Oncogene (2004) 23, 9289–9294 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $30.00 www.nature.com/onc SHORT REPORT Isolation and characterization of a novel CLUAP1 whose expression is frequently upregulated in colon cancer

Meiko Takahashi1, Yu-Min Lin2, Yusuke Nakamura1 and Yoichi Furukawa*,1

1Laboratory of Molecular Medicine, Center, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai Minato-ku, Tokyo 108-8639, Japan; 2Department of Internal Medicine, Shin Kong Wu Ho-Su Memorial Hospital, 95 Wen Chang Road, Taipei 11160, Taiwan

To disclose mechanisms of colorectal carcinogenesis and appear to be upregulated frequently in the cancer tissues identify novel diagnostic markers and drug targets for compared with the corresponding noncancerous cells. treatment of these tumors, we previously analysed the Since carcinogenesis involves activation of oncogenes expression profiles of 11 colorectal cancers using a and/or inactivation of tumor suppressor , en- genome-wide cDNA microarray containing 23 040 genes. hanced expression of at least some of these upregulated Among the genes commonly transactivated in the cancers, genes may reflect oncogenic properties. we identified a novel human gene, which we termed Among the genes with frequently elevated expression CLUAP1 (clusterin-associated protein 1). It encodes a in cancer tissues in our microarray data, an EST with an nuclear protein of 413 amino acids containing a coiled-coil in-house accession number of B9223 corresponding to domain. To investigate its function, we searched for KIAA0643, Hs. 155995 in UniGene cluster (http:// CLUAP1-interacting proteins using yeast two-hybrid www.ncbi.nlm.nih.gov/UniGene/), was upregulated in system and identified nuclear Clusterin. Expression of the cancer tissues compared to their corresponding CLUAP1 was gradually increased in the late S to G2/ noncancerous mucosa in a magnification range between M phases of cell cycle and it returned to the basal level in 2.1 and 3.5 in five out of seven cases that passed the the G0/G1 phases. Suppression of this gene by short cutoff filter (data not shown). To clarify the results of the interfering RNAs resulted in growth retardation in the microarray, we carried out semiquantitative RT–PCR transfected cells. These data provide better understanding and revealed that expression of B9223 was increased in of colorectal carcinogenesis, and inactivation of CLUAP1 14 of additional 20 colon cancers (70%) compared with may conceivably serve in the future as a novel therapeutic their corresponding normal mucosae (Figure 1a). intervention for treatment of colon cancer. Homology searches with the sequence of B9223 in Oncogene (2004) 23, 9289–9294. doi:10.1038/sj.onc.1208100 public databases using BLAST program in National Published online 11 October 2004 Center for Biotechnology Information (http:// www.ncbi.nlm.nih.gov/BLAST/) identified a gene that Keywords: CLUAP1; clusterin; colon cancer had been annotated as KIAA0643 protein, clone MGC:9638 (GenBank Accession number BC017070), and a genomic sequence with GenBank Accession number of NT_010552.13 assigned to chromosomal Colorectal carcinomas are leading causes of cancer band 16p13. To determine the coding sequence of the death worldwide. In spite of recent progress in gene, we predicted candidate–exon sequences in the diagnostic and therapeutic strategies, prognosis of genomic sequence using GENSCAN (http://genes.mit. patients with advanced cancers remains very poor. edu/GENSCAN.html) and Gene Recognition and Although molecular studies have revealed that altera- Assembly Internet Link (GRAIL, http://compbio.ornl. tions of tumor suppressor genes and/or oncogenes are gov/Grail-1.3/) program, and performed exon-connec- involved in colorectal carcinogenesis, the precise me- tion experiments. As a result, we obtained an assembled chanisms remain to be fully elucidated. To disclose sequence of 1681 nucleotides (GenBank Accession mechanisms underlying these tumors from a genome- number AB089691) containing an open-reading frame wide point of view and to discover target molecules for of 1239 nucleotides encoding a putative 413-amino-acid diagnosis and for development of novel therapeutic protein, and termed the gene CLUAP1 (clusterin- drugs, we have been analysing their expression profiles associated protein 1) because of the reason described by cDNA microarray representing 23 040 genes (Kita- below. The first ATG was flanked by a sequence hara et al., 2001; Lin et al., 2002). These efforts have (AGCGTTATGT) that agreed with the consensus pinpointed a number of genes, including ESTs, which sequence for the initiation of translation in eukaryotes, with an in-frame stop codon upstream. Comparison of *Correspondence: Y Furukawa; E-mail: [email protected] CLUAP1 cDNA and the genomic sequence disclosed Received 19 February 2004; revised 29 July 2004; accepted 2 August that this gene consisted of 11 exons. The amino-acid 2004; published online 11 October 2004 sequence of the predicted CLUAP1 protein showed 89, CLUAP1 is upregulated in colon cancer M Takahashi et al 9290 a N T N T N T N T N T N T N T N T N T N T B9223 GAPDH

N T N T N T N T N T N T N T N T N T N T B9223

GAPDH

b

Figure 1 (a) Semiquantitative RT–PCR showing that B9223 expression was increased in 14 of 20 colon cancers compared to their corresponding normal mucosae. Extraction of RNA, cDNA synthesis, and PCR reaction were carried out as reported previously (Shimokawa et al., 2003). Amplification proceeded for 4 min at 941C for denaturing, followed by 21 (for GAPDH) and 32 (for CLUAP1), cycles of 941C for 30 s, 601C for 30 s, and 721C for 60 s, in the GeneAmp PCR system 9700 (Perkin-Elmer, Foster City, CA, USA). Primer sequences were as follows: GAPDH, forward 50-ACAACAGCCTCAAGATCATCAG-30 and reverse 50-GGTCCAC- CACTGACACGTTG-30; CLUAP1, forward 50-GAGTGGAAGTAACGATGACTC-30 and reverse 50-GTCATTGTCACTCT- CATCCAG-30.(b) Alignment of CLUAP1 (GenBank Accession number AB089691), a mouse RIKEN cDNA 2610111M03 (BC024910), a zebra fish unknown protein for MGC: 56115 (AAH45921), A. ganbiae ENSANGP00000008052 (XP_310880), and a C. elegans putative protein for 4E162 (NP_500339). Boxed region corresponds to the coiled-coil motif, and shading indicates residues that are conserved in at least three proteins

62, 38, 38, and 31% identities to a mouse RIKEN expressed abundantly in human testis, thyroid and cDNA 2610111M03 (NP_084014), a zebra fish unknown trachea, and moderately in spinal cord and adrenal protein for MGC:56115 (AAH45921), Anopheles gan- gland (Figure 2a). To examine the expression and biae ENSANGP00000008052 (XP_310880), a Caenor- explore the function of CLUAP1, we prepared poly- habditis elegans hypothetical protein (BAD 23996), and clonal against CLUAP1. Western blot analysis Drosophila melanogaster CGI7599-PA (NP_608470), using whole extracts of colon cancer cells, including respectively. A search for protein motifs with the Simple HCT116, SNU-C4, and SW480, showed a 53 kDa band Modular Architecture Research Tool (SMART, http:// that corresponded to CLUAP1 (Figure 2b). As a smart.embl-heidelberg.de) revealed that the predicted negative control for the Western blot analysis, we used protein contained a single coiled-coil region (codons extracts from TE8 esophageal cancer cells that showed 195–267), which was conserved in the homologues. In low level of CLUAP1 expression by semiquantitative addition to the coiled-coil motif, these homologues also RT–PCR (Figure 2b). shared similarities in amino acids between codons 1 and To reveal its subcellular localization, fluorescent 184 of CLUAP1 (Figure 1b). immunocytochemical staining of endogenous CLUAP1 Multiple-tissue Northern blotting with CLUAP1 was carried out in HCT116 cells. Cells were stained with cDNA as a probe showed that a 1.6-kb transcript was anti-CLUAP1 and visualized by fluorescein-conjugated

Oncogene CLUAP1 is upregulated in colon cancer M Takahashi et al 9291

a Ovary Lung Thymus Lymph node Lymph Prostate Thyroid Trachea Spinal cord Spinal Liver Spleen Kidney Pancreas Testis Heart Brain Placenta Peripheral blood leukocytes blood Peripheral Colon Adrenal gland Adrenal Small intestine Bone marrow Bone Stomach Skeletal muscle

CLUAP1 1.6 kb ß-actin

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Figure 2 (a) Human multiple-tissue blots (Clontech) were hybridized with a 32P-labeled PCR product of CLUAP1. Prehybridization, hybridization, and washing were performed according to the supplier’s recommendations. A 1.6-kb transcript was expressed abundantly in human testis, thyroid, and trachea. Expression of b-actin served as an internal control. (b) Western blot analysis of CLUAP1 in cancer cell lines. HCT116, SW480, and SNU-C4 were established from colon cancer tissues, while TE8 was from esophageal cancer tissue. The band indicated with an arrow corresponded to CLUAP1. Expression of b-actin served as an internal control. (c) Immunocytochemical staining using anti-CLUAP1 antibody in HCT116 cells showing the nuclear localization of endogenous CLUAP1. Recombinant His-tagged CLUAP1 protein was produced in Escherichia coli and inoculated for the immunization of rabbits. The polyclonal antibody to CLUAP1 was purified from the sera as described elsewhere (Shimokawa et al., 2003). Immunohistochemical staining of HCT116 cells with rabbit anti-CLUAP1 antibody was performed as reported previously (Okabe et al., 2003). The reaction was visualized after incubation with fluorescein-conjugated anti-rabbit second antibody (Leinco and ICN). Nuclei were counter-stained with 40,60-diamidine-20-phenylindole dihydrochloride (DAPI). (d) Immunohistochemical staining of CLUAP1 in paraffin-embedded cancer tissues using anti-CLUAP1 antibody. We analysed a total of 24 clinical samples including 14 cancer and 10 adenoma tissues, and assessed the reactivity semiquantitatively using the following grading system; À, no staining; þ , slight staining; 3 þ , strong staining; and 2 þ , staining between þ and 3 þ . For positive controls (3 þ ), sections of normal testis were utilized. If the score of the cancer cells was greater than that of corresponding epithelial cells in noncancerous mucosae in the same tissue section, we defined that CLUAP expression was elevated in the cancer by immunohistochemistry. Two pathologists independently reviewed the sections. (e) Western blot analysis of CLUAP1 using three colon cancer tissues (T) and their corresponding noncancerous mucosa (N). The band indicated with an arrow corresponded to CLUAP1 secondary antibody. Signals were observed mainly in the interaction between CLUAP1 and CLU in the yeast nuclei (Figure 2c). To compare the expression levels of cells. Apart from the secreted form of CLU, another CLUAP1 protein between noncancerous epithelial cells form is synthesized from a second AUG and expressed and tumor cells, formalin-fixed paraffin-embedded in the cytoplasm and nucleus (Reddy et al., 1996; clinical tissues including 14 advanced cancers and 10 Leskov et al., 2003). Since subcellular localization of adenomas of the colon were subjected to immunohisto- CLUAP1 was nucleus, we focused on the nuclear form chemical staining. Cancerous cells were more strongly of CLU (nCLU). The positive CLU clones contained stained with anti-CLUAP1 antibody than noncancerous codons between 252 and 449, indicating that the region epithelial cells in six tumors out of the 14 cancers and responsible for the interaction was in nCLU. To prove two out of the 10 adenomas (Figure 2d). Further the association between CLUAP1 and nCLU in vivo,we Western blot analysis using three clinical tissues transfected COS7 cells with plasmids expressing myc- demonstrated that its expression was augmented in tagged CLUAP1 (pcDNA-myc-CLUAP1) with or with- one out of the three cancer tissues compared to the out plasmids expressing FLAG-tagged C-term nCLU noncancerous mucosa (Figure 2a). (p3XFLAG-Clusterin) and carried out co-immunopre- To clarify further the function(s) of CLUAP1, we cipitation assay. Immunoprecipitation with anti-FLAG searched for CLUAP1-interacting proteins using yeast antibody and Western blot using anti-myc antibody two-hybrid screening system. Among a total of 70 showed a single band corresponding to CLUAP1, and positive clones that were picked and sequenced, 48 immunoprecipitation with anti-myc antibody and Wes- sequences matched a known protein called Clusterin tern blot using anti-FLAG showed a band correspond- (CLU). Subsequent simultaneous transformation of ing to nCLU, suggesting that CLUAP1 associates with pAS2-CLUAP1 with pACT2-CLU corroborated an nCLU in vivo (Figure 3a).

Oncogene CLUAP1 is upregulated in colon cancer M Takahashi et al 9292 IP: α-myc α-FLAG p3XFLAG-Clusterin and double staining with the a revealed colocalization of these proteins in pcDNA-myc-CLUAP1 - + - + - + - + the nucleus (Figure 3e), supporting the view that p3XFLAG-Clusterin - - + + - - + + CLUAP1 and nCLU interact in the cells. We performed cell-cycle analysis and immunoblot IB: α-myc analysis of CLUAL1 using HCT116 cells synchronized at the G1 phase by aphidicolin treatment. After with- drawal of aphidicolin, the cells entered S phase around 2–4 h, G2/M at 6–8 h and completed their cycle in 12 h. IB: α-FLAG Western blot showed the expression of CLUAP1 increased from G1 phase, peaked at S phase and gradually decreased after G2M phase (Figure 4a). b d To investigate the function of CLUAP1 in cancer cells, we constructed plasmids expressing CLUAP1- short interfering RNA (siRNAs) and examined their effect on CLUAP1 expression. Transfection of SNU-C4 or HCT116 colon cancer cells with psiH1BX-CLUAP1, psiH1BX-EGFP or psiH1BX-mock revealed that psiH1BX-CLUAP1 significantly suppressed the expres- ce sion of CLUAP1 in the cells compared to psiH1BX- EGFP or psiH1BX-mock (Figure 4b, and data not shown for HCT116). To test whether the suppression of CLUAP1 may affect the growth of colon cancer cells, we transfected these cells with psiH1BX-CLUAP1 or psiH1BX-mock. Viable cells transfected with psiH1BX-CLUAP1 were markedly reduced compared Figure 3 (a) Interaction between CLUAP1 and nCLU in vivo. to those transfected with psiH1BX-mock, suggesting Yeast two-hybrid assays were performed with the MATCH- MAKER GAL4 Two-Hybrid System according to the manufac- that decreased expression of CLUAP1 suppressed turer’s protocols (BD Biosciences). A total of 1 Â 106 clones from a growth of SNU-C4 cells as well as that of HCT116 human testis MATCHMAKER cDNA library were screened using cells (Figure 4c, and data not shown for HCT116). pAS2-CLUAP1 expressing the entire coding region of CLUAP1 as In this paper, we described the isolation and bait (BD Biosciences). The C-terminal region of nuclear Clusterin characterization of a novel gene, CLUAP1, whose from the isolated positive clones was subcloned into p3XFLAG- CMV vector (p3XFLAG-Clusterin). The coding region of expression was significantly increased in colon cancer CLUAP1 was amplified by RT–PCR using gene specific primer tissues compared to the corresponding noncancerous sets 50-GAGGAATTCCGACCCTGGGCTCCTGGGGAC-30 and mucosa. The high identities of the putative CLUAP1 50-AAGCTCGAGAAGTCATTGTCACTCTCATCCAG-30, and protein with its homologues in other species suggest that cloned into appropriate cloning site of pcDNA3.1myc/His vector (pcDNA-myc-CLUAP1). COS7 cells were transfected with CLUAP1 may play a crucial role in the cell. In addition pcDNA-myc-CLUAP1 expressing myc-tagged CLUAP1 and/or to the coiled-coil domain between amino acids 195 and p3XFLAG-Clusterin as indicated, and their lysates were subse- 267, it contained a conserved N-terminal region, which quently immunoprecipitated with anti-myc (BD Biosciences) or may be essential for the function of this protein. anti-FLAG M2 (SIGMA) antibody. The precipitated protein was Although cytogenetic analysis of colorectal tumors separated by SDS–PAGE and immunoblot analysis was carried out using with anti-myc antibody (upper panel) or rabbit anti-FLAG using comparative genomic hybridization have so far antibody (lower panel). (b) Colocalization of CLUAP1 and nCLU identified a set of amplified chromosomal regions protein in cells. COS7 cells were transfected with pcDNA-myc- including 3q, 5p, 6p, 7p, 7q, 8q, 12p, 13q, 17q, 19p, CLUAP1 and p3XFLAG-Clusterin, and stained with mouse anti- 19q, 20q, and X (Nakao et al., 1998; De Angelis et al., myc antibody and visualized with anti-mouse antibody IgG conjugated with FITC. (c) The cells were stained with rabbit 1999; Korn et al., 1999; He et al., 2003), amplification of anti-FLAG antibody and visualized with anti-rabbit antibody IgG 16p has not been reported to our knowl- conjugated with Rhodamine. (d) Nuclei were counter-stained by edge. On the contrary, frequent LOHs were shown in DAPI. (e) Merged image of (b–d) the chromosomal region of 16p13. Therefore, its elevation may not be a result from genetic alterations, but from other mechanisms such as transcriptional activation or enhanced RNA stability. Future studies To confirm the nuclear localization of these proteins, concerning the mechanism(s) of its up-regulation we cotransfected COS7 cells with pcDNA-myc- including promoter assay are necessary. CLUAP1 and p3XFLAG-Clusterin, and examined their The identification of nCLU as a CLUAP1-interacting subcellular localization by immunocytochemical stain- protein suggested a possible involvement of CLUAP1 in ing. Staining with anti-myc antibody revealed that the signal transduction of growth and/or survival. CLU was tagged CLUAP1 protein localized in the nucleus, while originally identified as SP-40,40, a secreted glycoprotein that with anti-FLAG antibody demonstrated that the in ram rete testis enhancing cell aggregation in vitro tagged nCLU was also in the nucleus (Figure 3b–d). (Murphy et al., 1988). It is also known as complement Cotransfection with both pcDNA-myc-CLUAP1 and lysis inhibitor, testosterone-repressed prostate message-2,

Oncogene CLUAP1 is upregulated in colon cancer M Takahashi et al 9293

d te Time after withdrawal of aphidicolin a a e tr n u 0h 2h 4h 6h 8h 10h 12h

CLUAP1

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GAPDH Relative absorbance 0 mock siCLUAP1 Figure 4 (a) Cell-cycle-dependent expression of CLUAP1. To examine whether CLUAP1 has a role in cell-cycle progression, HCT116 cells were growth arrested in G1 phase by incubation with 5 mg/ml aphidicolin (Sigma Chemical Co.) for 24 h and released from G1 by washing five times with PBS. Cells were collected by trypsinization at 2-hour intervals for a total of 12 h and fixed in 70% cold ethanol. Cells treated with RNase and propidium iodide (50 mg/ml) in PBS were analysed by FACS analysis (Becton Dickinson, San Jose, CA, USA). Western blot was also carried out at the same time points to see if the expression levels of CLUAP1 altered during the cell cycle. Time after the withdrawal of aphidicolin is indicated at the top. (b) Effect of psiH1BX-CLUAP1 on the expression of CLUAP1 in SNU-C4 cells. We constructed plasmid vector, psiH1BX, expressing siRNA as described elsewhere (Shimokawa et al., 2003). Plasmids expressing CLUAP1-siRNAs were prepared by the cloning of double-stranded oligonucleotides into psiH1BX vector. The oligonucleotides used for CLUAP1-siRNA (psiH1BX-CLUAP1) were 50-TCCCGACCATCATAGGATGGAGCTTCAAGAGAG CTCCATCCTATGATGGTC-30 and 50-TTTTGACCATCATAGGATGGAGCTCTCTTGAAGCTCCATCCTATGATGGTC-30. psiH1BX-CLUAP1, psiH1BX-EGFP, or psiH1BX-mock plasmids were transfected into SNU-C4 cells, using FuGENE6 reagent (Roche) or Nucleofector reagent (Amaxa) according to the supplier’s recommendations. Total RNA was extracted from the cells 48 h after the transfection. Semiquantitative RT–PCR was carried out using RNA extracted from cells transfected with psiH1BX-mock, psiH1BX-EGFP, or psiH1BX-CLUAP1. (c) MTT assay showing the effect of psiH1BX-CLUAP1 on the viability of SNU-C4 cells. Cells were cultured in the presence of 400–800 mg/ml geneticin (G418) for 14 days before the assay was carried out. The data were subjected to analysis of variance (ANOVA) and the Scheffe´ ’s F-test

sulfated glycoprotein 2 or apolipoprotein J. CLU is CLUAP1 may not affect the apoptotic activity, but reportedly implicated in diverse physiological processes modulate other functions of nCLU. Notably, since such as sperm maturation, lipid transportation, comple- codons between 256 and 448 in nCLU are responsible ment inhibition, tissue remodeling, membrane recycling, for its interaction with Ku70, CLUAP1 may affect the cell adhesion, stress responses, senescence, proliferation association and result in altered DNA repairing capacity and apoptosis (Trougakos and Gonos, 2002). However, in cancer cells. Alternatively, sharing structural and its precise role and regulation remain poorly under- functional similarities with heat-shock proteins, CLU stood. Nuclear CLU was first identified as a Ku70 may affect the function of CLUAP1 through formation associating protein and it was shown to act as a of aggresomes. In agreement with this notion, a recent prodeath signal inhibiting cell growth and survival report has demonstrated that nCLU can indeed initiate (Yang et al., 1999, 2000). Only nCLU proteins with aggresome formation and this led to a severe disruption both the C-terminal coiled-coil domain and a functional of the mitochondrial distribution pattern (Debure et al., NLS could kill MCF7 breast cancer cells, and altera- 2003). Expression of CLU is reportedly enhanced in sex tions in, or deletions of, the NLS or coiled-coil domain hormone-induced prostate carcinogenesis (Ouyang et al., was reported to abrogate cell death (Leskov et al., 2003). 2001), ovarian cancer (Hough et al., 2001), lymphomas Although we tested whether transduction of CLUAP1 (Wellmann et al., 2000), and breast tumor (Redondo interfered with nCLU-induced apoptosis in MCF7 cells et al., 2000). Another recent report has revealed that or not, FACS analysis detected unchanged sub-G1 CLU was also upregulated in murine intestinal neopla- population between cells transfected with nCLU alone sias and human colorectal tumors (Chen et al., 2003). and those transfected with nCLU and CLUAP1, and Therefore, elevated expression of CLUAP1 and CLU similar results were observed by colony formation assay may additively or synergistically enhance their function (data not shown). Hence, elevated expression of and play a significant role in colon tumors.

Oncogene CLUAP1 is upregulated in colon cancer M Takahashi et al 9294 To clarify the role of CLUAP1 in cancer cells, we essential role for proliferation in collaboration with constructed siRNA designed to suppress the expression unidentified factor(s) or for maintenance of proliferating of CLUAP1. Consequently, the suppression of cells. Although the precise molecular mechanism by CLUAP1 resulted in the decrease in colony number of which enhanced expression of CLUAP1 is involved in cells, suggesting that decreased levels of CLUAP1 lead cell growth needs to be clarified, CLUAP1 may be a to growth suppression of the cancer cell lines tested. good candidate for the development of effective drugs to Consistently, expression of CLUAP1 was induced in treat colorectal tumors, because downregulated expres- S phase of cell-cycle progression, suggesting that its sion of CLUAP1 suppressed the growth of colon cancer elevated expression was relevant to cellular prolifera- cells in our experiments. tion. Since its expression was greatly enhanced in more than half of the colon cancers we examined, our results raised a hypothesis that activated CLUAP1 may lead to Acknowledgements We are grateful to Ms Yuka Yamane-Tanaka, Tae Makino- neoplasms by constitutively accelerating proliferation of Nagao, Yumi Nakajima, and Yoshika Sakamoto for technical colonic epithelial cells. This notion prompted us to assistance, and Drs Ryuji Hamamoto, Kazutaka Obama, and examine oncogenic activity of CLUAP1 by colony Natini Jinawath for helpful discussions. This work was formation assay, which resulted in unaltered colony- supported in part by Research for the Future Program Grant forming activity. Hence, CLUAP1 may exert an (00L01402) from the Japan Society for the Promotion of Science.

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