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Tumor Biology |
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
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Many epithelial cancers have a high incidence and are very much resistant to current therapies. In particular, lung cancer is one of the most frequent and most malignant diseases because we have no efficient way to suppress tumor growth and kill tumor cells in patients. In trials to search for tumor-specific antigens of human lung cancer cells, various carbohydrate antigens have been defined. Expression of sialyl Lewis X in NSCLCs3 was reported (17) , whereas SCLC cells mainly expressed gangliosides, such as GM1 (18) , fucosyl-GM1 (19, 20, 21) , GD3 (21 , 22) , 9-O-acetyl-GD3 (22) , and GD2 (23) . Some of them have been used as targets of immunotherapy (24, 25, 26) or immunolocalization (27) .
Recently, a number of glycosyltransferase genes were isolated, and their substrate specificities and expression patterns were investigated. Availability of these transferase genes and information about their structures and functions enable further analysis of the implications of those tumor-associated carbohydrate structures in malignant phenotypes of lung cancer cells. We are now able to directly analyze the roles of glycoconjugates expressed on tumor cells and the regulatory mechanisms for their expression.
In the present study, we analyzed the expression of gangliosides and the glycosyltransferase genes responsible for their syntheses in human lung cancer cell lines. Because b-series gangliosides were specifically found in SCLC cells with up-regulation of the GD3 synthase gene and GM2/GM1 were broadly detected in the majority of lung cancer cell lines, we tried remodeling of a ganglioside profile by introducing the GD3 synthase gene into a b-series-negative SCLC cell line. Using newly established transfectant cells, we have demonstrated the important role of ganglioside GD2 in the cell proliferation of SCLC cells. Moreover, the binding of anti-GD2 mAbs could induce apoptotic death of GD2-expressing SCLC cells, suggesting the usefulness of anti-GD2 antibodies or their derivatives in the therapy of SCLC patients.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Flow Cytometry.
Cell surface expression of gangliosides was analyzed by FACScan (Becton Dickinson) using various antiganglioside mAbs. The mAbs used were as follows: anti-GM3, M2590 (mouse IgM; Japan Biotest Research 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 Reagents kit (PE Biosystems). The TaqMan probe contained a reporter dye at the 5' end (FAM) and a quencher dye at the 3' 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 guanidine isothiocyanate/cesium chloride ultracentrifugation method, and real-time RT-PCR was performed using gene-specific primers in each sample 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), 5'-CATTCCAGCTGCCATTGAAGA-3', and a reverse primer (nucleotides 582–561), 5'-CTTGACAAAGGAGGGAGATTGC-3'. The primers for ß1,4N-acetylgalactosaminyltransferase (GM2 synthase; Ref. 30
) were a forward primer (GenBank NM-001478, nucleotides 1163–1184), 5'-GGACATGAGGCTGCTTTCACTA-3', and a reverse primer (nucleotides 1306–1286), 5'-CCGATCATAACGGAGGAAGGT-3'. The TaqMan probes used were 5'-FAM-ATCTATTTGACGGCCACAGCCACTCTTCT-TAMRA-3' for GD3 synthase and 5'-FAM-AGCCCAGTACAACATCAGCGCTCTAGTCAC-TAMRA-3' for GM2 synthase. For glyceraldehyde-3-phosphate dehydrogenase, Predeveloped TaqMan Assay Reagents Control kit (PE Biosystems) was used according to manufacturer’s protocol. RT-PCR reactions were performed on an ABI PRISM 7700 Detector (PE Biosystems). The relative expression level was deduced from a standard curve constructed using the positive control sample and normalized against the expression level of glyceraldehyde-3-phosphate dehydrogenase in each sample.
Gene Transfection and Selection.
Human 2,8-sialyltransferase cDNA clone pD3T-31 (29)
was subcloned into pMIKneo vector (Maruyama; Tokyo Medical Dental University) to obtain the pMIKneo/D3T-31. SCLC SK-LC-17 cells used for cDNA transfection were plated in a 60-mm plastic tissue culture plate (Falcon) at a density of 6 x 105 cells/4 ml/plate. After 24 h, the medium was removed, and the cells were washed twice with serum-free DMEM. The pMIKneo/D3T-31 vector (4 µg) was mixed with Lipofectamine (Life Technologies, Inc.) and then added to the cells in the plate. After a 6-h incubation, the medium was changed to the regular one as described above. Stable transfectants were selected with 250 µg/ml G418 (Life Technologies, Inc.).
Glycolipid Extraction and TLC.
Glycolipids were extracted as described previously (31)
. Briefly, glycolipids were extracted from 
400 µl of packed cells using chloroform/methanol (2:1, 1:1, and 1:2), sequentially. TLC was performed on high-performance TLC plates (Merck, Darmstadt, Germany) using a solvent system of chloroform:methanol:2.5 N NH4OH (60:35:8) and sprayed by resorcinol. For standards, acid glycolipids from human melanoma SK-MEL-37 cells were used.
Antibody Purification.
The anti-GD2 mAb (220-51) was purified using a protein G affinity column, and the concentration of the protein was determined by Lowry’s method (32)
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MTT Assay.
For cell proliferation assay, transfectant cells and control cells (1 x 104 cells/well) were prepared in 48-well plates in serum-containing medium and cultured for 6 days. For cell growth inhibition assay, transfectant cells and control cells (5 x 104 cells/well) were seeded in 48-well plates in serum-containing medium and treated with various anti-GD2 mAbs diluted to the indicated concentrations for 3 days. Freshly prepared medium containing antibodies was used for medium exchange on days 1 and 2. The anti-GD2 mAbs used were as follows: 220-51 (mouse IgG3); KM666 (mouse IgG3; provided by Kyowa Hakko Kogyo Co. Ltd., Tokyo, Japan); and KM1138 (chimeric IgG3; provided by Kyowa Hakko Kogyo Co. Ltd., Tokyo, Japan). To quantify the cell proliferation, MTT (Sigma Chemical Co.) was added to each well (0.5 mg/ml). After incubation for 2 h at 37°C, the supernatants were aspirated, and 100 µl of n-propyl alcohol containing 0.1% NP40 and 4 mM HCl 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 x 104 cells/well) were prepared in 96-well plates in serum-containing medium and treated with 40 µg/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 µg/ml), added to each filter (190 µg/filter; polyethylene terephthalate membrane, 8-µm pore size, 23.1 mm in diameter; Falcon 3093), and left to polymerize and dry overnight. The polymerized and dried Matrigel membranes 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 x 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 x200 magnification, and the mean of the number in each field was calculated. Assays were carried out in triplicate.
Western Immunoblotting.
Cells (8 x 105 cells) were plated in 60-mm tissue culture plates in serum-containing medium and then treated with either 40 or 60 µg/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, 1 mM phenylmethylsulfonyl fluoride, and 20 units of aprotinin]. Soluble proteins (50 µg/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 (PharMingen). 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 x 105 cells) were plated in 60-mm tissue culture plates in serum-containing medium and then treated with either 20 µg/ml of various anti-GD2 mAbs or 20 µg/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 µg/ml PI (Annexin V-FITC kit; Bender Medsystems), according to the manufacturer’s protocol. The numbers of apoptotic cells were monitored with a FACScan and determined using the CELLQuest program. For analysis of sub-G1 DNA content, cells (1.5 x 106 cells) were cultured with or without 100 µM z-VAD-fmk (Peptide Institute, Inc., Osaka, Japan) in the presence of mAb 220-51 (30 µg/ml). After 48 h, cells were fixed with 70% ethanol and incubated with 4 mM phosphate-citrate buffer (4 mM citric acid, 192 mM Na2 HPO4) for 30 min at room temperature. After centrifugation, cells were resuspended in PBS containing PI/RNase (10 µg/ml each) and incubated for 20 min at room temperature. Quantification of sub-G1 DNA content was determined using the CELLQuest program.
DNA Fragmentation Assay.
Cells (3 x 106 cells) were treated with 60 µg/ml of mAb 220-51. After 48 h, cells were harvested and lysed in 100 µl of 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 µl of RNase (10 mg/ml) and 2 µl of Proteinase K (10 mg/ml) were added. After incubation for 1 h at 37°C, the fragmented DNA was precipitated in 2-propanol and electrophoresed at 50 V for 1.6 h on a 2% agarose gel containing 0.2 µg/ml ethidium bromide in TAE buffer (40 mM Tris-acetate, 1mM EDTA). The gel was observed under UV light.
Statistical Analysis.
Significance was determined using the Student t test.
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RESULTS |
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. GM2 and GM1, belonging to the a-series 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)
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and Fig. 3A
). As for the GM2/GD2 synthase gene, its expression levels were well correlated with those of complex gangliosides (Fig. 3B)
. This gene was broadly expressed in all of the lung cancer lines examined, whereas SCLC lines showed slightly higher expression levels.
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. 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 1400-fold increase in the expression level, compared with that of the vector control, was observed (data not shown).
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. Cell growth, as presented by absorbance at 590 nm, was 2-fold or greater after 4–6 days of culture. Invasion activity as analyzed by the Boyden chamber method was markedly increased in the transfectant lines, i.e., 483 ± 117 cells were found for the transfectants and 98.7 ± 26.3 cells in the controls, which had penetrated the Matrigel layer and come out of the reverse side of the filter (Fig. 5, B and C)
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. Activation (phosphorylation) levels of MAPK were also reduced along with the time course after antibody addition (Fig. 6B)
. In vector control cells, neither suppression of cell proliferation nor reduction in MAPK activation were observed (Fig. 6, A and B)
. These suppression effects of anti-GD2 mAb on the cell growth were also detected in SCLC lines (NCI-N417 and ACC-LC-171; Fig. 6C
), which primarily expressed high levels of GD2 but not in a non-GD2-expressing line, ACC-LC-170 (data not shown). Other anti-GD2 mAbs than 220-51, including KM666 (mouse mAb) and KM1138 (mouse-human chimeric mAb), also showed significant and similar growth suppression activity for the transfectant cells (Fig. 7)
. The nonspecific mouse IgG3 fraction showed no effects (data not shown).
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, indicating the induction of apoptotic cell death. A vector control line, C-8, showed no staining for both reagents. The transfectant cells treated with anti-GD2 mAb showed marked shrinkage of cytoplasm and formation of spaces between cells, whereas vector control cells showed no change (Fig. 8B)
. When other anti-GD2 mAbs were added, similar morphological changes (data not shown) and double staining with Annexin V and PI were found (Fig. 9A)
. However, nonrelevant antibodies, such as anti-GM2 mAb or anti-GD3 mAb, did not induce these changes (Fig. 9B)
. During the time course, the Annexin V-positive population increased after 0.5 h, and then the PI-positive fraction expanded after 1 h incubation (Fig. 10, A and B)
. This apoptosis induction depended on the antibody concentration (Fig. 10C)
. To investigate the sensitivity to the apoptosis induction with the anti-GD2 mAb in cell lines with various levels of GD2 expression, GD2 expression levels and the percentage of positive Annexin V and PI after the antibody treatment were compared in six lung cancer cell lines. Not only high GD2 expressants such as ACC-LC-171 and NCI-N417 but also those with moderate GD2 levels showed apparent apoptosis (Fig. 11)
. To confirm the DNA degradation in the anti-GD2 mAb-treated cells, cytoplasmic DNA was prepared from 3 x 106 cells and used for agarose gel electrophoresis. Only cells treated with the mAb showed definite DNA ladder formation (Fig. 12A)
and also activation of caspase-3 (Fig. 12B)
. This apoptotic process was partially inhibited by a caspase inhibitor, z-VAD-fmk (Fig. 12C)
, which suggested that apoptosis induced by anti-GD2 mAb was mediated via the caspase-3-dependent pathway and some independent pathway.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Remodeling of ganglioside profiles in cultured cells has shown their roles in the regulation of cell proliferation and differentiation (36 , 37) . Then, gene targeting of galactosyl-ceramide synthase revealed GalCer functions in the stability of myelin (38) and spermatogenesis (39) . GM2/GD2 synthase gene knockout mice lacking complex gangliosides exhibited their roles in the regulation of the nervous system (40) , spermatogenesis (41) , and T-cell functions (42) . However, 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 I-infected lymphocytes under regulation of p40ras transactivator (14)
. Despite a number of studies indicating the tumor-specific expression or malignant phenotype-associated expression of GD2, no definite studies to demonstrate the significant roles of GD2 in tumor cell proliferation have been reported. The findings demonstrated in the present study strongly suggest that GD2 actually exerts important roles to enhance cellular proliferation signals as shown in Fig. 6B
.
Kasahara et al. (49) reported recently that GD3 on primary cultured cerebellar neurons mediated activation of a Src family tyrosine kinase, lyn, in cooperation with the TAG-1 molecule. Fukumoto et al. (37) reported that introduction of the GD3 synthase gene into the rat pheochromocytoma cell line PC12 resulted in the continuous activation of nerve growth factor receptor TrkA and its downstream signal molecule, MAPK. These alterations in the signal molecules appeared to induce a marked enhancement of cell proliferation. These findings suggest that GD2 on the cell surface of SK-LC-17 is associated with some growth factor receptors and modulates their functions, resulting in an observed increase in cell growth. Identification of GD2-associated receptors or signal molecules remains to be investigated.
Apoptosis induced by anti-GD2 mAbs is very interesting and provides a possibility for the application in immunotherapy of SCLC. Gangliosides, including GD2, are expressed on the outer layer of plasma membrane; therefore, it is unclear how GD2 can mediate the apoptosis signal triggered by the binding of anti-GD2 mAb. Binding of anti-GD2 mAb to GD2 might cause clustering of GD2 molecules, resulting in the modulation of unknown neighboring molecules and in the activation of subsequent intracellular molecules that emit a signal for apoptosis. No studies have been performed on the induction of apoptosis of tumor cells using antiglycolipid mAbs except for anti-GM2 mAb by Nakamura et al. (50) . However, they showed just a decrease of tumor cell layer volume in multicellular heterospheroids after treatment with anti-GM2 mAb. Apoptosis of SCLC cells induced by anti-GD2 mAb, as demonstrated in the present study, should be the first example in which apoptosis induction was definitely shown using antiglycolipid mAbs. The molecular mechanisms for this apoptosis are now under investigation in our laboratory and should provide information necessary for the application of anti-GD2 mAbs in therapies against drug-resistant malignant tumors.
Although this study was performed with lung cancer cell lines, GD2 expression in tumor specimens of SCLC has been reported (23) , and successful in vivo detection of SCLC tumor sites with anti-GD2 mAbs 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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by Grants-in-Aid 12217127 and 10178104 for Scientific Research on 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 of Svennerholm (52)
. 
2 To whom requests for reprints should be addressed, at Department of Biochemistry II, Nagoya University School of Medicine, 65 Tsurumai, Showa-ku, Nagoya, 466-0065, Japan. Fax: 81-52-744-2069; E-mail: koichi{at}med.nagoya-u.ac.jp
3 The abbreviations used are: NSCLC, non-small cell lung cancer; mAb, monoclonal antibody; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MAPK, mitogen-activated protein kinase; PI, propidium iodide; RT-PCR, reverse transcription-PCR; FAM, 6-carboxyfluorescein; TAMRA, 6-carboxy-N,N,N',N'-tetramethylrhodamine; z-VAD-fmk, z-Val-Ala-Asp-fluoromethyl ketone.
Received 10/23/00. Accepted 3/15/01.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-2,8-sialyltransferase cDNA using anti-GD2 monoclonal antibody. Proc. Natl. Acad. Sci. USA, 91: 10455-10459, 1994. cells. J. Biol. Chem., 269: 30451-30456, 1994.This article has been cited by other articles:
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, data not determined.
) or without (
) 40 µg/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 performed in triplicate. *, P < 0.05; **, P < 0.01 compared with the control (-).
, the positive percentages of PI. B, two-dimensional presentation of double staining with Annexin V and PI. Borders were set to make double-negative cells 95% at 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 with Annexin V and PI. Results are presented as in A.
