Globoside-dependent Adhesion of Human Embryonal Carcinoma Cells, Based on Carbohydrate-Carbohydrate Interaction, Initiates Signal Transduction and Induces Enhanced Activity of Transcription Factors AP1 and CREB*

Yu SongDagger , Donald A. Withers, and Sen-itiroh Hakomori

From the Pacific Northwest Research Foundation, Seattle, Washington 98122 and the Departments of Pathobiology and Microbiology, University of Washington, Seattle, Washington 98195

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Undifferentiated human embryonal carcinoma cells are characterized by high expression of lactoneotetraosylceramide (nLc4), globoside (Gb4), and extended globo-series glycosphingolipids (GSLs) termed "stage-specific embryonic antigens 3 and 4" (SSEA-3 and -4). Expression of these GSLs declines in association with a decline of homotypic adhesion during the differentiation process. Therefore, these GSLs may play an essential role in adhesion among these cells. As an example, human embryonal carcinoma 2102 cells display strong adhesion to plates coated with Gb4 ("Gb4-dependent cell adhesion"). This adhesion, which simulates homotypic 2102 cell aggregation, is based on interaction between Gb4 and nLc4, or between Gb4 and GalGb4 (IV3GalGb4; the major SSEA-3 epitope), as indicated by the following observations: (i) adhesion of 2102 cells or GSL-liposomes to GSL-coated plates in various combinations; (ii) inhibition of Gb4-dependent 2102 cell adhesion by preincubation of cells with anti-SSEA-3 or anti-nLc4 antibodies, or by pretreatment of Gb4-coated plates with aqueous micellar solution of nLc4 or GalGb4; (iii) decline of the cell adhesion in association with retinoic acid-induced differentiation, whereby SSEA-3 and nLc4 levels are reduced. Since cell adhesion is an essential prerequisite for induction of differentiation, as observed at each step of embryogenesis, expression of seven transcription factors following adhesion of 2102 cells to Gb4-coated plates, and to detergent-insoluble substrate adhesion matrix prepared from 2102 cells, were studied. In both types of adhesion, a strong enhancement of AP1 and CREB site binding activity was observed during the early stage (15-60 min following initial adhesion). Although 2102 cells showed strong adhesion to Gg3-coated plates, based on interaction between Gg3 and Gb4, adhesion of the cells to Gg3 did not cause changes of AP1 and CREB activity. No other transcription factors showed changes induced by Gg3- or Gb4-dependent adhesion.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Expression of adhesion receptor families (e.g. integrins, immunoglobulin analogs, cadherins, selectins, galectins, etc.) and their respective ligands plays a pivotal role in the cell adhesion process (1-5) (see "Discussion"). Carbohydrate-carbohydrate interaction has been shown to play an accessory role in initiating cell adhesion prior to involvement of integrins and other adhesion receptors as above (Refs. 6-8; see, for review, Ref. 9). Carbohydrate-carbohydrate interaction displays remarkable synergism with function of integrins (10), and perhaps of other adhesion receptors. The first cell adhesion event during the process of embryogenesis is the compaction of morula, in which Lex is involved in mouse (11).1 A model of embryogenesis is autoaggregation of mouse embryonal carcinoma F9 cells, which is based on Lex-to-Lex interaction in the presence of Ca2+ (6, 12). Involvement of uvomorulin or E-cadherin in the compaction process and in F9 cell autoaggregation has been clearly documented (3, 13). Recent studies indicate that E-cadherin and Lex cooperate in adhesion of F9 cells, and presumably in the compaction process (14). Globoside (Gb4) and extended globo-series GSLs,2 in addition to lacto-series structures, are expressed highly in preimplantation mouse embryo and mouse and human embryonal carcinoma cells, and show dramatic changes associated with differentiation of these cells (15-18).

We now find that nLc4, the precursor of Lex, is expressed highly, together with Gb4 and other globo-series GSLs, in human embryonal carcinoma 2102 cells, and that all these structures are involved in adhesion among these cells. The same adhesion process may be involved in compaction of human embryos. Since cell adhesion is a prerequisite for induction of differentiation, occurrence of signal transduction following carbohydrate-dependent cell adhesion is expected. This study was designed to answer two basic questions. (i) Are globo-series GSLs and nLc4 involved in adhesion of human embryonal carcinoma cells based on carbohydrate-carbohydrate interaction, and, if so, what is the adhesion mechanism? (ii) Is signal transduction triggered by Gb4-dependent cell adhesion? Our results indicate that adhesion of 2102 cells to Gb4-coated plates, which simulates self-aggregation of 2102 cells, is indeed based on carbohydrate-carbohydrate interaction, and induces signal transduction in terms of activation of transcription factors.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Cells, GSLs, and Antibodies-- Human embryonal carcinoma cell line 2102 Ep was derived from a surgical specimen of a primary testicular germ cell tumor containing embryonal carcinoma (19). The cells were maintained in DMEM supplemented with 10% FBS. All GSLs used were prepared and purified in this laboratory as described previously (20). Gb4, nLc4, and GM3 3 were prepared from human erythrocytes, Gg3 from guinea pig erythrocytes, sialosylparagloboside (IV3NeuAcnLc4) from human placenta, Gal-globoside (GalGb4; IV3GalGb4) and sialosyl-Gal-globoside (IV3NeuAcalpha 2right-arrow3GalGb4) from pig and human kidney, and GM1 from bovine brain. Lex pentaosyl-Cer (III3FucnLc4) was prepared by enzymatic fucosylation of nLc4 by fucosyltransferase (FucT III) from Colo205 cells in the presence of GDP-fucose (21). Lc3 was prepared by treatment of nLc4 with jack bean beta -galactosidase, followed by purification on C18 column and TLC. Polyclonal rabbit anti-Gb4 antibody (22), anti-Gg3 mAb 2D4 (23), and anti-nLc4 mAb 1B2 (24) were prepared in this laboratory. Anti-Gb4 mAb 9G7 (25) was kindly donated by Drs. Baiba Gillard and Donald M. Marcus (Baylor College of Medicine, Houston, TX). Anti-SSEA-3 mAb MC631 was purchased from Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA). The major epitope of MC631 is GalGb4, but the mAb cross-reacts with fucosyl-GalGb4 and weakly with Gb4 (17).

Expression of Carbohydrate Epitopes at the Surface of Human Embryonal Carcinoma 2102 Cells-- Carbohydrate epitopes expressed at the surface of 2102 cells were determined using flow cytometry as described previously (26, 27) with specific polyclonal and mAbs as described above. Briefly, 5 × 105 cells were incubated with the specific primary antibody with gentle shaking for 2 h at 4 °C, washed with washing buffer (1% BSA, PBS, 0.1% NaN3), treated with fluorescence-labeled goat (Fab)2 anti-mouse IgM + G (Tago, Camarillo, CA) or anti-rabbit Ig antibody for 1 h on ice, washed twice with washing buffer, fixed in 2% paraformaldehyde solution, and analyzed by flow cytometer (Coulter Electronics Corp., Hialeah, FL).

Gb4-Liposome Interaction with Various GSL-coated Surfaces-- Liposomes labeled with [3H]cholesterol containing Gb4 were prepared, and their interactions with plastic surfaces coated with various GSLs were studied as described previously (6), with slight modification. Briefly, 35 µg of dimyristoylphosphatidylcholine, 20 µg of cholesterol containing 3H-labeled material, and 12.5 µg of Gb4 were dissolved in 200 µl of chloroform-methanol (2:1), and the solution was evaporated to dryness in a rotary evaporator. The residue was mixed with 2 ml of PBS with or without bivalent cation, vigorously mixed with a Vortex mixer, sonicated for 30 min (Branson 5200), and allowed to stand at room temperature for 30 min. Liposome suspensions obtained after three repetitions of the sonication/incubation procedure were used. In most experiments, PBS containing 0.9 mM CaCl2 and 0.5 mM MgCl2 was used as a solution for suspension. Liposomes were also prepared in the absence of any bivalent cation, and in the presence of 15 mM EDTA in PBS.

To prepare solid phase polystyrene plastic surfaces coated with GSLs, 30 nmol/ml ethanol solutions of various GSLs were prepared, and a 50-µl aliquot (containing 1.5 nmol of GSL) or its appropriately diluted solution was added to each well of 96-well flat bottom plastic plates (Falcon 3912, flexible assay plate), dried at 37 °C, and blocked with 1% BSA in PBS. GSLs tested were nLc4, sialosyl-nLc4, Lc3, GM1, Gg3, GM3, Gb3, Gb4, GalGb4, sialosyl-GalGb4, Lex pentaosyl-Cer, Lea pentaosyl-Cer, and Leb hexaosyl-Cer.

To each GSL-coated well on the 96-well plate, 100 µl of liposome containing Gb4 and 3H-labeled cholesterol (1.0 × 105 cpm/well) was added and incubated in PBS at room temperature for 16 h. Wells were washed with PBS by seven repetitions of the following special procedure. To each well of the plate, 200 µl of PBS was added, after which the plate was held upside-down and shaken gently to remove the PBS. The 3H-labeled liposomes remaining on the plate were counted by scintillation counter.

2102 Cell Adhesion to GSL-coated Plates-- 2102 cells were cultured in DMEM containing 10% FBS in 15-cm plates. Cells were detached from confluent cultures in 0.52 mM EDTA, collected, and suspended in 1% BSA in DMEM at a density of 1 × 106 cells/ml. Aliquots (100 µl) of the cell suspension were placed in each well of a 96-well plate coated with various GSLs as described above, and the plates were incubated for 30 min to 2 h at 37 °C in a CO2 incubator. After incubation, the plates were washed once with PBS by the following special procedure. The plate was immersed in PBS(+) in a large container, and suspended upside down above the the bottom of the container for 15-20 min with gentle shaking to allow nonadherent cells to sediment (fall) out of the plates. The plate was then turned rightside up, removed from the container, and PBS in each well was removed by gentle aspiration. The adherent cells remaining after this procedure were fixed with 150 µl of 3.7% paraformaldehyde for 2 h at room temperature and stained with 0.1% Toluidine Blue O for 30 min. The remaining cells were lysed with 10% acetic acid, and absorbance was measured at 630 nm.

Effects of Treatment with Retinoic Acid or Bromodeoxyuridine (BrdUrd), or Preincubation with Various Anti-GSL Antibodies, on Gb4-dependent 2102 Cell Adhesion-- 2102 cells were seeded at a density of 1 × 106 cells/75-cm2 flask in DMEM supplemented with 10% FBS, and differentiation was induced by either 10 µM retinoic acid or 3 µM BrdUrd. All trans-retinoic acid and BrdUrd were purchased from Sigma. After 7 days of incubation, cells were harvested and expression of carbohydrate epitopes on the cell surface was determined. Next, binding to Gb4-coated solid phase was performed as described above.

For determination of the effect of mAbs on 2102 cell adhesion, cells (1 × 106/ml) were separately incubated in control medium or medium containing IgM mAbs 2D4 (anti-Gg3) (23), 1B2 (anti-nLc4) (24), MC631 (anti-SSEA-3) (17), or 9G7 (anti-Gb4) (25) for 1 h at 37 °C in a CO2 incubator. The concentrations of mAb in medium ranged from 1 to 40 µg/ml. After washing with serum-free medium, the cells were resuspended in 1% BSA in DMEM and used for binding assay. The effect of anti-Gb4 mAb 9G7 on Gb4-dependent 2102 cell adhesion was also studied by pretreatment of Gb4-coated plates with 9G7 (concentrations ranged from 0 to 1 µg/well), followed by addition of 2102 cells.

Inhibition of Gb4-dependent 2102 Cell Adhesion in the Presence of a Micellar Solution of nLc4 or GalGb4-- Each well of a 96-well plate was coated with 1.0 nmol of Gb4 by evaporation of ethanol solution at 37 °C. Each well was first incubated with 1% BSA in PBS (for blocking nonspecific binding), after which was added 100 µl of aqueous micellar solution of nLc4 or GalGb4 containing various quantities (5, 10, 20, 30, 40, or 50 nmol), and incubated 2 h at 37 °C. As a negative control, wells were incubated with BSA as above and 100 µl of aqueous micellar solution of Gb4 containing the same quantity range (5-50 nmol) was added. Next, aqueous micellar solution of GSL in each well was removed by aspiration, and 105 2102 cells were added to each well. After incubation for 1 h, non-adherent cells were washed out of the plate, and quantity of adherent cells was determined by absorbance of stained cells in each well as described above.

Large Scale Cell Adhesion Designed for Determination of Effect of Cell Adhesion on Signal Transduction-- Large quantities of 2102 cells (approximately 1 × 107) were adhered simultaneously to 10-cm cell culture plates coated with GSL or containing DISAM. GSL-coated plates were prepared by adding 6 ml of ethanol solution containing 300 nmol of Gb4 or Gg3 and air-drying inside a sterile hood. Ethanol solutions without GSL were treated under the same conditions, as controls. DISAM was prepared as follows. 2102 cells were cultured in 10-cm plates. 0.1% Triton X-100 in PBS was added to cell monolayer after confluence, and the plates were shaken in a gyratory shaker for 15 min at 4 °C and aspirated. All cells were detached or dissolved, and no cells remained on the plates. The residue remaining on the plate (i.e. DISAM) was washed five times with PBS and prepared for cell binding. DISAM thus prepared contained the majority of GSL at cell adhesion sites and membrane-associated pericellular matrix, as described previously (28, 29).

2102 cells from confluent culture were detached by 0.52 mM EDTA and centrifuged, the cell pellet was suspended in 1% FBS/DMEM, and 6 ml of cell suspension containing 107 cells was placed in Gb4- or Gg3-coated or DISAM-containing plates. Cell adhesion was initiated by centrifugation. Several plates were stacked in a hanging holder ("microplate carrier") designed for use in a centrifuge, and centrifuged at 600 rpm for 5 min. The time at the end of centrifugation was defined as the start of adhesion. After incubation (which began immediately after centrifugation) for 15, 30, or 60 min at 37 °C in CO2 incubator, cells adhering to the bottom of the plates were subjected to nuclear protein extraction.

Nuclear Protein Extraction-- Cells, adhered and incubated as above, were harvested by scraping with rubber policeman, centrifuged, and washed once with 1 ml of PBS and once with 1 ml of lysis buffer (10 mM HEPES, 10 mM KCl, and 1.5 mM MgCl2, pH 7.9, 4 °C). Cells were lysed by suspending the cell pellet in 30 µl of lysis buffer containing 0.1% Nonidet P-40 for 10 min on ice, by the mini-preparation method described previously (30). To isolate nuclei, the lysis buffer suspension was vigorously mixed and centrifuged in a microcentrifuge (5 min at 15,000 rpm, 4 °C). Nuclear proteins were extracted by resuspending the nuclear pellet in 20 µl of protein extraction buffer (420 mM NaCl, 20 mM HEPES, 1.5 mM MgCl2, 0.2 mM EDTA, and 25% glycerol, pH 7.9) for 10 min at 4 °C. After vigorous mixing, the nuclear suspension was microcentrifuged (5 min, 15000 rpm, 4 °C), the pellet was discarded, and the supernatant was microcentrifuged under the same conditions as above. The supernatant from the second centrifugation was diluted with 30 µl of diluting buffer (50 mM KCl, 20 mM HEPES, 0.2 mM EDTA, and 20% glycerol, pH 7.9) and the protein concentration was measured (Micro BCA protein assay, Pierce). Protease inhibitors, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 10 µg/ml leupeptin were added to both lysis and extraction buffers just before use. The diluting buffer contained the same amounts of dithiothreitol and leupeptin, but only 0.2 mM phenylmethylsulfonyl fluoride. The nuclear protein samples were stored at -80 °C until use.

Labeling Oligonucleotides for Gel Shift Assay-- Oligonucleotides containing consensus binding sites for AP1, AP2, CREB, E2F1, Egr, NFkappa B, and SP1 were purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, CA. The oligonucleotide probes were end-labeled using [gamma -32P]ATP and T4 polynucleotide kinase (30, 31). Briefly, 25 ng of each oligonucleotide, 20 units of T4 polynucleotide kinase, and 5 µCi of [gamma -32P]ATP were mixed in a microcentrifuge tube, and brought to 20 µl with T4 kinase buffer. The mixture was incubated at 37 °C for 30 min, and the labeled probe was separated from unincorporated [gamma -32P]ATP using QIAquick nucleotide removal kit (QIAGEN).

Gel Shift Assay-- Equal amounts of protein from each sample were mixed with a reaction buffer (1 mM EDTA, 50 mM NaCl, 10 mM Tris, and 5% glycerol) containing 1 µg of poly(dI-dC), 1 mM dithiothreitol, and 0.5 ng of end-labeled oligonucleotide probe. For competition experiments, 40 ng of each cold probe was added to each reaction. The reaction mixtures were incubated for 30 min at room temperature. For supershift assay, 2 µl of specific mAb directed to AP1 family transcription factors c-Jun and c-Fos, or anti-CREB mAb ATF-1, was first added to each reaction mixture. After 30 min of incubation at room temperature, 32P-labeled probe was added to the reaction mixture, which was incubated for another 30 min at room temperature. For each type of assay, samples were then loaded onto a 4% polyacrylamide gel (prerun at 150 V, 4 °C, 2 h) and run at 500 V, 4 °C. Dried gels were allowed to expose film (Kodak, X-Omat) for 12-16 h at room temperature with intensifying screens.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Expression of Carbohydrate Epitopes at the Surface of Human Embryonal Carcinoma 2102 Cells-- Cytofluorometric patterns of a few carbohydrate epitopes expressed at the surface of undifferentiated human embryonal carcinoma 2102 cells are shown in Fig. 1. The patterns are characterized by high expression of nLc4 and SSEA-3 (principal epitope, GalGb4) and moderate expression of Gb4. Gg3, defined by mAb 2D4, was not expressed (data not shown).


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Fig. 1.   Flow cytometric patterns of human embryonal carcinoma 2102 cells. Cells were treated with polyclonal anti-Gb4 antibody, anti-nLc4 mAb 1B2, and anti-SSEA-3 mAb MC631, followed by fluorescent-labeled second antibody. See "Experimental Procedures" for conditions. Name of primary antibody is shown at the top of each panel. Ordinate, count (approximate cell number).

Interaction of Gb4 with Various GSLs, Based on Adhesion of Gb4-Liposomes to GSL-coated Plates-- Binding of liposomes containing Gb4 to 96-well plates coated with various GSLs indicated strong interaction of the liposomes with nLc4 (Fig. 2A), weaker interaction with GalGb4 and Lex pentaosyl-Cer (Fig. 2B), and no interaction with Lc3, GM1, sialosylparagloboside (Fig. 2A), or with Gb4, Gb3, or GM3 (Fig. 2B). Gb4-liposomes also did not interact with mono- or disialosyl-GalGb4 (SSEA-4), Lea pentaosyl-Cer, or Leb hexaosyl-Cer (data not shown). Gb4-liposomes interacted strongly with Gg3-coated plates (data not shown); however, Gg3 and other ganglio-series GSLs are absent in 2102 cells. Interaction of Gb4-liposome with nLc4-, GalGb4-, or Lex GSL-coated plates was clearly dependent on the quantity of these GSLs coated on plates, in contrast to the absence of adhesion and the lack of dose-dependent interaction with Gb3, Lc3, Lea, Leb, GM1, GM3, sialosylparagloboside, monosialosyl GalGb4, and Gb4. Significantly, Gb4-liposome interaction with nLc4, GalGb4, and Lex was observed even in the absence of bivalent cation or in the presence of 15 mM EDTA.4


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Fig. 2.   Binding of Gb4-liposomes to plates coated with various GSLs. Gb4-liposomes labeled with (3H)cholesterol were prepared and incubated in 96-well plates coated with various GSLs as described under "Experimental Procedures." Wells were coated with GSLs in various amounts (0-1.5 nmol/well). The amount of radioactivity in Gb4-liposome bound to GSL-coated plate was measured by scintillation counter. A, binding of Gb4-liposomes to plates coated with nLc4 (bullet ), sialosyl nLc4 (down-triangle), Lc3 (square ), and GM1 (triangle ). B, binding of Gb4-liposomes to plates coated with GalGb4 (bullet ), Lex (black-square), GM3 (), Gb4 (triangle ), and Gb3 (down-triangle). Each point represents the mean of triplicate experiments. Standard variation is ~1% of value. Similar results were obtained in three separate experiments; data from one experiment are shown here.

Adhesion of 2102 Cells to Gb4-coated Plates (Gb4-dependent Cell Adhesion)-- 2102 cells, characterized as high expressors of nLc4 and SSEA-3, adhered strongly to Gg3- and Gb4-coated plates, but not to Gb3-, Lc3-, Lea-, Leb-, GM1-, or GM3-coated plates so far tested. This adhesion depended on the quantity of GSL coated (Fig. 3A), and occurred rapidly, reaching maximal value within 30 min. There was no significant increase during prolonged incubation time (data not shown). 2102 cell adhesion to Gg3-coated plates was clear and strong, but has no physiological significance since Gg3 is absent in these cells.


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Fig. 3.   Binding of 2102 cells to plates coated with various GSLs, and the effect of anti-GSL antibodies on Gb4-dependent 2102 cell adhesion. A, 2102 cell binding to GSL-coated plates. 2102 cells (1 × 105/well) were incubated in wells coated with Gg3 (bullet ), Gb4 (black-square), GM1 (triangle ), GM3 (down-triangle), Lea (), and nLc3 (diamond ) (0-1.5 nmol of GSL/well). Adherent cells were stained with 0.1% Toluidine Blue O and measured at 630 nm. B, effect of anti-GSL antibodies on Gb4-dependent adhesion. 2102 cells (1 × 105) were incubated in control medium or in medium containing mAbs 2D4 (anti-Gg3) (5 µg/ml), 9G7 (anti-Gb4) (20 µg/ml), MC631 (anti-SSEA-3) (20 µg/ml), or 1B2 (anti-nLc4) (2 µg/ml) for 1 h at 37 °C (see Footnote 5). Cells were washed and plated in wells coated with various quantities of Gb4 (abscissa). bullet , control (no treatment of cells); black-down-triangle , 2D4; black-square, 9G7; black-diamond , MC631; black-triangle, 1B2. Adherent cells were stained and measured as in A. Each point represents the mean of triplicate experiments. C, effect of anti-Gb4 mAb 9G7 on 2102 cell adhesion to Gb4-coated plates preincubated with various quantities of 9G7. Standard variation is indicated by bar.

Effect of Micellar GSL Solutions and Anti-GSL Antibodies on Gb4-dependent 2102 Cell Adhesion-- The partner ligand expressed at the 2102 cell surface that binds to Gb4 coated on plates might be nLc4 or GalGb4, but not Gb4 per se, as indicated by liposome binding assay (Fig. 2B). To clarify this point, the effect of micellar GSL solutions and anti-GSL antibodies on Gb4-dependent 2102 cell adhesion was studied. The adhesion was inhibited when plates were preincubated with micellar aqueous solution of nLc4 (Fig. 4A), and to a slightly lesser extent with micellar solution of GalGb4 (Fig. 4B). The adhesion was not inhibited (on the contrary, it was slightly enhanced) when plates were preincubated with Gb4 (Fig. 4C). These results indicate that nLc4 and GalGb4 expressed at the surface of 2102 cells are both involved in Gb4-dependent 2102 cell adhesion. Involvement of nLc4 and GalGb4 in this process was further confirmed by the fact that the adhesion was inhibited when cells were preincubated with mAb 1B2 (anti-nLc4) or with mAb MC631 (anti-SSEA-3), but not with mAb 2D4 (anti-Gg3) or with mAb 9G7 (anti-Gb4) (Fig. 3B).5 2102 cell adhesion to Gb4-coated plates was strongly inhibited when the plates were preincubated in the presence of anti-Gb4 mAb 9G7 (Fig. 3C), but not when the cells were preincubated with 9G7 (Fig. 3B).


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Fig. 4.   Effect of micellar aqueous solutions of nLc4 and GalGb4 on Gb4-dependent 2102 cell adhesion. Ethanol solution (100 µl) containing 1.0 nmol of Gb4 was added to each well of 96-well polystyrene plates and air dried. Micellar aqueous solution of nLc4, GalGb4, or Gb4 in PBS was prepared, and aliquots containing 0, 5, 10, 20, 30, 40, or 50 nmol of GSL were added to each well. Plates were incubated for 2 h at 37 °C, 2102 cells were added, and adhesion was measured as described under "Experimental Procedures" and Fig. 3 legend. A, effect of nLc4; B, effect of GalGb4; C, effect of Gb4. Each bar represents mean value of triplicate experiments. Standard variation is indicated by bar.

Down-regulation of SSEA-3 and nLc4 Expression in 2102 Cells by Incubation with Retinoic Acid or BrdUrd, and Resulting Reduction of Gb4-dependent Adhesion-- Human embryonal carcinoma cells are generally characterized by high expression of globo-series GSLs (16-18, 32); their expression declines during differentiation (17, 33, 34) (see "Discussion"). To study the involvement of GalGb4 or nLc4 in Gb4-dependent adhesion of 2102 cells, we examined the correlation of this adhesion with down-regulation of GalGb4 and nLc4 expression induced by retinoic acid or BrdUrd. The high expression of SSEA-3 on 2102 cells (Fig. 1) was significantly reduced upon retinoic acid- or BrdUrd-induced differentiation (Fig. 5, upper panel). A similar reduction of reactivity with mAb 1B2 was also observed for 2102 cells (Fig. 5, middle panel). In association with the reduction of SSEA-3 and nLc4 expression, Gb4-dependent adhesion was significantly reduced (Fig. 5, lower panel).


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Fig. 5.   Effect of retinoic acid and BrdUrd on SSEA-3 and nLc4 expression in 2102 cells, and on associated Gb4-dependent adhesion. Upper and middle panels, differentiation-dependent reduction of SSEA-3 and nLc4 expression induced by retinoic acid and BrdUrd. Upper four panels, SSEA-3 expression determined by flow cytometry with mAb MC631. Middle three panels, nLc4 expression determined with mAb 1B2. Non, cells cultured in regular DMEM supplemented with 10% FBS for 7 days. RA, cells incubated with 10 µM retinoic acid for 7 days. BUdR, cells incubated with 3 µM BrdUrd for 7 days. Control, cells cultured in regular DMEM and not treated with primary antibody. Ordinate, count (relative cell number); scale is identical in all panels. Lower panel, reduction of Gb4-dependent adhesion by retinoic acid and BrdUrd. After 7 days of incubation with 10 µM retinoic acid (black-square), 3 µM BrdUrd (black-triangle), or regular DMEM (bullet ), 2102 cells were plated in Gb4-coated wells (0-1.5 nmol/well), and adherent cells were stained and measured as described under "Experimental Procedures". Each point represents the mean of triplicate experiments. Standard variation is indicated by bar.

Expression of Various Transcription Factors in 2102 Cells, and Changes Induced by Gb4-or Gg3-dependent Adhesion-- Following 2102 cell adhesion to Gb4-coated plates, the activity levels of seven transcription factors (AP1, AP2, CREB, E2F1, Egr, NFkappa B, and SP1) were determined and compared with levels of the same transcription factors in cells plated on non-coated plates as control (Fig. 6). Activity levels of the same seven transcription factors were also determined and compared following 2102 cell adhesion to Gg3-coated plates (Fig. 7). Activities of all seven transcription factors were detected in 2102 cells. Cells attached to Gb4-coated plates showed significant enhancement of binding activity for one AP1 band, and for the major band representing CREB, whereas such enhancement was barely detected for other transcription factors. No changes in activity were found for any of the seven transcription factors when 2102 cells were attached to Gg3-coated plates (Fig. 7), despite the fact that this Gg3-dependent cell adhesion was strong. Furthermore, when 2102 cells were plated and forced to adhere by centrifugation on GM1-coated plates, there was no significant binding and no enhancement of CREB relative to control (Fig. 8). These results indicate that the activity changes of AP1 and CREB were induced by Gb4-dependent cell adhesion, but activity was not affected by adhesion to Gg3- or GM1-coated plates. AP1 and CREB bands, detected by 32P-labeled probe, were abolished by addition of 40 ng of each non-labeled probe (Fig. 8). This confirms that binding to AP1 and CREB consensus sites is sequence-specific. The activity changes in these bands induced by cell adhesion are therefore specific for AP1 and CREB.


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Fig. 6.   Activity levels (by gel shift analysis) of various transcription factors in 2102 cells, and the effect of Gb4-dependent adhesion. 2102 cells were plated on non-coated 10-cm tissue culture plates (Control) or on 10-cm plates coated with Gb4 (300 nmol/plate; labeled as Gb4), and incubated at 37 °C for 1 h. Nuclear extracts were incubated with labeled oligonucleotide probe containing consensus binding site for AP1, AP2, CREB, E2F1, Egr, NFkappa B, or SP1. Autoradiographs were obtained after separation on 4% polyacrylamide gel. Arrows indicate the location of AP1 or CREB specific bands.


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Fig. 7.   Activity levels (by gel shift analysis) of various transcription factors after incubation on Gg3-coated plates. 2102 cells were plated on non-coated 10 cm tissue culture plates (Control) or on plates coated with Gg3 (300 nmol/plate) and incubated at 37 °C for 1 h. Nuclear extracts were incubated with labeled oligonucleotide probe containing consensus binding site for AP1, AP2, CREB, E2F1, Egr, NFkappa B, or SP1. Autoradiographs were obtained after separation on 4% polyacrylamide gel.


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Fig. 8.   Abolition of AP1 and CREB activity by cold probes. Extracts from 2102 cells attached to control plates, Gb4-coated plates (labeled as Gb4), or GM1-coated plates (labeled as GM1) were incubated with (+) or without (-) addition of cold (non-labeled) AP1 and CREB probes (80 × chemical quantity of 32P-labeled probes) at room temperature for 20 min and subjected to gel shift assay. Arrows indicate the location of AP1 or CREB specific bands. No significant change of CREB activity was detected for cells attached to GM1 compared with control extract.

Changes in CREB Activity Associated with 2102 Cell Adhesion to 2102 Cell DISAM, Which Mimics Homotypic Adhesion of 2102 Cells-- 2102 cells were adhered to DISAM prepared from 2102 cell monolayer, and time-course changes of activity of various transcription factors were determined, as described under "Experimental Procedures." A remarkable enhancement of CREB activity of DISAM-adherent cells was observed consistently, in all experiments, from 15 to 30 min after initial adhesion (Fig. 9). On further prolonged adhesion (60 min or longer), CREB activity of these cells was significantly reduced, to a level the same or lower than that of cells adhered to control plates (data not shown). No other transcription factor in 2102 cells showed a similar magnitude of activity changes in association with DISAM-dependent cell adhesion.


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Fig. 9.   Time-dependent change in CREB activity associated with adhesion of 2102 cells to 2102 cell monolayer. 2102 cells were placed on DISAM for 15, 30, or 60 min. Nuclear protein extracts of attached cells were assayed for CREB activity. The competition experiment with cold CREB was carried out using 30-min extract. Arrows indicate the level of the CREB specific band.

Effect of Anti-transcription Factor Antibodies on Transcription Factor Expression Associated with Gb4-dependent or DISAM-dependent 2102 Cell Adhesion-- To further confirm the specificity of the enhanced expression of AP1 and CREB associated with Gb4-dependent or DISAM-dependent 2102 cell adhesion, we studied the effect of antibodies to these transcription factors. Supershift of enhanced CREB expression associated with both DISAM-dependent and Gb4-dependent cell adhesion was clearly observed (Fig. 10, A and B). Addition of antibody mixture directed to Fos or Jun family did not cause supershift of enhanced AP1 expression, but did inhibit binding to the AP1 site probe (data not shown; see "Discussion"). Specificity of the enhanced AP1 binding activity was clearly demonstrated by the competition with excess unlabeled AP1 probe (see "Discussion").


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Fig. 10.   Effect of anti-CREB antibody ("supershift") on CREB expression associated with DISAM-dependent or Gb4-dependent 2102 cell adhesion. A, DISAM-dependent cell adhesion. B, Gb4-dependent cell adhesion. Lane 1, cells added on noncoated plates. Lane 2, cells added on DISAM- or Gb4-coated plates without addition of anti-CREB antibody ATF-1. Lane 3, cells added on DISAM- or Gb4-coated plates with addition of ATF-1.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The specificity of cell adhesion is defined by the types of receptors expressed, and availability of ligands at the cell surface, i.e. binding of a specific integrin receptor to a defined ligand such as fibronectin, laminin, collagen, fibrinogen, etc. (1, 35, 36), and interaction of Ig family receptors (2, 37) and cadherin family receptors (3) among themselves. Carbohydrate-binding protein receptors such as galectins (38), selectins (5, 39), sialoadhesins (40, 41), and CD44 (4) play an additional essential role in adhesion of certain types of cells.

Adhesion based on carbohydrate-carbohydrate interaction initiates a quick, selective process prior to involvement of other receptor systems with stronger binding affinity. For example, adhesion of B16 melanoma cells to mouse endothelial cells under dynamic flow conditions is based initially on interaction of GM3 (expressed on B16 cells) with Gg3 or LacCer (expressed on endothelial cells) (8), with subsequent involvement of selectin- or integrin-dependent adhesion (42). Aggregation of F9 embryonal carcinoma cells, which mimics compaction, may take place cooperatively with Lex- and E-cadherin (uvomorulin)-dependent adhesion (14).

Many human embryonal carcinoma cells, particularly at the undifferentiated stage, show high expression of globo-series structures including SSEA-3 and -4, which are down-regulated upon differentiation in parallel with a decrease in cell adhesion (15, 18, 32, 43). These previous findings suggest that Gb4 and globo-series GSLs may be involved in cell adhesion, analogous to the involvement of Lex in compaction of mouse embryo and autoaggregation of F9 cells. The present study addresses two major questions. (i) Do Gb4, extended globo-series GSLs, or any other GSLs highly expressed in embryonal carcinoma cells function as adhesion molecules? If so, what is the mechanism? (ii) Does signal transduction occur following Gb4-dependent cell adhesion?

To answer the first question, we studied adhesion of human embryonal carcinoma 2102 cells which express high levels of GalGb4 and nLc4 and moderate level of Gb4 at the surface (Fig. 1), and found that these cells adhere strongly to plates coated with Gb4 or Gg3, and did not adhere at all to plates coated with Lc3, Gb3, Lea, Leb, GM3, GM1, or sialyl-nLc4 (sialylparagloboside) (Fig. 3). Among these tested GSL epitopes, Lc3, Gg3, Lea, and Leb are absent in 2102 cells and should not be considered in terms of physiological involvement.

Adhesion of 2102 cells to Gb4-coated plates, termed "Gb4-dependent adhesion," simulates aggregation of 2102 cells among themselves. The process is based primarily on carbohydrate-carbohydrate interaction between nLc4 or GalGb4 (expressed on cells) and Gb4 (coated on plates), as indicated by the facts that: (i) the adhesion is inhibited by preincubation of 2102 cells with anti-nLc4 mAb 1B2, anti-GalGb4 mAb SSEA-3 (Fig. 3B), and by preincubation of Gb4-coated plates with anti-Gb4 mAb 9G7 (Fig. 3C) or with micellar aqueous solution of nLc4 or GalGb4 (Fig. 4, A and B); (ii) liposomes containing Gb4 bind to plates coated with nLc4 or GalGb4, but not other GSLs; (iii) Gb4-dependent adhesion is reduced during retinoic acid- or BrdUrd-induced differentiation of 2102 cells, whereby GalGb4 expression is also greatly reduced (Fig. 5). Previous studies indicate that a decline in SSEA-3, SSEA-4, and nLc4 is associated with increased expression of Lex in 2102 cells upon differentiation induction (17, 33). Differentiation-dependent regulation of glycosyltransferase levels associated with changes of these epitopes has been studied extensively (34). A strong interaction between Gb4 and Gg3 was observed in the present study, but probably has no physiological significance in this cell adhesion system since Gg3 is absent in 2102 cells. 2102 cells adhered strongly to DISAM, which contains essentially all GSLs and pericellular matrix. This event mimics adhesion among 2102 cells. Binding of mAb 1B2 is also reduced during 2102 cell differentiation. This is in striking contrast to the TERA-2 cell differentiation induced by retinoic acid or BrdUrd (34).

The second question is in response to the general observation that cell adhesion is a prerequisite for induction of differentiation. To answer this question, we studied changes of transcription factor activity in 2102 cells following Gb4-dependent adhesion. This adhesion rapidly (within 30-60 min) induces strong enhancement of two transcription factors, AP1 and CREB, out of seven factors examined. Similar enhancement of CREB was observed within 15-30 min when 2102 cells were adhered to DISAM. Interestingly, there was no change of activity of any transcription factor when 2102 cells were adhered to Gg3-coated plates. These results indicate that carbohydrate specificity is required for signaling but not for adhesion per se. The signal transduction occurring in 2102 cells may be initiated through nLc4 or GalGb4, but not through Gb4, at the surface of these cells. This concept is illustrated in Fig. 11.


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Fig. 11.   Schematic model of homotypic 2102 cell adhesion based on Gb4-nLc4 or Gb4-GalGb4 interaction (left) and heterotypic 2102 cell adhesion to Gg3-expressing cells based on Gb4-Gg3 interaction (right). Aggregation of 2102 cells among themselves, which presumably mimics the compaction process of human embryo, is based on carbohydrate-carbohydrate interaction of nLc4-Gb4 or Gb4-GalGb4, as indicated by liposome interaction experiments and effects of aqueous micellar GSL solutions and anti-GSL antibodies on Gb4-dependent 2102 cell adhesion. Homotypic interactions of Gb4-Gb4, nLc4-nLc4, and GalGb4-GalGb4 are not involved in this process. Cell adhesion based on the mechanism shown in the left panel produces signals in the form of enhanced expression of AP1 and CREB, possibly through nLc4 or GalGb4, but not through Gb4. 2102 cell adhesion to Gg3-coated plates (a model of Gg3-containing membrane), based on the mechanism shown in the right panel, is very strong, but does not produce signals. This is probably because Gb4 per se is involved in the adhesion, and does not give rise to signaling. Solid lines, strong interaction and signaling; dotted lines, weaker interaction and signaling.

A "supershift" upon addition of anti-CREB mAb ATF-1 was clearly demonstrated in CREB expression associated with both Gb4-dependent and DISAM-dependent 2102 cell adhesion (Fig. 10). However, we were unable to definitively identify the protein subunits bound to the AP1 site that were induced upon 2102 cell adhesion to Gb4. Supershift experiments with antibodies that recognize all known Fos or all known Jun family proteins inhibited binding to the AP1 site probe, suggesting that antibodies interact with the DNA binding domain of these factors. The possible presence of a still unidentified member of the AP1 family cannot be ruled out. In any case, specificity of the AP1 site binding activity is demonstrated by competition with unlabeled excess AP1 oligonucleotide (Fig. 8).

GSLs at the cell surface are organized in clusters and form GSL microdomains (44, 45). Recent studies indicate that GSL microdomains are organized with various transducer molecules such as c-Src, Ras, Fak, and Rho in close association with specific GSLs, and that these microdomains can be isolated as detergent-insoluble low density vesicles (46). The effectiveness of GSLs to bring about cell adhesion and to induce signaling may well depend on the quantity and the quality of GSLs organized in microdomains. A relatively weak adhesion occurring between Gb4 and GalGb4 may still cause significant cell adhesion and signaling. Dounce homogenization of 2102 cells in Tris buffer containing 1% Triton X-100, followed by sucrose density-gradient centrifugation, results in separation of low density GSL-enriched vesicles which contain various transducer molecules.6 Further extensive studies are needed regarding types of transducer molecules elicited in response to Gb4-dependent adhesion of 2102 cells leading to enhanced expression of AP1 and CREB, and consequent phenotypic changes.

AP1 and CREB transcription factors are, in this case, functionally identifed by virtue of specific binding to the AP1 or CREB consensus binding site, respectively. Both of these transcriptional activators encompass a heterogeneous family of potential homo- or heterodimers between numerous members of their respective gene families. Each functionally defined factor could, therefore, be composed of a myriad of possible dimers (47). The specific AP1 and CREB subunits expressed in 2102 cells are yet to be determined; indeed, three or four specific AP1 site-bound complexes are seen in Figs. 6 and 8. Only one of these, however, is enhanced upon Gb4 binding. Interestingly, some AP1 and CREB family members form interfamily heterodimers, which bind preferentially to CREB sites (47). The functional consequences of interfamily dimerization on transcriptional regulation, however, are currently unknown.

Numerous studies have demonstrated activation of AP1 by factors binding to a cell surface receptor tyrosine kinase and activating the Ras or protein kinase C signaling pathway (reviewed in Ref. 48). These pathways result in transcriptional activation of c-Jun or c-Fos (AP1 subunit encoding genes) as well as post-translational modification of AP1 subunit phosphorylation state. Phosphorylation of c-Jun at two serine residues in the activation domain allows binding to the transcriptional coactivator CBP (49). The ultimate response can be mitogenic or differentiative, depending on cell type.

CREB activity is induced by stimuli that elevate intracellular cAMP levels (reviewed in Ref. 48). This pathway involves protein kinase A, which phosphorylates serine 133 of CREB, thus allowing binding to the transcriptional coactivators CBP and p300 (50, 51). Since AP1 and CREB subunits can dimerize in complex ways and both factors bind to the same transcriptional coactivators, the induction of both factors in Gb4-bound 2102 cells may elicit an "additive" AP1/CREB response. Alternatively, these factors could competitively bind to the transcriptional coactivator as is the case for nuclear hormone receptors and AP1 (52).

Gb4, originally found as a birefringent, globular, crystalline-like precipitate and so termed "globoside" (53), together with its extended form (16), are now known to be adhesion molecules. nLc4 and GalGb4, which are widely distributed in many types of human and animal cells, are now identified as counterpart adhesion molecules for Gb4. The data presented in this report are only one example of a physiological adhesion system based on this interaction. There are presumably many more examples, since Gb4 and nLc4 are common and abundant molecules expressed on the surface of animal cells.

Whether cell surface events influence gene activation has been a long-standing major question in cell biology (see, e.g., Ref. 54). A positive answer to this question is indicated by the fact that integrin-dependent adhesion of cells to matrix protein induces typical signal transduction. For example, fibronectin-dependent adhesion of smooth muscle cells and fibroblasts activates NFkappa B p50/p65 heterodimer (55), although the mechanism is unclear. A similar process should be considered for various types of cell surface receptors which control adhesion and motility, as listed in the first paragraph of this "Discussion." This idea can be applied to cell adhesion based on carbohydrate-carbohydrate interaction. Adhesion of B16 melanoma to Gg3- or LacCer-coated plates induces enhanced motility as determined by phagokinetic assay (7). The observed enhancement of transcription factors AP1 and CREB (out of various others) shortly after adhesion of 2102 cells to Gb4-coated plates supports the concept that cell adhesion mediated by carbohydrate-carbohydrate interaction influences nuclear events. Phenotypic changes triggered by changes of transcription factor activity in response to Gb4-dependent cell adhesion remain to be studied.

    ACKNOWLEDGEMENTS

We thank Drs. Kazuko Handa and Soichiro Yamamura for helpful advice and discussion during performance of the study, Drs. Baiba Gillard and Donald M. Marcus (Baylor College of Medicine, Houston, TX) for kind donation of anti-Gb4 mAb 9G7, and Dr. Stephen Anderson for scientific editing and preparation of the manuscript.

    FOOTNOTES

* This work was supported by National Institutes of Health NCI Outstanding Investigator Grant CA42505 (to S. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Lab Cell Biochem, NIDDK, NIH, MSC 0850, Bldg. 8, Rm. 402, Bethesda, MD 20892-0850.

1 Glycosphingolipids are abbreviated according to the recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature ((1977) Lipids 12, 455-463); however, the suffix -OseCer is omitted. Gb3, Galalpha 1right-arrow4Galbeta 1right-arrow4Glcbeta 1-Cer; Gb4, GalNAcbeta 1right-arrow3Galalpha 1right-arrow4Gal beta 1right-arrow4Glcbeta 1-Cer; GalGb4, Galbeta 1right-arrow3GalNAcbeta 1right-arrow3Galalpha 1right-arrow4Galbeta 1right-arrow4Glcbeta 1-Cer; Gg3, GalNAcbeta 1right-arrow4Galbeta 1right-arrow4Glcbeta 1-Cer; LacCer, Galbeta 1right-arrow4Glcbeta 1-Galbeta 1right-arrow4Glcbeta 1-Cer; Lc3, GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4Glcbeta 1-Cer; nLc4, Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4Glcbeta 1-Cer; Lea, Galbeta 1right-arrow3(Fucalpha 1right-arrow4)GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4Glcbeta 1-Cer; Leb, Fucalpha 1right-arrow2Galbeta 1right-arrow3(Fucalpha 1right-arrow4) GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4Glcbeta 1-Cer; Lex, Galbeta 1right-arrow4(Fucalpha 1right-arrow3)GlcNAc beta 1right-arrow3Galbeta 1right-arrow4Glcbeta 1-Cer.

2 The abbreviations used are: GSL, glycosphingolipid; BSA, bovine serum albumin; BrdUrd, bromodeoxyuridine; Cer, ceramide; DISAM, detergent-insoluble substrate adhesion matrix; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; mAb, monoclonal antibody; PBS, phosphate-buffered saline.

3 Gangliosides are abbreviated according to L. Svennerholm ((1977) Eur. J. Biochem. 79, 11-21).

4 Dependence of Lex-Lex, GM3-Gg3, and GalCer-sulfatide interactions on bivalent cation (Ca2+, Mg2+) has been well documented (6, 7, 10, 56). In contrast, the strong interaction of KDN-GM3 with Gg3, the basis of sperm-egg adhesion in rainbow trout, does not require bivalent cation (Y. Song, K. Kitajima, S. Inoue, S. Kudo, N. Kojima, S. Hakomori, and Y. Inoue, unpublished data). Similarly, interaction of Gb4 with nLc4 or with GalGb4 does not require bivalent cation. The molecular basis of bivalent cation dependence versus independence in carbohydrate-carbohydrate interaction is currently unknown.

5 Preincubation of 2102 cells with anti-SSEA-3 mAb MC631 inhibits Gb4-dependent cell adhesion only at high concentration (10-20 µg/ml), but not at lower concentration (1-2 µg/ml). The inhibitory effects of anti-carbohydrate antibodies on cell adhesion varied extensively depending on antibody affinity and quantity of antigen expressed at the cell surface. Compaction of morula is inhibited, or once-compacted embryo is decompacted, by multivalent lacto-N-neotetraose III (Lex) structure (11). Thus, Lex is obviously involved in the compaction process, and the mechanism is through Lex-Lex interaction (6), albeit anti-Lex (anti-SSEA-1) mAb did not inhibit compaction (B. A. Fenderson, D. Solter, and B. B. Knowles, unpublished data).

6 Y. Song, S. Yamamura, and S. Hakomori, unpublished data.

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Abstract
Introduction
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Results
Discussion
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