTGTAGTAACTGACACATCCTCTCTTTGC-3′ (probe); (b) for UbcH1B, 5′-AAGCGGTTACAAGAGGACCCA-3′ (forward primer), 5′-GGTGTCCCTTCTGGTCCAAA-3′ (reverse primer), and 5′-TGGGTGTCAGTGGCGCACCATCT-3′ (probe); (c) for UbcH2, 5′-ACCTCCACCGACCAGAAGAA-3′ (forward primer), 5′-GTCCCCGGTACCCTCTTCC-3′ (reverse primer), and 5′-AAGAGTACATCCAGAAATACGCCACGGAGG-3′ (probe); (d) for UbcH3, 5′-CGATGAGGATGACTCTGGCA-3′ (forward primer), 5′-TCTGTCGTCTAAGGAGCCACG-3′ (reverse primer), and 5′-CCTGACACCACCAGAATAAACTTGCCGA-3′ (probe); (e) for UbcH5A, 5′-CGATCCACCTGCTCACTGTT-3′ (forward primer), 5′-TGAGAAAGAAGACTCCACCTTG-3′ (reverse primer), and 5′-TGGGAGATGACTTGTTCCACTGGCAAG-3′ (probe); (f) for UbcH5B, 5′-ATTGAATGATCTGGCACGGG-3′ (forward primer), 5′-TGTCATTTGGCCCCATTATTG-3′ (reverse primer), and 5′-TCCAGCACAGTGTTCAGCAGGTCCTGT-3′ (probe); (g) for UbcH5C, 5′-AGAGTGAGGAGCCAGACGACA-3′ (forward primer), 5′-CTGCAGAACATTGTGCTGGAG-3′ (reverse primer), and 5′-ACACACTATGGCGCTGAAACGGATTAATAAGG-3′ (probe); (h) for UbcH6, 5′-CAACCAGCAAACCGAGAAAG-3′ (forward primer), 5′-GGTGGAGGGTCTAAAGTGATG-3′ (reverse primer), and 5′-CCCAAGAAGAAGGAGAGTAAAGTCAGCATGAGC-3′ (probe); (i) for UbcH7, 5′-GGCAAGGGCTTATTGTTCCT-3′ (forward primer), 5′-AATGTGATCTTCGGTGGTTTG-3′ (reverse primer), and 5′-TCCATATGATAAGGGAGCCTTCAGAATCGAA-3′ (probe); (j) for UbcH8, 5′-TGCCCATCATCAGCAGTGAG-3′ (forward primer), 5′-GGGCTCCCTGATATTCGGTC-3′ (reverse primer), and 5′-TGGAAGCCTTGCACCAAGACTTGCC-3′ (probe); (k) for UbcH9, 5′-GAGTCCCGCTTTGACTATTTC-3′ (forward primer), 5′-TCGTGTTCTGCTCTGTTGGT-3′ (reverse primer), and 5′-TGCTGTCTATTTGTTCCCTTTTGACAGACTGC-3′ (probe); (l) for UbcH10, 5′-TGGTCTGCCCTGTATGATGT-3′ (forward primer), 5′-AAAAGCTGTGGGGTTTTTCC-3′ (reverse primer), and 5′-TCCATCCAGAGCCTTCTAGGAGAACCCA-3′ (probe); (m) for UbcH12, 5′-ATCCAGACGACCTCCTCAAC-3′ (forward primer), 5′-CCATTGTCTCACACTTCACCT-3′ (reverse primer), and 5′-CTGTCCTGATGAGGGCTTCTACAAGAGTGG-3′ (probe); (n) for UbcH13, 5′-AAGATAAGTGGTCCCCAGCA-3′ (forward primer), 5′-GGCTTCGTTGGTCTTCCAC-3′ (reverse primer), and 5′-ACAGTTCTGCTATCGATCCAGGCCTTGTT-3′ (probe); (o) for UbcH16, 5′-TGGGCTTTGATAGGAATGCAG-3′ (forward primer), 5′-GCTCTCTATGCCTCAGTTACTCAGAA-3′ (reverse primer), and 5′-TAGTGGCCTTGTCTTCAAAATCATGGGATG-3′ (probe); (p) for UbcH17, 5′-ATTCAGCTGCTCATGTCAGAACC-3′ (forward primer), 5′-CTGGCATTCTTGAGGAAGGC-3′ (reverse primer), and 5′-CCCTGATGACCCGCTCATGGCT-3′ (probe); and (q) for UbcH19, 5′-TGCAGAACCACATCCTGAGG-3′ (forward primer), 5′-GGAGACTGGTGGAAGCCACA-3′ (reverse primer), and 5′-TCTCAGCTTATCCTGGAGGGAATTGGGA-3′ (probe).

Expression of 17 Ubiquitin-conjugating Enzyme (E2) Genes in Human Normal Tissues and Cancer Cell Lines.

To search for genes encoding ubiquitin-conjugating enzymes (E2s), we performed a BLAST analysis of nucleotide and protein sequence database (DDBJ) using both full-length and amino acid residues 60–91 of UbcH5A as query. As listed in Table 1, 17 clones, including UbcH5A, matched this search. To examine the tissue distributions of 17 E2 genes, the primer/probe set was designed for each E2 gene, and total RNA prepared from 25 different human normal tissues was subjected to the quantitative real-time RT-PCR analysis. The expression level of each E2 mRNA was normalized to that of GAPDH. Fig. 1 A shows the expression patterns for these genes in each of the normal tissues. First, we observed that all of the E2 mRNAs that we examined were undetectable in skeletal muscle and expressed at extremely low levels in colon and heart. Among them, UbcH1A, UbcH1B, and Ubc5C were expressed at high levels in placenta and pancreas, and UbcH1B, UbcH5B, UbcH12, UbcH13, and UbcH17 were highly expressed in testis. In addition, the expression levels of UbcH6, UbcH16, and UbcH19 were high in pancreas. Of note, expression levels of UbcH10 and UbcH17 in the majority of normal tissues were significantly lower than those of the other E2 genes.

We next measured the expression levels of the above-mentioned E2 gene family in 24 human cell lines derived from cancers of multiple origins using quantitative real-time RT-PCR. As seen in Fig. 1 B, UbcH1A, UbcH10, and UbcH16 were expressed at high levels in osteosarcoma OST cells. Additionally, neuroblastoma TGW cells showed high levels of UbcH6, UbcH10, and UbcH17 mRNA. In contrast to the results obtained from the expression analysis in normal tissues, UbcH10 was expressed at a high level in the majority of cancerous cell lines examined as compared with the other E2 genes.

UbcH10 mRNA Is Expressed at High Levels in Primary Cancerous Tissues of the Lung, Stomach, Uterus, and Bladder.

The elevated level of UbcH10 expression in certain cancerous cell lines suggests that UbcH10 might be expressed at high levels in human primary tumor tissues. Therefore, we performed quantitative real-time RT-PCR to examine the expression level of UbcH10 in various human tumor tissues derived from six lung adenocarcinomas, three gastric adenocarcinomas, two uterine carcinomas, and six bladder carcinomas as well as their corresponding normal tissues. Our results showed that UbcH10 mRNA expression was significantly increased in all of the tumor tissues we examined as compared with the matched normal tissues, suggesting that up-regulation of UbcH10 expression contributes to the occurrence or progression of various human tumors (Fig. 2,A). To validate the expression changes detected by quantitative real-time RT-PCR analysis, we performed Northern blot analysis with the same RNA samples used for the quantitative real-time RT-PCR analysis. As shown in Fig. 2 B, UbcH10 mRNA (approximately 2.0 kb) was highly expressed in all of the investigated tumor tissues. In contrast, UbcH10 mRNA was expressed at extremely low levels in matched adjacent normal tissues, correlating well with the results obtained by the quantitative real-time RT-PCR analysis for UbcH10.

Generation of Stable Transfectants Overexpressing UbcH10.

To examine a possible role of UbcH10 in the cell growth regulation, mouse fibroblast NIH3T3 cells were stably transfected with a cytomegalovirus promoter-driven expression plasmid pcDNA3 (containing resistance to G418) encoding human full-length UbcH10 tagged with the FLAG peptide on its NH2 terminus (pcDNA3-FLAG-UbcH10). The transfected cells were selected in the presence of G418. After 2 weeks of selection, we obtained several drug-resistant transfectants. Western blot analysis using a monoclonal anti-FLAG antibody revealed that UbcH10-2, UbcH10-12, UbcH10-17, UbcH10-20, UbcH10-40, and UbcH10-59 cells expressed relatively larger amounts of UbcH10 than UbcH10-7 and UbcH10-15 cells, whereas FLAG-UbcH10 was not detected in the parental NIH3T3 cells and NIH3T3 cells transfected with an empty plasmid (V-15 and V-29; Fig. 3,A). The equal protein loading was confirmed by reprobing the anti-FLAG blot with an antibody against actin. No obvious morphological changes could be observed in UbcH10-overexpressing NIH3T3 cells (data not shown). To visualize subcellular distribution of UbcH10, immunofluorescence microscopic analysis was performed. V-15 or UbcH10-17 cells were stained with the anti-FLAG antibody followed by incubation with a rhodamine-conjugated secondary antibody. In agreement with the previous observations (16), ectopically overexpressed FLAG-UbcH10 was largely perinuclear in UbcH10-17 cells, and we did not detect any immunoreactivities in V-15 cells (Fig. 3 B). A similar result was obtained in a separate experiment in which UbcH10-20 cells were used. Therefore, we selected UbcH10-17 and UbcH10-20 cells for further analysis.

Overexpression of UbcH10 Stimulates Growth and Colony-forming Activity in NIH3T3 Cells.

We next examined the possible effect(s) of UbcH10 on the initiation of DNA synthesis. To this end, V-15 and UbcH10-17 cells were grown in the presence of BrdUrd (at a final concentration of 10 μm), and the number of cells that were stained with an antibody against BrdUrd was scored. As shown in Fig. 4, UbcH10-17 cells incorporated BrdUrd much more than the control transfectants. On average, 52.3% of UbcH10-17 cells and 28% of V-15 cells entered into S phase as indicated by incorporation of BrdUrd, respectively. These results strongly suggest that overexpression of UbcH10 stimulated an ability of cells to proceed through the S phase.

Next, we examined the growth of parental NIH3T3, V-15, UbcH10-17, and UbcH10-20 cells in different serum culture conditions (10% and 2% serum). The growth rate of each cell line was determined by counting the number of cells daily. As shown in Fig. 5,A, UbcH10-overexpressing transfectants grew at a much faster rate than NIH3T3 or the control transfectants. Similar results were obtained under a low serum culture condition (Fig. 5,B). In addition, cells overexpressing UbcH10 overgrew a monolayer by doubling the saturation density of control cells (Table 2). The accelerated growth rate of UbcH10-overexpressing transfectants raised the possibility that they might have become transformed. To this end, we examined their ability to grow in soft agar medium. As shown in Fig. 5 C, UbcH10-17 and UbcH10-20 cells formed colonies in soft agar medium (13 ± 1 and 14 ± 2 colonies/dish, respectively) more efficiently than the parental and the empty plasmid-transfected V-15 cells (4 ± 1 and 2 ± 1 colonies/dish, respectively). Under our experimental conditions, NIH3T3 transfectants overexpressing UbcH10 created distinct colonies in soft agar medium after 3 weeks; however, we could detect only small colonies after 2 weeks of culture. Taken together, our present results provide the first evidence that the E2 ubiquitin-conjugating enzyme gene, UbcH10, is highly expressed in various human primary tumors compared with their corresponding normal tissues and that UbcH10 has an ability to promote cell growth and transformation.

In the present study, we evaluated the expression level of the E2 gene family in a wide variety of human normal tissues and 24 cell lines derived from multiple tumor origins by quantitative real-time RT-PCR analysis. We found that, among the 17 E2 genes examined, UbcH10 was expressed at very low to undetectable levels in normal tissues, whereas numerous cancerous cell lines expressed it at high levels. Of note, UbcH10 was highly expressed in primary tumors of the lung, stomach, uterus, and bladder compared with the matched normal tissues. Consistent with the above-mentioned observations, overexpression of UbcH10 caused an efficient incorporation of BrdUrd, an accelerated growth rate, an increase in saturation density, and a promotion of anchorage-independent growth. These data provide evidence that the deregulated overexpression of UbcH10 may lead to growth promotion as well as malignant transformation and that UbcH10 may be an essential oncogenic factor in a variety of tumors.

In our experiments, the expression of UbcH10 in tumor tissues as well as cancerous cell lines was examined by RNA levels. As described by Shekhar et al.(21), the expression levels of HR6B protein, which is the mammalian homologue of yeast ubiquitin-conjugating enzyme Rad6, in mouse mammary tumor cells were in good agreement with those detected by RT-PCR. Additionally, Arvand et al.(18) reported that endogenous UbcH10 was up-regulated at both the mRNA and protein level in transformed NIH3T3 cells, suggesting that the expression level of UbcH10 mRNA might reflect in part the intracellular level of UbcH10 protein. To confirm our present results, it is necessary to analyze UbcH10 protein levels in tumor tissues as well as cancerous cell lines.

UbcH10 has been identified as a human homologue of the cyclin-selective E2 (E2-C) that is required for the destruction of mitotic cyclins (14, 15, 16). Townsley et al.(16) described that enforced expression of the dominant-negative type of UbcH10 inhibited the ubiquitination and the subsequent degradation of mitotic cyclins. In accordance with the above-mentioned observations, UbcH10 is functionally associated with the APC, which acts as the E3 ubiquitin ligase to catalyze the transfer of ubiquitin to mitotic cyclins (9, 14, 15). APC is activated during mitosis, remains active throughout the G1 phase, and is degraded at the G1-S boundary (22, 23). Intriguingly, Arvand et al.(18) reported that UbcH10 protein was highly expressed in G2-M phase, but its expression level was extremely low in G0-G1 phase. Yamanaka et al.(24) found that UbcH10 that contains the destruction box (D box) underwent APC-dependent degradation at early G1 phase, suggesting that the cell cycle-dependent expression of UbcH10 might be a unique autoregulatory feedback loop for the regulation of APC activity. Considering that the dominant-negative UbcH10 arrests mammalian cells in M phase and inhibits the onset of anaphase, it is possible that the function of UbcH10 is closely linked to cell cycle progression (16). Thus, the enforced overproduction of UbcH10 might disrupt the autoregulatory feedback loop and thereby lead to deregulated cell growth; however, the precise molecular mechanisms by which UbcH10 promotes cell growth are unclear.

Recently, Shekhar et al.(21) found that overexpression of Ubc2/Rad6 induced anchorage-independent growth of recipient cells, indicating that deregulated expression of Ubc2/Rad6 is involved in malignant transformation. In addition, McDoniels-Silvers et al.(19) reported that the expression levels of Ubc9 were increased in human lung adenocarcinomas compared with those of their corresponding normal tissues. These findings imply that certain E2 proteins might be closely linked to tumorigenesis. As described previously, overexpression of EWS/FLI1 in NIH3T3 cells induced anchorage-independent growth in soft agar medium and generated tumors in nude mice (25, 26, 27, 28). Of note, Arvand et al.(18) reported that endogenous UbcH10 was up-regulated in NIH3T3 cells transformed with EWS/FLI1 but not in nontransformed NIH3T3 cells, indicating that UbcH10 could play an important regulatory role in the transformation. According to their results, however, stable NIH3T3 transfectants overexpressing mouse UbcH10 did not form colonies in soft agar medium under their experimental conditions. They suggested that other gene products in addition to UbcH10 might be required for cellular transformation. Their findings differ from our present results, which showed UbcH10-induced colony formation in soft agar medium. Intriguingly, they also demonstrated that YAL-7 cells expressing EWS/FLI1 did not grow in soft agar medium, and there existed a significant difference in the EWS/FLI1-mediated induction level ofUbcH10 between NIH3T3 and YAL-7 cells, raising the possibility that a certain threshold level of UbcH10 protein might be required to render cells for being transformed. Although the underlying cause of this discrepancy is not clear, it could be explained in part by the differences in intracellular expression levels of UbcH10 between our stable transfectants and their clones.

Pagano et al.(17) reported that Ubc2/Rad6 and Ubc3/CDC34 were specifically involved in the ubiquitination-dependent degradation of cyclin-dependent kinase inhibitor p27. UbcH7/E2-F1 was reported to support E6-AP-dependent ubiquitination and function in the conjugation and subsequent degradation of tumor suppressor p53 (29, 30, 31). Thus, it is possible that UbcH10, together with a particular E3 protein(s), might recognize and break down substrate proteins with growth-regulatory function. In this connection, identification of target proteins of UbcH10 should help promote understanding of its role in malignant transformation and tumor cell growth.

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

Supported by a Grant-in-Aid from the Ministry of Health and Welfare for a New 10-Year Strategy for Cancer Control, a Grant-in-Aid for Scientific Research on Priority Areas, and a Grant-in-Aid for Scientific Research (B) from the Ministry of Education, Science, Sports and Culture, Japan.

3

The abbreviations used are: APC, anaphase-promoting complex; BrdUrd, 5-bromo-2′-deoxyuridine; CS, calf serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT-PCR, reverse transcription-PCR.

Fig. 1.

The expression of UbcH10 mRNA is up-regulated in cancerous cell lines. The expression levels of human ubiquitin-conjugating enzyme (E2) genes as listed in Table 1 were examined by quantitative real-time RT-PCR analysis. Total RNA prepared from 25 different human normal tissues [brain, liver, heart, kidney, trachea, colon, small intestine, stomach, bone marrow, thymus, spleen, mammary gland, testis, uterus, prostate, skeletal muscle, cerebellum, fetal brain, spinal cord, fetal liver, placenta, adrenal gland, pancreas, salivary gland, and thyroid (A)] or 24 different human cancerous cell lines [OST, SAOS-2, RMS-MK, ASPS-KY, COLO320, SW480, LoVo, HepG2, MB453, MB231, G-361, G32TG, TTC2, Kato-III, EC-GI-10, ASPC-1, A549, SKN-AS, SMS-KAN, SMS-KCN, IMR32, SKN-DZ, TGW, and NB-1 (B)] was subjected to quantitative real-time RT-PCR as described in “Materials and Methods.” Relative expression level of each E2 mRNA was determined by calculating the ratio between E2 and GAPDH.

Fig. 1.

The expression of UbcH10 mRNA is up-regulated in cancerous cell lines. The expression levels of human ubiquitin-conjugating enzyme (E2) genes as listed in Table 1 were examined by quantitative real-time RT-PCR analysis. Total RNA prepared from 25 different human normal tissues [brain, liver, heart, kidney, trachea, colon, small intestine, stomach, bone marrow, thymus, spleen, mammary gland, testis, uterus, prostate, skeletal muscle, cerebellum, fetal brain, spinal cord, fetal liver, placenta, adrenal gland, pancreas, salivary gland, and thyroid (A)] or 24 different human cancerous cell lines [OST, SAOS-2, RMS-MK, ASPS-KY, COLO320, SW480, LoVo, HepG2, MB453, MB231, G-361, G32TG, TTC2, Kato-III, EC-GI-10, ASPC-1, A549, SKN-AS, SMS-KAN, SMS-KCN, IMR32, SKN-DZ, TGW, and NB-1 (B)] was subjected to quantitative real-time RT-PCR as described in “Materials and Methods.” Relative expression level of each E2 mRNA was determined by calculating the ratio between E2 and GAPDH.

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Fig. 2.

Expression of UbcH10 mRNA in various primary tumors and their corresponding normal tissues. A, quantitative real-time RT-PCR analysis. Total RNA derived from the indicated tumors (T) and corresponding normal tissues (N) was subjected to quantitative real-time RT-PCR as described in “Materials and Methods.” Relative expression levels of UbcH10 mRNA were determined by calculating the ratio between UbcH10 and GAPDH. B, Northern blot analysis. Total RNA prepared from the indicated tumors (T) and corresponding normal tissues (N) was fractionated on 1% formaldehyde-agarose gel and transferred onto nitrocellulose membrane filter. The filter was hybridized with radiolabeled UbcH10 cDNA probe. The bottom panel shows the ethidium bromide-stained 18S and 28S rRNA bands (arrowheads) immediately after blotting.

Fig. 2.

Expression of UbcH10 mRNA in various primary tumors and their corresponding normal tissues. A, quantitative real-time RT-PCR analysis. Total RNA derived from the indicated tumors (T) and corresponding normal tissues (N) was subjected to quantitative real-time RT-PCR as described in “Materials and Methods.” Relative expression levels of UbcH10 mRNA were determined by calculating the ratio between UbcH10 and GAPDH. B, Northern blot analysis. Total RNA prepared from the indicated tumors (T) and corresponding normal tissues (N) was fractionated on 1% formaldehyde-agarose gel and transferred onto nitrocellulose membrane filter. The filter was hybridized with radiolabeled UbcH10 cDNA probe. The bottom panel shows the ethidium bromide-stained 18S and 28S rRNA bands (arrowheads) immediately after blotting.

Close modal
Fig. 3.

Overexpression of UbcH10 in NIH3T3 cells. A, Western blot analysis. NIH3T3 cells were stably transfected with the expression plasmid for FLAG-UbcH10 or control pcDNA3 plasmid alone, and drug-resistant colonies were isolated. Whole cell lysates prepared from the indicated cell clones were subjected to 15% SDS-PAGE, transferred onto a nitrocellulose membrane, and immunoblotted with a monoclonal anti-FLAG antibody (top panel). Loading and transfer conditions were verified by reprobing the blot with a polyclonal anti-actin antibody (bottom panel). B, immunofluorescent staining of UbcH10. V-15, UbcH10-17, and UbcH10-20 cells were stained with the monoclonal anti-FLAG antibody and rhodamine-conjugated goat antimouse IgG secondary antibody to visualize localization of UbcH10.

Fig. 3.

Overexpression of UbcH10 in NIH3T3 cells. A, Western blot analysis. NIH3T3 cells were stably transfected with the expression plasmid for FLAG-UbcH10 or control pcDNA3 plasmid alone, and drug-resistant colonies were isolated. Whole cell lysates prepared from the indicated cell clones were subjected to 15% SDS-PAGE, transferred onto a nitrocellulose membrane, and immunoblotted with a monoclonal anti-FLAG antibody (top panel). Loading and transfer conditions were verified by reprobing the blot with a polyclonal anti-actin antibody (bottom panel). B, immunofluorescent staining of UbcH10. V-15, UbcH10-17, and UbcH10-20 cells were stained with the monoclonal anti-FLAG antibody and rhodamine-conjugated goat antimouse IgG secondary antibody to visualize localization of UbcH10.

Close modal
Fig. 4.

Effect of forced expression of UbcH10 on DNA synthesis in NIH3T3 cells. A, exponentially growing V-15 (top panels) and UbcH10-17 (bottom panels) cells were exposed to BrdUrd (10 μm) for 10 min and then stained with a monoclonal anti-BrdUrd antibody, followed by a FITC-conjugated goat antimouse IgG secondary antibody (left panels). DNA was stained with propidium iodide (PI; right panels). A minimum of 300 cells/dish was examined. B, the number of BrdUrd-positive cells was counted and expressed as a percentage of the total number of cells examined. Data are the means ± SE from three independent experiments.

Fig. 4.

Effect of forced expression of UbcH10 on DNA synthesis in NIH3T3 cells. A, exponentially growing V-15 (top panels) and UbcH10-17 (bottom panels) cells were exposed to BrdUrd (10 μm) for 10 min and then stained with a monoclonal anti-BrdUrd antibody, followed by a FITC-conjugated goat antimouse IgG secondary antibody (left panels). DNA was stained with propidium iodide (PI; right panels). A minimum of 300 cells/dish was examined. B, the number of BrdUrd-positive cells was counted and expressed as a percentage of the total number of cells examined. Data are the means ± SE from three independent experiments.

Close modal
Fig. 5.

Effect of UbcH10 overexpression on cell growth characteristics. The parental NIH3T3, V-15, UbcH10-17, and UbcH10-20 cells were plated on day 0 and cultured in the presence of 10% (A) or 2% (B) CS. Cells were trypsinized at 24-h time intervals, and the number of cells was counted in triplicate. The data represent the means ± SE from three independent experiments. C, soft agar colony formation assay. The parental NIH3T3 cells and the indicated stable transfectants (2.5 × 103 cells/dish) were grown in soft agar medium. After 3 weeks, cells were examined by phase-contrast microscopy, and the number of colonies with a diameter of >100 μm was counted. Data are the means ± SE from three independent experiments.

Fig. 5.

Effect of UbcH10 overexpression on cell growth characteristics. The parental NIH3T3, V-15, UbcH10-17, and UbcH10-20 cells were plated on day 0 and cultured in the presence of 10% (A) or 2% (B) CS. Cells were trypsinized at 24-h time intervals, and the number of cells was counted in triplicate. The data represent the means ± SE from three independent experiments. C, soft agar colony formation assay. The parental NIH3T3 cells and the indicated stable transfectants (2.5 × 103 cells/dish) were grown in soft agar medium. After 3 weeks, cells were examined by phase-contrast microscopy, and the number of colonies with a diameter of >100 μm was counted. Data are the means ± SE from three independent experiments.

Close modal
Table 1

Human ubiquitin-conjugating enzymes (E2s) and yeast homologues

E2sAlternative nameChromosomal locusGenBank accession no.Nucleotide length (bp)Amino acid length (aa)aYeast homologue
UbcH1A HHR6A Xq24-25 M74524 1743 152 Ubc2/Rad6 
UbcH1B HHR6B 5q23-31 M74525 2591 152 Ubc2/Rad6 
UbcH2 E2H 7q32 Z29330 1579 183 Ubc8 
UbcH3 CDC34 19p13.3 L22005, NM_004359 1462 236 Ubc3/cdc34 
UbcH5A E2D1 10q11.2-21 X78140 459 147 Ubc4/5 
UbcH5B E2D2 5q31.1-33.3 L40146 509 147 Ubc4/5 
UbcH5C E2D3 4q24-26 U39318 724 147 Ubc4/5 
UbcH6 E2E1 3p24.3 X92963 582 193 Ubc4/5 
UbcH7 E2L3 22q11.2 X92962, AJ000519 2845 154  
UbcH8 E2L6 11q12 AF031141, NM_004223 1223 153  
UbcH9 E2E3 2q32.1 AB017644 1216 207 Ubc4/5 
UbcH10 E2-C 20q12-13 U73379 783 179 Ubc4 
UbcH12 E2M 19q13 AF075599 751 183 Ubc12 
UbcH13 E2N 12q21-22 D83004 1203 152 Ubc13 
UbcH16 HIP2 4p14 U58522 2149 200  
UbcH17 HSPC150 1p36.1-32.1 AF161499 928 197  
UbcH19  7p13 AF125045 1315 109  
E2sAlternative nameChromosomal locusGenBank accession no.Nucleotide length (bp)Amino acid length (aa)aYeast homologue
UbcH1A HHR6A Xq24-25 M74524 1743 152 Ubc2/Rad6 
UbcH1B HHR6B 5q23-31 M74525 2591 152 Ubc2/Rad6 
UbcH2 E2H 7q32 Z29330 1579 183 Ubc8 
UbcH3 CDC34 19p13.3 L22005, NM_004359 1462 236 Ubc3/cdc34 
UbcH5A E2D1 10q11.2-21 X78140 459 147 Ubc4/5 
UbcH5B E2D2 5q31.1-33.3 L40146 509 147 Ubc4/5 
UbcH5C E2D3 4q24-26 U39318 724 147 Ubc4/5 
UbcH6 E2E1 3p24.3 X92963 582 193 Ubc4/5 
UbcH7 E2L3 22q11.2 X92962, AJ000519 2845 154  
UbcH8 E2L6 11q12 AF031141, NM_004223 1223 153  
UbcH9 E2E3 2q32.1 AB017644 1216 207 Ubc4/5 
UbcH10 E2-C 20q12-13 U73379 783 179 Ubc4 
UbcH12 E2M 19q13 AF075599 751 183 Ubc12 
UbcH13 E2N 12q21-22 D83004 1203 152 Ubc13 
UbcH16 HIP2 4p14 U58522 2149 200  
UbcH17 HSPC150 1p36.1-32.1 AF161499 928 197  
UbcH19  7p13 AF125045 1315 109  
a

aa, amino acid(s).

Table 2

Growth properties of the transfectants

Cell linesGrowth in monolayersa
Doubling time (h)Saturation density (104 cells)
NIH3T3 19.5 7.7 ± 0.6 
V-15 19.8 7.0 ± 2.1 
UbcH10-17 15.7 14.9 ± 0.7 
UbcH10-20 14.9 18.0 ± 2.7 
Cell linesGrowth in monolayersa
Doubling time (h)Saturation density (104 cells)
NIH3T3 19.5 7.7 ± 0.6 
V-15 19.8 7.0 ± 2.1 
UbcH10-17 15.7 14.9 ± 0.7 
UbcH10-20 14.9 18.0 ± 2.7 
a

Doubling time is determined by calculating the growth rate of exponentially growing cells. Saturation density is the number of cells/well after the culture had reached confluence (means ± SE). Each experiment was carried out at least twice.

We thank Shigeru Sakiyama for critical reading of the manuscript and Maya Sakamoto for excellent technical assistance.

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