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1 IP Characterization of a Monoclonal Antibody pecific to Human iah-1 Interacting Protein un Young Yoon 1, Joung Hyuck Joo 1, Joo Heon Kim 2, Ho Bum Kang 1, Jin ook Kim 1, Younghee Lee 1, Do Hwan Kwon 1, Chang Nam Kim 3, In eong Choe 1 and Jae Wha Kim 1 1 Laboratory of Cell Biology, Korea Research Institute of Bioscience and Biotechnology (KRIBB), P.O. Box 115, Daejeon, Republic of Korea, 2 Department of Pathology, Eulji University chool of Medicine, Daejeon, Republic of Korea, 3 Department of urgery, Eulji University chool of Medicine, Daejeon, Republic of Korea ABTRACT Background: A human orthologue of mouse 100A6-binding protein (CacyBP), iah- 1-interacting protein (IP) had been shown to be a component of novel ubiquitinylation pathway regulating -catenin degradation. The role of the protein seems to be important in cell proliferation and cancer evolution but the expression pattern of IP in actively dividing cancer tissues has not been known. For the elucidation of the role of IP protein in carcinogenesis, it is essential to produce monoclonal antibodies specific to the protein. Methods: cdna sequence coding for ORF region of human IP gene was amplified and cloned into an expression vector to produce His-tag fusion protein. Recombinant IP protein and monoclonal antibody to the protein were produced. The N-terminal specificity of anti-ip monoclonal antibody was conformed by immunoblot analysis and enzyme linked immunosorbent assay (ELIA). To study the relation between IP and colon carcinogenesis, the presence of IP protein in colon carcinoma tissues was visualized by immunostaining using the monoclonal antibody produced in this study. Results: His-tag-IP (NIP) recombinant protein was produced and purified. A monoclonal antibody (Korea patent pending; # ) to the protein was produced and employed to analyze the expression pattern of IP in colon carcinoma tissues. Conclusion: The data suggested that anti-ip monoclonal antibody produced here was valuable for the diagnosis of colon carcinoma and elucidation of the mechanism of colon carcinogenesis. (Immune Network 2004;4(1):23-30) Key Words: iah-1 interacting protein (IP), 100A6, monoclonal antibody, colorectal cancer Tel:, Fax: choerncbg@kribb.re.kr Immune Network 23

2 24 un Young Yoon, et al.

3 Monoclonal Antibody pecific to iah-1 Interacting Protein 25

4 26 un Young Yoon, et al.

5 Monoclonal Antibody pecific to iah-1 Interacting Protein 27 kda IPTG induction M NIP Con CIP CIP NIP Figure 1. Production of IP recombinant proteins. cdna fragments of N-terminal (amino acid 1-60) and C-terminal (amino acid ) of IP were subcloned into pet28a plasmid vector and named as pet28a-nip for a vector containing N-terminal sequence and pet28a-cip for the one containing C-terminal. Each vector was transformed into E. coli (BL21, DE3) and transformed cells were treated with 1 mm IPTG to induce the production of the recombinant proteins. Cell lysates of the treated cells were loaded onto 12% D-PAGE gel, runned and stained with Coomassie blue. Lane 1 and 2 were lysates of pet28a-nip transformants, lane 3 and 4 were lysates of control pet28a transformants, and lane 5 and 6 were lysates of pet28a-cip transformants. Lane 2, 4, and 6 were the lysates of transformed cells treated with 1 mm IPTG for 3 h. Location of NIP and CIP were marked by arrows. Lane M was size markers. A kda Marker E1 E2 E3 E4 E5 E6 NIP Recombinant protein B kda Marker Purified human NIP recombinant protein Purified human CIP recombinant protein CIP Recombinant protein Figure 2. Purification of IP recombinant proteins. His-tag-IP recombinant proteins were purified by NTA-agarose affinity column and analyzed by 12% D-PAGE gel. pet28a-nip or pet28a-cip transformed cells were treated with 1 mm IPTG for 3 h and recombinant proteins were purified using the NTA- Agarose affinity column. The proteins were eluted stepwise from the column by buffers containing various concentrations of immidazole (E1-E6). Each elution product was loaded and separated on PAGE gel (A). Locations of purified recombinant NIP, upper panel, and CIP, lower panel, were marked by arrows. The proteins were eluted and concentrated by electro-elution method and loaded on 12% D-PAGE again to confirm the purity (B). Lane M was size markers.

6 28 un Young Yoon, et al. A Absorbance at 490nm 0.40 Anti-human IP 1G3 mab 0.35 Normal mouse serum Anti-human IL-5 mab Dilution factor B Immunoglobulin Isotype (Ig): M A G3 G2b G2a G1 Anti-human IP mab Isotyping strip Figure 3. Characterization of monoclonal antibody (mab) specific to IP recombinant protein. Titration curve of anti-ip mab was determined by ELIA method. Each micro-titer plate well was coated with recombinant IP and reacted with diluted anti-ip antibody, Anti-human IL-5 antibody, and normal mouse serum were used as controls (A). The isotype of the monoclonal antibody produced by IP60 IG3 hybridoma cells was IgG 1 (B). Absorbance at 490nm Histag-NIP recombinant protein Histag-CIP recombinant protein Histag-CIL5 recombinant protein Dilution Factor (human IP hybridoma culture sup) Figure 4. Binding specificity of the anti-ip mab. The anti-ip mab did not show any reactivity with His-tag-CIP and Histag-IL-5 proteins. It only reacted with His-tag-NIP recombinant protein containing N-terminal region of IP. Each micro- titer plate well was coated with a relevant recombinant protein and reacted with diluted culture supernatant of IP60 IG3 hybridoma cells.

Monoclonal Antibody pecific to iah-1 Interacting Protein 29 A His tag-nip His tag-cip α-t7 tag His tag-nip His tag-cip α-ip1g3 His tag-cip His tag-nip B 35 29 21 6.

7 Monoclonal Antibody pecific to iah-1 Interacting Protein 29 A His tag-nip His tag-cip α-t7 tag His tag-nip His tag-cip α-ip1g3 His tag-cip His tag-nip B His tag- 100A6 HepG2 Hep3B Chang liver IP Figure 5. Binding specificity of the anti-ip mab determined by Western bolt analysis. Recombinant NIP and CIP were detected by T7-tag antibody (Novagen) which could recognize proteins marked by His-tag but only NIP was detected by the antibody produced by IP60 IG3 hybridoma (A). The reactivity of the antibody to IP native protein of 30 kda produced in the cells of HepG2, Hep3B and Chang was also confirmed (B). His-tag-100A6 was used as negative control. #14 #16 #17 #18 #19 #30 #31 #32 #33 #34 #7 #12 #20 #21 #22 #23 #24 #25 #26 #27 IP GAPDH IP A NC NC #37 #38 #39 #40 #41 #42 #43 #44 #45 IP GAPDH GAPDH B CC NC 50µm Figure 6. Expression of IP in human colon carcinoma tissues. RT-PCR method was employed to reveal the comparative expression levels of IP mrna in 29 normal and colorectal carcinoma tissue samples. The expression level of reference gene (GAPDH) was also determined to compensate the variation in the amount of loaded RNAs from each sample. N; normal tissue, C; colon carcinoma tissue, numeric; patient number. CC 50µm Figure 7. Expression of IP in human colon carcinoma immunohistochemical staining using monoclonal antibody to IP produced in this study. The positive immunoreactivity of IP was revealed in the cytoplasm of cancer when compared to adjacent normal cells. NC; normal colon, ; stroma, CC; colon cancer.

8 30 un Young Yoon, et al. 1. Matsuzawa I, Reed JC: iah-1, IP, and Ebi collaborate in a novel pathway for beta-catenin degradation linked to p53 responses. Mol Cell 7; , Donato R: Functional roles of 100 proteins, calcium-binding proteins of the EF-hand type. Biochim Biophys Acta 1450; , Donato R: 100: a multigenic family of calcium-modulated proteins of the EF-hand type with intracellular and extracellular functional roles. Int J Biochem Cell Biol 33; , Jastrzebska B, Filipek A, Nowicka D, Kaczmarek L, Kuznicki J: Calcyclin (100A6) binding protein (CacyBP) is highly expressed in brain neurons. J Histochem Cytochem 48; , Zimmer DB, Cornwall EH, Landar A, ong W: The 100 protein family: history, function, and expression. Brain Res Bull 37; , Filipek A, Wojda U: p30, a novel protein target of mouse calcyclin (100A6). Biochem J 320 (Pt 2); , Filipek A, Jastrzebska B, Nowotny M, Kwiatkowska K, Hetman M, urmacz L, Wyroba E, Kuznicki J: Ca2+ -dependent translocation of the calcyclin-binding protein in neurons and neuroblastoma NB-2a cells. J Biol Chem 277; , Xia ZB, Dai M, Magoulas C, Broxmeyer HE, Lu L: Differentially expressed genes during in vitro differentiation of murine embryonic stem cells transduced with a human erythropoietin receptor cdna. J Hematother tem Cell Res 9; , Pircher TJ, Geiger JN, Zhang D, Miller CP, Gaines P, Wojchowski DM: Integrative signaling by minimal erythropoietin receptor forms and c-kit. J Biol Chem 276; , Nowotny M, Bhattacharya, Filipek A, Krezel AM, Chazin W, Kuznicki J: Characterization of the interaction of calcyclin (100A6) and calcyclin-binding protein. J Biol Chem 275; , Filipek A, Jastrzebska B, Nowotny M, Kuznicki J: CacyBP/ IP, a calcyclin and iah-1-interacting protein, binds EF-hand proteins of the 100 family. J Biol Chem, 277; , Matsuzawa, Li C, Ni CZ, Takayama, Reed JC, Ely KR: tructural analysis of iah1 and its interactions with iahinteracting protein (IP). J Biol Chem 278; , Boni R, Heizmann CW, Doguoglu A, Ilg EC, chafer BW, Dummer R, Burg G: Ca (2+) -binding proteins 100A6 and 100B in primary cutaneous melanoma. J Cutan Pathol 24; 76-80, Breen EC, Tang K: Calcyclin (100A6) regulates pulmonary fibroblast proliferation, morphology, and cytoskeletal organization in vitro. J Cell Biochem 88; , Maelandsmo GM, Florenes VA, Mellingsaeter T, Hovig E, Kerbel R, Fodstad O: Differential expression patterns of 100A2, 100A4 and 100A6 during progression of human malignant melanoma. Int J Cancer 74; , Yang Q, O Hanlon D, Heizmann CW, Marks A: Demonstration of heterodimer formation between 100B and 10-0A6 in the yeast two-hybrid system and human melanoma. Exp Cell Res 246; , Komatsu K, Murata K, Kameyama M, Ayaki M, Mukai M, Ishiguro, Miyoshi J, Tatsuta M, Inoue M, Nakamura H: Expression of 100A6 and 100A4 in matched samples of human colorectal mucosa, primary colorectal adenocarcinomas and liver metastases. Oncology 63; , Komatsu K, Kobune-Fujiwara Y, Andoh A, Ishiguro, Hunai H, uzuki N, Kameyama M, Murata K, Miyoshi J, Akedo H, Tatsuta M, Nakamura H: Increased expression of 100A6 at the invading fronts of the primary lesion and liver metastasis in patients with colorectal adenocarcinoma. Br J Cancer 83; , Komatsu K, Andoh A, Ishiguro, uzuki N, Hunai H, Kobune-Fujiwara Y, Kameyama M, Miyoshi J, Akedo H, Nakamura H: Increased expression of 100A6 (Calcyclin), a calcium-binding protein of the 100 family, in human colorectal adenocarcinomas. Clin Cancer Res 6; , 2000

Can032.hwp

Can032.hwp Chromosomal Alterations in Hepatocellular Carcinoma Cell Lines Detected by Comparative Genomic Hybridization Sang Jin Park 1, Mahn Joon Ha, Ph.D. 1, Hugh Chul Kim, M.D. 2 and Hyon Ju Kim, M.D. 1 1 Laboratory