Ganglioside GD2 in Small Cell Lung Cancer Cell Lines: Enhancement of Cell Proliferation and Mediation of Apoptosis1

[CANCER RESEARCH 61, 4244–4252, May, 15, 2001]

Shoko Yoshida, Satoshi Fukumoto, Haruhiko Kawaguchi, Shigeki Sato, Ryuzo Ueda, and Koichi Furukawa2 Department of Biochemistry II, Nagoya University School of Medicine, Nagoya 466-0065 [S. Y., S. F., K. F.]; Department of Internal Medicine II, Nagoya City University School of Medicine, Nagoya 467-8601 [S. Y., H. K., S. S., R. U.]; and Department of Pediatric Dentistry, Nagasaki University School of Dentistry, Nagasaki 852-8102 [S. F.], Japan

ABSTRACT photrophic virus type I-infected cells under the regulation of the p40tax transactivator (14). However, the implications of the expression Expression levels of gangliosides and glycosyltransferase genes respon- of these tumor-associated glycolipids have never been clarified to sible for their syntheses in human lung cancer cell lines and a normal date, except for a few studies describing indirect evidence about the bronchial epithelial cell line were analyzed. Both non-small cell lung cancers and small cell lung cancers (SCLCs) mainly expressed G and role of GD3 in the growth of melanoma cells (15, 16). M2 Many epithelial cancers have a high incidence and are very much GM1, whereas only SCLCs expressed b-series gangliosides, such as GD2, resistant to current therapies. In particular, lung cancer is one of the GD1b, and GT1b. Accordingly, many SCLC cell lines showed up-regulation of the GD3 synthase gene. Consequently, we introduced GD3 synthase most frequent and most malignant diseases because we have no cDNA into a SCLC line with low expression of b-series gangliosides and efficient way to suppress tumor growth and kill tumor cells in patients. analyzed the effects of newly expressed gangliosides on tumor phenotypes. In trials to search for tumor-specific antigens of human lung cancer The transfectant cells expressing high levels of GD2 and GD3 exhibited cells, various carbohydrate antigens have been defined. Expression of markedly increased growth rates and strongly enhanced invasion activi- sialyl Lewis X in NSCLCs3 was reported (17), whereas SCLC cells ties. Addition of anti-G monoclonal antibodies into the culture medium D2 mainly expressed gangliosides, such as G (18), fucosyl-G (19– of these cells resulted in the marked growth suppression of G -expressing M1 M1 D2 21), G (21, 22), 9-O-acetyl-G (22), and G (23). Some of them cell lines with reduced activation levels of mitogen-activated protein ki- D3 D3 D2 nases but not of nonexpressants, suggesting that G plays important roles have been used as targets of immunotherapy (24–26) or immunolo- D2 calization (27). in cell proliferation. Moreover, GD2-expressing cells treated with anti-GD2 antibodies showed features of apoptotic cell death at 30 min after addition Recently, a number of glycosyltransferase genes were isolated, and of antibodies, i.e., shrinkage of cytoplasm, binding of Annexin V, and their substrate specificities and expression patterns were investigated. staining with propidium iodide, followed by DNA fragmentation. This Availability of these transferase genes and information about their

GD2-mediated apoptosis was associated with caspase-3 activation and structures and functions enable further analysis of the implications of partly inhibited by a caspase inhibitor, z-Val-Ala-Asp-fluoromethyl ke- those tumor-associated carbohydrate structures in malignant pheno- tone. The finding that anti-GD2 antibodies suppressed the cell growth and types of lung cancer cells. We are now able to directly analyze the induced apoptosis of SCLC cells strongly suggested the usefulness of G D2 roles of glycoconjugates expressed on tumor cells and the regulatory as a target for the therapy of disastrous cancer, although the precise mechanisms for their expression. mechanisms for apoptosis remain to be clarified. In the present study, we analyzed the expression of gangliosides and the glycosyltransferase genes responsible for their syntheses in INTRODUCTION human lung cancer cell lines. Because b-series gangliosides were specifically found in SCLC cells with up-regulation of the G Acidic glycosphingolipids and gangliosides are widely expressed in D3 synthase gene and GM2/GM1 were broadly detected in the majority of many tissues and organs in vertebrates (1) and have been suggested to lung cancer cell lines, we tried remodeling of a ganglioside profile by be involved in the regulation of development and differentiation as introducing the GD3 synthase gene into a b-series-negative SCLC cell recognition molecules or signal modulators (2). However, there are line. Using newly established transfectant cells, we have demonstrated some ganglioside species with relatively simple structures that show the important role of ganglioside G in the cell proliferation of SCLC very restricted expression in normal tissues and markedly enhanced D2 cells. Moreover, the binding of anti-GD2 mAbs could induce apoptotic expression in particular malignant tumors, i.e., tumor-associated an- death of G -expressing SCLC cells, suggesting the usefulness of tigens. In the analysis of ganglioside expression in various human D2 anti-GD2 antibodies or their derivatives in the therapy of SCLC tumors, characteristic patterns have been demonstrated depending on patients. individual tumor types, such as melanomas (3, 4), sarcomas (5), astrocytomas (6), and epithelial cancers (7). Among tumor-associated glycolipids, disialosyl ganglioside GD3 has been considered a human MATERIALS AND METHODS melanoma-specific antigen (8) and has been used as a target molecule in antibody immunotherapy (9). Disialosyl ganglioside GD2 has also Cell Lines. All human lung cancer cell lines were provided by Dr. T. been considered a neuroblastoma-associated antigen (10). It has also Takahashi (Aichi Cancer Center Research Institute, Aichi, Japan) and were been used as a target of antibody therapy (11–13). We reported maintained in RPMI 1640 containing 10% FBS in a humidified 5% CO2 previously that G was specifically expressed in human T-cell lym- atmosphere at 37°C. The immortalized human bronchial epithelial cell, BEAS- D2 2B, was obtained from the American Type Culture Collection and was cultured on human fibronectin-coated plastic dishes in serum-free small airway cell Received 10/23/00; accepted 3/15/01. growth medium (Clonetics) in a humidified 5% CO atmosphere at 37°C. The costs of publication of this article were defrayed in part by the payment of page 2 charges. This article must therefore be hereby marked advertisement in accordance with Flow Cytometry. Cell surface expression of gangliosides was analyzed by 18 U.S.C. Section 1734 solely to indicate this fact. FACScan (Becton Dickinson) using various antiganglioside mAbs. The mAbs 1 Supported by Grants-in-Aid 12217127 and 10178104 for Scientific Research on used were as follows: anti-GM3, M2590 (mouse IgM; Japan Biotest Research Priority Areas and by the Center of Excellence Research from the Ministry of Education, Science, Sports and Culture, Japan. This work was also supported by Grants-in-Aid from the Ministry of Health and Welfare of Japan. Ganglioside nomenclature is based on that 3 The abbreviations used are: NSCLC, non-small cell lung cancer; mAb, monoclonal of Svennerholm (52). antibody; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MAPK, 2 To whom requests for reprints should be addressed, at Department of Biochemistry mitogen-activated protein kinase; PI, propidium iodide; RT-PCR, reverse transcription- II, Nagoya University School of Medicine, 65 Tsurumai, Showa-ku, Nagoya, 466-0065, PCR; FAM, 6-carboxyfluorescein; TAMRA, 6-carboxy-N,N,NЈ,NЈ-tetramethylrhoda- Japan. Fax: 81-52-744-2069; E-mail: [email protected]. mine; z-VAD-fmk, z-Val-Ala-Asp-fluoromethyl ketone. 4244

Institute, Tokyo, Japan); anti-GM2, 10-11 (mouse IgM; provided by Dr. P. O. Livingston, Memorial Sloan-Kettering Cancer Center, New York, NY); anti-

GD1a, 92-22 (mouse IgM); anti-GD3, R-24 (mouse IgG3; provided by Dr. L. J. Old, Memorial Sloan-Kettering Cancer Center); anti-GD2, 220–51 (mouse IgG3); anti-GD1b, 370 (mouse IgM); anti-GT1b, 549 (mouse IgM); anti-GA2, 2D4 (mouse IgM; American Type Culture Collection); and anti-GA1, 229 (mouse IgM). Unless described otherwise, the antibodies were generated in our laboratory (28). The cells were incubated with mAbs for 45 min on ice and then stained with FITC-conjugated goat antimouse IgM or IgG (Cappel,

Durham, NC). To analyze GM1, cells were incubated with a biotin-conjugated cholera toxin B subunit (List Biological Laboratories, Campbell, CA) for 45 min on ice and then stained with FITC-conjugated avidin (EY Laboratories, San Mateo, CA). Control cells for flow cytometry were prepared using the second antibody alone. For quantification of positive cells, the CELLQuest program was used. Quantitative Real-Time RT-PCR Analysis. The quantitative RT-PCR analysis was performed using TaqMan One-Step RT-PCR Master Mix Re- agents kit (PE Biosystems). The TaqMan probe contained a reporter dye at the 5Ј end (FAM) and a quencher dye at the 3Ј end (TAMRA; PE Biosystems). Target-specific PCR amplification results in cleavage and release of the reporter dye from the quencher-containing probe by nuclease activity of AmpliTaq Gold (PE Biosystems). Thus, the fluorescence signal generated from released reporter dye is proportional to the amount of PCR product. To compare the relative expression levels of glycosyltransferase genes in various ✽ lung carcinoma cells, total RNA was extracted from each cell line with the Fig. 2. Synthetic pathway of gangliosides. , point where GD3 synthase works. guanidine isothiocyanate/cesium chloride ultracentrifugation method, and real- Lac-Cer, lactosyl ceramide; Gal, galactose; GalNAc, N-acetylgalactosamine; Glc, glu- time RT-PCR was performed using gene-specific primers in each sample cose; SA, sialic acid. according to the manufacturer’s protocol. The primers used for ␣2,8-sialyltransferase (GD3 synthase; Ref. 29) were a forward primer (GenBank L32867, nucleotides 456–476), 5Ј-CATTCCAGCTGCCATTGAAGA-3Ј, and a re- reactions were performed on an ABI PRISM 7700 Detector (PE Biosystems). The verse primer (nucleotides 582–561), 5Ј-CTTGACAAAGGAGGGAGATTGC- relative expression level was deduced from a standard curve constructed using Ј ␤ 3 . The primers for 1,4N-acetylgalactosaminyltransferase (GM2 synthase; the positive control sample and normalized against the expression level of Ref. 30) were a forward primer (GenBank NM-001478, nucleotides 1163– glyceraldehyde-3-phosphate dehydrogenase in each sample. 1184), 5Ј-GGACATGAGGCTGCTTTCACTA-3Ј, and a reverse primer Gene Transfection and Selection. Human ␣2,8-sialyltransferase cDNA (nucleotides 1306–1286), 5Ј-CCGATCATAACGGAGGAAGGT-3Ј. The clone pD3T-31 (29) was subcloned into pMIKneo vector (Maruyama; Tokyo TaqMan probes used were 5Ј-FAM-ATCTATTTGACGGCCACAGCCAC- Medical Dental University) to obtain the pMIKneo/D3T-31. SCLC SK-LC-17 Ј Ј TCTTCT-TAMRA-3 for GD3 synthase and 5 -FAM-AGCCCAGTACAA- cells used for cDNA transfection were plated in a 60-mm plastic tissue culture Ј ϫ 5 CATCAGCGCTCTAGTCAC-TAMRA-3 for GM2 synthase. For glyceralde- plate (Falcon) at a density of 6 10 cells/4 ml/plate. After 24 h, the medium hyde-3-phosphate dehydrogenase, Predeveloped TaqMan Assay Reagents Control was removed, and the cells were washed twice with serum-free DMEM. The kit (PE Biosystems) was used according to manufacturer’s protocol. RT-PCR pMIKneo/D3T-31 vector (4 ␮g) was mixed with Lipofectamine (Life Tech-

Fig. 1. Ganglioside typing of lung cancer cell lines. Cell surface expression of gangliosides in 44 lung cancer cell lines (A, lung small cell carcinoma; B, lung adenocarcinoma; C, lung squamous cell carcinoma; and D, lung large cell carcinoma) and a bronchial epithelial line, BEAS-2B (E), were analyzed by flow cytometry using antiganglioside mAbs as described in “Materials and Methods.” The ganglioside expression level was classified into five groups based on the percentages of positive cells (A, bottom). ✽, data not determined. 4245

Fig. 3. Correlation between the expression levels of gangliosides and those of glycosyltransferases in the lung cancer cell lines. Surface expression of gangliosides was analyzed as described in Fig. 1. Left, numbers are individual cell lines presented in Fig. 1. Percentages of positive cells are shown. Expression levels of the GD3 synthase gene and the GM2/GD2 synthase gene were measured with a quantitative real-time RT-PCR analyzer system as described in “Materials and Methods.” A: a, SCLC; b, NSCLC. Percentages of b-series positive cells (left) were the sum of those of GD3-, GD2-, GD1b-, and GT1b-positive cells. The relative expression level of the GD3 synthase gene (right) is presented as a percentage of that of melanoma SK-MEL-37 cells, which were regarded as 100%. B: a, SCLC; b, NSCLC. The percentages of complex ganglioside-positive cells (left) were the sum of those of GM2-, GM1-, GD1a-, GD2-, GD1b-, and GT1b-positive cells. The relative expression level of the GM2/GD2 synthase gene (right) is presented as a percentage of that of the SCLC line ACC-LC-171 cells, which were regarded as 100%.

nologies, Inc.) and then added to the cells in the plate. After a 6-h incubation, Antibody Purification. The anti-GD2 mAb (220-51) was purified using a the medium was changed to the regular one as described above. Stable protein G affinity column, and the concentration of the protein was determined transfectants were selected with 250 ␮g/ml G418 (Life Technologies, Inc.). by Lowry’s method (32). Glycolipid Extraction and TLC. Glycolipids were extracted as described MTT Assay. For cell proliferation assay, transfectant cells and control cells previously (31). Briefly, glycolipids were extracted from ϳ400 ␮l of packed (1 ϫ 104 cells/well) were prepared in 48-well plates in serum-containing cells using chloroform/methanol (2:1, 1:1, and 1:2), sequentially. TLC was medium and cultured for 6 days. For cell growth inhibition assay, transfectant performed on high-performance TLC plates (Merck, Darmstadt, Germany) cells and control cells (5 ϫ 104 cells/well) were seeded in 48-well plates in using a solvent system of chloroform:methanol:2.5 N NH4OH (60:35:8) and serum-containing medium and treated with various anti-GD2 mAbs diluted to sprayed by resorcinol. For standards, acid glycolipids from human melanoma the indicated concentrations for 3 days. Freshly prepared medium containing

SK-MEL-37 cells were used. antibodies was used for medium exchange on days 1 and 2. The anti-GD2

Fig. 4. Remodeling of ganglioside expression by gene manipulation. A, surface expression of gangliosides in transfectant cells. A vector control cell line (C-8; top) transfected with pMIKneo vector alone and a GD3 synthase gene transfectant cell line (D-18; bottom) transfected with pMIKneo/D3T-31 were used for flow cytometry as described in “Materials and Methods.” Filled gray lines, the controls with the second antibody alone. B, TLC of glycolipids extracted from SK-LC-17 cells (LC-17), C-8, and D-18 are presented. TLC was performed as described in “Materials and Methods.” 4246

were added. The color reaction was quantitated using an automatic plate reader, Immuno-Mini NJ-2300 (Nihon InterMed, Tokyo, Japan), at 590 nm with a reference filter of 620 nm as reported previously (33). MTT assays were carried out in triplicate. To analyze the growth-suppression effects of mAb 220-51 in SCLC lines, cells (4 ϫ 104 cells/well) were prepared in 96-well plates in serum-containing medium and treated with 40 ␮g/ml of mAb 220-51 for 3 days and then used for the MTT assay. In this case, lysis buffer (n-propylalcohol with 0.1% NP40 and 4mM HCL) was added to each well without aspiration of the medium. In Vitro Invasion Assay. The Boyden chamber in vitro invasion assay was performed as described previously (34) with some modifications. In brief, Matrigel (Becton Dickinson) was diluted with ice-cold PBS (250 ␮g/ml), added to each filter (190 ␮g/filter; polyethylene terephthalate membrane, 8-␮m pore size, 23.1 mm in diameter; Falcon 3093), and left to polymerize and dry overnight. The polymerized and dried Matrigel mem- branes were reconstituted with serum-free medium. The lower chamber (6-well plate; Falcon 3502) was filled with serum-free medium (3 ml) before the chamber was assembled. Cells (1 ϫ 106 cells/well) were added to serum-free medium in the upper chamber and incubated for 18 h in a

humidified 5% CO2 atmosphere at 37°C. After incubation, the cells on the upper surface of the filter were removed completely by wiping with a cotton swab. The filters were fixed in ethanol and stained with Giemsa (Wako). The number of cells migrating to the lower surface of the filters was counted in 10 fields under ϫ200 magnification, and the mean of the number in each field was calculated. Assays were carried out in triplicate. Western Immunoblotting. Cells (8 ϫ 105 cells) were plated in 60-mm tissue culture plates in serum-containing medium and then treated with either 40 or 60 ␮g/ml of mAb 220-51 for the appropriate times in the individual experiments. After treatment, cells were harvested with 0.02% EDTA, washed twice with ice-cold PBS, and solubilized in lysis buffer [20 mM Tris-HCl (pH

7.4), 1% NP40, 10% glycerol, 50 mM NaF, 1 mM Na3VO4,1mM phenylmeth- ylsulfonyl fluoride, and 20 units of aprotinin]. Soluble proteins (50 ␮g/lane) were subjected to either 10 or 15% SDS-PAGE and then transferred onto a polyvinylidene difluoride membrane (Millipore). The membrane was incubated with 5% dry milk in PBS overnight at 4°C, washed with PBS containing 0.05% Tween 20, and then incubated with antibodies reactive with either MAPK, phospho-MAPK (New England Biolabs), or active caspase-3 (Phar- Mingen). Bands were detected with peroxidase-conjugated antirabbit IgG (New England Biolabs) combined with an ECL kit (Amersham Pharmacia Biotech). Analysis of Apoptotic Cells. Cells (6 ϫ 105 cells) were plated in 60-mm tissue culture plates in serum-containing medium and then treated with either ␮ ␮ 20 g/ml of various anti-GD2 mAbs or 20 g/ml of irrelevant mAbs (10-11 and R24) for 24 h at 37°C. For analysis of the time course and dose dependency of the antibody effects, cells were treated with mAb 220-51 diluted appropriately. After treatment, cells were harvested with trypsin/EDTA, resuspended in binding buffer [10 mM HEPES/NaOH (pH 7.4), 140 mM NaCl, and 2.5 mM ␮ CaCl2], and then incubated with FITC-conjugated Annexin V and 1 g/ml PI (Annexin V-FITC kit; Bender Medsystems), according to the manufacturer’s Fig. 5. Proliferation and invasion activity of the GD3 synthase gene transfectant cells. protocol. The numbers of apoptotic cells were monitored with a FACScan and A, MTT assay. Three GD3 synthase gene transfectants (D-8, D-13, and D-18) and two vector controls (C-2 and C-8) were cultured in serum-containing medium and were used determined using the CELLQuest program. For analysis of sub-G1 DNA for MTT assay for 6 days as described in “Materials and Methods.” The absorbances (590 content, cells (1.5 ϫ 106 cells) were cultured with or without 100 ␮M z-VAD- nm) were measured on days 1–6. Data are means of three independent experiments; bars, fmk (Peptide Institute, Inc., Osaka, Japan) in the presence of mAb 220-51 (30 P Ͻ 0.01 compared ,ءء ;P Ͻ 0.05 ,ء .SD. Each experiment was performed in triplicate ␮g/ml). After 48 h, cells were fixed with 70% ethanol and incubated with 4 mM with all three of the GD3 synthase gene transfectants. B, invasion activity of three GD3 synthase gene transfectants (D-8, D-13, and D-18) and three vector control lines (C-2, phosphate-citrate buffer (4 mM citric acid, 192 mM Na2 HPO4) for 30 min at C-6, and C-8) was analyzed using the Boyden chamber method as described in “Materials room temperature. After centrifugation, cells were resuspended in PBS con- and Methods.” The number of cells migrating to the lower surface of the filter is shown. taining PI/RNase (10 ␮g/ml each) and incubated for 20 min at room temper- Data are means of triplicate samples in a representative experiment; bars, SD. **, Ͻ ature. Quantification of sub-G1 DNA content was determined using the P 0.01 compared with all three of the GD3 synthase gene transfectants. Similar results were obtained in three independent experiments. C, micrographs at ϫ200 magnification CELLQuest program. of D-13 (a) and C-8 (b), which migrated to the lower surface of the filter. They were fixed DNA Fragmentation Assay. Cells (3 ϫ 106 cells) were treated with 60 and stained with Giemsa reagent. ␮g/ml of mAb 220-51. After 48 h, cells were harvested and lysed in 100 ␮lof lysis buffer [10 mM Tris-HCl (pH 7.4), 10 mM EDTA, and 0.5% Triton X-100] for 10 min at 4°C. After centrifugation, the supernatants were collected, and 2 mAbs used were as follows: 220-51 (mouse IgG3); KM666 (mouse IgG3; ␮l of RNase (10 mg/ml) and 2 ␮l of Proteinase K (10 mg/ml) were added. provided by Kyowa Hakko Kogyo Co. Ltd., Tokyo, Japan); and KM1138 After incubation for1hat37°C, the fragmented DNA was precipitated in (chimeric IgG3; provided by Kyowa Hakko Kogyo Co. Ltd., Tokyo, Japan). 2-propanol and electrophoresed at 50 V for 1.6 h on a 2% agarose gel To quantify the cell proliferation, MTT (Sigma Chemical Co.) was added to containing 0.2 ␮g/ml ethidium bromide in TAE buffer (40 mM Tris-acetate, each well (0.5 mg/ml). After incubation for2hat37°C, the supernatants were 1mM EDTA). The gel was observed under UV light. aspirated, and 100 ␮lofn-propyl alcohol containing 0.1% NP40 and 4 mM HCl Statistical Analysis. Significance was determined using the Student t test. 4247

Fig. 6. Suppression of cell growth by anti-GD2 mAb. A, MTT assay of D-18 and C-8 treated with mAb 220-51 in serum-containing medium on days 1–3. Right, concentration of mAb 220-51 used. Data are means of three independent experiments; bars, SD. Each experiment was performed in P Ͻ 0.01 compared with the ,ءء ;P Ͻ 0.05 ,ء .triplicate control (0 ␮g/ml). B, time course of phosphorylation levels of MAPK in the transfectants after addition of anti-GD2 mAb. D-18 (a) and C-8 (b) were treated with 40 ␮g/ml of mAb 220-51 for the times indicated and then used for the 10% SDS-PAGE and immunoblotting with an antiphosphorylated MAPK (ERK 1/2) antibody (upper) or an anti-MAPK antibody (lower) as described in “Materials and Methods.” C, suppression of cell growth of GD2-expressing cells. GD2- expressing SCLC lines NCI-N417 and ACC-LC-171 were treated with (o) or without (f)40␮g/ml of mAb 220-51 for 3 days and used for the MTT assay. Data are means of three independent experiments; bars, SD. Each experiment was P Ͻ 0.01 compared ,ءء ;P Ͻ 0.05 ,ء .performed in triplicate with the control (Ϫ).

RESULTS examined, whereas SCLC lines showed slightly higher expression levels. Ganglioside Typing of Lung Cancer Cell Lines. Expression of Remodelling of Ganglioside Expression by Gene Manipulation. gangliosides in 44 lung cancer cell lines was analyzed by flow To analyze the effect of b-series gangliosides dominantly expressed in cytometry (Fig. 1, A–D). GM2 and GM1, belonging to the a-series the SCLC cell lines on malignant cancer phenotypes, a cDNA expres- gangliosides, were expressed at significant levels in almost all of the lung cancer cell lines, whereas the b-series gangliosides, such as GD2,

GD1b, and GT1b, were found characteristically in SCLC lines. These b-series gangliosides were not expressed or were expressed at very low levels, if present, in non-SCLC lines. A bronchial epithelial cell line, BEAS-2B, expressed no or very low levels of gangliosides except for GM1 (Fig. 1E). Gene Expression of Glycosyltransferases in the Lung Cancer Cell Lines. To analyze the correlation between the expression levels of gangliosides and those of glycosyltransferase genes, expression levels of the GD3 synthase gene and the GM2/GD2 synthase gene were measured with a quantitative real-time RT-PCR analyzer system (PE

Biosystems). The expression level of the GD3 synthase gene was well correlated with that of b-series gangliosides, i.e., high levels of the Fig. 7. Comparison of growth-suppressive effects of various anti-GD2 mAbs in the GD3 synthase gene expression were shown in the SCLC cell lines (Fig. transfectant cells. D-18 was prepared as described in “Materials and Methods” and then treated with 20 ␮g/ml of mAb 220-51, KM666, or KM1138 for 3 days and used for the 2 and Fig. 3A). As for the GM2/GD2 synthase gene, its expression MTT assay. Right, symbols for each antibody. Data are means of three independent ,ءء ;P Ͻ 0.05 ,ء .levels were well correlated with those of complex gangliosides (Fig. experiments; bars, SD. Each experiment was performed in triplicate 3B). This gene was broadly expressed in all of the lung cancer lines P Ͻ 0.01 compared with the control (none). 4248

sion vector of GD3 synthase was introduced into a SCLC line, SK- LC-17, which did not express b-series gangliosides. Newly established transfectant cell lines showed definite and high expression of b-series gangliosides in the flow cytometric analysis (Fig. 4A). In

contrast, GM2 and GM1 levels were reduced dramatically because of the shift of the synthetic flow from a-series to b-series. Control lines transfected with a vector alone showed no change in the ganglioside

profile compared with the parent cell, i.e., GM2 and GM1 expressions were observed (Fig. 4A). These findings were confirmed by TLC of ganglioside fractions extracted from the individual cell lines (Fig. 4B).

As for GM3, its expression could not be detected in the flow cytometry, presumably because of the low accessibility of the antibody. However, high expression in the vector control and the parent cell and marked reduction in the transfectant lines, because of the consumption as a substrate of the transfected enzyme, were exhibited in TLC.

When the expression levels of the GD3 synthase gene in the transfectants were analyzed by real-time RT-PCR, an ϳ1400-fold increase in the expression level, compared with that of the vector control, was observed (data not shown).

Changes in Phenotypes of GD3 Synthase Gene Transfectant Cells. In the MTT assay, the transfectant cells expressing b-series gangliosides exhibited significantly enhanced proliferation compared with control lines (Fig. 5A). Cell growth, as presented by absorbance at 590 nm, was ϳ2-fold or greater after 4–6 days of culture. Invasion Fig. 8. Apoptosis induction in the GD3 synthase gene transfectant cells by anti-GD2 activity as analyzed by the Boyden chamber method was markedly mAb. A, double staining of Annexin V and PI in the transfectants after anti-GD2 mAb increased in the transfectant lines, i.e., 483 Ϯ 117 cells were found for treatment. D-18 (left) and C-8 (right) were treated with 20 ␮g/ml of mAb 220-51 for 24 h and stained with FITC-conjugated Annexin V (top) and PI (bottom) and then analyzed by the transfectants and 98.7 Ϯ 26.3 cells in the controls, which had flow cytometry as described in “Materials and Methods.” Filled gray lines, negative penetrated the Matrigel layer and come out of the reverse side of the controls with neither Annexin V nor PI. B, morphological changes of transfectants treated with (b and d) or without (a and c)40␮g/ml of mAb 220-51 for 12 h were observed under filter (Fig. 5, B and C). a phase-contrast microscope with ϫ100. a and b, D-18; c and d, C-8. Suppression of Cell Growth by Anti-GD2 Antibodies. Effects of anti-GD2 mAbs on cell growth were then examined by adding antibodies to the culture medium. The increased cell growth after GD3 positive population increased after 0.5 h, and then the PI-positive synthase gene transfection was strongly suppressed in the presence of fraction expanded after 1 h incubation (Fig. 10, A and B). This

an anti-GD2 mAb 220-51. The suppression effects were dependent on apoptosis induction depended on the antibody concentration (Fig. the concentration of the added antibody and became significant, even 10C). To investigate the sensitivity to the apoptosis induction with the ␮ at 5 g/ml on day 3 (Fig. 6A). Activation (phosphorylation) levels of anti-GD2 mAb in cell lines with various levels of GD2 expression, GD2 MAPK were also reduced along with the time course after antibody expression levels and the percentage of positive Annexin V and PI addition (Fig. 6B). In vector control cells, neither suppression of cell after the antibody treatment were compared in six lung cancer cell

proliferation nor reduction in MAPK activation were observed (Fig. 6, lines. Not only high GD2 expressants such as ACC-LC-171 and A and B). These suppression effects of anti-GD2 mAb on the cell NCI-N417 but also those with moderate GD2 levels showed apparent growth were also detected in SCLC lines (NCI-N417 and ACC-LC- apoptosis (Fig. 11). To confirm the DNA degradation in the anti-GD2 6 171; Fig. 6C), which primarily expressed high levels of GD2 but not mAb-treated cells, cytoplasmic DNA was prepared from 3 ϫ 10 cells in a non-GD2-expressing line, ACC-LC-170 (data not shown). Other and used for agarose gel electrophoresis. Only cells treated with the anti-GD2 mAbs than 220-51, including KM666 (mouse mAb) and mAb showed definite DNA ladder formation (Fig. 12A) and also KM1138 (mouse-human chimeric mAb), also showed significant and activation of caspase-3 (Fig. 12B). This apoptotic process was par- similar growth suppression activity for the transfectant cells (Fig. 7). tially inhibited by a caspase inhibitor, z-VAD-fmk (Fig. 12C), which

The nonspecific mouse IgG3 fraction showed no effects (data not suggested that apoptosis induced by anti-GD2 mAb was mediated via shown). the caspase-3-dependent pathway and some independent pathway.

Apoptosis Induction by Anti-GD2 mAbs. To clarify the induction of apoptosis during the growth suppression of SCLC cell lines with DISCUSSION anti-GD2 mAbs, double staining of cells with Annexin V (FITC) and PI was performed. Transfectant lines treated with anti-GD2 mAb were Despite much effort to elucidate the biological functions of glyco- 72.2% positive for Annexin V binding and 76.9% positive for PI sphingolipids, few definite molecular functions have been demon- staining at 24 h after the addition of mAbs (Fig. 8A), indicating the strated to date. Current ways to modulate carbohydrate structures on induction of apoptotic cell death. A vector control line, C-8, showed cells have provided only indirect information about the roles of no staining for both reagents. The transfectant cells treated with glycoconjugates, because methods to modulate carbohydrate chains

anti-GD2 mAb showed marked shrinkage of cytoplasm and formation harbor substantial limitations in the specificity of their effects. Suc- of spaces between cells, whereas vector control cells showed no cessful cloning of glycosyltransferase genes dramatically changed this

change (Fig. 8B). When other anti-GD2 mAbs were added, similar situation and improved the limitations in the analysis of carbohydrate morphological changes (data not shown) and double staining with functions. In particular, application of gene knockout technology has Annexin V and PI were found (Fig. 9A). However, nonrelevant clearly demonstrated in vivo functions of products of individual gly-

antibodies, such as anti-GM2 mAb or anti-GD3 mAb, did not induce cosyltransferases (35), although the expected outcome has not neces- these changes (Fig. 9B). During the time course, the Annexin V- sarily been obtained because of various unknown reasons. 4249

Fig. 9. Anti-GD2 mAb-specific apoptosis induction in the transfectant cells. A, double staining of the transfectants treated with various anti-GD2 mAbs using Annexin V and PI. D-18 was treated with 20 ␮g/ml of mAb 220-51 (left), KM666 (middle), or KM1138 (right) for 24 h and stained with Annexin V (top) and PI (bottom) and then analyzed by flow cytometry as described in “Materials and Methods.” Filled gray lines, negative controls with neither Annexin V nor PI. B, D-18 was treated with 20 ␮g/ml of mAb 220-51 (left), mAb 10-11 (anti-GM2 mAb; middle), or mAb R24 (anti-GD3 mAb; right) for 24 h. After the treatments, they were stained as described in A.

Fig. 10. The time course and dose dependency of the anti-GD2 mAb effects in the Remodeling of ganglioside profiles in cultured cells has shown transfectant cells. D-18 was treated with 20 ␮g/ml of mAb 220-51 for the times indicated their roles in the regulation of cell proliferation and differentiation and used for double staining with Annexin V and PI. A: f, the positive percentages of Annexin V; u, the positive percentages of PI. B, two-dimensional presentation of double (36, 37). Then, gene targeting of galactosyl-ceramide synthase re- staining with Annexin V and PI. Borders were set to make double-negative cells 95% at vealed GalCer functions in the stability of myelin (38) and spermat- 0 h, and the percentages of individual fractions are shown. C, D-18 was treated with mAb 220-51 diluted at the concentrations indicated for 12 h and then used for double staining ogenesis (39). GM2/GD2 synthase gene knockout mice lacking com- with Annexin V and PI. Results are presented as in A. plex gangliosides exhibited their roles in the regulation of the nervous system (40), spermatogenesis (41), and T-cell functions (42). How- ever, roles of gangliosides in various malignant phenotypes in tumors have never been clearly demonstrated by glycosyltransferase gene manipulation, although many glycolipid antigens have been considered as tumor markers or have been used as target molecules of antibody therapy (9, 10).

GD2 studied in this paper is one of the melanoma-associated glycolipid antigens (8). In particular, GD2 is up-regulated in advanced and vertically metastatic melanoma cells (43), suggesting that GD2 plays roles in malignant features of melanomas such as metastasis

(44). Actually, GD2 and GD3 have been considered to modify integrin functions (45). Moreover, an anti-GD2 immune reaction against melanoma cells in melanoma patients was reported by Watanabe et al.

(46) and Cahan et al. (47). These findings suggest that GD2 is one of the target molecules for immunotherapy of melanoma patients. In addition to melanoma, GD2 has been considered to be a neuroblastoma-associated antigen, and anti-GD2 mAb has been tried in a therapy for neuroblastoma patients (48). We also reported previously the specific expression of GD2 on human T-cell lymphotrophic virus type ras Fig. 11. Apoptosis induction in lung cancer cell lines with various levels of GD2 I-infected lymphocytes under regulation of p40 transactivator (14). ϫ 5 expression by an anti-GD2 mAb. Cells (8 10 cells/well) were seeded in a six-well plate Despite a number of studies indicating the tumor-specific expression in serum-containing medium and treated with 40 ␮g/ml of mAb 220-51 for 24 h and used for double staining with Annexin V and PI. A, two-dimensional presentation of the double or malignant phenotype-associated expression of GD2, no definite staining of ACC-LC-171 cells with Annexin V and PI (left, nontreated cells; right, treated studies to demonstrate the significant roles of GD2 in tumor cell cells). B, GD2 expression levels (left) and positive percentages (right) stained with proliferation have been reported. The findings demonstrated in the Annexin V (f) and PI (u) of six lung cancer cell lines are shown. 4250

was also reported (27), suggesting that GD2 in SCLC is worth being focused on as a target of chemoimmunotherapy. Of course, many devices in the modification of antibodies, in the modes of antibody treatment, e.g., in combination with vaccines in the adjuvant setting (11), or in the controlling of side effects (51) remain to be achieved.

ACKNOWLEDGMENTS

We thank Dr. T. Takahashi at Aichi Cancer Center for kindly providing cell

lines, Dr. K. Shitara at Kyowa Hakko Kogyo Co. Ltd. for providing anti-GD2 monoclonal antibodies, and Dr. H. Kimura at the Department of Pediatrics in Nagoya University School of Medicine for valuable suggestion about real-time RT-PCR. We also thank T. Ando and E. Kondo for technical and secretarial assistance, respectively.

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Shoko Yoshida, Satoshi Fukumoto, Haruhiko Kawaguchi, et al.

Cancer Res 2001;61:4244-4252.

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