ANTICANCER RESEARCH 30: 1573-1578 (2010) Generation of an Antibody against the Protein Phosphatase 1 Inhibitor KEPI and Characterization of the Epitope KATJANA DASKALOW1,2*, PRISCA BOISGUERIN3, BURKHARD JANDRIG2, FRANK K.H. VAN LANDEGHEM5, RUDOLF VOLKMER3, BURKHARD MICHEEL1 and JÖRG A. SCHENK1,4 1Institute of Biochemistry and Biology, University of Potsdam, Golm, Germany; 2Max-Delbrück-Center for Molecular Medicine (MDC), Berlin, Germany; 3Institute of Medical Immunology, Charité - Universitätsmedizin, Berlin, Germany; 4UP Transfer GmbH, Hybrotec, Potsdam, Germany; 5Institute of Neuropathology, University of Bonn Medical Center, Bonn, Germany Abstract. A monoclonal antibody against the potential heart and skeletal muscle (1). The gene of human KEPI tumor suppressor kinase-enhanced protein phosphatase 1 consists of four exons and is located on chromosome 6q24- (PP1) inhibitor KEPI (PPP1R14C) was generated and q25. KEPI expression is regulated by morphine, and characterized. Human KEPI was expressed in Escherichia phosphorylated KEPI inhibits PP1, which modulates the coli and used to immunize Balb/c mice. Using hybridoma physiological activities of several other proteins (1). technology, one clone, G18AF8, was isolated producing Recently, a deletion in this region in a patient was found to antibodies which bound specifically to the KEPI protein in cause growth failure, cardiac septal defect, thin upper lip and ELISA, immunoblotting and flow cytometry. The antibody asymmetric dysmorphic ears (2). A frequent loss of was also successfully applied to stain KEPI protein in heterozygosity (LOH) in 6q23-q25 in breast and other types paraffin sections of human brain. The epitope was mapped of cancer suggested the presence of genes involved in tumor using peptide array technology and confirmed as development. Microcell-mediated chromosome transfer GARVFFQSPR. This corresponds to the N-terminal region studies showed that the introduction of a normal human of KEPI. Amino acid substitution analysis revealed that two chromosome 6 fragment encompassing the region 6q23.3- residues, F and Q, are essential for binding. Affinity of q25 can suppress the neoplastic phenotype of breast cancer binding was determined by competitive ELISA as 1 μM. In cell lines (3). In microarray studies on melanoma cell lines, Western blot assays testing G18AF8 antibody on brain the KEPI gene was found to be highly methylated and samples of several species, reactivity with hamster, rat and therefore down-regulated compared to normal cells (4). chicken samples was found, suggesting a broad homology of Expression studies of KEPI in breast cancer cell lines, breast this KEPI epitope in vertebrates. This antibody could be used tumors, and metastases by reverse transcriptase-polymerase in expression studies at the protein level e.g. in tumor tissues. chain reaction (RT-PCR) showed a reduced or complete loss of KEPI expression (5). Therefore, expression studies on the The human kinase-enhanced protein phosphatase 1 (PP1) protein level would help to identify KEPI as a tumor inhibitor KEPI, also known as protein phosphatase 1 suppressor protein. We describe here the generation of a regulatory subunit 14C (PPP1R14C) is expressed in brain, KEPI-specific monoclonal antibody, the characterization of the epitope bound by this antibody and the use of the antibody to identify the KEPI protein in tissue samples. *Present address: AJ Innuscreen GmbH, Berlin, Germany. Materials and Methods Correspondence to: Jörg A. Schenk, UP Transfer GmbH, Hybrotec, Cloning of KEPI cDNA, protein production and purification. The c/o Fraunhofer Institute for Biomedical Engineering, Am cDNA of kinase C-enhanced PP1 inhibitor (KEPI) (GenBank Mühlenberg 13, D-14476 Potsdam, OT Golm, Germany. Tel: +49 AL096708) was cloned in pET-30 Xa/LIC and pET-32 Xa/LIC 33158187231, Fax: +49 3319771143, e-mail: joerg.schenk@up- vectors (Novagen, Schwalbach, Germany) according to the transfer.de manufacturer’s protocol. Escherichia coli BL21-Gold(DE3)pLysS (Stratagene, Amsterdam, the Netherlands) were transformed with Key Words: Hybridoma, monoclonal, KEPI, peptide array, the generated plasmids by heat shock. For induction of KEPI fusion substitutional analysis. protein, an overnight culture was grown (37˚C, 250 rpm) in LB 0250-7005/2010 $2.00+.40 1573 ANTICANCER RESEARCH 30: 1573-1578 (2010) media containing appropriate antibiotic to an optical density according to standard protocols. Stable cell lines were generated by OD680nm of 0.4-0.6 before induction with isopropyl-thio-beta-D- selection in 600 μg/ml G418 containing medium for approximately 2 galactopyranoside (IPTG) to a final concentration of 100 μM. weeks. For protein detection in Western blot, the cells were washed Maximum concentration of pET30 and pET32 fusion protein was twice in phosphate-buffered saline (PBS) and lysed in 300 mM NaCl, obtained after 16 h and 4 h, respectively. The osmotic shock fraction 50 mM Tris-HCl pH 7.6, 0.5% Triton X-100 and 1 mM of whole cell extract (OS) was isolated from pelleted bacteria by phenylmethylsulfonylfluoride (PMSF). FACS analysis was performed adding 5 mM MgSO4 (1/5 volume of the culture) and extracting the on a FACScalibur (Becton-Dickinson, Heidelberg, Germany). Cells clarified supernatant by centrifugation for at least 30 min. OS of were incubated with 4% formaldehyde for 10 min and intracellular non-transformed E. coli were used as control. The KEPI protein was staining was performed with 10 μg/ml G18AF8 or murine anti-GFP purified using TALONspin columns (BD Biosciences, Heidelberg, (1 μg/ml, Roche Diagnostics, Penzberg, Germany), followed by Germany) according to the manufacturer’s instructions. For biotinylated goat anti-mouse-Ig (Dianova, Hamburg, Germany) and mammalian expression of KEPI and KEPI-GFP fusion proteins, streptavidin-phycoerythrin (Pharmingen Biosciences, San Diego, CA, plasmids pEGFP-C2 (Clontech, Heidelberg, Germany) or pcDNA3.1 USA). All reagents were diluted in 5% (w/v) saponin. (Invitrogen, Heidelberg, Germany) were used. Immunohistochemical and immunofluorescence examinations. The Generation and characterization of monoclonal antibodies. study was performed with approval of the Institutional Review Board Hybridoma technology was applied for the generation of murine of the Charité, in accordance with Berlin law. Tissue samples were monoclonal KEPI-specific antibodies. Female Balb/c mice were obtained from ten female patients with an intracranial metastasis of immunized three times with unpurified heat-denatured KEPI pET30 a known ductal mamma carcinoma. They were fixed in buffered 4% fusion protein. The booster immunization was performed with formaldehyde overnight and paraffin embedded. Four-μm paraffin purified KEPI pET32 fusion protein. Five days later, electrofusion of sections were used for hematoxylin and eosin staining, alcian blue- spleen cells with myeloma cells (Sp2/0, ATCC CRL-1581) in the PAS reaction and immunohistochemical staining with antibodies presence of polyethylene glycol 8000 was performed as described against cytokeratins (Lu5; DAKO, Hamburg, Germany) and Ki-67 elsewhere (6). Selected hybrids were cultivated in RPMI-1640 (MIB-1; DAKO) to identify the tumor region. According to a medium (containing 10% (v/v) fetal calf serum (FCS), 2 mM standard protocol for immunohistochemistry, sections were dewaxed glutamine, 50 μM β-mercaptoethanol) and subcloned by limiting and subjected to microwave pretreatment (3×4 min in 10 mM citrate dilution on mouse peritoneal feeder cells. Culture supernatants of buffer, pH 6.5, 600 W; Bosch, Berlin, Germany). Endogenous clones and subclones were tested in an enzyme immunoassay peroxidase activity was blocked by incubating sections with 0.6% (ELISA) for antigen binding with KEPI pET32 fusion protein bound hydrogen peroxide for 15 min at room temperature. Subsequently, to the solid phase. Class and subclass of monoclonal antibodies were sections were incubated with normal goat serum diluted 1 to 20 in determined as described elsewhere (7). The antibodies were purified PBS for 20 min and then incubated with the antibody against KEPI from cell culture supernatant by affinity chromatography using overnight (diluted 1 to 100, in 10% FCS in PBS). Goat anti-mouse Protein A columns (GE Healthcare, Munich, Germany). IgG labelled with horseradish peroxidase (diluted 1 to 100, 90 min; DAKO) was applied and after detection with Vectastain ABC Elite SDS-PAGE and Western blot. Samples were separated in a gradient kit (Vector Laboratories, Wertheim, Germany) immunopositive cells (12.5 to 20%) SDS-polyacrylamide gel according to Laemmli (8). were visualized with 3,3’-diaminobenzidine (Sigma, Germany). Proteins were stained in 0.25% Coomassie Brilliant Blue R-250 Nuclei were counterstained with hematoxylin. For negative control, (Serva, Heidelberg, Germany) and destained. For Western blot, the the primary antibody was omitted. separated proteins were blotted on nitrocellulose membranes (PROTRAN NC BA3; Schleicher & Schuell, Dassel, Germany) Peptide array synthesis and binding studies. Cellulose membrane- using a semi-dry blotting apparatus. After staining with Ponceau S, bound peptides were automatically prepared according to standard the membrane was blocked in Western blocking reagent (Roche, SPOT synthesis protocols using a MultiPep synthesizer (Intavis AG, Mannheim, Germany) (diluted in PBS, pH 7.4, 0.1% Tween 20), Cologne, Germany) as described in detail previously (11). For incubated for 1 h in 0.5 μg/ml purified antibody G18AF8 and generation