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Human Cancer Biology Overexpression of Peptidyl-Prolyl Isomerase-Like 1 Is Associated with the Growth of Colon Cancer Cells Kazutaka Obama,1Tatsushi Kato,3 Suguru Hasegawa,3 Seiji Satoh,3 Yusuke Nakamura,1and Yoichi Furukawa2 Abstract Purpose and Experimental Design: To discover novel therapeutic targets for colon cancers, we previously surveyed expression patterns among 23,000 genes in colon cancer tissues using a cDNA microarray. Among the genes that were up-regulated in the tumors, we selected for this study peptidyl-prolyl isomerase-like1 (PPIL1) encoding PPIL1, a cyclophilin-related protein. Results: Western blot analysis and immunohistochemical staining using PPIL1-specific antibody showed that PPIL1protein was frequently overexpressed in colon cancer cells compared with noncancerous epithelial cells of the colon mucosa. Colony formation assay showed a growth- promoting effect of wild-type PPIL1 on NIH3T3 and HEK293 cells. Consistently, transfection of short-interfering RNA specific to PPIL1 into SNUC4 and SNUC5 cells effectively reduced expres- sion of the gene and retarded growth of the colon cancer cells.We further identified two PPIL1- interacting proteins, SNW1/SKIP (SKI-binding protein) and stathmin. SNW1/SKIP is involved in the regulation of transcription and mRNA splicing, whereas stathmin is involved in stabilization of microtubules. Therefore, elevated expression of PPIL1may play an important role in proliferation of cancer cells through the control of SNW1/SKIPand/or stathmin. Conclusion: The findings reported here may offer new insight into colonic carcinogenesis and contribute to the development of new molecular strategies for treatment of human colorectal tumors. Colon cancer is one of the most common causes of cancer Peptidyl-prolyl isomerase-like 1 (PPIL1) was first identified as a death throughout the world. Molecular investigations have cyclophilin-related gene encoding a protein that shares 46.0% provided evidence that multiple genetic alterations are involved identity in amino acid sequence with human cyclophilin A (8). in colorectal tumorigenesis. Early in those studies, loss of It is expressed in heart, skeletal muscle, and liver (8). However, heterozygosity was observed frequently at loci on chromo- its biological function remains unclear. Cyclophilins and somes 1p, 2p, 3p, 5q, 8p, 11q, 17p, 18q, and 22q (1, 2). Later, FK506-binding proteins are two families of peptidyl-prolyl alterations in genes encoding K-ras, adenomatous polyposis isomerases that are extensively characterized. Cyclophilins form coli protein (APC), h-catenin (CTNNB1), p53 (TP53), and/or complexes with cyclosporin A, whereas FK506-binding proteins conductin (AXIN2) were shown to be involved in colonic bind the immunosuppressant drug FK506. A third family of carcinogenesis (3–7). In spite of intensive study at the peptidyl-prolyl isomerases includes parvulin and Pin1. These molecular level, however, the roles of a large number of genes proteins do not bind immunosuppressants but are involved that show altered expression in colorectal cancers remain in cell cycle progression and cell survival (9, 10). Pin1 is a unclear. phosphorylation-dependent peptidyl-prolyl isomerase that catalyzes the cis-trans isomerization of phosphoserine-proline and phosphothreonine-proline bonds in multiple proteins such as CDC25, RNA polymerase II, cyclin D1, p53, and Tau Authors’Affiliations: 1Laboratory of Molecular Medicine; 2Promotion of Genome- (11, 12). It interacts with phosphorylated-p53 when the latter Based Medicine Project, Human Genome Center, Institute of Medical Science,The is activated in response to DNA damage, altering the stability of University ofTokyo,Tokyo, Japan; and 3Department of Gastroenterological Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan p53 through isomerization of a motif involving a serine/ Received 3/16/05; revised 10/7/05; accepted 10/10/05. threonine residue followed by a proline (Ser/Thr-Pro; Grant support: Research for the Future Program grant 00L01402 from the Japan refs. 10, 13). All of these peptidyl-prolyl isomerase families Society for the Promotion of Science. carry out cis-trans isomerization of the peptide bond on the The costs of publication of this article were defrayed in part by the payment of page NH -terminal side of Pro residues, forming a tight bend in the charges. This article must therefore be hereby marked advertisement in accordance 2 with 18 U.S.C. Section 1734 solely to indicate this fact. polypeptide backbone that substantially changes the confor- Note: K. Obama and T. Kato contributed equally to this work. mation of the protein. Through this isomerization and Requests for reprints: Yoichi Furukawa, Promotion of Genome-Based Medicine consequent conformational change, peptidyl-prolyl isomerases Project, Human Genome Center, Institute of Medical Science, The University of alter the functions of their target proteins (14). Tokyo, 4-6-1 Shirokanedai, Minato-ku, 108-8639 Tokyo, Japan. Phone: 81-3- 5449-5373; Fax: 81-3-5449-5124; E-mail: [email protected]. To discover target molecules for development of novel F 2006 American Association for Cancer Research. diagnostic strategies and/or therapeutic drugs, we previously doi: 10.1158/1078-0432.CCR-05-0588 carried out a genome-wide analysis of gene expression in Clin Cancer Res 2006;12(1) January 1, 2006 70 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2006 American Association for Cancer Research. PPIL1 as a Novel MolecularTarget for Colon Cancer human colon cancer tissues by means of cDNA microarray siB; and 5V-TCCCCCAGCTTCTAGATGACATATTCAAGAGATATGT- consisting of 23,040 genes (15). In this report, we show that CATCTAGAAGCTGG-3V and 5V-AAAACCAGCTTCTAGATGACA- PPIL1, a gene that is frequently up-regulated in colon cancers, TATCTCTTGAATATGTCATCTAGAAGCTGG-3V for siC. psiH1BX-PPIL1 promotes growth of cancer cells. In addition, we document or psiH1BX-EGFP (17) plasmids were transfected into SNUC4 and SNUC5 cells, which constitutionally express abundant PPIL1. Silencing interactions between PPIL1 and two known proteins, SNW1/ of PPIL1 by the plasmids was examined by semiquantitative reverse SKIP and stathmin, in vitro and in vivo. These data may not only transcription-PCR 48 hours after transfection. Cells transfected with lead to a more profound understanding of colorectal carcino- the plasmids were cultured in the presence of appropriate concentra- genesis but also provide clues for the development of novel tion of geneticin (G418) for 10 days, when viability of transfected cells therapeutic strategies. was examined by cell counting kit according to the protocol of the supplier (Dojindo Laboratories, Kumamoto, Japan). We additionally analyzed sub-G1 and G0-G1,S,andG2-M populations in cells Materials and Methods transfected with psiH1BX-PPIL1 or psiH1BX-EGFP using flow cytom- eter according to the recommendations of the manufacturer (Becton Cell lines and tissue specimens. Human colon cancer cell lines Dickinson, San Jose, CA). SNUC4 and SNUC5 were obtained from the Korean cell line bank. Bacterial two-hybrid experiment. A bacterial two-hybrid system HEK293 (human embryonic kidney), COS-7 (monkey kidney), and (Stratagene) was done with the BacterioMatch Two-Hybrid System NIH3T3 (mouse fibroblasts) cells were purchased from the American according to the protocols of the manufacturer. We cloned the entire Type Culture Collection (Rockville, MD). All tumor tissues and coding sequence of PPIL1 into the BamHl-Xhol site of pBT vector as a corresponding nontumorous mucosae were excised with informed bait and screened a human fetal-brain cDNA library (Stratagene). consent from 79 patients who underwent colectomy or endoscopic Immunoprecipitation assay. The entire coding region of the polypectomy at the Department of Surgery, Tokyo Hitachi Hospital. human stathmin gene (STMN1) and SNW1/SKIP was amplified by Real-time PCR (TaqMan) assay. Extraction of RNA, preparation of reverse transcription-PCR using primers as follows: 5V-ATTAGAT- cDNA, and real-time PCR were done as described previously (16). The CTTCACCATGGCTTCTGATATCC-3V (forward) and 5V-AATGGT- h-actin gene (ACTB) served as a quantitative control. The sequences ACCTTAGTCAGCTTCAGTCTCGTCAGC-3V(reverse) for stathmin, and of primers and probes are as follows: PPIL1 forward primer, 5V-TGGGAATTCCGGAAGAAGATGGCGCTCACCAGC-3V (forward) and 5V-TGACCCAACAGGGACAGGTC-3V and reverse, 5V-GTTCATCTT- 5V-GTGCCTCGAGCTTCCTCCTCTTCTTGCCTTCATGC-3V (reverse) for CAAACTGTTTGCCAT-3V; probe, 5V-FAM-AGGTGGTGCATCTAT-MGB- SNW1/SKIP. The PCR products were cloned into pCMV-HA or 3V; ACTB forward primer, 5V-GGCACCCAGCACAATGAAG-3V and pcDNA3.1/Myc-His (Invitrogen), respectively. COS-7 cells transfected reverse, 5V-ACACGGAGTACTTGCGCTCA-3V; probe, 5V-FAM-TCAAGAT- with pFLAG-PPIL1 (expressing FLAG-tagged PPIL1), pCMVHA-STMN1 CATTGCTCCTC-MGB-3V. (expressing HA-tagged stathmin), pcDNA-SNW1/SKIP-myc (expressing Immunoblot analysis and immunohistochemical staining using poly- myc-tagged SNW1/SKIP), or a combination of the plasmids were clonal antibody against PPIL1. The polyclonal antibody to PPIL1 was washed with PBS and lysed in TNE buffer. Immunoprecipitation and purified from sera of immunized rabbit with recombinant glutathione immunoblotting were carried out using anti-HA (3F10; Roche, S-transferase–PPIL1 protein prepared in Escherichia coli. Immunohis- Mannheim, Germany), anti-myc (9E10: Santa Cruz Biotechnologies, tochemical staining was carried out using affinity-purified
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  • Early Growth Response 1 Regulates Hematopoietic Support and Proliferation in Human Primary Bone Marrow Stromal Cells

    Early Growth Response 1 Regulates Hematopoietic Support and Proliferation in Human Primary Bone Marrow Stromal Cells

    Hematopoiesis SUPPLEMENTARY APPENDIX Early growth response 1 regulates hematopoietic support and proliferation in human primary bone marrow stromal cells Hongzhe Li, 1,2 Hooi-Ching Lim, 1,2 Dimitra Zacharaki, 1,2 Xiaojie Xian, 2,3 Keane J.G. Kenswil, 4 Sandro Bräunig, 1,2 Marc H.G.P. Raaijmakers, 4 Niels-Bjarne Woods, 2,3 Jenny Hansson, 1,2 and Stefan Scheding 1,2,5 1Division of Molecular Hematology, Department of Laboratory Medicine, Lund University, Lund, Sweden; 2Lund Stem Cell Center, Depart - ment of Laboratory Medicine, Lund University, Lund, Sweden; 3Division of Molecular Medicine and Gene Therapy, Department of Labora - tory Medicine, Lund University, Lund, Sweden; 4Department of Hematology, Erasmus MC Cancer Institute, Rotterdam, the Netherlands and 5Department of Hematology, Skåne University Hospital Lund, Skåne, Sweden ©2020 Ferrata Storti Foundation. This is an open-access paper. doi:10.3324/haematol. 2019.216648 Received: January 14, 2019. Accepted: July 19, 2019. Pre-published: August 1, 2019. Correspondence: STEFAN SCHEDING - [email protected] Li et al.: Supplemental data 1. Supplemental Materials and Methods BM-MNC isolation Bone marrow mononuclear cells (BM-MNC) from BM aspiration samples were isolated by density gradient centrifugation (LSM 1077 Lymphocyte, PAA, Pasching, Austria) either with or without prior incubation with RosetteSep Human Mesenchymal Stem Cell Enrichment Cocktail (STEMCELL Technologies, Vancouver, Canada) for lineage depletion (CD3, CD14, CD19, CD38, CD66b, glycophorin A). BM-MNCs from fetal long bones and adult hip bones were isolated as reported previously 1 by gently crushing bones (femora, tibiae, fibulae, humeri, radii and ulna) in PBS+0.5% FCS subsequent passing of the cell suspension through a 40-µm filter.