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Cell Growth & Differentiation Vol. 12, 623-630, December 2001
© 2001 American Association for Cancer Research

Fusion Hybrids with Macrophage and Melanoma Cells Up-Regulate N-Acetylglucosaminyltransferase V, ß1–6 Branching, and Metastasis1

Ashok K. Chakraborty2, John Pawelek, Yoshitaka Ikeda, Eiji Miyoshi2, Natalia Kolesnikova, Yoko Funasaka, Masamitsu Ichihashi and Naoyuki Taniguchi

Department of Dermatology, Yale University School of Medicine, New Haven, Connecticut 06510 [A. K. C., J. P., N. K.]; Department of Biochemistry, Osaka University Medical School, Osaka 565-0871, Japan [Y. I., E. M., N. T.]; and Department of Dermatology, Kobe University School of Medicine, Kobe 650-0017, Japan [Y. F., M. I.]


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
It was shown previously that a majority of hybrids produced by in vitro fusion of normal macrophages with Cloudman S91 melanoma cells displayed enhanced metastatic potential in vivo, increased motility in vitro, increased ability to produce melanin, and responsiveness to melanocyte stimulating hormone compared with the parental Cloudman S91 melanoma cells. These hybrids also showed altered N-glycosylation consistent with a slower migration pattern of lysosome-associated membrane protein (LAMP-1) on electrophoretic gels. Because LAMP-1 is the major carrier of polylactosamine sugar structures, and synthesis of this complex sugar moiety indicates the extent of ß1,6 branch formation by ß1,6-N-acetyl-glucosaminyltransferase V (GnT-V), we analyzed the expression of GnT-V and ß1,6 branching in highly metastatic macrophage-fusion hybrids and compared with poorly metastatic ones. GnT-V was up-regulated in regard to both mRNA levels and enzymatic activity specifically in metastatic hybrids as well as parental macrophages compared with weakly metastatic hybrids and parental melanoma cells. Macrophages and metastatic hybrids also showed increased binding of the lectin L-phytohemagglutinin, which specifically binds to the ß1,6-branched sugar moiety. In addition, in metastatic hybrids there was increased cell surface expression of LAMP-1 and ß1 integrin, two prominent substrates for GnT-V also known to be associated with metastasis. Finally, exposure of metastatic hybrids in vitro to L-phytohemagglutinin or LAMP-1 completely eliminated melanocyte stimulating hormone/ isobutylmethyl xanthine-induced motility, suggesting a role for GnT-V in the motility of these cells. In summary, macrophage fusion with melanoma cells often increased metastatic potential, which was associated with enhanced expression of GnT-V and ß1,6-branching in glycoproteins. It is suggested that the known correlation with elevated GnT-V in both human and animal metastasis could, at least in some cases, reflect previous fusion of tumor cells with tumor-infiltrating macrophages, which, similar to malignant cells, show elevated expression of GnT-V and ß1,6-branched polylactosamines.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
In vitro fusion of weakly metastatic mouse Cloudman S91(6neo) melanoma cells with peritoneal macrophages from DBA/2J mice produced hybrids, a majority of which displayed enhanced metastatic potential in vivo (1) . In addition, hybrids with higher metastatic potential tended to be more pigmented, more motile, and more dendritic than parental melanoma cells, with higher expression of MSH3 receptors and markedly increased responsiveness to cAMP induction by MSH combined with IBMX, or cholera toxin (2, 3, 4) . Recently, a large spontaneous melanoma metastasis to the lung was found to be comprised of host/tumor hybrids derived from Cloudman S91 melanoma cells (DBA/2J origin) implanted s.c. in the tail of a BALB/c nude mouse (5) . All of the cells cultured from this metastasis appeared to be hybrids and showed the same phenotypes described above for artificial macrophage/melanoma hybrids (5) . Two other spontaneous melanoma/host hybrids in mice were described earlier (6 , 7) , and one of them, PADA, was found to be highly metastatic and again resembled artificial macrophage/melanoma hybrids in phenotype (1 , 3) . In both artificial and spontaneous hybrids, additional analyses revealed altered N-glycosylation patterns compared with parental melanoma cells (2 , 8) . Using [3H]glucosamine as a marker of N-glycosylation, incorporation into tyrosinase and LAMP-1 was significantly elevated in metastatic hybrids compared with parental melanoma cells and was suppressed by N-glycosylation inhibitors. Furthermore, tyrosinase and LAMP-1 from hybrids migrated more slowly on gels compared with the same proteins from parental melanoma cells, consistent with increased glycosylation. The slower electrophoretic gel migration of LAMP-1 from metastatic hybrids was similar to LAMP-1 of peritoneal macrophages (2 , 8) . Studies with swainsonine, a glycosylation inhibitor that prevents ß1,6 branch formation and metastasis (9, 10, 11, 12, 13) , indicated that the slower mobility of LAMP-1 from metastatic hybrids and macrophages was likely attributable to increased ß1,6 branched structures (2 , 8) .

LAMPs-1 and 2 are the most densely N-glycosylated of cellular proteins and major carriers of N-acetylpolylactosamine antennae (14) . N-acetylpolylactosamine synthesis is initiated by GnT-V (EC 2.4.1.155; ß1,6-N-acetylglucosaminyltransferase V), which, in the trans Golgi, catalyzes the formation of ß1,6 branches on the trimannosyl terminus of Asn-linked oligosaccharides (15 , 16) , allowing for the enzymatic addition of ß1,6-GlcNAc-linked poly-N-acetyllactosaminyl chains by UDPGlcNAc:N-acetyllactosaminide ß1,3-N-acetylglucosaminyl transferase (17) . In this regard, the activity of GnT-V independently regulates the polylactosamine contents of N-linked oligosaccharides and acts as a marker for the level of ß1,6-branching constructs (18 , 19) , which can be recognized by their binding to the lectin L-PHA (20) . Because elevated GnT-V activity is characteristic of both macrophages and metastatic cells (see "Discussion"), we sought to determine whether elevated GnT-V activity could account for at least some of the enhanced glycosylation in the metastatic macrophage/melanoma hybrids described above.

Here we report our results on assays of GnT-V mRNA levels and enzyme activity, L-PHA binding (specific for ß1,6 branching; Ref. 20 ), and cell surface expression of LAMP-1 and ß1 integrin, two substrates of GnT-V known to be involved in metastasis (10 , 14 , 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31) , in peritoneal macrophages, Cloudman S91(6neo) cells, and macrophage/melanoma hybrids of high and low metastatic potential. By all of these assays used, the results indeed correlated an elevated GnT-V activity, a common characteristic of both macrophages and metastatic cells, with enhanced metastatic potential of macrophage-fusion hybrids.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
LAMP-1 in Metastatic Hybrids.
LAMP-1 isolated from parental Cloudman S91(6neo) melanoma cells typically migrated in electrophoretic gels in a broad band with an average molecular mass of 97 kDa, whereas LAMP-1 from normal DBA/2J mouse peritoneal macrophages migrated more slowly, with an average molecular mass of 132 kDa (Fig. 1A)Citation . In a panel of hybrids, LAMP-1 from hybrids with high metastatic potential (95-H1, 94-H48, PADA, and LM6) migrated on gels similar to macrophage LAMP-1 compared with LAMP-1 from poorly metastatic hybrids (95H-3 and 95-H12), of which the gel mobility was closer to that of parental Cloudman S91 cells (Fig. 1B)Citation . Stimulation with MSH, however, showed no additional effect on LAMP-1 mobility on gels (Fig. 1, A and B)Citation , suggesting that its net glycosylation was unchanged by this treatment, as expected from previous studies (2 , 8) . Because LAMP-1 is a key substrate for GnT-V (14 , 21) , and swainsonine treatment [which prevents ß1,6 branch formation (9) ] increases the mobility of LAMP-1 from metastatic hybrids and macrophages (2 , 8) , the increased glycosylation of LAMP-1 in metastatic hybrids suggested that GnT-V expression might be up-regulated in these cells.



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Fig. 1. Immunoblot analyses of LAMP-1. A, LAMP-1 from parental Cloudman S91 melanoma line [S91(6neo)] compared with that from isogenic thioglycollate-induced DBA/2J peritoneal macrophages (). B, LAMP-1 from the same parental melanoma line S91(6neo) compared with macrophage/melanoma artificial fusion hybrids of low (hybrids 95-H3 and 95-H12) and high (hybrids 94-H4, 94-H48, and 95-H1) metastatic potential. PADA and LM6 were two spontaneous in vivo Cloudman S91 hybrids of high metastatic potential (see "Materials and Methods"). Metastatic potentials for these cell lines are shown in Table 1Citation .

 
GnT-V Enzymatic Activity.
Direct enzymatic assays revealed that the specific activity of GnT-V in cell lysates was 2–3-fold higher in highly metastatic hybrids and peritoneal macrophages compared with parental Cloudman S91 melanoma cells and weakly metastatic hybrids (Table 1)Citation . This was true for hybrids of high metastatic potential whether they were artificially generated with polyethylene glycol in vitro (hybrids 94-H48, 95-H4, and 95-H1; Ref. 1 ), a spontaneous host/tumor hybrid isolated from within a Cloudman S91 melanoma primary tumor in vivo (PADA; Ref. 6 ), or host/tumor hybrid cells from a spontaneous pulmonary metastasis of a Cloudman melanoma tumor (LM6; Ref. 5 ). The increased enzymatic activity was significant (P < 0.001) and consistent with previous reports from other laboratories, showing from 2- to 10-fold elevations in GnT-V-specific activity in metastatic cells, including some that were transformed with oncogenic viruses (9 , 18 , 32 , 33) .


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Table 1 GnT-V activity in macrophage-fusion hybrid cells exhibiting high and low metastatic potential

 
GnT-V mRNA.
Northern blot analyses of the same cells used for the enzyme assay for GnT-V activity revealed that the GnT-V mRNA was detected as a major band corresponding to ~7.5 kb and a minor band to ~3.5 kb as described (34) . Densitometric scanning of the bands and normalization with control 28S and 18S RNA indicated that both species of GnT-V mRNA were elevated several-fold in highly metastatic hybrids and peritoneal macrophages, compared with poorly metastatic hybrids and parental melanoma cells. The results from multiple pooled experiments on GnT-V mRNA were summarized in Table 2Citation , with a comparison of mRNA level to metastatic potential (1) . It was apparent that whereas constitutive expression of GnT-V mRNA was elevated in metastatic hybrids as seen in Fig. 2Citation , MSH/IBMX treatment had no effect on mRNA levels in any of the cell lines (Table 2)Citation .


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Table 2 Comparison of GnT-V mRNA expression in artificial and spontaneous hybrid cells of high and low metastatic potential as quantitated densitometry of gels

 


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Fig. 2. Northern blot analysis of hybridizable GnT-V mRNA in various cell lines. A, total RNA (30 µg) was analyzed by hybridization with 32P-labeled GnT-V cDNA ("Materials and Methods"). B, ethidium bromide staining of RNA samples showing the relative concentration of rRNA, added as an internal control for each lane. C, densitometric scanning of both the 7.5-kb and 3.5-kb mRNA species, and normalization of the band intensity with 18S total RNA. Cell lines were: artificial macrophage/melanoma fusion hybrids of high (hybrids 94-H4, 94-H48, and 95-H1), and low (hybrid 95-H3) metastatic potential, spontaneous in vivo hybrid PADA, parental S91 melanoma cells, and DBA/2J peritoneal macrophages (). A summary of results with GnT-V mRNA levels in this and an additional experiment including MSH/IBMX treatment is given in Table 2Citation .

 
Flow Cytometric Detection of L-PHA Binding, LAMP-1, and ß1-Integrin.
To assess GnT-V activity in vivo we measured cell surface ß1,6-branched oligosaccharides via their specific binding to L-PHA and detection by flow cytometry (Fig. 3)Citation . As with GnT-V mRNA levels and enzymatic activity, there was increased L-PHA binding (3–6-fold) in highly metastatic hybrids, both artificial and spontaneous, as well as in peritoneal macrophages compared with parental melanoma cells and poorly metastatic hybrids. The enhanced L-PHA binding was presumably restricted to the cell surface, because the binding and wash procedures were carried out on ice with no previous lysis of cells (see "Materials and Methods").



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Fig. 3. Flow cytometric analyses of L-PHA binding, and expression of LAMP-1 and ß1-integrin on the cell surface of high and low metastatic hybrids, parental Cloudman melanoma cells, and normal peritoneal macrophages. A, histogram of fluorescent intensity with (---) or without (----) FITC-conjugated L-PHA (left), rat antimouse LAMP-1 (middle), and rat antimouse CD29 for ß1-integrin (right). Cell lines are indicated on the right. B, quantitation of data from flow cytometric analyses. Fluorescent intensity relative to negative controls, representing mean from five pooled experiments. *P < 0.01; **P < 0.001; bars, ±SD.

 
Flow cytometry was also used to analyze cell surface expression of LAMP-1 and ß1 integrin, two major protein substrates for GnT-V (10 , 14 , 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31) , using monoclonal antibodies (Figs. 3Citation and 4). These proteins were overexpressed on the cell surface in highly metastatic hybrids (94-H48, 95-H4, 95-H1, PADA, and LM6) and peritoneal macrophages compared with parental Cloudman S91 cells and weakly metastatic hybrids (95-H3 and 95-H12). However, it should be pointed out that the specificity of the antibodies for these proteins might be directed in part to the ß1,6-branched polylactosamine content as opposed to the core protein structure per se, because the specificity of the antibodies in this regard were unknown, and such polylactosamines are highly immunogenic (35) . Thus, the apparent overexpression of LAMP-1 and ß1 integrin might be attributable to increased glycosylation of the proteins as opposed to an increased number of protein molecules on the cell surface. This question could not be resolved by the assays used.

Effects of LAMP-1 and L-PHA on Cell Motility in Vitro.
Studies on chemotactic motility using a two-chambered assay system demonstrated that when highly metastatic hybrid 95H-1 was exposed to either L-PHA (5–50 µg/ml) or anti-LAMP-1 (1:50 dilution), both completely eliminated MSH/IBMX-inducible motility (i.e. that above basal levels), whereas some inhibition of basal motility (that in the absence of MSH/IBMX) has been observed with anti-LAMP-1 but not with L-PHA (Table 3)Citation . Motility of parental Cloudman S91 cells, which was negligible and was not affected by MSH/IBMX treatment compared with metastatic hybrid, 95-H1, (1 , 3) , was unaffected by either L-PHA or anti-LAMP-1 under the same conditions (not shown).


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Table 3 Effects of LAMP-1 and L-PHA on migration of metastatic macrophage x melanoma hybrid, 95-H1, with or without pretreatment with MSH/IBMX (48 h)

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Through several approaches we have shown that fusion hybrids of macrophages and Cloudman S91 melanoma cells displayed macrophage-like elevated GnT-V expression that correlated with their high metastatic potential. These studies together with previous work have revealed that such fusion hybrids express at least two classes of regulatory systems that differ from those of parental Cloudman cells in responses to elevated cAMP. Some cAMP-related phenotypes, including tyrosinase activity, melanization, chemotactic motility, dendricity, and the incorporation of [3H]glucosamine as an indicator of N-glycosylation, were all up-regulated in the basal state in metastatic hybrids and could be additionally stimulated by MSH/IBMX. However, the levels of GnT-V mRNA, GnT-V enzymatic activity, and the slow gel mobility of LAMP-1, though up-regulated in the basal state of metastatic hybrids, could not be additionally increased by MSH/IBMX. Similarly, it was shown previously that MSH receptor (MC1-R) mRNA and cell surface binding activity were elevated in the basal state of metastatic hybrids but not increased by MSH/IBMX (4) . Although there could be several explanations for the above observations, it appears that separate but interacting pathways exist for the gene expression in the hybrids. The pathways are presumably expressed as the balance between the macrophage and melanoma genetic expression programs, and it is tempting to speculate that up-regulation of GnT-V activity and ß1,6 branching in hybrids reflects dominant regulation by macrophage elements such as transcription factors and repressors. However, little is known about these questions.

Nonetheless, GnT-V-generated, ß1,6-branched polylactosamines are a shared common feature between normal granulocytes, monocytes, and a variety of malignant cells (9 , 16 , 18 , 19 , 28 , 36, 37, 38, 39) . Fukuda (40) demonstrated that the majority of antibodies raised versus monocytes or granulocytes recognized part of their polylactosamine structure, and the first structure of a sialylated fucosyl polylactosaminoglycan was deduced from material isolated from granulocytes (37) . In a number of studies, both rodent (18) and human (19) tumor progression to a metastatic phenotype is directly associated with increased levels of ß1,6-branched Asn-linked oligosaccharides. Rodent cells transformed with polyoma or Rous sarcoma virus, or transfected with H-ras oncogenes have been attributed to increased ß1,6-GlcNAc branching of complex-type oligosaccharides as well as increased polylactosamine content (41) . Transformation of rat2 fibroblast with either T24H-ras, v-K-ras, or with the tyrosine kinase oncogene v-fps induces both increased GnT-V activity and invasiveness, and metastatic potential (42) . In addition, the loss of GnT-V activity in a glycosylation mutant of a highly metastatic cell line, MDAY-D2, was associated with a loss of metastatic potential in mice (9) . Similar transformed phenotypes, along with stimulation of GnT-V expression, have been documented in our macrophage fusion hybrids.

The functional significance of increased ß1,6-branching of Asn-linked oligosaccharides and the identity of glycoproteins bearing these structures have not been well established. However, there is evidence that the presence of such oligosaccharides on various cell adhesion molecules such as integrins, cadherins, and carcinoembryonic antigen may influence tumor cell adhesion (10 , 21) , invasion (10) , and motility (16) . Polylactosamine structures are carriers of Lex and sialyl-Lex moieties, which, in humans, are used by monocytes, granulocytes, and malignant cells in selectin binding (35 , 43, 44, 45, 46, 47, 48, 49, 50, 51, 52) . B16 mouse melanoma cells expressing moderate amounts of human sialyl-Lex on polylactosamines showed marked increases in pulmonary metastases (48) . In human cancer, increased expression of sialyl-Lex and/or sialyl-Lea antigens on poly-N-acetyllactosamine antenna is associated with poor patient prognosis (49, 50, 51) .

In our experiment, artificial and spontaneous melanoma hybrids of high metastatic potential showed enhanced expression of GnT-V enzymatic activity and mRNA accompanied by increased ß1,6 branching and enhanced cell surface expression of two prominent GnT-V protein substrates: LAMP-1 and ß1 integrin. LAMPs with increased proportion of poly-N-acetyllactosamine in their ß1,6-branching construct often overexpressed in malignant cells (18 , 22 , 23) probably stabilize the lysosomes longer, and, thus, the latter can efficiently be transported to the cell surface and secrete more lysosomal enzymes to degrade surrounding tissues (14) . LAMPs also function as adhesion molecules, and it has been demonstrated that the extent of adhesion to E-selectin and cell surface sialyl-Lex determinants was proportional to the amount of cell surface LAMP-1 in colonic carcinoma cells (24) . It has been suggested that specific accumulation and localization of LAMPs could facilitate both the adhesion of tumor cells to extracellular matrix proteins and the invasion process itself by interactions between LAMPs and E-selectin, and between LAMPs and galectins (endogenous galactoside-binding lectins; Refs. 22 , 24 , 53 ). ß1-Integrin, a substrate of GnT-V with an extended polylactosamine sugar chain, is essential for cell adhesion to fibronectin (25) and is probably involved in metastasis of melanoma cells (26) . ß1,6-Branched polylactosamine structures in integrins, like {alpha}5ß1 and {alpha}vß3, results in reduced adhesion to fibronectin and enhanced motility (11 , 16) . It has also been suggested that GnT-V-dependent glycosylation regulates cell motility of both normal leukocytes and metastatic tumor cells in mice by reducing the stability of integrin receptor aggregates that maintain firm cell-substratum attachment (11 , 31 , 38 , 54) . Furthermore, ß1 integrin binds the cytosolic protein paxillin, which also binds to the signaling molecules focal adhesion kinase (FAK), csk, and c-src at discrete sites of cell attachment in spreading and motile cells (55) . GnT-V-deficient tumor cells have 20-fold reduced lung metastatic capability and demonstrate impaired focal adhesion formation (56) . In regard to the above facts, our observations of increased expression of LAMP-1 and ß-1 integrin subunit having ß1,6-branched polylactosamine sugar structure in our metastatic fusion hybrids may be consistent.

In addition, up-regulated GnT-V activity was at least in part responsible for increased chemotactic motility of hybrids in vitro, because L-PHA, which exhibits high affinity binding to the ß1,6-branched structures, inhibited motility of hybrids but not of parental melanoma cells. A previous treatment of the metastatic hybrids with swainsonine, a nontoxic inhibitor that blocks the formation of ß1–6-branched oligosaccharids and inhibits the invasion of human melanoma cells in vitro (10, 11, 12, 13) , increased the mobility of LAMP1 on SDS-PAGE gels (2 , 8) , decreased L-PHA binding, and reduced motility in vitro (data not shown). Interestingly, anti-LAMP-1 antibodies also inhibited motility of metastatic hybrids, possibly indicating a role for LAMP-1 in motility.

A number of important questions concerning the relationship among ß1,6-branched glycans, the malignant phenotype of the hybrids, and contributions from the macrophage remain to be answered. The other enzymes, such as core 2GlcNac-T, GlcNAc-T(i), or GlcNAc-TIV, that contribute to lactosamine content of surface glycoproteins, may have an effect similar to GnT-V in cancer biology, and, therefore, may be worth pursuing in our fusion hybrids. GnT-V gene expression in oncogenic transformation is regulated by the Ets family including Ets-1, a transcriptional factor, which also regulates several enzymes associated with cell invasion and metastasis, like cyclin D (cell cycle progression); Rho/Cdc42/rac-1/Taim 1 (motility); MMP-2, -3, and -9 (metalloproteinases); and vascular endothelial growth factor and basic fibroblast growth factor (growth factors; reviewed in Ref. 16 ). Investigations of such factors, in our metastatic macrophage-fusion hybrids, are of our future interest. Furthermore, in some of our nonmetastatic hybrids, the apparent lack of correlation of GnT-V enzymatic activity with its mRNA level (Fig. 2Citation ; Table 2Citation ) could suggest the multiple mechanisms of regulation of GnT-V expression that include the stability of mRNA, the transcription rate of the gene, post-translational interactions, and the protein stability as well. Only additional experiments can reveal the answer.

To understand the functional consequences of the changes in GnT-V activity in macrophage-fusion hybrids, investigations warrant the production of transgenic mice that lack ectopic GnT-V expression, as well as mice that show ectopic overexpression of this enzyme. Macrophages from these mice can be used for generating hybrids. The results of these experiments should significantly increase our understanding of the function of Asn-liked ß1,6-branched oligosaccharides in regulating invasiveness and metastatic potential, and the role of macrophage fusion in such characteristic cellular transformation. In addition, reagents such as antisense cDNA or some selective inhibitor of GnT-V expression in hybrid cells could determine whether inhibition of ß1,6 branching of cell surface glycoproteins can affect the invasiveness and metastatic potential of tumor cells.

However, our present report is unique in presenting evidence that the macrophage-associated traits of increased GnT-V activity, ß1–6 branching, and N-acetylpolylactosamine content in human and animal metastases could reflect previous fusion of tumor-associated macrophages with cells of the primary tumor. This notion is additionally supported by our recent findings on characterization of spontaneous metastases in vivo that were comprised of host x tumor hybrid containing higher DNA content than the parental cells (5) .


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Cells and Cell Culture.
The preparation of mouse peritoneal macrophages from syngenic DBA/2J mice, and fusion with the G418-resistant Cloudman S91 cell line, PS1- HGPRT-1 (also referred to as "6neo"), to generate hybrids were as described before (1) . For this study, we used some of the high (94-H4, 94-H48, and 95-H1) and low (95-H3 and 95-H12) metastatic macrophage fusion hybrids as representative cell lines from a larger panel described recently (1) . Spontaneous in vivo host/tumor hybrids were PADA (6) , isolated from within a primary Cloudman S91 tumor growing s.c. in a DBA2/J mouse, and LM6 (5) , isolated from a spontaneous pulmonary metastasis from Cloudman S91 tumor cells implanted s.c. in the tail of a BALB/c nu/nu mouse. Hybrid cells as well as parental cells were cultured in HAM’s F10 or DMEM nutrient medium with 10% horse or fetal bovine serum in a gassed (5% CO2 in air), humidified incubator at 37°C.

Immunoblotting.
Cells were solubilized in 1% NP40, 0.01% SDS, 0.1 M Tris-HCl (pH 7.2), 100 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin, and proteins were separated on 7% SDS gels and then transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore Corp., Bedford, MA). The membranes were blocked with 5% BSA and incubated with rat monoclonal antibodies (1:1000) directed against murine LAMP-1 (1D4B; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) followed by goat antirat immunoglobulins (1:1000) conjugated with horseradish peroxidase (Chemicon, Temecula, CA). Visualization of antibody binding was carried out with Enhanced Chemiluminescence (NEN Life Science Products, Boston, MA) according to the manufacturer’s instructions.

GnT-V Enzymatic Assay.
Cell pellets were pulse sonicated in 3 volume of PBS(-) at 4°C for 10' at intervals of on 10 s, off 10 s. After centrifugation at 900 x g for 10', the supernatants were collected and used as crude enzyme preparations for the GnT-V assay using a fluorescence- labeled pyridylaminated biantennary sugar chain as an acceptor substrate (52) . In brief, the reaction mixture consisted of 62.5 mM 4-morpholinepropanesulfonic acid; 2-(N-morpholino)ethanesulfonic acid buffer (pH 6.25) containing 5 mM EDTA, 20 mM UDP-GlcNAc, 100 mM N-acetylglucosamine, 0.25% (w/v) Triton X-100, 10 µl (500 pmol) acceptor substrate, and 10–15 µl enzyme preparation in a total volume of 50 µl. The other GlcNAc transferases acting on the same substrate, GnT-III and GnT-IV, were inhibited by the presence of 5 mM EDTA (52) . The reaction was carried out at 37°C for 5 h and stopped by heating at 100°C for 2 min. Samples were then centrifuged to remove insoluble materials, and an aliquot (10 µl) of the supernatant was subjected to an high-performance liquid chromatography equipped with a TSK gel ODS-80TM column (4.6 x 150 mm; Tosoh, Tokyo, Japan). Elution was performed at 55°C with a 20 mM acetate buffer (sodium acetate 20 mM, glacial acetic acid to adjust pH 4.0) containing 0.17% n-butyl alcohol at a flow rate 1.0 ml/min, and fluorescence was detected using excitation and emission wavelengths of 320 and 400 nm, respectively. Reaction products were identified via retention time and were compared with authentic products generated from GnT-V cDNA-transfected COS cell extracts, which exhibit no detectable GlcNAc transferase activities except GnT-V (57) . The enzyme activity is expressed as pmol of N-acetylglucosamine transferred per h per mg of protein. Protein was determined with a bicin-choninic acid kit (Pierce, Rockford, IL) using BSA as a standard. The detectable limit of the product was found to <0.1 pmol/assay.

RNA Extraction and Northern Blot Analysis.
Total cellular RNA (30 µg), extracted from the cultured cells using the ISOGEN solution (Nippon Gene Corp., Toyoma, Japan) following the manufacturer’s instructions, were size fractionated on a 1% agarose gel containing 2.2 M formaldehyde and then blotted onto a Zeta probe (Bio-Rad) membrane through capillary action. The membrane filter was prehybridized for 2 h and then hybridized with 32P-labeled GnT-V cDNA (34) at 42°C in the hybridization buffer for 24 h. The filter was washed at 55°C, first with 2 x SSC [1 x SSC is 15 mM sodium citrate and 150 mM NaCl (pH 7.0)] plus 0.1% SDS twice, followed by once with 0.2 x SSC plus 0.1% SDS. The filter was then exposed to X-ray film (Kodak, Tokyo, Japan) with an intensifying screen at -80°C for 2 days. RNA in the gel was stained with ethidium bromide. RNA bands were scanned by densitometry, and the amounts of GnT-V mRNA quantitated by normalization to 18S and 28S RNAs as internal controls.

Flow Cytometry.
Binding assays were carried out for 1 h on ice. For LAMP-1, cells (104) were incubated 1 h on ice with rat antimouse LAMP-1 monoclonal antibody, 1D4B, (1:25; DSHB, University of Iowa) followed by FITC-conjugated F(ab')2 fragment of goat antirat IgG (1:50; Chemicon, Inc., Temecula, CA) as the secondary antibody. For ß1-integrin, cells (104) were incubated with rat antimouse CD29 monoclonal antibody (1:30; PharMingen, San Diego, CA) followed by goat antirat IgG-FITC (1:25; PharMingen) as the secondary antibody. L-PHA binding to cells was carried out by incubating with FITC-conjugated L-PHA (10 µg/ml; Vector, Burlingame, CA) in PBS(-) with 0.1% BSA and 0.05% sodium azide. Cells were washed 3–4 times with PBS(-) and then fixed with 1% paraformaldehyde. Flow cytometry was carried out with a FACS Vantage Flow cytometer (Becton Dickinson). Because the antibody and lectin incubations were carried out with intact cells on ice, it was assumed that predominantly cell surface expression of the target ligands were measured.

Motility Assay.
To assess motility, cells (5 x 104/0.5 ml culture medium without serum) were seeded into Coster Transwell cell culture chamber inserts (12-µ pore diameter) and placed into wells containing 1.5 ml of DMEM nutrient medium without serum and with 3T3 fibroblast conditioned medium as a chemoattractant (3) , in a gassed, humidified incubator. At various times up to 6 h, the inserts were withdrawn, cells on the upper surface were removed with a cotton swab, and the cells on the underside were fixed with methanol and stained with hematoxylin. The membrane filters were cut with a scalpel and mounted on slides. Migration was expressed as the total cells counted in 10 microscopic fields with a light microscope at x430 magnification.


    Acknowledgments
 
We thank Dr. Jean Bolognia and James Platt for their helpful comments.


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

1 Supported in part by a grant from Vion Pharmaceuticals, New Haven, CT. Back

2 To whom requests for reprints should be addressed, at Department of Dermatology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520. Phone: (203) 785-4963; Fax: (203) 785-7637; E-mail: ashok.chakraborty{at}yale.edu. Back

3 The abbreviations used are: MSH, melanocyte-stimulating hormone; IBMX, isobutylmethyl xanthine; Lex, Lewisx; L-PHA, leukoagglutinating phytohematoagglutinin; LAMP-1, lysosome-associated membrane protein-1; GnT-V, ß1,6-N-acetylglucosaminyltransferase V; PBS(-): Ca+2/Mg+2- free PBS. Back

Received for publication 8/ 2/01. Revision received 9/17/01. Accepted for publication 9/25/01.


    References
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 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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