Fragments of Human Oncoprotein MDM2 Reveal Variable Distribution within and on Cultivated Human Hepatoma Cells

Biochemical and Biophysical Research Communications 283, 956 –963 (2001) doi:10.1006/bbrc.2001.4868, available online at http://www.idealibrary.com on

Fragments of Human Oncoprotein MDM2 Reveal Variable Distribution within and on Cultivated Human Hepatoma Cells Thilo Schlott,* ,1 Jens-Gerd Scharf,† Afsaneh Soruri,‡ Afshin Fayyazi,§ Christian Griesinger, ¶ Christian Albrecht, ¶ Helmut Eiffert,储 and Manfred Droese* *Department of Cytopathology, †Division of Gastroenterology and Endocrinology, ‡Division of Immunology, §Department of Pathology, and 储Division of Medical Microbiology, Georg-August-University, Goettingen, Germany; and ¶ Institute of Organic Chemistry, Johann-Wolfgang-von-Goethe-University, Frankfurt, Germany

Received March 26, 2001

Human oncoprotein MDM2 reveals a MHC class I binding motif HMDM441 characterizing MDM2 as a potential tumor antigen. To analyze the distribution of MDM2 proteins containing this motif in liver cancer cells we produced rabbit anti-HMDM441 serum. The novel antibodies bound to an MDM2 fragment of approximately 55 kDa which lacked the N-terminal region and was present in lysate and supernatant of a human hepatoma cell line overexpressing normal 90kDa MDM2. The 55-kDa fragment was detected in the cytoplasm and nucleoli and at the nuclear envelope of hepatoma cells, whereas normal hepatocytes were negative. Double-fluorescence labeling indicated that the MDM2 fragments and MHC class I molecules were coexpressed on the surface of the hepatoma cells. Further studies must clarify whether MDM2 fragments containing motif HMDM441 are novel targets of immunotherapy and immunochemical tumor diagnosis. © 2001 Academic Press

Key Words: MDM2; MHC class I; tumor antigen; confocal laser scanning.

The MDM2 oncoprotein is an essential regulator of eukaryotic cell division, apoptosis, and DNA repair. By interacting with several proteins such as tumor suppressors pRb, p53 and transcriptional factors E2F1/ DP1 and TFIID, MDM2 influences basic molecular reactions (1, 2). Abnormalities concerning MDM2 expression and MDM2 genetics have been described. MDM2 overexpression is an important tumorigenic mechanism immortalizing rat fibroblasts and transforming rat astrocytes into astrocytomes (3, 4). Over1 To whom correspondence and reprint requests should be addressed at Department of Cytopathology, Georg-August-University, Robert-Koch-Strasse 40, D-37075 Goettingen, Germany. Fax: 0049551398641. E-mail: [email protected].

0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

expression of MDM2 blocks transregulatory functions of p53, promotes degradation of bound p53, represses accumulation of mutant p53 and inhibits E2F1dependent apoptosis (5– 8). In different tumors, MDM2 overexpression is associated with genetic changes such as gene amplification (9), point mutations (10, 11), and MDM2 RNA splicing (12). Besides genetic analysis, the MDM2 protein was screened immunologically for MHC class binding pattern. Two MDM2 peptides, MDM100 and MDM441, were tested in cultivated rat cells and found to induce allo-restricted cytotoxic T lymphocytes (13). Further in vitro assay proved that cytotoxic T cells activated by MDM100 could specifically kill MHC class I-expressing mouse cancer cells (14). In the present study, polyclonal antibodies were produced against human MDM2 peptide containing a potential MHC class I binding pattern and used for investigating the distribution of MDM2 proteins in human hepatoma cell line and hepatocytes. Furthermore, the cell line was analyzed for surface coexpression of MDM2 protein fragment containing the HMDM441 sequence and of MHC class I molecules. MATERIALS AND METHODS Tumor samples. Human hepatoma cell lines (SK-Hep1, Hep-G2, Hep-3B, PLC) were cultivated as previously described (15). Human hepatocytes cultivated in collagen matrix (16) and normal human hepatocytes obtained from TEBU GmbH (Frankfurt, Germany) served as controls. Synthesis of peptide HMDM441 and immunization. Peptide HMDM441, which correlated to amino acid position 441 to 452 in the published MDM2 sequence (17), was synthesized on a 9050 Plus PepSynthesizer (Milligen, Milford, MA) using Fmoc/tBu protection and Dic/HOBt activation. The synthesized peptide was deprotected and cleaved from the solid support upon treatment with Reagent K (82.5% trifluoroacetic acid, 5% thioanisol, 5% water, 5% phenol, and 2.5% ethandithiol). The peptide was purified by RP-HPLC using standard AcCN/H 2O gradients (99% purity). Identification of syn-

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thetic peptide was carried out using MALDI-TOF-MS. N-terminal cysteine residue served to couple the peptide to carrier KLH-SMCC. Immunization of rabbits was performed by EUROGENTEC (Herstal, Belgium). In each case, 20 ␮g of coupled peptide was used for primary injection and boostering. Preimmune serum was supplied as a negative control. Immunocytochemistry. Immunocytochemistry was performed with serum HMDM441 and antibody SMP 14 (Santa Cruz Technologies, Santa Cruz, CA) as described previously (10). SMP14 is specific for the epitope corresponding to amino acids 154 –167 of MDM2 of human origin. Hepatoma cell line SK-Hep1 was grown on glass slides and fixed in ice-cold methanol (100%, ⫺20°C) for 5 min and in ice-cold acetone (100%, ⫺20°C) for 10 s. The cells were washed three times with 1⫻ TRIS buffer. Primary antibody SMP14 (diluted 1:500) or serum HMDM441 diluted 1:50 in human serum (drug-free serum, BIO-RAD, Germany) was pipetted onto the cells. The SK-Hep1 cells were incubated at RT for 1 h, and washed three times with 1⫻ Tris buffer. Rabbit anti-mouse antibody (DAKO, Denmark) diluted 1:20 in human serum or swine anti-rabbit antibody (DAKO) was added to the samples and incubated for 1h at RT. Cells were washed three times with 1⫻ Tris buffer. APAAP complex (APAAP, mouse, monoclonal, DAKO) diluted 1:20 in human serum was pipetted onto the hepatocytes. After incubating the cells with APAAP complex for 1 h at RT, reaction product was detected (18). To block potential endogenous alkaline phosphatase activity the substrate solution was saturated with levamisole (Sigma, Munich, Germany). Dot blot. For dot blot analysis, 1 ␮g peptide HMDM441 was diluted 1:2, 1:5, 1:10, 1:20, 1:50, and 1:100 in PBS, blotted onto nitrocellulose membrane (Schleicher and Schuell, Dassel, Germany), and saturated with BSA (5%) for 1 h. Membrane was incubated with preimmune serum and postimmune serum from rabbit (1:100 diluted in PBS) for 2 h. After washing three times in TBS, incubation with anti-rabbit immunoglobulin coupled with HRP (DAKO) and diluted 1:500 in TBS was performed for 1 h. After three washings in TBS, secondary antibody was detected with DAB liquid substrate system (Sigma). Immunoblot with serum HMDM441. Cells pelleted by centrifugation were washed three times in PBS (pH 7.4) and heated in 100 ␮l cracking buffer (Load 1, Roth, Karlsruhe, Germany) to 95°C for 3 min. Lysate and supernatant were separated by denaturating standard polyacrylamide gel electrophoresis (SDS–PAGE). Electroblotting was performed applying polyvinylidene difluoride membrane (PVDF; Milipore, Bedford, MA). Immunoblotting was performed as described (15). For detection of serum HMDM441, secondary swine anti-rabbit antibody (P0217, HRP conjugate, DAKO) was used. Secondary antibody was detected by applying the ECL detection kit according to the manufacturer’s instructions (RPN2106, Amersham, Braunschweig, Germany). N-terminal protein sequencing. Following immunoblotting, Coomassie-stained bands correlating with the labeled bands were excised from the PVDF blotting membrane and sequenced by Edman degradation using the Applied Biosystems protein sequencer Model 470A. Confocal laser-scanning microscopy. For immunofluorescence microscopy cell cultures were grown on sterile coverslips in petri dishes and analyzed by confocal laser-scanning microscopy. All samples were analyzed on a LEICA TCS confocal laser-scanning system with microscope DM IRB. In detail, scanning modus x–y axis was used to put horizontal sections in the cells to study the distribution of fluorescence signals. Two different labeling techniques were used. For single fluorescence labeling of cells with serum HMDM441, coverslips were placed into cold methanol (100%, ⫺20°C) for 5 min and into cold acetone (100%, ⫺20°C) for 10 s. Afterward, cells were immediately put into a glass container with PBS (pH 7.2) so that the slides were not allowed to dry. Cells were washed three times in PBS for several minutes. Excess PBS was removed by touching with dry

filter paper. The coverslips were placed on parafilm and then put into wet chamber. Unspecific binding was blocked by incubation in rabbit IgG (DAKO) for 30 min. Serum HMDM441 was diluted 1:20 in rabbit serum and applied onto the coverslips. Cells were incubated for 12 h and washed three times in PBS for 5 min on a lab shaker. Afterwards secondary swine antibody anti rabbit labeled with fluorescence dye FITC (F205, DAKO) was diluted in rabbit serum 1:10 and added to the cells. Cells were incubated for 30 min and washed three times in PBS. For mounting of the cover slips, DAKO R fluorescent mounting medium (DAKO) was used. In negative control experiments, 5 ␮g peptide HMDM441 was incubated with serum HMDM441 for 3 h before adding the antibodies. Double fluorescence labeling of hepatoma cells was performed using serum HMDM441 and MHC class I antibody. Cells were either fixed in cold formalin (4%) at ⫺20°C for 10 s or nonfixed. In each case, the cells were washed three times in PBS and incubated with rabbit serum (DAKO) to block unspecific antibody binding. Undiluted supernatant which contained primary mouse anti-human MHC class I antibody, was obtained from hybridoma culture HB95 (ATCC, U.S.A.) and then added to the cultures. After 3 h incubation with primary antibody cells were washed three times in PBS. Secondary rabbit anti-mouse antibody labeled with fluorescence dye PE (R0439, DAKO) was diluted in PBS 1:10 and pipetted onto the slides. After 2 h, samples were washed and mouse IgG (DAKO) was added for blocking unspecific binding (1:20, 20 min incubation). Serum HMDM441 was diluted 1:10 in PBS and incubated with the cells for 3 h. Secondary swine anti-rabbit antibody labeled with FITC (F205, DAKO) was diluted 1:50 and applied to the cells. Slides were incubated for 2 h and washed three times with PBS on a rocking platform. Cells were embedded in DAKO R fluorescent mounting medium (DAKO). Cells with cofluorescence were further tested by the x–z axis scanning modus to put a vertical section through the cells and to analyze whether fluorescence signals were located only on the surface of cells and not within the cells. The following negative controls were performed: (a) serum HMDM441 was blocked with peptide HMDM441 for 2 h at RT before adding the antibodies to the cells; (b) MHC class I molecules on the surface of SK Hep1 cells were blocked by incubation with monoclonal mouse anti-human HLAABC antibody (Pharmingen, U.S.A.) diluted 1: 50 in PBS for 2 h at RT before adding anti-human MHC class I antibody HB95. One microgram of antibody was used for blocking 1 ⫻ 10 5 cells; (c) serum HMDM441 was blocked with peptide HMDM441 and MHC class I antigens were blocked by monoclonal mouse anti-human HLA-ABC antibody.

RESULTS Peptide HMDM441 was synthesized containing a potential HLA-binding pattern previously described for rat MDM2 (13). Sequence alignment indicated that this pattern is also conserved in human MDM2. Polyclonal immune serum against peptide HMDM441 was produced in rabbits and tested by dot blotting. One of the rabbits contained serum reacting with peptide HMDM441 diluted 1:2 to 1:100 while preimmune serum gave a negative result (Fig. 1). Serum HMDM441 was tested with protein lysate from different human hepatoma cell lines using immunoblotting technique. Protein sequencing was then performed to identify each protein band detected by serum HMDM441. Protein bands revealing signals of unknown proteins/proteins differing from MDM2 as well as protein bands revealing block of N-terminus referred to cell lines which were excluded from further

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FIG. 1. Dot blotting with serum HMDM441. Peptide HMDM441 was diluted 1:2, 1:5, 1:10, 1:20, 1:50, and 1:100 in PBS. Five microliters of each solution was transferred onto nitrocellulose membrane and detected with rabbit postimmune serum (lanes A–F; ⫹). Rabbit preimmune serum did not react with the peptide (lanes A–F; ⫺).

localization experiments because of potential immunological cross-reactions. In lysate obtained from hepatoma cell line SK-Hep1, serum HMDM441 detected just one intense band of about 55 kDa (Fig. 2, lane A).

To compare the cell line SK-Hep1 with normal liver cells, lysate obtained from normal hepatocytes was separated by SDS–PAGE and incubated with postimmune serum. As a result of immunoblotting, serum HMDM441 gave negative results (Fig. 2, lane B). Furthermore, rabbit preimmune serum serving as negative control neither reacted with the lysate from hepatoma cell line nor with lysate obtained from normal hepatocytes (Fig. 2, lanes C and D). The 55-kDa protein band was also found in supernatant of SK-Hep1 culture (Fig. 2, lane I). N-terminal sequence analysis of the 55-kDa band present in lysate and supernatant revealed strong signals that indicated the amino acid sequence AGVSEH- (Fig. 3). Sequence alignment with EMBL data bank demonstrated that the protein represented an MDM2 fragment lacking the first 225 amino acid residues including the p53 binding site and the so called first inhibitory domain of cell division ID1 (19). Considering that the molecular mass of full-size MDM2 carrying posttranslational modifications (90 kDa) is equivalent to 491 amino acid residues, the 266-amino-acid fragment of MDM2 theoretically corre-

FIG. 2. Western immunoblotting with serum HMDM441. Cell lysates obtained from human hepatoma cell line SK-Hep1, and human hepatocytes were separated on denaturing 10% polyacrylamide gel and blotted onto nylon membrane. Lanes A and B: Reactions of postimmune serum; lanes C and D: Reactions of preimmune serum. The analysis shows that serum HMDM441 detects a 55-kDa protein in SK-Hep1 lysate (lane A). The antibodies do not react with proteins obtained from human hepatocytes (lane B). Preimmune serum neither detects protein bands in SK-Hep1 lysate (lane C) nor in human hepatocytes (lane D). Additionally, cell lysates obtained from human hepatoma cell line SK-Hep1 were separated on denaturing 10% polyacrylamide gel and blotted onto nylon membrane. The reactions of postimmune serum with supernatant of cell culture (lane I) and with whole lysate (lane II) are shown. Data suggest that the 55-kDa fragment of MDM2 is present in the medium of the cell culture. 958

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FIG. 3. Data of N-terminal sequencing of 55-kDa protein band which was identified by serum HMDM441 in the immunoblot. Correlating Coomassie-stained protein bands were cut out of the membrane and analyzed by Edman degradation. The pattern of peaks indicates the amino acid sequence A-G-V-S-E-H– which correlates with MDM2 protein lacking the first 225 amino acids.

sponds to 54.1% of the total protein. This calculation is in accordance with the results of immunoblotting analysis which had identified a protein band of 55 kDa. Immunocytochemistry was performed with antibody SMP 14 and serum HMDM441 on hepatoma cell line SK Hep1 cultivated on glass slides and on hepatocytes. The cell line showed a dense nuclear staining using antibody SMP14, indicating overexpression of MDM2 (Fig. 4A). In contrast, using serum HMDM441 a staining of nucleoli and of the cytoplasm was found (Fig. 4B). Additionally, some of the hepatoma cells showed MDM2 fragment accumulation at the nuclear envelope. In all cases, preimmune serum gave negative results. Normal human hepatocytes did not reveal any nuclear expression of MDM2 protein or MDM2 fragments and were not further analyzed in the following experiments (data not shown). These data were reaffirmed by localization experiments performed on the confocal laser-scanning microscope with high resolution. In detail, MDM2 protein fragments were present in the nucleoli and the cytoplasm of hepatoma cells (Fig. 5A). As hepatoma cells of the mono-

layer enter mitosis, they round up and become only tenuously attached. MDM2 fragments were located around the nucleus of these detachable cells (Fig. 5B). In each experiment, negative controls were performed successfully: Postimmune serum was incubated with peptide HMDM441 and used for immunocytochemistry, resulting in an absence of labeling. Furthermore, the use of rabbit preimmune serum instead of postimmune serum led to negative results (data not shown). Finally, the hepatoma cells were examined for coexpression of MDM2 proteins with HMDM441 epitope and MHC class I molecules by applying double fluorescence labeling and confocal laser-scanning microscopy. The hepatoma cells were either nonfixed or fixed. For fixation, short incubation times in neutral buffered paraformaldehyde (4%) were used to reduce permeabilization of cell membrane and to prevent antibody diffusion into the cytoplasm or the nucleus. Briefly, the data show that both types of molecules were expressed on the cell surface of fixed and nonfixed cells, as could be concluded by the intensity of the fluorescence signals. In the first channel, a fine granular labeling of MDM2 fragments was ob-

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FIG. 4. Detection of MDM2 proteins in hepatoma cells by immunocytochemistry. (A) Antibody SMP 14 detects nuclear overexpression of MDM2. (B) Following immunization with synthetic peptide HMDM441, postimmune serum reacts with the cytoplasm, the nucleoli, and the nuclear envelope of hepatoma cells. Bar: 25 ␮m.

served covering the entire cytoplasmic membrane (Figs. 6A1 and 6B1). In contrast, MHC class I molecules were arranged in spot-like clusters as observed in the second channel (Figs. 6A2 and 6B2). Overlay of both channels indicated fluorescence concentrated at some of the MHC class I clusters (Figs. 6A3 and 6B3). Especially the nonfixed tumor cells revealed a fine granular staining of particles with interspersed intense signals as is demonstrated in this study for a cluster of twisted and folded hepatoma cells (Figs. 6B1– 6B3). x–z axis scanning of these cells proved that the signals were located on the cell surface and not within the cells: A corona was detected covering the cell lumen which consisted of MHC class I signals, diffuse HMDM441 signals, and signals resulting from local coexpression (Figs. 7A–7C). In all confocal studies, control experiments were performed successfully and resulted in an absence of fluorescence. DISCUSSION Identifying antigenic targets on human tumor cells has become an exciting field of immunological research. For example, mutant tumor suppressor p53

has been shown to induce high-affinity cytotoxic T cells and thus to be suited for specific tumor immunotherapy (20). Moreover, the effects of MDM2—an important binding partner of p53— on in vitro T-cell response was investigated (13). As a result, two peptides of murine MDM2 protein, MDM100 and MDM441, were characterized as potential tumor antigens stimulating cytotoxic T cells. The MDM100 peptide was used in the mouse model for specifically destroying melanoma and lymphoma cells by cytotoxic T lymphocytes (14). These findings prompted us to select a short peptide from the human sequence containing the conserved MHC class I binding pattern of MDM441 for rabbit immunization. The first aim of the present study was to produce novel MDM2 antibodies directed against the C-terminal domain containing the HMDM441 epitope at MDM2 position 442– 454 and to test the antibodies with normal human hepatocytes and human hepatoma cell lines. In cell line SK-Hep1, serum HMDM441 reacted with human MDM2 protein fragments of about 55 kDa. Fulllength MDM2 with a molecular mass of 90 kDa was not detected in hepatoma lysate by immunoblotting technique using serum HMDM441. Two arguments may explain these variations in antibody reactivity: First, normal MDM2 may not be expressed in the hepatoma cell line. The immunocytochemical data, however, show that the cells expressed large amounts of full-size MDM2 protein in the nucleus as detected by commercially available antibody SMP 14. Second, due to altered protein conformation, the C-terminal HMDM441 epitope was accessible for serum HMDM441 only in the human 55-kDa MDM2 fragments. This plausible hypothesis suggests different conformation for the RING finger domain for human MDM2 full-size protein and MDM2 fragments arising from MDM2 disintegration. The MDM2 frag-

FIG. 5. Confocal laser-scanning microscopy for localization of proteins containing the HMDM441 epitope in hepatoma cells. (A) Clustering of MDM2 proteins in the nucleoli (arrow; bar: 12 ␮m). (B) A corona around the nuclear envelope was detected in rounded, detachable tumor cells entering the mitotic phase (arrow; bar: 8 ␮m). The fluorescence indicates an association of MDM2 with the nuclear envelope. Notably, this accumulation can also be seen in some of the cells presented in Fig. 4B.

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FIG. 6. Coexpression of MDM2 fragments containing the HMDM441 epitope and MHC class I molecules on the surface of hepatoma cell overexpressing MDM2. For this experiment, the x–y scanning modus of the confocal laser-scanning microscope was used (cells viewed from above). The cells were sectioned automatically at fixed levels and the levels were stacked by the software. Immunolabeling of single cells fixed in 4% neutral-buffered formalin (upper row; A1–A3; bar: 10 ␮m) and a cluster of nonfixed cells (lower row; B1-B3; note that the monolayer of about 20 nonfixed cells was twisted and folded during sample preparation. Bar: 50 ␮m). A1 and B1 contain fluorescence of MDM2 protein fragments. A2 and B2 show fluorescence of MHC class I molecules concentrated in spots or in a fine staining, as demonstrated for the nonfixed cells (B2). A3 and B3 present channel overlay. Local concentrations of MDM2 fragments and MHC class I molecules are indicated by the fluorescence of some spots in A3 and by the intense signals in B3. However, there are still clusters of MHC class I proteins that are not associated with MDM2 molecules.

ments were present in different subcellular parts of hepatoma cells, a finding which is in accordance with the suggestion that MDM2 protein variants may function at different locations before they are degraded. In fact, MDM2 protein was found clustered at the golgi apparatus and endoplasmatic reticulum of nonneoplastic mucosa cells of bronchi but not in the cell nuclei (21). Moreover, staining of the cytoplasma was also reported for a 60-kDa isoform of MDM2 resulting from caspase cleavage (22). All of these cell compartments are known to be closely associated with proteasomes (23) and revealed dense labeling in the present study. Proteolytic complexes are also found in close proximity to or associated with the nuclear membrane (24), which supports our observation that a corona of molecules containing the HMDM441 epitope was clustered around the nucleus of SK-Hep1 cells growing in a monolayer and round-shaped detachable tumor cells entering the mitotic phase. There-

fore, we suggest that the signals observed in each cell of nonsynchronized hepatoma cultures may reflect a “snapshot” of MDM2 protein turnover. Since the 55-kDa fragment is quite large for a degradation product, it may be the result of one of the initial steps of MDM2 cleavage: the activity of 26 S proteasomal complexes cleaving whole antigen into intermediate-sized fragments that are degraded by PA28/20 S proteasomal complexes to produce peptides of 8 –10 residues in length (25). In the context of protein turnover, it has been reported that the small ubiquitin-related modifier SUMO-1 targets a lysine residue in MDM2 at position 446 (26) which is located in the center of peptide HMDM441 and which forms a part of the antibody epitope. Since the authors demonstrated that SUMO-1 conjugation results in inhibition of MDM2 selfubiquitination, the 55-kDa MDM2 fragments detected in the present study cannot be protected by SUMO-1

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FIG. 7. x–z scanning modus of the confocal laser-scanning microscope. A hepatoma cell is presented in a side view. A vertical section was put through signals of cofluorescence presented in Fig. 6A3. Data show that MDM2 fragments (A, bar: 6 ␮m) and MHC class I molecules (B, bar: 6 ␮m) are clustered around the cytoplasm of the cell. Local concentrations of both proteins are indicated by spots located on top and under the cell (C, bar: 3 ␮m). The cofluorescence is located on and underneath the surface of the tumor cells. The faint horizontal lines in the pictures represent background fluorescence located on the surface of the glass slide.

and may reveal a higher rate of turnover than sumoylated MDM2. However, the detection of MDM2 cleavage products in the nucleoli of malignant and benign cells is puzzling. Though some studies have shown that MDM2 is transported into the nucleolus and bound there by tumor suppressor p19 ARF for inactivation (27, 28), nothing has been reported so far on MDM2 degradation in the nucleoli. We speculate that nucleolar inactivation of MDM2 by p19 may be associated with MDM2 protein instability, partial MDM2 disintegration and formation of the 55-kDa variant in the transcriptional active domains of the nucleus. Furthermore, the labeling may be caused by MDM2 fragments that are exported into the cytoplasm through the nucleolus. Notably, the 55-kDa MDM2 fragment was also

present in the supernatant of the cell culture. The MDM2 fragments may be released in vivo by tumor lysis or other mechanisms, especially since MDM2 autoantibodies were detected in sera derived from cancer patients (29) and since MDM2 is supposed to be part of the secretory machinery of the cell (21). If the release of the 55-kDa fragment is a common feature of malignant tumors, then ELISA based on the HMDM441 peptide might prove helpful for cancer diagnostics. Although MDM2 is ubiquitously expressed in human cells at low levels, carcinoma cells overexpressing large amounts of tumor antigen MDM2 might be suited for immunotherapy. In fact, former in vitro experiments proved that MDM2 peptide stimulates cytotoxic CTL which lyse cultivated mouse tumor cells (13). These data prompted us to analyze in the second part of the study the expression of MHC class I molecules and MDM2 containing HMDM441 on the surface of a human hepatoma cell line. As a result of confocal laserscanning microscopy, a spot-like arrangement of MHC class I protein and MDM2 fragments was observed for SK-Hep1 cells which had been either nonfixed or fixed in neutral-buffered formalin for seconds. x–z scanning proved that most of the signals were located on the cell surface. To our knowledge, this is the first reference to surface expression of MDM2 in human tumor cells. The significant MHC class I signals are in contrast to former investigations indicating low expression of MHC class I molecules in HCC cells (30). However, since some of the antibodies might have penetrated the fixed hepatoma cells despite short-time formalin fixation, the significant spot-like arrangement on the fixed cells could reflect both MDM2–MHC class I complexes transported in the golgi apparatus and complexes already localized on the surface. It should be noted that nonfixed, nonpermeable hepatoma cells revealed a more granular surface staining with less intense MHC class I signals. On the basis of these data one can only speculate that peptide HMDM441 and the MHC class I molecules form complexes since confocal laserscanning microscopy technique is not suited for this kind of binding analysis. If both protein types actually form complexes on the cell surface, MDM2 fragments might mediate immunological response in tumor therapy of human hepatoma. In summary, we describe the production of 55-kDa MDM2 fragments which are present in different parts of SK-Hep1 hepatoma cells and which lack the 225 N-terminal amino acids. The formation of these degradation products was closely associated with overexpression of full-size MDM2, a common feature of human tumors. Moreover, the MDM2 fragments and MHC class I molecules were coexpressed on the surface of the cells although a direct interaction between both types of molecules could not be proven. Since the present study had to be restricted to just one hepatoma cell line giving specific MDM2 signals when using poly-

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