[CANCER RESEARCH 59, 234 –240, January 1, 1999]
Sialidase Gene Transfection Enhances Epidermal Growth Factor Receptor Activity in an Epidermoid Carcinoma Cell Line, A4311 Emmanuelle J. Meuillet, Roger Kroes, Hirotaka Yamamoto, Thomas G. Warner, Jeffry Ferrari, Barbara Mania-Farnell, David George, Abdelhadi Rebbaa, Joseph R. Moskal, and Eric G. Bremer2 The Brain Tumor Research Program, Children’s Memorial Hospital and The Chicago Institute for Neurosurgery and Neuroresearch, Chicago, Illinois 60614 [E. J. M., R. K., H. Y., B. M-F., D. G., H. R., J. R. M., E. G. B.], and Genentech, Inc., South San Francisco, California 94080 [T. G. W., J. F.]
ABSTRACT Glycosphingolipids expressed in cancer cells have been implicated in the modulation of tumor cell growth through their interaction with transmembrane signaling molecules such as growth factor receptors. For glycosphingolipids to interact with growth factor receptors, the presence of sialic acid seems to be essential. Stable transfection of a gene encoding a soluble Mr 42,000 sialidase into a human epidermoid carcinoma cell line (A431) provided an approach by which the level of terminal lipid-bound sialic acid on the cell surface could be altered. In the sialidase-positive clones, the level of ganglioside GM3 was diminished, and little change was observed in protein sialylation. Sialidase-transfected cells grew faster than control cells. Sialidase expression did not modify the binding of epidermal growth factor (EGF) to its receptor but enhanced EGF receptor (EGFR) tyrosine autophosphorylation as compared to that of parental cells or cells transfected with the vector (pcDNA3) alone. Moreover, the phosphorylation of the EGFR, as well as other protein substrates, was observed at low EGF concentrations, suggesting an increase in the receptor kinase sensitivity. These data provided evidence that changes in ganglioside expression in cancer cells by appropriate gene transfection can dramatically affect EGFR kinase activity. Hence, the modulation of ganglioside expression may represent an approach to alter tumor cell growth.
INTRODUCTION Changes in GSL3 expression in cancer cells have been correlated with changes in cell proliferation, migration, and adhesion (1). Gangliosides are sialic acid-containing GSLs that are generally localized on the outer leaflet of the plasma membrane in mammalian cells (2). Gangliosides and sphingolipids have been described to affect transmembrane signaling essential for tumor cell growth, invasion, and metastasis. For example, gangliosides have been shown to modulate the growth rate and cell phenotype of a murine primitive neuroectodermal tumor (3). One hypothesis is that gangliosides may modulate GFR signaling (4). GFRs are often overexpressed in cancer cells. For instance, overexpression and autocrine activation of EGFR have been demonstrated to cause transformation of cultured cells and correlate with tumor progression in cancer patients (5, 6). Therefore, changes in GSL composition could contribute to the transformed phenotype by their influence on GFRs. To better understand the role of GSLs in cell growth and the effects of ganglioside interaction with GFR function, several different apReceived 7/27/98; accepted 10/29/98. The costs of publication of this article were 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. 1 Supported in part by a grant from the Falk Foundation and NIH Grant NS33383 (to E. G. B.). E. J. M. has been supported by postdoctoral fellowships from AFRP (Foundation for Retinitis Pigmentosa, France) and by the Gus Foundation (Chicago, IL). 2 To whom requests for reprints should be addressed, at Brain Tumor Research Program, Children’s Memorial Institute for Education and Research - Neurobiology, Children’s Memorial Medical Center, 2300 Children’s Plaza M/C 226, Chicago, IL 60614. Phone: (773) 868-8082; Fax: (773) 868-8066; E-mail:
[email protected]. 3 The abbreviations used are: GSL, glycosphingolipid; 4-MU, 4-methyl umbelliferone; ECL, enhanced chemiluminescence; EGF, epidermal growth factor; EGFR, EGF receptor; GFR, growth factor receptor; PNA, peanut agglutinin; MAL-II, Macckia amurensis lectin II; PDMP, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol; DOTAP, N-[1(2,3-dioleoyloxyl)propyl]-N,N,N-trimethylammoniummethyl sulfate; FBS, fetal bovine serum; PVDF, polyvinylidene difluoride; TLC, thin-layer chromatography.
proaches have been used to alter GSL levels. One approach has been to examine cell growth in the presence and absence of exogenously added gangliosides. For instance, Bremer et al. (7) reported that GM3 inhibits the growth of cultured cells and regulates EGFR function. Gangliosides have been shown to modulate several additional GFR systems. GM3 inhibits basic fibroblast GFR autophosphorylation (7, 8), GM1 inhibits platelet-derived GFR (9, 10), and GT1b stimulates Trk B (nerve GFR; Ref. 11). In retinal glial cells, GM3 differentially alters the autophosphorylation of both EGFR and basic fibroblast GFR, thereby affecting the corresponding signaling pathways (12). These studies suggest that ganglioside modulation of GFR function by gene transfection may represent a general phenomenon, and specific gangliosides can interact with specific receptors. A second approach to modulate GSL content is to add inhibitors or differentiating inducers of GSL metabolism. For example, the use of the ceramide analogue PDMP, which inhibits UDPglucose-ceramide glucosyltransferase, induced a large depletion in GSLs and reduced the synthesis of all GSLs derived from glucosylceramide (13). PDMP has been used extensively to evaluate the role of GSLs in tumor growth and metastasis (13, 14). For example, the ability of murine lung carcinoma cells to invade reconstituted basement membranes in vitro was reduced in the presence of PDMP. The reduction in invasiveness correlated with the degree of GSL depletion. These results suggested that GSLs in tumor cell membranes are essential for the metastatic spread of tumor cells through the basement membranes (14). Another study demonstrated that exogenously added neuraminidase converted GM3 into lactosylceramide and increased cell proliferation in human skin fibroblasts (15, 16). The authors suggested that endogenous regulation of GM3 content might release the cells from inhibition by the tyrosine kinase activity of the EGFR and enable prereplicative mechanisms to occur. Treatment of A431 cells with endoglycoceramidase, which causes the hydrolysis of cell surface GSLs, also suggested that GM3 may be an important constituent for EGFR autophosphorylation and signaling (17). The removal of sugar chains from GSLs reduced EGF-dependent EGFR phosphorylation in the cells, confirming that endogenous gangliosides, possibly GM3, might be one of the integral constituents supporting EGFR phosphorylation. A third approach, the transfection of enzymes involved in ganglioside biosynthesis, may be used to test the possibility of ganglioside involvement in cell proliferation. For example, it was reported that the transfection of GD3 synthase cDNA into Neuro2a cells, a neuroblastoma cell line, caused cell differentiation with neurite sprouting (18). The treatment of the human promyelocytic leukemia cell line HL-60 with antisense oligodeoxynucleotide to UDP-N-acetylgalactosamine: b-1,4-N-acetylgalactosaminyl-transferase (GM2-synthase) and CMPsialic acid:a-2,8-sialyltransferase (GD3-synthase) sequences increased the content of GM3 concomitantly with a decrease in more complex gangliosides (19). In another example, Tokuyama et al. (20) reported a marked suppression of metastasis by B16 murine melanoma cells transfected with a sialidase gene. This sialidase reduced ganglioside content without affecting glycoprotein sialylation, suggesting a role for gangliosides in the formation of metastasis.
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In this study, to investigate the effects of the endogenous ganglioside levels on EGFR function, we transfected a sialidase gene into the A431 cell line. This gene, which was recently purified and cloned (21, 22), encodes a cytosolic enzyme and is similar to that used by Tokuyama et al. (20). Several lines of evidence have suggested that GM3 interacts with EGFR and thereby inhibits its kinase activity (7). In this study, we modulated the global ganglioside content (especially GM3) by transfection of the sialidase gene. We suggest that the observed increase in cell proliferation may be due, in part, to the activation of EGFR as a result of decreased GM3. Because overexpression of GSLs is considered to be an important contributor to tumorigenesis, our data may provide a possible explanation for their role in tumor biology via the modulation of GFR responses.
Detection of Sialidase Protein by Western Blot. Cells were seeded in 6-well plates containing DMEM and 10% FBS. When the cells were near confluence, monolayers were washed twice with cold PBS and solubilized by the addition of 200 ml of lysis buffer [50 mM HEPES (pH 7.4), 150 mM NaCl, 100 mM NaF, 1 mM MgCl2, 1.5 mM EGTA, and 1% Triton X-100]. Protein levels were quantified by the method of Bradford (23) using BSA as a standard. Protein (40 mg) was applied to a 7.5% polyacrylamide gel. Western blots were performed as described previously (24). The membrane was incubated with antisialidase serum generated in a rabbit for 1 h. The polyclonal antibody was generated, and the IgG fraction was purified by protein A column chromatography as described by Warner et al. (21). Secondary antibodies coupled to peroxidase allowed the detection of the sialidase protein using ECL reagents. Sialidase Activity. Measurement of sialidase activity was carried out by the fluorescence of 4-MU. This compound is released by the sialidase at pH 6.5 after a 20-min incubation with 4-MU-5-neuraminic acid substrate (21, 22). Briefly, cells were prepared as described above. Monolayers were lysed in MATERIALS AND METHODS lysis buffer (20 mM Tris, 1% Triton X-100, 137 mM NaCl, and 1 mM Na3VO4). Reagents. 125I-labeled EGF (specific activity, 6640 kBq/mg; 179 mCi/mg), The lysates (40 ml) were incubated at 37°C for 15 min in an extraction buffer horseradish-linked peroxidase antibodies, and the ECL detection kit were (pH 6.5). Carbonate buffer was added to stop the reaction, and the fluorescence obtained from Amersham (Arlington Heights, IL). pcDNA3 expression vector was measured with a fluorometer (DyNAquant 200; Hoefer). Measurement for was obtained from Invitrogen (San Diego, CA). Pyrococcus furiosus DNA lysosomal sialidase activity was also performed using the same conditions, except at pH 4. A specific sialidase inhibitor, 2,3-dehydro-3-deoxy-N-acetylpolymerase, Duralon nylon membrane, QuikHyb solution, and the random priming kit were purchased from Stratagene (La Jolla, CA). DOTAP was neuraminic acid (80 mM), was incubated for 24 h with the cells to determine the obtained from Boehringer Mannheim (Indianapolis, IN). FBS, Geneticin specificity of the sialidase activity assay. Glycosylation State of Whole Cell Extracts and EGFR. Cells were (G418; antibiotic), and DMEM were purchased from Life Technologies, Inc. 2 (Grand Island, NY). Antiphosphotyrosine (monoclonal antibody; clone 4G10) cultivated in 75-cm flasks and washed once in PBS. Monolayers were lysed in lysis buffer as described above. Protein (50 mg) was applied to a 7.5% antibodies were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). X-OMAT films were obtained from Kodak (Rochester, NY). PNA and polyacrylamide gel. After electrotransfer to a PVDF membrane, nonspecific Mal-II were obtained from Vector Laboratories (Burlingame, CA). 4-MU, sites were blocked by incubation in 3% BSA for 30 min at room temperature. 4-MU-5-neuraminic acid, protein A-agarose, monoclonal mouse anti-EGFR The membrane was washed three times with PBS containing 0.2% Tween-20 antibodies (clone F4), and orcinol were obtained from Sigma (St. Louis, MO). and incubated for 1 h at room temperature in the same buffer containing 2 PVDF membrane was purchased from Millipore (Bedford, MA). All other mg/ml biotinylated PNA or biotinylated MAL-II in PBS supplemented with MgCl2 (1 mM) and CaCl2 (1 mM). After washing, the membrane was incubated chemicals and reagents were of analytical grade. Cell Culture. Cells were grown in 75-cm2 plastic tissue culture flasks or in for 45 min with streptavidin peroxidase-coupled antibodies, and the sialic multiwell plates for all experiments in DMEM supplemented with 10% heat- acid-containing proteins were detected by ECL reagents. Changes in EGFR glycosylation were analyzed by immunoprecipitation of inactivated FBS, 4.5 g/liter glucose, 100 units/ml penicillin, and 100 mg/ml the receptor and detection of sialic acid residues on the receptor. Cell lysates streptomycin under a 5% CO2 atmosphere at 37°C. Transfections. A431 human epidermoid carcinoma cells were transfected (500 mg) were incubated at room temperature for 2 h with 5 mg of anti-EGFR with a 1.2-kb sialidase cDNA. For the production of stable transfectants of antibody complexed with rabbit antimouse IgG and protein A-agarose. After washing the pellet, 30 ml of Laemmli buffer (25) were added, the mixture was Chinese hamster ovary sialidase in A431 cells, a 1.2-kb cDNA was inserted into the pcDNA3 expression vector at the BamHI and XbaI sites. The 1.2-kb boiled for 5 min, and the proteins in the supernatant were separated by sialidase cDNA (22) was amplified by the PCR with Pyrococcus furiosus DNA SDS-PAGE. Sialic structures on the precipitated EGFR were detected by polymerase. The sense primer contained an artificial BamHI site at the 59 end incubation with biotinylated PNA or MAL-II. Reactive sialic acid residues on (59-AGGATCCCATGGCGACTTGCCCTGTC-39; bp 184 –204), and the an- EGFR were detected with streptavidin peroxidase-coupled antibodies and ECL tisense primer contained an artificial XbaI site at the 39 end (39-TCTAGAAG- reagents. Ganglioside Analysis. For ganglioside analysis, confluent cell monolayers CACTTTGGGCCGCATGC-59; bp 1354 to 1333). The amplified DNA was digested with BamHI and XbaI, separated by agarose gel electrophoresis, and grown in 225-cm2 flasks were rinsed three times in PBS. Equal amounts of ligated into pcDNA3. The orientation of the cDNA insert was confirmed by protein were pooled together for a total protein content of 6 mg/clone. GanDNA sequencing. The pcDNA3/sialidase construct or pcDNA3 alone as a gliosides were extracted in chloroform/methanol [chloroform/methanol, 1:1 control was then transfected into the cells using a cationic liposome system, (v/v)] according to methods described previously (26). These extracts were run DOTAP. Putative transfectants were selected by antibiotic resistance in cell on a TLC plate in a chloroform/methanol/CaCl2 (55:45:9, v/v) solvent system medium containing 500 mg/ml G418. After 4 weeks in culture in the presence (27). The CaCl2 used was a 0.2% solution (g/v). Gangliosides and other GSLs of G418, the surviving clones were tested for the presence of sialidase mRNA were visualized with orcinol spray reagent (28). Cell Proliferation Assay. Cells were seeded into 24-well plates at 104 and sialidase protein expression. All experiments were performed near the end cells/well in DMEM and 10% FBS. Cells were counted every day in a of the exponential phase of growth. The results of all experiments were hemocytometer. Briefly, the cells were washed in PBS. Monolayers were normalized according to the protein content of the cell extracts. Detection of Sialidase mRNA in Transfectants. Northern analysis was trypsinized for 2 min and transferred into Eppendorf tubes. Aliquots (10 ml) of the cells were counted. Each day represents a measure of three different wells. performed to detect the expression of sialidase mRNA in the transfectants. Briefly, cells were seeded in 100-mm Petri dishes containing DMEM supple- The data represent the values of three separate experiments. Determination of 125I-labeled EGF Binding Activity. Measurement of mented with 10% FBS. When cells were near confluence, monolayers were EGFR binding was determined by competition displacement curves as dewashed with cold PBS. Total RNA was isolated from parental A431 cells and transfectants using isothiocyanate followed by CsCl2 centrifugation. Twenty scribed previously (12). All points were determined in triplicate for a minimum mg of total RNA per lane were electrophoresed in a 1% formaldehyde-agarose of three separate experiments. Determination of EGFR Autophosphorylation. Cells were seeded into gel and transferred to Duralon nylon membranes. After UV cross-linking, blots were hybridized with a 32P-radiolabeled sialidase cDNA probe synthesized 12-well plates in DMEM and 10% FBS for 24 h and transferred to DMEM with a random priming kit and QuikHyb solution. After washing at 60°C, the overnight. Cells were then stimulated with EGF (10 nM) for different times or blot was exposed to Kodak X-OMAT film for 16 h, and the film was with different concentrations of EGF for 10 min. The incubations were stopped by aspirating the medium and extracting proteins in a lysis buffer (20 mM Tris, developed. 235
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1% Triton X-100, 137 mM NaCl, and 1 mM Na3VO4). After protein determination, Laemmli sample buffer (25) was added. Lysates were boiled for 5 min and analyzed by electrophoresis on a 7.5% SDS-PAGE. Proteins were electrophoretically transferred to nitrocellulose membranes, preincubated in binding buffer (PBS and 5% nonfat dry milk), and incubated with antiphosphotyrosine antibodies (clone 4G10; 400 ng/ml). Immunoreactive bands were detected using antimouse antibodies coupled to peroxidase, the ECL system, and Kodak film.
RESULTS Characterization of Sialidase-transfected Cells Sialidase Gene Transfection. A431 cells were transfected with a hamster sialidase gene using a cationic liposome system (DOTAP). After transfection and selection in G418 media, RNA from individual clones was extracted as described in “Materials and Methods.” Total RNA was run on an agarose gel to verify RNA quality (Fig. 1A). By Northern blot, a 1.2-kb transcript was detected in several sialidasetransfected cells (Fig. 1B). Thirty-one clones were tested for the expression of sialidase mRNA, and 12 clones were sialidase mRNApositive. These 12 clones, labeled 1 to 12, were considered for further analysis. Expression of Sialidase Activity in the mRNA-positive Clones. The expression of sialidase protein and its activity were examined in the 12 positive clones chosen by Northern blot. Sialidase activity was measured in parental cells (A431), in pcDNA3-transfected cells (A431/pcDNA3), and in cells transfected with the sialidase gene. Among the 12 selected clones, enhanced sialidase activity at pH 6.5 was detected in clones 1, 4, 5, 7, 9, and 10 (Fig. 2, p). There was no change in lysosomal sialidase activity (which was measured at pH 4) in any of the clones tested (data not shown). A specific sialidase inhibitor, 2,3-dehydro-3-deoxy-N-acetylneuraminic acid (80 mM), was also incubated for 24 h with the cells to determine the specificity of the sialidase activity assay. A strong inhibition of the enhanced sialidase activity, which was present in all of the positive clones, was observed as compared to nontreated cells (data not shown). The expression of the sialidase protein was also checked by Western blot. A Mr 42,000 band corresponding to the sialidase protein was detected in the same clones that showed increased pH 6.5 sialidase activity (data not shown). Ganglioside Composition of A431, pcDNA3, and Sialidasetransfected Cells. Sialidase is a cytosolic enzyme that has been reported to act on GSLs and modulate lipid-bound sialic acid content
Fig. 2. Expression of sialidase activity in A431, pcDNA3-, and sialidase-transfected cells. Sialidase activity was measured in parental cells (A431, s), pcDNA3-transfected cells (M), and the 12 positives clones using a fluorescent probe (4-MU-N-acetyl-neuraminic acid). Among the 12 clones expressing the message for the protein, only 6 showed enhanced protein activity (p). Clones and control that are negative for the activity are represented by M. The data presented on each clone are the average of five separate experiments (each done in quadruplicate) 6 SD. P , 0.05 (pp) and P , 0.1 (p) by Student’s t test when compared to parental cells (A431).
in whole cells (21). In this study, ganglioside composition was determined in parental cells (A431), pcDNA3-transfected cells, and sialidase-transfected cells (six positive clones). Fig. 3, left panel, Lane 2 shows the ganglioside content in parental cells (A431). GM3 is the major ganglioside in this cell line as described previously by Barbour et al. (29) and as compared to Lanes 1 and 9, which contain 5 and 10 mg, respectively, of GM3 and GM1 and GT1b standards (std). Ganglioside content was not changed in pcDNA3-transfected cells (Lane 3) as compared to parental cells. However, clones 1, 4, 5, 7, and 10 showed a diminution of GM3. The right panel of Fig. 3 shows another TLC plate. Clones 7 and 9 showed a great diminution in GM3 content as compared to pcDNA3-transfected cells. Other orcinol-positive bands were detected with migrations similar to that of the GM1 and GT1b standards. The identity of these bands is unknown. They may correspond to neutral GSLs increased upon sialidase overexpression.
Fig. 1. Screening of A431/sialidase-transfected cells by Northern blot. A, RNA was extracted and run on a 3% agarose gel. rRNAs were detected at 18S and 28S as indicated by the arrows. B, selected clones (30) were checked for their ability to express sialidase mRNA. A band at 1.2 kb was detected on the membrane (the arrow indicates the mRNA). Among the clones tested, 12 expressed sialidase mRNA (numbers below the panel).
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MAL-II binding was observed on EGFR from parental cells (A431), pcDNA3-transfected cells, or sialidase-transfected cells (data not shown). The EGFR from A431 cells appeared to contain very little a2,3-linked sialic acid, which is consistent with previously published reports (32, 33). Fig. 3. Ganglioside analysis of sialidase-transfected cells. Gangliosides were extracted according to the protocol described in “Materials and Methods.” Gangliosides were run on a TLC plate in a chloroform/methanol/CaCl2 solvent [55:45:9 (v/v)] and revealed by orcinol stain. The horizontal arrows indicate the migration of standards. A mixture of GM3 and GM1 and GT1b standards (5 mg) was run in Lanes 1 and 9. GM3 standard was run in Lane 10 (10 mg). Clones 1, 4, 5, 7, 9, and 10 showed decreases in GM3 content as compared to parental cells (A431) or pcDNA3-transfected cells.
Cell Growth Behavior of Sialidase-transfected Cells Exogenous GM3 has already been demonstrated to inhibit cell proliferation in A431 cells (7). Hence, when diminishing the total GM3 content in the same cells, an increase in cellular growth is expected. Fig. 5 shows the cell growth curves for parental cells and pcDNA3 (average of three clones)- and sialidase-transfected cells (an average of six clones). Growth curves were plotted for 5 days in culture. Sialidase-transfected cells grew significantly faster than either parental cells or pcDNA3-transfected cells. Effects of Sialidase on EGFR Function
Fig. 4. Lectin binding to sialidase-transfected glycoproteins. This figure illustrates the glycosylation pattern of whole cell extracts from A431 cells (control, C) and pcDNA3 (vector only-transfected cells, PC)- and sialidase-transfected cells (clones 1, 4, 5, 7, 9, and 10). Whole cell extracts were run on a 7.5% SDS-PAGE, and Western blots were probed with PNA (A) and MAL-II (B). No significant differences were observed when comparing the glycosylation of the proteins obtained from sialidase-transfected cells to parental cells (C) or pcDNA3-transfected cells (PC).
Effect of Sialidase on EGF Binding. Exogenously added GM3 has been shown to inhibit EGFR signaling in A431 cells (7). These data suggested that GM3 depletion may enhance EGFR activity. The first step through which EGF initiates cellular activity is by binding to its receptor. The increased cell proliferation due to sialidase overexpression could result from an effect of this enzyme on the EGFR ability to bind its natural ligand. To examine this point, specific 125 I-labeled EGF binding was determined for parental cells, pcDNA3transfected cells (an average of three clones), and sialidase-transfected cells (an average of six clones). Competition binding curves were performed for 4 h at room temperature, allowing for time to reach equilibrium (Fig. 6). The curves obtained were similar and, after Scatchard analysis (34), did not reveal any modification in the binding parameters. Sialidase overexpression had no effect on EGF binding to the receptor. Effect of Sialidase on EGFR Autophosphorylation. EGFR autophosphorylation in A431 cells has previously been shown to be inhibited by exogenous GM3 (7, 8). Hence, diminution of endogenous GM3 by overexpression of sialidase activity in sialidase-transfected
Overall, the overexpression of sialidase altered ganglioside distribution primarily by reducing the amount of GM3. Sialylation State of Whole Cell Extracts and EGFR Was Unchanged in Sialidase-transfected Cells. The activity of sialidase on glycoproteins obtained from whole cell extracts was also checked to determine and verify the specificity of the transfected sialidase. Total cell lysates were run on a 7.5% SDS-PAGE, transferred to a PVDF membrane, and probed with MAL-II. This lectin recognizes a broad spectrum of glycosylated proteins and is specific for a-2,3-linked sialic acid (30). With this lectin, no significant binding was observed in the molecular weight range for the EGFR (Mr 180,000), suggesting very little sialylation of the receptor in A431 cells (Fig. 4B). The major MAL-II bands at Mr 120,000 and Mr 80,000 remained in the transfectants. Other minor differences were noted in MAL-II binding between the transfectant and the controls, but they were not consistent with the expression of sialidase activity. A similar pattern was observed for PNA that binds galactosyl-b-1,3-N-acetylgalactosamine (31), a common substrate for a2,3-linked sialic acid. These data suggest that additional PNA binding sites were not revealed by the presence of increased sialidase activity (Fig. 4A). Because EGFR was not detected on the lectin blots, we examined lectin binding to the immunoprecipitated EGFR. The receptor was purified by immunoprecipitation and by visual examination, and no
Fig. 5. Cell proliferation of sialidase-transfected cells. Cells were seeded in 24-well plates and counted daily. The pcDNA3 curve (E) is an average of three different pcDNA3-transfected cells. The curve with ƒ is an average of six sialidase-positive clones. Sialidase overexpression enhanced cell proliferation. The effects of sialidase overexpression were observed after 3 days. The graph is an average of four separate experiments, each of which was done in triplicate. p, P , 0.05 when compared to parental cells. Data represent the means, and bars represent SEs.
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Fig. 6. Sialidase effect on 125I-labeled EGF binding to the EGFR. Competition binding curves were obtained with increasing cold EGF concentrations. The pcDNA3 curve (F) is an average of three pcDNA3 clones. The sialidase curve (ƒ) is an average of the six positive clones. No significant differences were observed when comparing the binding of 125 I-labeled EGF at the surface of sialidase-transfected cells (ƒ) to that of parental cells (A431, E) or pcDNA3-transfected cells (pcDNA3, F). Data are the means, and bars represent SEs.
cells could activate EGFR autophosphorylation. To test this hypothesis, EGFR autophosphorylation was analyzed in parental cells and pcDNA3- and sialidase-transfected cells. Sialidase-transfected cells expressed the same amount of EGFR protein as compared to parental
or pcDNA3-transfected cells, as verified on Western blots probed with anti-EGFR antibodies (clone F4; data not shown). Briefly, cells were incubated with 10 nM EGF for 10 min. Tyrosinephosphorylated proteins were visualized on Western blots with specific antiphosphotyrosine antibodies (clone 4G10). EGF stimulated the phosphorylation of a major band at Mr ;180,000, consistent with the EGFR (Fig. 7A, arrow). The phosphorylation state of EGFR was enhanced in sialidase-transfected cells as compared to control or pcDNA3-transfected cells. EGFR autophosphorylation was greatly increased in clones 5, 7, 9, and 10 (Fig. 7). Densitometric analysis of Western blots is illustrated in Fig. 7B. Data are the means from approximately 10 different experiments. In clones 5, 7, 9, and 10, EGFR autophosphorylation was significantly increased (i.e., P 5 0.015 for clone 9 and P 5 0.05 for clone 7) as compared to parental cells. EGFR autophosphorylation was not affected in pcDNA3-transfected cells as compared to parental cells (P 5 0.8). EGFR autophosphorylation was not affected by the overexpression of the sialidase in clones 1 and 4 (data not shown for clone 4). Clones 1 and 4 did express high enzyme activity (Fig. 2) and decreased ganglioside content (Fig. 3). Sialidase-transfected cells showed increased autophosphorylation in response to EGF and at lower doses of EGF (0.01 and 0.1 nM) as compared to control and pcDNA3-transfected cells (1–10 nM; Fig. 7C). For example, clone 9 showed a higher EGFR autophosphorylation state as compared to control cells (A431), even at lower doses of EGF. Upon EGF stimulation, the binding of the ligand leads to the activation of the tyrosine kinase and, through a cascade of transductional events, allows cell proliferation and differentiation (35). Numerous intracellular substrates become phosphorylated on tyrosine residues. Fig. 7C shows a panel of proteins phosphorylated with increasing doses of EGF. In particular, note that the bands at Mr
Fig. 7. Effects of sialidase transfection on EGFR autophosphorylation. A, sialidase activity altered EGFR autophosphorylation. Cells were stimulated for 10 min in the presence of 10 nM EGF and lysed, and phosphotyrosine-containing proteins were detected with antiphosphotyrosine antibodies (clone 4G10; Upstate Biotechnology, Inc.). A band at Mr 175,000 was consistent with EGFR. EGFR autophosphorylation was increased in sialidase-transfected cells (clones 5, 7, 9, and 10) as compared to control cells (A431) or pcDNA3-transfected cells (pcDNA3). B, the graph shows the relative EGFR autophosphorylation state from vector only-transfected cells (an average of three clones; pcDNA3) and sialidase-transfected cells (clones 1, 5, 7, 9, and 10) as compared to the autophosphorylation of the receptor in parental cells (A431) upon stimulation with EGF. A significant increase in EGFR autophosphorylation in clones 5, 7, 9, and 10 was observed as compared to parental cells (A431) or pcDNA3-transfected cells. The graph represents the average of 10 different experiments. P , 0.1 (p) and P , 0.05 (pp) according to the parametric Student’s t test and when compared to pcDNA3-transfected cells. Data represent the means, and the bars represent SEs. C, tyrosine phosphorylation in parental cells and sialidase-transfected cells (clone 9) upon EGF stimulation. Cells have a basal phosphorylation state (Lane NS, nonstimulated A431 cells). A dose curve for the phosphorylation of EGFR and its substrates was performed by a 10-min stimulation with increasing doses of EGF. Note the increased EGFR autophosphorylation state at low concentrations of EGF (0.01 and 0.1 nM) in clone 9 as compared to control cells (maximum observed at 1 and 10 nM). Some additional substrates also appeared to be phosphorylated with lower doses of EGF stimulation as compared to control cells.
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84,000 and Mr 59,000 required lower concentrations of EGF to be phosphorylated in clone 9 as compared to parental cells. These data are consistent with the observation that overexpression of sialidase results in increased sensitivity to EGF. Moreover, some signaling pathways initiated by EGF involving these putative substrates could be affected by the diminution of GM3 content as a result of sialidase expression. DISCUSSION Gangliosides are important for normal cell function. There are many studies that support the involvement of gangliosides in transmembrane signaling and in both cell to cell and cell to matrix interactions (36, 37). In addition, changes in ganglioside composition have been described as a feature of virtually all tumor types including brain tumors (38 – 41). Polysialylated gangliosides (e.g., GD3) have been shown to be overexpressed in patients with tumors of either neuroectodermal or epithelial origin (42), suggesting that the paracrine signal(s) from tumor cells of epithelial origin (e.g., carcinoma of the cervix, lung, prostate, breast, head and neck, colon, and ovary) may stimulate overexpression and shedding of gangliosides from tumor-infiltrating mesenchymal cells (43). In general, changes in GSLs often accompany the malignant transformation of cells (1, 43), and alterations in their expression have been correlated with tumor grade, metastatic potential, and prognosis (44, 45). A recent study by Tokuyama et al. (20) has demonstrated the suppression of metastasis in B16 murine melanoma cells transfected with a sialidase gene. One mechanism explaining the ganglioside effects in tumorigenesis may reside in the adhesion-promoting action of gangliosides on basement membrane components. Through this characteristic, gangliosides play an important role in invasive processes (45, 46). Another mechanism explaining how gangliosides can contribute to tumor cell behavior is their possible interaction with GFRs. The presence of sialic acid seems to be essential for GSLs to interact with GFRs. The involvement of sialidase in proliferative processes has been suggested by a number of investigators. For example, Ogura and Sweeley (47) suggested the importance of sialidase activity in the proliferation of cultured fibroblasts. The extracellular addition of neuraminidase, which decreased the amount of GM3, increased the proliferation of the treated fibroblasts. Sato and Miyagi (48) found that a cytosolic sialidase was expressed and highly active during myotube formation. In this study, we have shown that overexpression of sialidase by gene transfection similarly resulted in increased cell proliferation. Taken together, these data suggest that the regulation of sialic acid levels on the cell surface can modulate cell proliferation. Previous studies have shown that the ganglioside GM3 can modulate EGFR function. Exogenously added GM3 inhibits cell proliferation and EGFR signaling in A431 cells (7, 8). This study presents a new approach, via gene transfection, to regulate GSL composition and thereby monitor the interaction of ganglioside (e.g., GM3) with EGFR. Sialidase activity was increased in sialidase-transfected cells, resulting in GM3 depletion and increased cell proliferation. Sialidase effects on cell proliferation seemed to be mediated, at least in part, by the activation of the EGFR. Indeed, decreased GM3 levels induced the activation of EGFR autophosphorylation but did not modify the binding of EGF to its receptor. These data correlate with observations that the addition of exogenous GM3 to culture media leads to the inhibition of EGFR autophosphorylation (8, 12, 49) without affecting EGF binding. Several studies support the hypothesis that GM3 interacts with EGFR. GM3 inhibits EGFR tyrosyl kinase activity in detergent micelles, plasma membrane vesicles, and whole cells. Immunoaffinity-purified EGFR preparations contain GM3, implying that the GSL is intimately associated with the receptor kinase in cell
membranes. Moreover, GM3 but not de-N-acetyl GM3 interacts specifically with the EGFR in intact membranes (50). Other data have suggested that GM3 does not directly inhibit the EGFR kinase domain in vitro (51) but needs to interact with the extracellular domain of the receptor to produce its inhibitory effect. Among the six clones overexpressing the sialidase gene, four showed an increased EGFR autophosphorylation, and two did not differ from that of controls. The decrease in GM3 content suggests one mechanism by which EGFR signaling may be modulated in sialidase-transfected cells. The discrepancies observed in two sialidase-transfected clones (clones 1 and 4) might reflect some other regulatory pathways. The discovery of gangliosides in caveolae membrane domains (52) suggests an indirect way that gangliosides could modulate membrane protein activity. By altering the ganglioside content of the cell, the distribution of receptors within specialized membranes could be affected, thereby decreasing the efficiency of receptor activation and signaling. Another possibility is that the sialidase could be affecting other enzymes that regulate EGFR activity such as a tyrosine-specific phosphatase (53) or serine/threonine kinases (54). In addition to GM3 modulation of EGFR, glycosylation of the receptor itself may also contribute to the regulation of its function (32, 33). Therefore, we analyzed glycoprotein sialylation with lectins to identify the glycosylation branching structure on EGFR and other glycoproteins (32, 33). PNA from Arachis hypogaea and MAL-II from Macckia amurensis seeds are known to recognize galactosyl-b1,3-N-acetylgalactosamine and a2,3-linked sialic acids, respectively. Lectin binding data did reveal minor changes in the sialylation of lower molecular weight proteins, but no change was apparent for the EGFR. This observation was consistent with the results obtained by Tokuyama et al. (20). It should be noted that the natural substrates available to cytosolic sialidases are not well characterized. Because gangliosides are localized on the cell surface, sialylated glycoconjugates are not expected to be present in the cytosol. Moreover, no sialidase activity has been detected in the culture medium of the positive clones (data not shown), indicating that the overexpressed enzyme is not secreted. Nevertheless, the specificity of sialidase purified from Chinese hamster ovary cells has been described in vitro (21, 55, 56). The activity of this sialidase has been described to be primarily effective on lipid-bound sialic acids (21) with an optimal activity at pH 6.5 for ganglioside substrates (21, 57–59). A liver cytosolic sialidase has also been reported to exhibit similar kinetic and physical properties (57). On the other hand, lysosomal sialidases have been described to be active only at pH 4 (60). At pH 4, no change in the sialidase activity in sialidase-transfected cells was detected as compared to parental (A431) or pcDNA3-transfected cells (data not shown). Moreover, in the presence of a specific sialidase inhibitor, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (14), the increased sialidase activity measured in our positive transfected clones was specifically inhibited at pH 6.5 (data not shown). Taken together, these observations suggest that we did not alter other sialidase activities present in the cell line. Moreover, in parental cells (A431) and pcDNA3-transfected cells, .90% of the ganglioside consisted of GM3. An almost complete diminution of GM3 content was observed in sialidase-transfected cells. In conclusion, our results showed that the overexpression of this specific sialidase activity resulted primarily in ganglioside desialylation with very little effect on the overall protein glycosylation. In conclusion, the diminution of GM3 content by the transfection of a sialidase gene resulted in an increase in EGFR kinase activity associated with increased cell proliferation. We suggest that ganglioside composition plays an important role in proliferation processes. In this report, we demonstrate that by decreasing the GM3 content of a
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Sialidase Gene Transfection Enhances Epidermal Growth Factor Receptor Activity in an Epidermoid Carcinoma Cell Line, A431 Emmanuelle J. Meuillet, Roger Kroes, Hirotaka Yamamoto, et al. Cancer Res 1999;59:234-240.
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