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Clinical Cancer Research Vol. 11, 2492-2501, April 2005
© 2005 American Association for Cancer Research

Human Cancer Biology

Cleavage of L1 in Exosomes and Apoptotic Membrane Vesicles Released from Ovarian Carcinoma Cells

Paul Gutwein1, Alexander Stoeck1, Svenja Riedle1, Daniela Gast1, Steffen Runz1, Thomas P. Condon4, Alexander Marmé2, Minh-Chau Phong3, Otwin Linderkamp4, Alexander Skorokhod1 and Peter Altevogt1

Authors' Affiliations: 1 Tumor Immunology Programme, D010, German Cancer Research Center; 2 University Hospital for Gynecology and Obstetrics, and 3 Pedriatics, University of Heidelberg, Heidelberg, Germany; and 4 ISIS Pharmaceuticals Carlsbad, California

Requests for reprints: Peter Altevogt, Tumor Immunology Programme, G0100, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. Phone: 49-6221-423725; Fax: 49-6221-423702; E-mail: P.Altevogt{at}dkfz-de..


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: The L1 adhesion molecule (CD171) is overexpressed in human ovarian and endometrial carcinomas and is associated with bad prognosis. Although expressed as a transmembrane molecule, L1 is released from carcinoma cells in a soluble form. Soluble L1 is present in serum and ascites of ovarian carcinoma patients. We investigated the mode of L1 cleavage and the function of soluble L1.

Experimental Design: We used ovarian carcinoma cell lines and ascites from ovarian carcinoma patients to analyze soluble L1 and L1 cleavage by Western blot analysis and ELISA.

Results: We find that in ovarian carcinoma cells the constitutive cleavage of L1 proceeds in secretory vesicles. We show that apoptotic stimuli like C2-ceramide, staurosporine, UV irradiation, and hypoxic conditions enhance L1-vesicle release resulting in elevated levels of soluble L1. Constitutive cleavage of L1 is mediated by a disintegrin and metalloproteinase 10, but under apoptotic conditions multiple metalloproteinases are involved. L1 cleavage occurs in two types of vesicles with distinct density features: constitutively released vesicles with similarity to exosomes and apoptotic vesicles. Both types of L1-containing vesicles are present in the ascites fluids of ovarian carcinoma patients. Soluble L1 from ascites is a potent inducer of cell migration and can trigger extracellular signal-regulated kinase phosphorylation.

Conclusions: We suggest that tumor-derived vesicles may be an important source for soluble L1 that could regulate tumor cell function in an autocrine/paracrine fashion.

Key Words: shedding • ascites • metalloproteinases • ADAM

L1 is the prototype of a neural subfamily of cell adhesion molecules structurally belonging to the immunoglobulin (Ig) superfamily (1). In humans, L1 is a 200- to 220-kDa transmembrane glycoprotein composed of six Ig-like domains and five fibronectin type III repeats. L1 plays a crucial role in axon guidance and cell migration in the developing nervous system (2, 3). L1 is not only expressed in the nervous system but is also found on different tumor cells like lung cancer (4), gliomas (5), melanomas (6, 7), and renal carcinoma (8). We recently reported that L1 is overexpressed in ovarian and endometrial carcinomas in a stage-dependent manner and that L1 expression was a predictor of poor outcome (9). It was proposed that L1 could promote carcinoma progression by augmenting tumor cell migration on extracellular matrix compounds (9, 10).

Although a transmembrane molecule, L1 can be cleaved and released in a soluble form into the extracellular space (10, 11). Importantly, soluble L1 was specifically detected in serum samples of ovarian and uterine cancer patients (9). The cleavage of the ectodomain of transmembrane molecules is termed ectodomain shedding (12), and it affects many cell surface molecules such as growth factors like membrane-anchored heparin-binding epidermal growth factor–like growth factor (proHB-EGF; refs. 13, 14), transforming growth factor-{alpha} (15), membrane receptors like Her2/neu (16), and adhesion molecules like L-selectin (17, 18) and ß-amyloid precursor protein (19, 20). Shedding can occur in a constitutive fashion or can be induced. Common unphysiologic activators of ectodomain shedding are phorbol esters (21–23), pervanadate treatment, and cholesterol extracting agents such as methyl-ß-cyclodextrin (24–26). Several physiologic activators of ectodomain shedding such as chemotactic peptides, cytokines, and growth factors have been also described (27–29). The activation of G-protein–coupled receptors can induce shedding of growth factors that in turn mediate the activation of tyrosine kinase receptors such as the epidermal growth factor (EGF) receptor (27, 30). We showed before that shedding of L1 can be induced through stimuli like phorbol 12-myristate 13-acetate, pervanadate, and methyl-ß-cyclodextrin (10, 11, 24). A recent study has shown that in renal carcinoma the shedding of L1 is enhanced by hepatocyte growth factor (8). Other physiologic stimuli that lead to enhanced L1 shedding have not been identified yet.

Another shedding event frequently observed in tumor cells is the ability to release intact membrane vesicles into the environment (31). The precise mechanism of membrane shedding and the origin of released vesicles are not known, however, recent work has characterized at least two distinct types of vesicles: apoptotic blebs and exosomes (32, 33). The release of apoptotic blebs is initiated shortly after the induction of apoptotic cell death (33), whereas exosomes represent membrane constituents that are released from live cells via the fusion of multivesicular endosomes with the plasma membrane (33, 34). We reported before that spontaneous vesicles released from cells transfected with the neural form of L1 contained L1 and the cleavage proteinase a disintegrin and metalloproteinase 10 (ADAM10; ref. 24). In the present communication we have investigated the shedding of L1 in ovarian carcinoma cells in vitro and in vivo. We show that the constitutive cleavage is mediated by ADAM10 and proceeds in secretory vesicles. Shedding is increased by apoptotic stimuli via the release of membrane blebs in which multiple metalloproteinases are active. We provide evidence that both types of L1-containing vesicles occur in the ascites of ovarian carcinoma patients and that soluble L1 in the ascites can modulate the function of carcinoma cells. Our results suggest an important role for vesicles in the release of soluble L1 from tumor cells.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cells. HeLa cells were obtained from the tumor bank of the German Cancer Research Center (Heidelberg, Germany). The ovarian carcinoma cell line OVMz and Chinese hamster ovary (CHO) cells stably transfected with human L1 (CHO-hL1) were described before (10). Cells were cultivated in DMEM supplemented with 10% fetal bovine serum at 37°C, 5% CO2, and 100% humidity.

Chemicals and antibodies. Antibodies to the ectodomain [L1-11A, subclone of monoclonal antibody (mAb) UJ 127.11] or the cytoplasmic domain (pcytL1) of human L1 were described (10). Antibodies #2547 to the ectodomain and #71 to the cytoplasmic tail of ADAM10 were described (24). Additional polyclonal antibodies to ADAM17 and ADAM10 or mAbs to integrins were obtained from Chemicon (Hofheim, Germany). MAb Syb-1 to human CD9 was a gift from Dr. Eric Rubinstein. Antibodies to extracellular signal-regulated kinase (ERK) and phospho-ERK were purchased from BD Transduction (Heidelberg, Germany). Secondary antibody Alexa 488–conjugated goat anti-mouse was purchased from Molecular Probes (Leiden, The Netherlands) and Cy3-conjugated goat anti-rabbit antibody was obtained from Dianova (Hamburg, Germany). 4',6-Diamidino-2-phenylindole, staurosporine, a cell-permeable C2-ceramide, and doxorubicin were purchased from Sigma (Taufkirchen, Germany). Metalloproteinase inhibitors tumor necrosis factor-protease inhibitor-0 (TAPI-0) and TAPI-1 were from Calbiochem (Bad Soden, Germany), Triton X-100 was from Gerbu (Gaiberg, Germany), and caspase inhibitor ZVAD-fmk was from BACHEM (Weil am Rhein, Germany). A stock solution of pervanadate was prepared as described before (11).

Antisense strategy. The selection of potent and specific antisense oligonucleotide inhibitors of human ADAM10 (ISIS 100750) and ADAM17 (ISIS 16337) together with the mismatched controls ISIS 108030 (for ADAM10) and ISIS 17337 (for ADAM17) has been described (35). Cells were transfected using oligofectamine (Invitrogen, Karlstuhe, Germany) and were analyzed 48 hours later.

Analysis of L1 shedding and induction of apoptosis. Cells (100,000 cells per well) were cultured in duplicate in six-well culture plates and after washing once with PBS incubated for 18 hours with or without apoptotic stimuli in serum-free medium. Staurosporine (2 µmol/L), C2-ceramide (40 µmol/L), pervanadate (200 µmol/L), doxorubicin (5 µg/mL), and UV irradiation were used as apoptotic reagents. Metalloproteinase inhibitors TAPI-1 and TAPI-0 (10 µmol/L) and caspase inhibitor ZVAD-fmk (25 µmol/L) were added 30 minutes before treatment. After 18 hours, the supernatant was removed and cells were scraped into Eppendorf tubes and centrifuged. Cell pellet was lysed in lysis buffer [20 mmol/L Tris-HCl (pH 8.0) containing 1% Triton X-100, 150 mmol/L NaCl, and 1 mmol/L phenylmethylsulfonyl fluoride], cleared by centrifugation, and mixed with 2-fold concentrated reducing SDS sample buffer. Supernatants of treated cells were collected and centrifuged for 10 minutes at 1,200 x g and for 20 minutes at 10,000 x g to remove cellular debris. Membrane vesicles were collected by centrifugation at 100,000 x g for 2 or 18 hours at 4°C using a Beckman SW 40 rotor. Vesicles were directly dissolved in SDS sample buffer or processed for gradient centrifugation (see below). For hypoxia studies cells were incubated under hypoxic conditions (1% O2) using a Reming Bioinstruments chamber and oxygen regulator (Reming Bioinstruments, Redfield, NY).

Assessment of apoptotic cell death. For the analysis of DNA fragmentation, the cell pellet was washed once in PBS (pH 7.4) and lysed in a hypotonic lysis buffer (0.1% sodium citrate, 0.1% Triton X-100, 50 µg/mL propidium iodide) at 4°C overnight. The nuclei were then analyzed for DNA content by flow cytometry (36).

Isolation of vesicles from ascites fluid. Analysis of patient tumor material was under approval of the ethics commission of the University of Heidelberg. Ascites from ovarian carcinoma patients were cleared from cells and debris by two rounds of centrifugation as described above. The cleared ascites was then overlaid on a 40% sucrose cushion and centrifuged at 100,000 x g for 90 minutes. The interphase was removed and pelleted by ultracentrifugation.

Sucrose density gradient fractionation. Pelleted vesicles were resuspended in PBS and loaded on top of a step gradient comprising layers of 2, 1.3, 1.16, 0.8, 0.5, and 0.25 mol/L sucrose as described (24). The gradients were centrifuged for 2.5 hours at 100,000 x g in a Beckman SW40 rotor. Twelve 1-mL fractions were collected from the top of the gradient and precipitated by chloroform/methanol. Samples were analyzed by SDS-PAGE and Western blotting as described below.

Immunofluorescence. For immunofluorescent staining, cells were grown on coverslips and fixed for 20 minutes with 4% paraformaldehyde-PBS at room temperature. Cells were washed in PBS and permeabilized with 0.1% Saponin in PBS containing 5% goat serum for 15 minutes at room temperature. Cells were then incubated for 1 hour with primary antibodies (L1-11A supernatant undiluted and #2547 1:500 dilution). After three washing steps with PBS, cells were incubated for 30 minutes in the dark with Alexa 488–conjugated goat anti-mouse IgG or Cy3-conjugated goat anti-rabbit IgG. After washing the cells twice with PBS, nuclei were stained with 4',6-diamidino-2-phenylindole. Stained cells were mounted on glass slides and examined with an epifluorescence microscope (Axioplan-2, Zeiss, Oberkochem, Germany).

Transmigration assays. This assay has been described before (10). Briefly, fibronectin or bovine serum albumin for control was coated on the backside of Transwell chambers (6.5 mm diameter, 5 µm pore size; Costar, Cambridge, MA). Cells in RPMI 1640 containing 0.5 % bovine serum albumin were seeded into the upper chamber and allowed to transmigrate to the lower compartment. For mAb blocking, the cells were preincubated at 10 µg/mL with the respective mAb. Transmigration was quantified using crystal violet staining. Each determination was done in quadruplicate and the data are given as mean values ± SE.

Biochemical analysis. The isolation of soluble L1 from culture supernatant was described before (37, 38) and was adapted here for ascites. The vesicle-cleared ascites fluid was adsorbed to Sepharose-linked mAb L1-11A and bound L1 was eluted with 0.1 mol/L glycine-HCl buffer (pH 2.8). Eluted fractions were neutralized and an aliquot of the samples was separated by SDS-PAGE under reducing conditions and transferred to an Immobilon membrane using semidry blotting. After blocking with 5% skim milk in TBS, the blots were developed with the respective primary antibody followed by peroxidase-conjugated secondary antibody and enhanced chemiluminescence (ECL) detection.

Statistical analysis. For the analysis of statistical significance the Student's t test was used.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Apoptotic stimuli enhance the cleavage of L1 adhesion molecule. Recent work has shown that adhesion molecules such as E-cadherin, L-selectin, and platelet/endothelial cell adhesion molecule 1 are cleaved off the membrane during apoptosis (39–41). We examined whether apoptotic stimuli could augment the release of L1. As shown in Fig. 1A, treatment of CHO-hL1 cells with the apoptotic inducers C2-ceramide or staurosporine significantly augmented the amount of released L1 in the medium. Parallel measurements of apoptosis confirmed that both compounds induced apoptosis (not shown). Doxorubicin did not induce L1 release and did not cause apoptosis under the assay conditions. TAPI-0 treatment partially reduced the amount of C2-ceramide– and staurosporine-released L1 (Fig. 1A). Preincubating with the caspase inhibitor ZVAD-fmk blocked the C2-ceramide–induced L1 shedding only weakly (~20%), but strongly reduced the staurosporine-induced L1 release (~65 %). As expected, ZVAD-fmk reduced the rate of apoptosis (data not shown).



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  		Fig. 1. Apoptotic stimuli induce metalloproteinase-mediated cleavage of L1. A, CHO-hL1 cells were incubated for 18 hours with 40 µmol/L C2-ceramide, 5 µg/mL doxorubicin, or 2 µmol/L staurosporine. The metalloproteinase inhibitor TAPI and caspase inhibitor ZVAD-fmk were incubated for 30 minutes before adding the apoptotic stimuli. Supernatants were trichloroacetic acid precipitated and analyzed for L1 using Western blot analysis with mAb L1-11A against the ectodomain of L1 followed by peroxidase-conjugated secondary antibody and ECL detection. B, OVMz cells were incubated for 18 hours with 200 µmol/L pervanadate, 40 µmol/L C2-ceramide, or 2 µmol/L staurosporine, or were UV irradiated. The metalloproteinase inhibitor TAPI-0 (10 µmol/L) was added 30 minutes before. Supernatants were trichloroacetic acid precipitated and analyzed for L1 by Western blotting with mAb L1-11A followed by peroxidase-conjugated secondary antibody and ECL detection.

 
In the ovarian carcinoma cell line OVMz essentially similar results were observed (Fig. 1B). In contrast to CHO-hL1 cells, the strongest inducer was staurosporine but the amount of soluble L1 was only increased 2.5-fold (Fig. 1B). Importantly, all apoptotic stimuli caused a metalloproteinase-mediated cleavage of L1 as it was blocked by TAPI (Fig. 1B).

C2-ceramide enhances the formation of membrane vesicles containing ADAM10 and L1. For CHO-hL1 cells the size heterogeneity and the partial inhibition by TAPI-0 suggested that L1 released through apoptosis was composed of two forms: a truly soluble L1-200 form (membrane cleaved) and a full-length form (L1-220) in membrane vesicles. Both forms are only poorly resolved by SDS-PAGE (24). We investigated whether membrane vesicle release was indeed enhanced in cells treated with apoptotic stimuli. When L1-expressing HeLa cells were treated with C2-ceramide for 6 hours we noticed that the tumor cells formed membrane blebs (Fig. 2A). Bleb formation is a well-known characteristic for apoptosis. We analyzed the location of L1 and ADAM10 in C2-ceramide–treated cells using fluorescence microscopy. As shown in Fig. 2B, in untreated control cells ADAM10 was expressed mostly within the cell whereas L1 staining was seen both inside the cell and at the plasma membrane. Treatment with C2-ceramide led to the accumulation of ADAM10 in the lumen of the membrane blebs (Fig. 2B, red arrows) and a prominent staining of L1 in membrane blebs.



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  		Fig. 2. C2-ceramide induces membrane blebbing in apoptotic cells. A, HeLa cells were plated on coverslips and incubated for 6 hours with 40 µmol/L C2-ceramide. Cells were fixed with paraformaldehyde and analyzed with an epifluorescence microscope. Red arrows, C2-ceramide induces membrane blebbing. B, ADAM10 and L1 are located in membrane blebs. Cells were stained with polyclonal antibody #2547 to the ectodomain of ADAM10 followed by Cy3-conjugated goat anti-rabbit IgG or with mAb L1-11A followed by Alexa 488–conjugated goat anti-mouse IgG secondary antibody. The nuclei were stained with 4',6-diamidino-2-phenylindole. In nonstimulated control cells ADAM10 staining is predominantly in the cytoplasm. L1 shows a more membranous staining. Treatment with C2-ceramide induces membrane blebbing and ADAM10 and L1 are now localized in membrane blebs (red and green arrows).

 
Apoptosis-induced L1 cleavage occurs in membrane vesicles. Membrane vesicles were isolated from the supernatant of live and apoptotic cells. As shown in Fig. 3A we detected L1-220 and the cleavage fragment L1-32 in membrane vesicles from CHO-hL1 cells. The cleavage was metalloproteinase mediated as TAPI pretreatment reduced the amount of the L1-32 fragment. The analysis of OVMz cells revealed essentially similar results (Fig. 3B).



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  		Fig. 3. Cleavage of L1 in released membrane vesicles. Cells were treated with the indicated apoptosis inducers in the absence or presence of TAPI-1 at 10 µmol/L for 18 hours and membrane vesicles were isolated from the supernatants. Vesicles were suspended in SDS sample buffer and analyzed by Western blotting with pcytL1 followed by peroxidase-conjugated secondary antibody and ECL detection. A, analysis of CHO-hL1 cells. B, analysis of OVMz cells.

 
Hypoxic conditions lead to apoptosis of tumor cells and augment the release of soluble L1. In malignant tumors the rate of apoptosis is high in undervascularized areas (42, 43). It is known that low oxygen pressure or hypoxia can directly induce apoptosis of tumor cells (44). As shown in Fig. 4A, incubation of CHO-hL1 under hypoxic conditions led to enhanced apoptosis (from 17% to 69 %). Interestingly, we observed that the presence of TAPI increased the apoptosis rate up to 88%. Similar effects could be observed in OVMz cells. Incubation under hypoxic conditions increased the rate of apoptosis in the latter cells from 11% to 25 %. As shown in Fig. 4B, the presence of TAPI rendered OVMz cells extremely sensitive to apoptosis under hypoxic conditions (65% apoptosis). The analysis of supernatants revealed that hypoxic conditions enhanced the level of soluble L1. In CHO-hL1 (Fig. 4C) and OVMz (Fig. 4D) cells, there was an ~2.5-fold increase of soluble L1 in the supernatant. Notably, the enhanced cleavage was again blocked by TAPI-0 to basal levels (Fig. 4C and D).



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  		Fig. 4. Inhibitors of metalloproteinases affect rate of apoptosis and L1 shedding under hypoxia. CHO-hL1 (A) and OVMz cells (B) were incubated for 60 hours under normoxic and hypoxic conditions in the presence or absence of 10 µmol/L TAPI-0. The rate of apoptosis was measured by Nicoletti staining. Note that the TAPI-0 solvent DMSO did not affect the rate of apoptosis. C and D, soluble L1 was analyzed using Western blot analysis with mAb L1-11A to the ectodomain of L1 followed by peroxidase-conjugated secondary antibody and ECL detection.

 
Cleavage of L1 under apoptosis is mediated by multiple metalloproteinases. ADAM10 has been identified as an important metalloproteinase for L1 cleavage (10, 24). To examine the role in apoptosis-induced shedding, we used antisense oligonucleotides specific for ADAM10 and ADAM17. Biochemical analysis indicated that the antisense oligonucleotides diminished the expression level of the respective proteinase (Fig. 5A). The ADAM10 antisense oligonucleotide reduced the constitutive L1 shedding by 80% as evidenced by a diminished release of L1-200 (Fig. 5B) whereas antisense oligonucleotides specific for ADAM17 did not (Fig. 5B). In the presence of C2-ceramide, the effect of ADAM10-specific antisense oligonucleotides was clearly abrogated (Fig. 5C). These results confirmed the role of ADAM10 in the constitutive cleavage of L1 but suggested that under apoptosis additional metalloproteinases are activated leading to enhanced L1 release.



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  		Fig. 5. Role of ADAM10 in the constitutive and apoptosis-induced L1 shedding. A, down-regulation of ADAM10 and ADAM17 after transfection with specific antisense oligonucleotides (ASO) using antibodies to ADAM10 and ADAM17 followed by secondary antibody and ECL detection. Actin was used as loading control. B and C, OVMz cells were transfected with antisense oligonucleotides specific for ADAM10, ADAM17, or control antisense oligonucleotide and the amount of soluble L1 in the presence or absence of C2-ceramide as apoptosis inducer was analyzed. Conditioned media were analyzed by Western blot with mAb L1-11A to the ectodomain of L1 followed by peroxidase-conjugated secondary antibody and ECL detection.

 
Two types of L1-containing vesicles are released from ovarian carcinoma cells. To allow a more refined analysis of L1-containing vesicles, we used sucrose density gradient centrifugation as previously described (24). Figure 6B shows an analysis of vesicles derived from OVMz cells cultivated in the absence or presence of C2-ceramide. Vesicles containing L1 and ADAM10 were present in the middle part of the gradient and also at higher density at the bottom of the gradient. Interestingly, an additional fragment L1-28 possibly generated by further cleavage of L1-32 was also observed. Strikingly, C2-ceramide led to an increase in vesicles with higher density appearing at the bottom of the gradient. As these vesicles are enhanced under apoptotic conditions, they most likely represent apoptotic blebs. In contrast, the amount of vesicles detected in the middle part of the gradient was unaffected by C2-ceramide. These vesicles are released constitutively and might be related to exosomes. To test this assumption, the gradient fractions were analyzed with a mAb to the tetraspan CD9, an established marker for exosomes from dendritic and tumor cells (34). Indeed, as shown in Fig. 6B, the middle fractions of both gradients were positive for CD9.



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  		Fig. 6. Analysis of apoptotic vesicles by sucrose gradient fractionation. A, schematic representation of vesicle fractionation using sucrose density gradient centrifugation. B, OVMz cells in serum-free medium were cultivated for 48 hours in the absence or presence of C2-ceramide to induce apoptosis. Vesicles were isolated from the supernatant and applied onto a sucrose density gradient. Proteins from each fraction were separated by SDS-PAGE under reducing conditions (L1 and ADAM10) or nonreducing conditions (CD9) followed by Western blotting with the indicated antibodies and ECL detection. Fractions of the gradient referred to as exosomes and membrane blebs are indicated.

 
L1-containing vesicles in ascites of ovarian carcinoma patients. Next we isolated vesicles from the ascites fluids of ovarian carcinoma patients. The histologic type of the analyzed tumors is given in the legend to Fig. 7. As shown in Fig. 7A, four of five ascites contained vesicles with full-length L1-220 and were positive for soluble L1 by ELISA (Fig. 7B). As revealed by sucrose density centrifugation, the ascites-derived vesicles contained L1-28 and floated in the bottom of the gradient similar in density to apoptotic blebs (Fig. 7C). The exosome marker CD9 was predominantly present in the middle part of the gradient (Fig. 7C). Interestingly, only in vesicles derived from patient 4 the L1-32 cleavage form was present. All other ascites were devoid of L1-32 in this part of the gradient. Therefore, the ascites clearly contained two types of vesicles, one containing L1-32 being related to exosomes and the other containing L1-28 being related to apoptotic membrane blebs.



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  		Fig. 7. Analysis of ascites vesicles from ovarian carcinoma patients. A, vesicles from the ascites of ovarian carcinoma patients were dissolved in SDS sample buffer and probed with biotinylated mAb L1-11A followed by peroxidase-conjugated streptavidin and ECL detection. Patient 1, endometroid cystadenocarcinoma; patients 2 to 5, serous or papillary-serous cystadenocarcinomas. B, soluble L1 in ascites fluids was measured using a sandwich ELISA specific for soluble L1. C, pelleted vesicles were suspended in sucrose solution and applied onto a sucrose density gradient. Proteins from each fraction were separated by SDS-PAGE followed by Western blotting with the indicated antibodies and ECL detection. *, indicated protein bands were also reactive with the secondary antibody and are therefore nonspecific.

 
Soluble L1 from ascites binds to cells and triggers cell migration and extracellular signal-regulated kinase phosphorylation. We investigated the putative biological activity of soluble L1 in ascites fluids. The affinity-purified soluble L1 was found to be intact and undegraded with a size of 150 kDa (L1-150) and 200 kDa (L1-200; Fig. 8A). As shown in Fig. 8B, ascites L1 was a potent dose-dependent inducer of cell migration. The enhanced cell migration was blocked in the presence of mAbs to the {alpha}5ß1 and {alpha}vß5 integrins, but only little effect was seen with the mAb to {alpha}vß3 (Fig. 8C). To obtain evidence for the direct binding of soluble L1 to cells, HEK293 cells were incubated with purified L1 (10 µg/mL). As shown in Fig. 8D, cell surface–bound L1 was readily detected. The binding to cells was blocked in part by preincubation of cells with mAbs to the integrins {alpha}5ß1, {alpha}vß5, and {alpha}vß3, respectively, but not to {alpha}6 integrin (Fig. 8D). In agreement with previously published data (37, 45), these results suggested that L1 can bind to several integrins at the cell surface.



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  		Fig. 8. Soluble L1 from the ascites fluid triggers cell migration. A, purification of soluble L1 from the ascites fluids of ovarian carcinoma patients by affinity chromatography using L1-11A sepharose. An aliquot of purified L1 was separated by SDS-PAGE. The membrane was stained with amido black to visualize protein and L1 was detected by mAb L1-11A followed by peroxidase-conjugated secondary antibody and ECL detection. B,soluble L1 at the indicated concentrations was added to HEK293 cells and haptotactic migration on fibronectin was examined. The experiment was done thrice in quadruplicates with similar results; a representative study is shown. *, P = 0.0038. C, HEK293 cells stimulated with soluble L1 were preincubated with the indicated mAbs at 10 µg/mL and haptotactic migration on fibronectin was examined. The mAbs were present during the assay time. The experiment was done thrice in quadruplicates with similar results; a representative study is shown. D, binding of soluble L1 to HEK293 cells in the absence or presence of the indicated mAbs to integrins (10 µg/mL). Bound L1 was detected with biotinylated mAb L1-11A followed by streptavidin-conjugated phycoerythrin. E, HEK293 cells were starved for 30 minutes under serum-free conditions and then stimulated with soluble L1 (10 µg/mL) for the indicated length of time with or without the addition of 5% fetal bovine serum before harvest to assess ERK activation. The experiments were done thrice with similar results; a representative study is shown.

 
Binding to {alpha}v integrins can induce activation of the mitogen-activated protein kinase pathway (46). Indeed, when added to HEK293 cells, the soluble L1 led to a transient stimulation of ERK phosphorylation (Fig. 8E).


   Discussion
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 Materials and Methods
 Results
 Discussion
References
 
Serum and ascites of ovarian carcinoma patients accumulate significant amounts of soluble L1 released from tumor cells. In a previous work using L1-transfected CHO and HEK293 cells, we observed that soluble L1 was released either directly from the cell surface or in secretory membrane vesicles in which subsequent cleavage occurred. In the present report we provide evidence that the latter mechanism of L1 release seems to be operative in ovarian carcinoma cells both in vitro and in vivo. We show that the release of L1 occurs in two types of vesicles: exosomes, which are released constitutively, and apoptotic membrane vesicles, which are increased in the presence of apoptotic stimuli.

The release of vesicles from living cells is considered to be a normal physiologic process (47, 48). It is particularly active in proliferating cells, such as cancer cells, where it can occur continuously. Here we show that cancer cells undergoing apoptosis augment the release of L1. In CHO-hL1 cells the released L1 was heterogeneous in size but was more homogeneous in OVMz cells. The degree of induction of L1 release in response to apoptotic stimuli was higher in CHO-hL1 when compared with ovarian carcinoma cells. This could be related to differences in the mode of L1 release. We observed that in CHO-hL1 cells the cleavage of L1 proceeds both at the cell surface and in released vesicles whereas in OVMz cells we observed mainly cleavage in vesicles.5 The vesicles released from OVMz cells could be divided into two major fractions: (a) spontaneously released vesicles that contained ADAM10, the cleavage fragment L1-32, and the tetraspan CD9. These vesicles are most likely related to exosomes; (b) a second fraction that was increased following apoptotic stimuli and that contained L1-32 and ADAM10 but was nearly negative for CD9. This pool contains most likely apoptotic membrane blebs. The formation of such apoptotic blebs containing ADAM10 and L1 could be shown by fluorescence microscopy. The observation that L1 and ADAM10 were seen in exosomes deserves a further notion. We previously reported that predominantly the neural form of L1 containing the RSLE form is recruited in spontaneously released vesicles (24). The RSLE motif facilitates interaction with the µ2 chain of the clathrin adaptor AP-2 and affects the phosphorylation and endocytosis of L1 by creating an endocytic motif allowing clathrin-mediated internalization (49, 50). Indeed, we found by reverse transcription-PCR that OVMz cells expressed the neural form of L1.6

Using antisense oligonucleotides specific for ADAM10 and ADAM17, we observed that under constitutive conditions ADAM10 was the dominant proteinase involved in L1 release whereas ADAM17 was not involved. This is consistent with our previous results showing that a dominant-negative ADAM10 construct could block the constitutive cleavage of L1 in several cell lines such as AR carcinoma cells, CHO cells, and HEK293 cells (10, 24). Under apoptotic conditions, the effect of ADAM10-specific antisense oligonucleotides was abrogated, indicating that besides ADAM10, other metalloproteinases can cleave L1. This is in agreement with a recent study showing that dying cells undergo a stress response leading to an up-regulated cleavage of the EGF receptor ligand HB-EGF involving multiple ADAMs (51).

L1-containing vesicles were also observed in vivo in the ascites of ovarian carcinoma patients. Although the number of patients studied is still small, several observations could be made: (a) in all patients investigated soluble L1 was accompanied by L1-containing vesicles; (b) ascites vesicles had similar density features as in vitro derived vesicles and could again be divided into a CD9-positive exosomal fraction and into a CD9 low containing fraction with a density similar to apoptotic blebs; (c) only in one ascites the L1-32 was detected in the CD9-positive exosomal fractions whereas the L1-28 fragment was present in the membrane bleb fraction. We suspect that the L1-28 fragment is a degradation product derived from L1-32 that could well be generated during vesicle isolation. A similar form was also seen in OVMz vesicles, ruling out the possibility that this fragment occurred only in vivo. (d) L1-containing vesicles and soluble L1 in the ascites fluid were variable in amount and composition between individual patients. This could be related to the individual status of the tumor and the treatment history of the patients. Apoptotic conditions in ascites could be caused by therapy or hypoxia leading to apoptotic vesicles. We conclude that the presence of vesicles in the ascites containing L1-220 and the cleavage products L1-32 and L1-28 strongly suggest that cleavage in released vesicles can proceed also in vivo. It is quite possible that soluble L1 in the serum and ascites fluid of carcinoma patients at least in part is generated by ADAM10. This is supported by the observation that L1-32, the product of ADAM10 cleavage, is present in ovarian carcinoma cell lysates and that ADAM10 is expressed in nearly all advanced tumors where soluble L1 was detected (9).

It is well established that tumor cells use proteolytic activity to modify their environment, which often includes tumor cell surface molecules (51). An intriguing question is whether the generation of soluble L1 is a mere byproduct or of advantage for the tumor. In experimental studies an important link was found between cell migration and L1 shedding: the enhanced migration of L1-expressing cells was dependent on metalloproteinase activity and the released L1 ectodomain was able to stimulate migration through autocrine/paracrine binding to {alpha}vß5 integrins (10). In the present study we show that released L1 remains intact and can be purified from ascites fluid. Purified L1 bound to cells via integrins, triggered cell motility on extracellular matrix proteins, and induced the phosphorylation of ERK. It is well possible that the migration of ovarian carcinomas towards components of the extracellular matrix is driven by L1 and other shed molecules. Not only the expression of L1 but also the proteolytic machinery for ectodomain shedding is therefore of paramount importance for tumor cells. Silletti et al. (52) reported recently that in L1-positive compared with L1-negative cells a sustained ERK phosphorylation was observed. The expression of L1 up-regulated mRNAs for the motility-related proteins rac and rho and a variety of other gene products including the ß3-integrin chain and the proteases B- and L-cathepsin (52). Thus, beyond the augmentation of cell motility, L1 expression may have other important effects such as the induction of a more invasive phenotype that could account for its association with bad prognosis. Whether or not soluble L1 plays a role in gene regulation remains to be investigated.

Recent analysis has shown that lysophosphatidic acid is present in high concentration in ascites and plasma of ovarian cancer patients, which stimulates cell migration via a Ras-mitogen-activated protein/ERK kinase pathway (53), induces the ectodomain shedding of HB-EGF (54), and causes transcriptional activity of the IL-8 gene (55). This work is related to our study as similar to L1, HB-EGF is cleaved by ADAM10 (30, 56). Lysophosphatidic acid could therefore well act as an upstream inducer of L1 shedding and allow the release of both growth factor receptor ligands (HB-EGF) as well as integrin ligands (L1) that might cooperate to ensure tumor cell growth and motility.

In summary, our results underline the important role of secretory vesicles in the constitutive release of L1 from carcinoma cells both in vitro and in vivo. The data suggest that ADAM10 has a dominant role in this process. We propose that the release via vesicles and the subsequent cleavage could be an important pathway of L1 release and that of other cell surface molecules. Soluble L1 could regulate tumor cell function in an autocrine/paracrine fashion.


   Acknowledgments
 
We thank Caroline Marth and Alexander Strecker for excellent technical assistance and Dr. Eric Rubinstein (Villejuif Cedex, France) for the gift of CD9 antibody.


   Footnotes
 
Grant support: Deutsche Krebshilfe grant 10-1307-3Al (P. Altevogt).

Note: P. Gutwein and A. Stoeck contributed equally to this work.

The costs of publication of this article defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

5 S. Riedle, unpublished observations. Back

6 A. Stoeck, unpublished observations. Back

Received 8/24/04; revised 10/13/04; accepted 12/ 7/04.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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