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Tumor Biology |
Institut für Pathologie, D-97080 Würzburg [F. H., R. H., C. S., N. A., K. S., A. F., H. K. M-H., H. P. V.], and European Molecular Biology Laboratory, D-69012 Heidelberg [A. S., M. M.], Germany
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In earlier publications, we have described the generation of an affinity-maturated human monoclonal antibody SC-1 by fusion of a B cell from a patient with a signet ring cell carcinoma of the stomach with a heteromyeloma (12) . Immunohistochemical studies have shown that SC-1 reacts with nearly all diffuse-type and about 20% of intestinal-type adenocarcinomas (13) , whereas thus far, only restricted reactivity has been shown on a number of other malignant cells. Some cross-reactivity was found with embryonal glandular tissue, indicating an oncofetal origin of this molecule (14) . Antibody SC-1 induces specific apoptosis of stomach carcinoma cells both in vitro and in experimental in vivo systems (12, 13, 14) . The antibody is successfully used in a clinical Phase II study, showing specific induction of regression and apoptosis in primary stomach cancers without any toxic cross-reactivity (15) . Here, we describe the SC-1 antigen as an isoform of CD55/DAF,4 a highly glycosylated, GPI-anchored protein, which protects cells from autologous complement (16, 17, 18) and exists in various isoforms (19 , 20) .
Aside from specific signals, induction of apoptosis requires an activation of specific membrane-bound, cytoplasmic, and nuclear proteins, which regulate the cell cycle, and an ordered degradation of cellular components. Fas-induced apoptosis can be enhanced by cross-linking and requires tyrosine kinase activity, leading to phosphorylation of proteins of 116, 97, 66–69, 50, and 44 kDa (21) . We found that SC-1-induced apoptosis also can be enhanced by cross-linking, but with different tyrosine kinase activity as compared to Fas-induced apoptosis, inducing phosphorylation of three proteins (of approximately 60, 75, and 110 kDa) at tyrosine residues and dephosphorylation of a serine residue of an approximately 35-kDa protein.
The central components of the apoptotic processes are the caspases, a growing family of at least 14 proteolytic enzymes, which irreversibly promote the apoptotic events (22) . Caspase precursors are constitutively expressed in all cells and are activated by proteolytic cleavage (23) . We investigated the expression pattern and activation of the well-characterized caspase-3 (CPP32; Ref. 24 ) and caspase-8 (FLICE; Ref. 25 ). We found a higher expression 15 or 60 min after incubating cells with SC-1 and a high caspase-8 activity 20 h after induction of apoptosis, whereas activity of caspase-3 was elevated to a minor extent.
Our results confirm that apoptosis is not restricted to a specific family of membrane proteins with death domains, but can also be induced specifically in tumor cells through carbohydrates and GPI-linked molecules, such as CD55/DAF variants, and that this approach can be used for specific adjuvant immunotherapy (15) .
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Purification of the SC-1 Antibody
The human monoclonal antibody was purified from mass cultures, using cation exchange chromatography followed by gel filtration as described elsewhere (27)
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Purification of the SC-1 Receptor
For preparation of membrane proteins, harvested cells were resuspended in hypotonic buffer (20 mM HEPES, 3 mM KCl, 3 mM MgCl2), incubated on ice (15 min), and sonicated (5 min), and the nuclei were pelleted by centrifugation (10,000 x g for 10 min). The membranes were pelleted by centrifugation (100.000 x g for 30 min) and resuspended in membrane lysis buffer (50 mM HEPES, pH 7.4, 0.1 mM EDTA, 1 M NaCl, 10% glycerol and 1% Triton X-100). Complete protease inhibitor (Roche Molecular Biochemicals, Mannheim, Germany) was added to all solutions.
The purification of the antigens was performed by column chromatography using an Amersham Pharmacia Biotech (Freiburg, Germany) fast protein liquid chromatography unit. For size exclusion chromatography, an Amersham Pharmacia Biotech Superdex 200 column (XK16/60) was loaded with 5 mg of membrane protein preparation and run with Buffer A (100 mM Tris/Cl, pH 7.5, 2 mM EDTA, 40 mM NaCl, 1% Triton X-100). The eluate was fractionated and examined in Western blot analysis for reaction with antibody SC-1. Positive fractions were loaded on a MonoQ (5/5) column using Buffer A. The bound proteins were eluated with a linear gradient using Buffer B (100 mM Tris/Cl, pH 7.5, 1 M NaCl, 2 mM EDTA, 40 mM NaCl, 1% Triton X-100), fractionized, and examined by Coomassie-stained SDS-PAGE and Western blot analysis. Positive bands were cut out from gel and sequenced.
Preparation of Cell Lysates after Induction with SC-1
Cell line 23132 was grown on 100-mm cell culture plates to subconfluency, and then the SC-1 antibody was added to a final volume of 30 µg/ml and incubated for the periods indicated. Then culture plates were washed once with PBS, and subsequently cells were directly lysed with SDS-buffer (50 mM Tris/Cl, pH 6.8, 10 mM DTT, 2% (w/v) SDS, 10% (v/v) glycerol). Cell debris was collected with a rubber policeman.
Gel Electrophoresis and Blotting
SDS-PAGE under reducing conditions and Western blotting of proteins were performed using standard protocols as described elsewhere (13)
. Briefly, blotted nitrocellulose membranes were blocked with PBS containing 0.1% (v/v) Tween-20 and 2% (w/v) low-fat milk powder or 3% (w/v) BSA (for determination of phosphorylation), followed by a 1-h incubation with primary antibody. The antibodies were used at the following concentrations: SC-1 (human), 10 µg/ml; goat anticaspase-3 and -8 (SantaCruz, Heidelberg, Germany), 5 µg/ml; strepavidin-coupled antiphosphotyrosine (clone PT-66), 1:20,000; and strepavidin-coupled antiphosphoserine (clone PSR-45), 1:30,000 (Sigma, Munich, Germany). The secondary antibodies (peroxidase-coupled rabbit antihuman IgM or rabbit antigoat antibody (Dianova, Hamburg, Germany) and peroxidase-coupled extravidin (Sigma)) were detected with the SuperSignal chemiluminescence kit from Pierce (KMF, St. Augustin, Germany).
Protein Sequencing
A protein band having an apparent molecular mass of 82 kDa was isolated by one-dimensional PAGE and visualized by Coomassie staining. P82 band was in-gel digested with trypsin (Roche Molecular Biochemicals, unmodified, sequencing grade) as described (28)
. Unseparated pool of tryptic peptides was sequenced by nanoelectrospray tandem mass spectrometry as described (29)
. Sequencing was performed on a API III triple quadrupole mass spectrometer (PE Sciex, Concord Ontario, Canada). Peptide sequence tags (30)
were assembled using tandem mass spectrometric data. Searching of comprehensive protein and expressed sequence tag databases was performed using PeptideSearch version 3.0 software, developed in-house.
Reverse Transcription-PCR
cDNA synthesis from total RNA from tumor cells 23132 was performed with 5 µg of total RNA using Life Technologies, Inc. (Eggenstein, Germany) Moloney murine leukemia virus reverse transcriptase according to the supplier’s manual. PCRs were carried out in a 25-µl volume with 1.75 mM MgCl2, 0.4 pM primer, 200 µM each dNTP, and 1 unit of Taq polymerase (MBI Fermentas, St. Leon-Rot, Germany). The PCR products for cloning the CD55 antisense vector were amplified using the following cycle profiles: CD55 (640-bp fragment spanning the sequence from bp 382 to 1022): 95°C for 2 min; followed by 35 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 60 s; and a final extension at 72°C for 4 min.
Cloning Procedures
Cloning for Sequencing.
PCR products were purified from agarose gel using a Jetsorb gel extraction kit (Genomed, Bad Oeynhausen, Germany). Cloning of PCR fragments was performed with pCR-Script Amp SK (+) cloning kit (Stratagene, Heidelberg, Germany).
Cloning of Antisense Vector pHook2-CD55anti.
The CD55-PCR product was blunted with Pfu-polymerase and directly cloned into the SmaI cut expression vector pHOOK-2 (Invitrogen, Leek, the Netherlands). One clone with antisense direction of the insert following the PCMV-promoter was selected for the antisense experiments.
DNA Sequencing
Eight positive clones were sequenced using the DyeDeoxy termination cycle sequencing kit (Applied BioSystems Inc., Weiterstadt, Germany) and analyzed with an automated DNA sequencer ABIPrism373. Both strands were sequenced using T3 and T7 primers. The sequences were analyzed using DNASIS for Windows software and the BLAST program from the National Center for Biotechnology Information.
Transfection
For transfection experiments, 2–5 x 107 detached cells were washed in TBS and resuspended in 400 µl of TBS. 10 µg of plasmid DNA, isolated with Endotox-free Maxi-Prep kit (Qiagen, Hilden, Germany), were added, and the cells were pulsed with 240 V, 960 µF, using a Bio-Rad (Munich, Germany) electroporation unit. 5 x 105 transfected cells were seeded on 60-mm cell culture and incubated for 24 h as indicated above. Apoptosis was induced by adding 50 µg/ml of purified SC-1 antibody to the growth medium. After 24 h, cells were trypsinized and used for preparing cytospins.
Phospholipase Assay
Detached and pelleted cells were resuspended in RPMI 1640 with supplements and incubated for 90 min at 37°C. After this recovery period, 20 milliunits/ml PI-PLC (Roche Molecular Biochemicals) were added, and cells were incubated for another 60 min. Finally, cells were washed and used for preparing cytospins.
Glycosidase Assay
Detached and washed cells were resuspended in RPMI 1640 containing 10% FCS and incubated for 1 h on ice and then counted, and cytospins were prepared. After air drying, cytospin preparations were acetone fixed (10 min), washed, and incubated with 20 microunits/ml O-glycosidase or 5 milliunits/ml N-glycosidase (Roche Molecular Biochemicals) for 4 h at 37°C.
Immunohistochemical Staining
The following antibodies were used for immunohistochemical staining: purified SC-1, anti-CEA (DAKO, Hamburg, Germany), anti-EMA (Loxo, Dossenheim, Germany), and anti-CD55 (Biozol, Eiching, Germany). Cytospin preparations were fixed with acetone and stained as described elsewhere (31)
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Apoptosis Assay
Cytospin preparations (5000 cells/slide) were fixed in acetone, followed by washings with TBS. Then the cytospins underwent staining with the FragEl-Klenow DNA fragmentation kit (Calbiochem-Novabiochem, Bad Soden, Germany), according to the supplier’s manual and as described elsewhere (15
, 27)
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MTT Assay
The MTT proliferation assay for measuring the apoptotic activity of antibody SC-1 on stomach cancer cells was performed as described elsewhere (14)
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Caspase-3 and -8 Assays
The activation of caspase-8 was measured with the ApoAlert caspase fluorescent assay kits (Clontech, Heidelberg, Germany). Briefly, 1 x 106 cells were incubated with 40 µg/ml SC-1 for 7 and 20 h. Then cells were collected and resuspended in cell lysis buffer, and caspase activity was measured according to supplier’s manual.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Lane 1). We isolated these proteins from membrane fractions and prepurified them by sequential size exclusion and anion exchange chromatography (Fig. 1a
, Lanes 2 and 3). The molecules were excised from Coomassie-stained SDS-PAGE and directly sequenced by nanoelectrospray tandem mass spectrometry after in-gel tryptic digest. The protein of approximately 50 kDa was identified as dihydrolipoamide succinyltransferase (E2K, GenBank accession no. L37418), and the protein of approximately 70 kDa as the human lupus p70 (Ku) autoantigen protein (GenBank accession no. J04611; data not shown). These reactivities are most likely due to nonspecific binding of SC-1 caused by protein denaturation in Western blot analysis, which is a common observation for monoclonal antibodies. Because the SC-1 antibody only binds cell surface antigens in immunohistochemical stainings, cytoplasmic and nuclear antigens, such as E2K and p70 (Ku), can be excluded from specificity.
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, panels 1 and 2), co-migrating with another protein, which was identified as hnRNP R (GenBank accession no. O43390). The molecular mass of hnRNP R is 71 kDa, but it is known to migrate at 82 kDa. We further investigated CD55, which exists in humans in two genetically determined isoforms (secreted DAF-A and membrane-bound DAF-B) caused by differential splicing (18)
. Through reverse transcription-PCR analysis, we found that the cell line 23132 expresses only the GPI-anchored (DAF-B) isoform of CD55 (data not shown).
Transient Transfection with the CD55 Antisense Vector.
To analyze whether inhibition of CD55/DAF expression influences SC-1-induced apoptosis, the cell line 23132 was transiently transfected with the CD55 antisense vector pHOOK-2-cd55anti and control vector pHOOK-2 by electrotransfection. Cytospins of transfected cells were immunohistochemically stained with SC-1, anti-CD55 (data not shown), and anti-CEA. Cells transfected with the control vector showed intensive staining with SC-1 and CEA (Fig. 2, a and c)
, but we observed hardly any staining with SC-1 in cells transfected with the CD55 antisense vector (Fig. 2b)
. The staining with anti-CEA antibody revealed that the expression pattern of CEA, also GPI-anchored, is not affected by the transfection with the antisense vector, but that expression of CD55 was specifically reduced due to expression of CD55 antisense RNA.
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, a clear loss of staining intensity can be observed on PI-PLC-treated cells stained with SC-1 (Fig. 4b)
. The cleavage of GPI anchor was confirmed by staining with anti-CD55, with a reduced staining after PI-PLC treatment (Fig. 4d)
, as compared to untreated cells (Fig. 4c)
. PI-PLC treatment did not have any effect on staining with anti-EMA (data not shown), which indicates that the PI-PLC treatment has no effect on non-GPI-anchored membrane proteins. In the MTT test, the treatment of cells with PI-PLC leads to a significant (P 
0.05, Student’s t test) decrease in apoptotic cells (Fig. 5d)
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, whereas staining with anti-CD55, which binds to the unglycosylated SCR3 region, was not affected by protein deglycosylation (Fig. 4e)
. Incubation with phosphate buffer and digestion with O-glycosidase (data not shown) had no effect on SC-1 binding (data not shown). This shows that the specificity of SC-1 binding must be located in sugar residues and not in the primary protein sequence.
MTT and Cross-linking of CD55/SC-1.
The apoptotic activity of SC-1 in vitro was measured in MTT assays, as shown here on two different stomach carcinoma cell lines (Fig. 5a)
. To investigate whether receptor complexing might be a part of a signaling pathway through CD55/DAF, we first incubated cells for 24 h with an increasing amount of SC-1, to evaluate the optimal apoptotic activity of SC-1 (Fig. 5b)
. Cross-linking was then performed with 40 µg/ml antibody with and without a rabbit antihuman IgM. After incubation for 48 h, we found a rate of death cells 47% higher than in control incubated with SC-1 (Fig. 5c)
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Protein Phosphorylation.
We investigated the phosphorylation pattern after inducing cells with 40 µg/ml purified SC-1 antibody by Western blots of cytoplasmic and membrane extracts. We found an early tyrosine phosphorylation of proteins of 110 and 60 kDa 30–60 s after apoptosis induction (Fig. 6a)
. These proteins were found only in the cytoplasm (60 kDa) or in membrane extracts and cytoplasm (110 kDa). Furthermore, we found a slow tyrosine phosphorylation of a 75-kDa cytoplasmic protein, with the highest level after 10 min (Fig. 6b)
, as well as the complete disappearance of a serine phosphorylation of a 35-kDa protein, also 10 min after induction.
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. Also, activation of these caspases was observed. This activation occurs through proteolytic cleavage of the procaspases (22)
. This cleavage was shown for caspase-3 by identification of the p20 cleavage product (Fig. 7a)
. Corresponding to the small amount of cleavage product, only minor activation of caspase-3 was observed with the ApoAlert caspase kit (data not shown). In contrast, we found a strong increase of caspase-8 activity 20 h after induction with SC-1, indicating the involvement of this caspase in the apoptotic process (Fig. 7b)
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Induction of specific apoptosis is the most effective and safest way to remove tumor cells from the organism. Because tumor cells have lost control of cell proliferation by genetic alteration, most apoptosis mechanisms known from normal counterparts are ineffective. This becomes extremely evident in the approach to kill tumor cells through the Fas/APO-1 receptor ligand family, which plays a key role in normal immune development, homeostasis, modulation, and function but fails in the specific killing of malignant cells. Although this is a very promising line of investigation, neither antibodies against Fas/APO-1 nor the more restricted Fas ligand approach will cause a breakthrough in cancer therapy.
Because the easiest way to attack tumor cells is through the cell membrane, the obvious need is to search for other receptor/ligand/antibody systems through which apoptosis in tumor cells can be induced. Therefore, in an extensive study of the humoral immunity of stomach cancer patients, we screened for antibodies that influence cell growth. The human antibody SC-1 inhibits in vitro and in vivo growth of stomach cancer cells by inducing apoptosis through binding to CD55/DAF receptor.
There are two genetically determined isoforms of CD55, termed DAF-A and DAF-B, in existence, and in addition. there are different glycosylated isoforms of CD55 that are expressed by erythrocytes, lymphocytes, and sperm and in glandular fluid. Their molecular masses vary from 50 to 70 kDa, due to different glycosylation patterns, and they are different in size to the isoform defined by the SC-1 antibody on stomach cancer cells (82 kDa). CD55 is not a transmembrane molecule, but the DAF-B isoform is linked to the cell surface by a GPI anchor and contains no death domain (32) . Interestingly, an up-regulation of CD55/DAF in the human gastric mucosa was found in Helicobacter pylori-infected individuals (33) , and there is strong evidence that H. pylori is one cause for the development of gastric cancer and was correctly classified as carcinogen by the WHO (34) .
Indirect evidence for the participation of CD55 in apoptotic events has been provided by Jones and Morgan (35) , who reported that reduced expression of CD55/DAF together with CD59 in human polymorphonuclear leukocytes correlates closely with the appearance of apoptotic morphology. On the other hand, Brodsky et al. (36) have shown that in paroxysmal nocturnal hemoglobinuria, which is a genetically determined hematopoietic stem cell disorder and results in the absence of GPI-linked molecules including CD55, the cells are also protected from apoptosis induced by ionized irradiation. Furthermore, on tumor cells a heterogeneous pattern of CD55 and often an up-regulation can be observed (37) . Several other GPI-linked molecules, such as CD14 (38) , are multispecific in function and are somehow involved in cellular regulation (39) , including apoptotic events.
Abnormal glycolipid and glycoprotein synthesis by tumor cells very often results in expression of these modified structures on the surface, and they are a preferred target for antibody-based immunotherapy, mostly performed with immunoglobulins bred in animals. A typical feature of diffuse-type stomach carcinoma (including signet ring cell carcinoma) is the strong production and intracellular deposition of mucin (40) , and alterations in these carbohydrate structures are a commonly observed phenomenon, e.g., MUC1–6 in mamma carcinoma (41) . These molecules are highly immunogenic (42) , and it is therefore likely that an already strongly glycosylated membrane molecule, such as CD55/DAF, could be further glycosylated and modified by tumor-specific glycosylation machinery to also become immunogenic.
Cross-linking of the CD55/DAF receptor by the antibody SC-1 increases cell death of stomach cancer cells. Cross-linking of membrane molecules upon signals is essential for or increases the effect of external signals. The apoptosis signal of the Fas/CD95 antibodies on Fas receptors, leading to the death-inducing signaling complex, can similarly be enhanced by cross-linking of the receptors (43) .
The antibody SC-1 induces a tyrosine phosphorylation of three different molecules with molecular masses of 60, 75, and 110 kDa and a decrease of serine phosphorylation of a molecule with 35 kDa. Tyrosine kinase activity is an early event after induction of apoptosis, e.g., treatment of cells with Fas antibody induces a tyrosine phosphorylation of 97-, 66-, and 50-kDa proteins (21) . A detailed analysis of these SC-1-associated molecules by protein sequencing is under way.
Furthermore, SC-1 increases levels of caspase-8 (FLICE) and caspase-3 (CPP-32) and leads to a strong activation of caspase-8, similar to other known apoptotic pathways. (24) . Caspases are a group of intracellular proteases and are responsible for the disassembly of the cell into apoptotic bodies (22) . The proteins are present as inactive pro-enzymes that are activated by proteolytic enzymes after apoptotic signals (44) .
The human monoclonal antibody SC-1 is currently being used successfully in a clinical Phase II trial on patients with intestinal- and diffuse-type adenocarcinoma of the stomach. In this study, patients who were tested positively for SC-1 expression receive one dose of purified SC-1 prior to gastrectomy. The effect of antibody SC-1 on the primary tumor and lymphnodes is measured. In 90% of cases, a measurable induction of apoptosis can be observed, and in 50%, a significant tumor regression as compared to the biopsies can be observed (15) . In addition, about 70% of the patients are free of lymphnode metastases. These data show that tumor cells can be selectively removed in vivo by a specific apoptotic signal.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This project was partly supported by the Deutsche Krebshilfe e.V. 
2 Current address: Center for Experimental BioInformatics, Odense University, Stærmosegaardsvej 16, DK-5230 Odense M, Denmark.
3 To whom requests for reprints should be addressed, at Institut für Pathologie, Universität Würzburg, Josef-Schneider Strasse 2, D-97080 Würzburg, Germany. Phone: 49-931-2013898; Fax.: 49-931-2013440; E-mail: path027{at}mail.uni-wuerzburg.de
4 The abbreviations used are: DAF, decay-accelerating factor; PI-PLC, glycosylphosphatidylinositol-specific phospholipase C; GPI, glycosylphosphatidylinositol; TBS, Tris-buffered saline; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; CEA, carcinoembryonic antigen; EMA, epithelial membrane antigen.
Received 3/11/99. Accepted 8/19/99.
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