Expression of polypeptide GalNAc-transferases in stratified epithelia and squamous cell carcinomas: immunohistological evaluation using monoclonal antibodies to three members of the GalNAc-transferase family

Ulla Mandel5, Helle Hassan, Marianne H.Therkildsen1, Jørgen Rygaard2, Mikkel H.Jakobsen3, Birgitte R.Juhl4, Erik Dabelsteen and Henrik Clausen

Department of Oral Diagnostics, School of Dentistry, Faculty of Health Sciences, University of Copenhagen, Nørre Alle 20, 2200 N, Denmark and 1Department of Pathology, Roskilde County Hospital, 2The Bartholin Institute, and 3Departments of Otolaryngology and Head and Neck Surgery and 4Pathology, Rigshospitalet, Denmark

Received on March 10, 1998; revised on May 18, 1998; accepted on May 20, 1998

Mucin-type O-glycosylation is initiated by a large family of UDP-GalNAc: polypeptide N-acetyl-galactosaminyltransferases (GalNAc-transferases). Individual GalNAc-transferases appear to have different functions and Northern analysis indicates that they are differently expressed in different organs. This suggests that O-glycosylation may vary with the repertoire of GalNAc-transferases expressed in a given cell. In order to study the repertoire of GalNAc-transferases in situ in tissues and changes in tumors, we have generated a panel of monoclonal antibodies (MAbs) with well defined specificity for human GalNAc-T1, -T2, and -T3. Application of this panel of novel antibodies revealed that GalNAc- transferases are differentially expressed in different cell lines, in spermatozoa, and in oral mucosa and carcinomas. For example, GalNAc-T1 and -T2 but not -T3 were highly expressed in WI38 cells, and GalNAc-T3 but not GalNAc-T1 or -T2 was expressed in spermatozoa. The expression patterns in normal oral mucosa were found to vary with cell differentiation, and for GalNAc-T2 and -T3 this was reflected in oral squamous cell carcinomas. The expression pattern of GalNAc-T1 was on the other hand changed in tumors to either total loss or expression in cytological poorly differentiated tumor cells, where the normal undifferentiated cells lacked expression. These results demonstrate that the repertoire of GalNAc-transferases is different in different cell types and vary with cellular differentiation, and malignant transformation. The implication of this is not yet fully understood, but it suggests that specific changes in sites of O-glycosylation of proteins may occur as a result of changes in the repertoire of GalNAc-transferases.

Key words: polypeptide GalNAc-transferases/O-glycosylation/ monoclonal antibodies/squamous cell carcinomas/epithelial differentiation

Introduction

Mucins are glycoproteins with high-density of O-glycosylation and are found in most secretions and on the cell surface of epithelial cells (Gendler and Spicer, 1995). Recently, membrane glycoproteins with mucin-like domains have attracted attention for their role in cell-cell adhesion in leukocyte extravasation and in metastasis (Springer, 1994; Lasky, 1995; Zhang et al., 1996). The expression of mucins is often altered in cancer: aberrant glycosylation with immature structures and exposure of peptide backbone are common features of tumor mucins (Devine and McKenzie, 1992; Kim, 1992; Taylor-Papadimitriou et al., 1993; Kim et al., 1996; Lloyd et al., 1996). One mechanism for differential O-glycosylation of mucins may be the differential expression of enzymes that initiate O-glycosylation (Clausen and Bennett, 1996), resulting in differences in the density or positions of attachment of O-linked oligosaccharides to the mucin core protein.

The initial key step in the regulation of O-glycosylation is the enzymatic transfer of GalNAc from UDP-GalNAc to serine and threonine residues, a reaction catalyzed by a family of UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferases (GalNAc-transferases) (EC 2.4.1.41) (Clausen and Bennett, 1996). The molecular processes governing the specificity and kinetics of the initiation step of O-glycosylation are not clarified. It is increasingly clear, however, that the substrate specificities of the large family of GalNAc-transferases are a major factor in determining sites of O-glycan attachments, and hence that the repertoire of GalNAc-transferases expressed in a cell may control the O-glycan attachment pattern (Wandall et al., 1997).

The human GalNAc-transferase family includes at least four distinct functional enzymes (Homa et al., 1993; White et al., 1995; Bennett et al., 1996; Clausen and Bennett, 1996; Hagen et al., 1997; Bennett et al., in preparation), and several additional GalNAc-transferase genes are under investigation. A large GalNAc-transferase family is found as early in evolution as in C.elegans (Hagen and Nehrke, 1998). Characteristics of the human GalNAc-transferase family include: (1) overall sequence similarities of approximately 40-45%, with some members showing higher similarities (Clausen and Bennett, 1996); (2) different chromosomal localizations and genomic organizations, however, some members share several intron/exon boundaries (Clausen and Bennett, 1996; Bennett et al., 1998); (3) the substrate specificities are different but there are partial overlaps in their specificities (Bennett et al., 1996; Hagen et al., 1997; Wandall et al., 1997); and (4) the expression patterns analyzed by Northern analysis in human cells and organs are different (Homa et al., 1993; White et al., 1995; Bennett et al., 1996; Zara et al., 1996; Hagen et al., 1997; Sutherlin et al., 1997). These features suggest that each of these GalNAc-transferases is unique and may have different functions. In order to gain further insight into the role of each member of the GalNAc-transferase family, it is important to define their expression pattern and subcellular location as well as potential changes in diseases.

Here, we report the production and characterization of a panel of monoclonal antibodies to three homologous human polypeptide GalNAc-transferases. Immunohistological analysis using this panel of MAbs clearly establishes that the repertoire of GalNAc-transferases in a cell is variable and changes with cell differentiation and in malignancy.

Results

Generation of monoclonal antibodies to human GalNAc-T1, -T2, and -T3

Monoclonal antibodies, designated UH3, UH4, and UH5 (all IgG1), which specifically react with GalNAc-T1, -T2, or -T3, respectively, were selected. The specificities of the three antibodies were confirmed by ELISA (Figure 1), immunocytology with Spodoptera frugiperda (Sf9) insect cells expressing different GalNAc-transferases (Table I), and differential immunoprecipitation of GalNAc-transferase activities from the medium of Sf9 cells expressing different GalNAc-transferases (Figure 2). None of the antibodies reacted by Western blot analysis. Antibodies displaying reactivity by Western blotting were found as well, but none of these reacted with the native enzymes by immunocytology or immunoprecipitation (Sutherlin et al., 1997; Rottger et al., 1998). It is desirable to select antibodies to continuous epitopes that may be applied for all procedures, however, our experience in this study as well as in a previous study (White et al., 1990) suggests that this may be a very difficult task. Thus, all antibodies that reacted with the native enzyme did not react by Western blotting, and antibodies selected by Western blotting did not react with the native enzymes. The reason for this is not known at present, but it may be related to a complex tertiary structure of the enzymes with potentially extensive intramolecular disulfide bridging (Clausen and Bennett, 1996). Antibodies to GalNAc-T2 and -T3 that Western blot were also selected and these may react with tissue sections after antigen retrieval attempts, however, preliminary studies have not confirmed this possibility (unpublished observations). All antibodies displayed the same differential specificity with HeLa cells transfected with expression constructs of the full coding sequence of the different GalNAc-transferases (Rottger et al., 1998).


Figure 1. Enzyme-linked immunoadsorbent assay analyses with MAbs to GalNAc-T1 (MAb UH3, A), GalNAc-T2 (MAb UH4, B) and GalNAc-T3 (MAb UH5, C) with purified recombinant GalNAc-T1, -T2, -T3 ,and AOSM. Antigen dilution with initial concentration of 10 µg/ml. Symbols: solid triangles, GalNAc-T1; solid squares, GalNAc-T2; open circles, GalNAc-T3; multiplication symbol, asialo ovine submandibular gland mucin.


Figure 2. Immunoprecipitation of recombinant human GalNAc-transferase activities by MAbs to GalNAc-T1 (A), -T2 (B), and -T3 (C). Secreted forms of human GalNAc-transferases were expressed in Sf 9 cells, and media were harvested 3 days postinfection and used as enzyme source. Protein G Sepharose was saturated sequentially with rabbit anti-mouse IgG and MAbs as culture supernatants. A 5% suspension of protein G beads was added to Sf9 medium containing either GalNAc-T1, -T2, -T3, or -T5. After incubation for 1 h at 4°C, beads were washed in PBS, and resuspended in 25 mM Tris (pH 7.4), 0.25% Triton X-100. GalNAc-transferase activities were measured in the supernatants (S) and the washed pellets (P) using a standard reaction mixture containing 25 mM Tris (pH 7.4), 10 mM MnCl2, 0.25% Triton X-100, 100 µM UDP-[14C]-GalNAc (2,000 c.p.m./nmol), and 25 µg acceptor peptide substrate.

Table I. Expression of GalNAc-T1, -T2 and -T3 in tumor cell lines and Sf9 cells
  GalNAc-T1 MAb UH3 GalNAc-T2 MAb UH4 GalNAc-T3 MAb UH5
A431 + +++ +++
WI38 ++ +++ -
MKN45 +++ + +++
Colo205 + ++ +++
HeLa + ++ -
HL60 +++ +++ +
Sf9-GalNAc-T1 +++ - -
Sf9-GalNAc-T2 - +++ -
Sf9-GalNAc-T3 - - +++
Sf9-A-enzyme - - -
Intensity grading: -, negative; +, weakly positive; ++, moderately positive; +++, strongly positive.

Immunolabeling of cell lines and spermatozoa

The expressed repertoire of GalNAc-transferases varied in six different tumor cell lines (Table I, Figure 3). GalNAc-T3 was strongly expressed in A431, MKN45, and Colo205 cancer cell lines, in contrast to no expression in WI38 and HeLa cells, and only weak expression in HL60 cells. GalNAc-T1 and -T2 were expressed in all the tested cell lines with variable intensity.


Figure 3. Immunocytological expression of GalNAc-T1 (MAb UH3), -T2 (MAb UH4), and -T3 (MAb UH5) in human fibroblast cell line WI38. GalNAc-T1 and T2 were strongly expressed in WI38 cells (A, B), respectively, in contrast to no expression of GalNAc-T3 (C) (450×).

Immunohistological expression patterns of GalNAc-transferases

Normal stratified squamous epithelium of the mouth, exocervix, and skin. GalNAc-T1, -T2, and -T3 were strongly expressed in different stratified squamous epithelia (Figure 4). A clear difference in the distribution was observed among the three enzymes with respect to the different cell layers in the epithelia.


Figure 4. Immunohistological expression of GalNAc -T1, -T2, and -T3 defined by MAbs UH3, UH4, and UH5, respectively, in neighboring sections of normal oral nonkeratinized stratified epithelium (150×) and skin (450×). GalNAc-T1 strongly in uttermost cell layers in oral mucosa (A) versus upper half-cell layers in skin (D). GalNAc-T2 in the lower half of the oral mucosa (B) versus basal cell layers of skin (E). GalNAc-T3 in all cell layers both in oral mucosa (C) and skin (F). Localization of basal membrane and epithelial surface assigned by dotted lines.

MAb UH3 (GalNAc-T1) was found to stain strongly in the most differentiated upper cell layers in the nonkeratinized epithelium of mouth (Figure 4A) and exocervix (not shown) and the intensity was gradually lost toward the lower half of the more undifferentiated basal cell layers, where extremely weak or no staining could be observed. This pattern of expression was also observed in skin; however, the difference between cell layers was less pronounced (Figure 4D). MAb UH4 (GalNAc-T2) showed an opposite staining pattern to that of GalNAc-T1 in all epithelia: the undifferentiated basal and parabasal cells stained strongly and reactivity was gradually lost towards the upper half of the cell layers, where weak (in nonkeratinized epithelium of mouth (Figure 4B) and exocervix (not shown)) or no (in skin (Figure 4E)) labeling was observed. In contrast MAb UH5 to GalNAc-T3 stained all the cell layers with the same intensity in all three epithelia (Figure 4C,F). Both GalNAc-T1 and -T2 were expressed in stromal cells, whereas GalNAc-T3 was not detected in these cells.

Oral squamous cell carcinomas. Although the staining intensity and distribution showed some variation from individual to individual, a distinct pattern of distribution was clearly observed with each of the three anti-GalNAc-transferase antibodies (Table II). The patterns observed for GalNAc-T2 and -T3 were largely comparable to the staining patterns found in non-neoplastic epithelium. In contrast the expression of GalNAc-T1 was different from that in non-neoplastic mucosa.


Table II. GalNAc-transferase expression in oral squamous cell carcinomas
R, recurrent tumor; ¤, radio therapy prior to surgery; ND, not determined. Percentage of tumor cells stained: 1 <25%, 2 25-75%, 3 >75%. Relative intensity: + weaker than normal; ++, similar to normal; +++, stronger than normal.

MAb UH4 (GalNAc-T2) generally stained the majority of the carcinoma cells in all the tumors, with variable intensity in the different tumor cells. Strong staining was seen in the cytologically poorly differentiated carcinoma cells that were found in small carcinoma islands (Figure 5H), and in the periphery of larger tumor islands. Weaker or no staining was found in the cytologically more differentiated carcinoma cells found in the central parts of the larger tumor islands (Figure 5B,E). MAb UH5 (GalNAc-T3) also stained the majority of the tumor cells, and the intensity within each tumor was more homogeneous compared to that of GalNAc-T2 (Figure 5C,F,I). In contrast to the widely expressed GalNAc-T2 and -T3 in the oral squamous cell carcinomas there was almost no staining with MAb UH3 (GalNAc-T1) in approximately one-fourth (7/29) of the tumors. In the remaining 22, weak or moderate staining was seen in a large fraction of both cytologically poorly and well differentiated carcinoma cells (Figure 5A,D,G). There was no specific correlation between the observed staining patterns and the histopathological grading or the clinical tumor stage, although this may be due to the limited number of specimens investigated.


Figure 5. Immunohistological expression of GalNAc -T1, -T2, and -T3 defined by MAbs UH3, UH4, and UH5, respectively, in oral squamous cell carcinomas. (A-C) illustrate case 19 by conventional fluorescence microscopy, whereas (D-F) (case 8) and (G-I) (case 6) are confocal microscope images (positive reaction is green and tumor cells are counter-stained red using a polyclonal antibody to keratin). Little or no GalNAc-T1 in carcinoma cells in contrast to strong expression in stromal cells (A, D, G). GalNAc-T2 in cytological poorly differentiated tumor cells (B, E, H) versus weak or no expression in well-differentiated carcinoma cells i.e. in the central part of tumor islands (B, E). Strong expression of GalNAc-T2 in stromal cells (B, E). GalNAc-T3 in both cytological poorly and well differentiated carcinoma cells versus absence in stromal cells (C, F, I). Note that GalNAc-T2 and -T3 occupied larger areas of cell cytoplasm in carcinoma cells than in normal epithelial cells (Figure 4). All panels 300×, except (F) (200×).

Both GalNAc-T1 and -T2 were expressed in stromal cells, whereas GalNAc-T3 was generally not detected in these cells. GalNAc-T1 and -T2 were observed in fibroblasts and in subsets of leukocytes. This expression pattern may in part explain the ubiquitous expression pattern of GalNAc-T1 and -T2 in Northern analysis of human organs (Homa et al., 1993; White et al., 1995; Bennett et al., 1996).

Subcellular localization of GalNAc-transferases

MAbs UH3, UH4, and UH5 to GalNAc-T1, -T2, and -T3 produced a fine granular, supranuclear staining in the normal epithelial cells, suggestive of Golgi localization (Figure 4). In tumor cells the labeling of GalNAc-T2 and -T3 quantitatively occupied larger areas of the cell cytoplasm (Figure 5). The putative localization of the GalNAc-transferases to the Golgi apparatus was further corroborated by simultaneous staining with a nucleus specific stain using conventional or confocal microscopy (Figure 6). Morphological changes of the Golgi apparatus are associated with pathological changes such as hypertrophy, atrophy, dilation, or collapse of Golgi elements (Morre, 1991).


Figure 6. Immunohistological expression of GalNAc-T3 (MAb UH5) in oral squamous cell carcinoma. Note juxta-nuclear localization of the enzyme. Positive reaction is green and tumor cells are counterstained red using a nucleus specific stain.

Immunolabeling of spermatozoa

Previously, Northern analysis demonstrated that GalNAc-T3 was strongly expressed in testis (Bennett et al., 1996). Preliminary immunohistological analyses of testis sections revealed that GalNAc-T3 expression was restricted to immature stages of spermatozoa, and there was no expression of GalNAc-T1 and -T2. In the present study, ejaculated spermatozoa were selected as an example of a cell type with no GalNAc-T1 and -T2 expression. As shown in Figure 7 GalNAc-T3 was found to be strongly expressed in ejaculated spermatozoa, while no detectable expression of GalNAc-T1 or -T2 was observed.


Figure 7. Immunohistological expression of GalNAc-T1 and -T3 defined by MAbs UH3 and UH5 respectively, in human spermatozoa. Strong expression of GalNAc-T3 (B), versus no expression of GalNAc-T1 (A).

Discussion

In the present study, we have generated a set of monoclonal antibodies to three members of the human polypeptide GalNAc-transferase family which have a central role in the biosynthesis of mucins. The panel of MAbs was shown by immunocytology and immunoprecipitation of activity to be specific for the individual GalNAc-transferases. Application of this set of antibodies demonstrated that the repertoire of GalNAc-transferases is different in different cell types, and that it may change in relation to cellular differentiation and in cancer.

Polyclonal antibodies to bovine GalNAc-T1 have previously been prepared (Wang et al., 1992; Homa et al., 1993), but the fine specificity of these has not been reported. One of these sera was used to evaluate the subcellular localization of GalNAc-T1 by immuno-EM (Roth et al., 1994); however, it is not clear to what extent the antiserum was specific for GalNAc-T1. Polyclonal sera may also include anti-carbohydrate antibodies as was found in antisera against the [beta]4galactosyltransferase (Childs et al., 1986). The availability of monoclonal antibodies to different isoforms should help clarify this problem, and the present application of these MAbs clearly indicated Golgi-associated localization of the enzymes in normal cells. This finding was supported by expression studies with tagged GalNAc-T1, -T2, and -T3 that suggest that these enzymes show distinct different localization in Golgi, but are present throughout the Golgi-stacks (Rottger et al., 1998).

The repertoire of GalNAc-transferases in cells is variable

The panel of cell lines tested revealed that some cells lack GalNAc-T3, whereas GalNAc-T1 and -T2 were expressed in all although in different levels (Table I). An example of lack of GalNAc-T1 and -T2 was, however, found in spermatozoa, that only expressed GalNAc-T3. Spermatozoa were tested because the initial Northern analysis of GalNAc-T3 expression revealed highest expression in testis and pancreas (Bennett et al., 1996). Preliminary immunohistological analysis of testis sections showed that GalNAc-T3 expression was confined to spermatozoa, whereas GalNAc-T1 and -T2 were expressed in other cell types (U. Mandel and A. Giwercman, unpublished observations).

The repertoire of GalNAc-transferases vary with differentiation

Stratified squamous epithelium of oral mucosa provides a good model for studies of changes in expression of antigens in relation to differentiation (Vedtofte et al., 1984; Clausen and Hakomori, 1989; Dabelsteen et al., 1991). Glycosylation in keratinized and nonkeratinized epithelia are characteristically different, and the glycosylation pattern increases in complexity as cells differentiate from the basal cell layer until exfoliation at the surface (Vedtofte et al., 1984; Mandel et al., 1991). We have previously shown that the expression of histo-blood group A/B antigens in mature cell layers is directly correlated to expression of the A/B glycosyltransferase (Mandel et al., 1990), and a characteristic loss of A/B antigens in oral carcinomas is followed by a coordinated loss of A/B enzyme expression (Mandel et al., 1992b). In the present study, the three GalNAc-transferases were found to be expressed in different cell layers; GalNAc-T2 in undifferentiated basal layers, GalNAc-T1 in differentiated superficial layers, and GalNAc-T3 in all layers.

The repertoire of GalNAc-transferases may change in cancer

The observed changes in GalNAc-transferase repertoire in oral carcinomas are likely to be related to the differentiation state of the cells. GalNAc-T3 was expressed in all cell layers of normal epithelium and in most carcinoma cells. GalNAc-T2 was mainly found in the undifferentiated cell layers in the normal epithelium, and it was expressed in most carcinoma cells except in well differentiated foci. In contrast, GalNAc-T1 expression was markedly changed in tumors. Over 25% of the cases lacked expression of GalNAc-T1, and the rest showed low levels of expression in some areas of the tumor. Expression of GalNAc-T1 was found in both poorly and well-differentiated carcinoma cells. Thus, there appeared to be no correlation to differentiation stage which may be expected from the expression pattern found in normal epithelium. While GalNAc-T3 was homogeneously expressed in both normal oral epithelium and cancer a previous study has shown that GalNAc-T3 expression is selectively lost in poorly differentiated pancreatic cancer cell lines (Sutherlin et al., 1997).

It is premature to attempt to correlate the GalNAc-transferase repertoire with an overall pattern of O-glycosylation of specific glycoproteins. Firstly, studies of the specificities of the GalNAc-transferases analyzed so far have not yet revealed defined acceptor peptide sequence motifs which could be used to predict the O-glycosylation capacity of each enzyme; secondly, several uncharacterized GalNAc-transferases are predicted to exist; and thirdly, the in vivo sites of O-glycosylation of mucins from normal or tumor tissues are largely unknown due to experimental constraints, although this has improved by new methodologies (Gooley and Williams, 1997), and the attachment sites of repeats of porcine submaxillary gland mucin and human MUC1 has recently been determined (Gerken et al., 1997; Muller et al., 1997).

The nature of the mucin-like molecules expressed in stratified squamous epithelia is still unclear. MUC1 and CD43 have been identified, but the expression levels are low (Nielsen et al., 1997). Recently, we identified a novel cell membrane associated mucin-like glycoprotein expressed in all stratified epithelia, but the molecular characteristics of this are not well defined (Nielsen et al., 1997). Interestingly, this membrane mucin was selectively expressed in differentiated cell layers, and lost in cancer cells. This suggests that the need for O-glycosylation of different mucin substrates may change during epithelial differentiation and hence potentially point to a role for variations in the repertoire of expressed GalNAc-transferases.

One of the only structurally established examples of a specific cancer-associated change in attachment sites of O-glycans is that of a single O-glycosylation site in the IIICS domain of fibronectin (Matsuura et al., 1988). A single Thr site in the IIICS domain of fibronectin in the peptide sequence, VTHPGY, is differentially utilized in carcinomas, and O-glycosylation in this site induces an antibody epitope defined by monoclonal antibodies FDC-6 and 5C10 (Matsuura and Hakomori, 1985; Matsuura et al., 1988; Kosmehl et al., 1996). Application of these antibodies has shown a restricted expression of this epitope in stroma surrounding invading squamous cell carcinomas and adenocarcinomas (Loridon-Rosa et al., 1990; Mandel et al., 1992a). Early studies by Matsuura et al. (Matsuura et al., 1989) demonstrated a novel GalNAc-transferase activity in hepatoma extracts capable of glycosylating the identified fibronectin sequence, and this activity was not present in normal tissues. This may have been the first evidence of cancer-associated changes in GalNAc-transferase activity although the genetic and enzymic basis therefore is still not clarified. In recent studies of the substrate specificities of GalNAc-T1, -T2, and -T3 we found that GalNAc-T3, and not the other two enzymes, was capable of glycosylating the fibronectin substrate (Wandall et al., 1997). Since GalNAc-T3 was only expressed in epithelial cells and not in connective tissue cells in stroma of oral carcinomas, it is unlikely to represent the enzyme responsible for the cancer-associated O-glycosylation of fibronectin. Recently, we have cloned and expressed a close homolog of GalNAc-T3, designated GalNAc-T5, and this enzyme has in preliminary studies been shown to have the same substrate specificity as GalNAc-T3 and studies of expression are undergoing (E. P. Bennett and H. Clausen, unpublished observations).

In summary, a novel panel of antibodies to polypeptide GalNAc-transferases was developed and used to show that the repertoire of GalNAc-transferases is variable in cells of different types and differentiation, and that the expression may change in cancer cells. The results suggest that incomplete glycosylation of mucins in cancer, specifically reduced molar ratios of O-glycan chains, may be related to specific cancer-associated changes of the expression of GalNAc-transferases. The availability of these antibodies will be valuable tools to provide insight into mucin biosynthesis and the regulation of O-glycosylation in normal and malignant cells.

Material and methods

Production of anti-GalNAc-transferase monoclonal antibodies

Purified recombinant soluble enzymes were used as immunogens. Previously, soluble forms of human GalNAc-T1, -T2, and -T3 were expressed in Sf9 cells using the Baculo-virus expression system as secreted proteins, and purified to near homogeneity with specific activities of 0.6 U/mg for GalNAc-T1, 0.5 U/mg for GalNAc-T2, and 0.5 U/mg for GalNAc-T3 measured using peptides derived from MUC2 and MUC1 tandem repeats (Wandall et al., 1997). Balb/c mice were immunized with one subcutaneous or intraperitoneal injection of 10 µg undenatured protein in Freund's complete adjuvant, followed by two injections with Freund's incomplete adjuvant, and finally an intravenous boost without adjuvant. Eyebleeds were taken 7 days after third immunization, and the titer and specificity of anti-GalNAc-transferase antibodies were evaluated. Fusion to NS-1 and the cloning procedure were as described previously (White et al., 1990).

Hybridomas were selected by three criteria: (1) differential reactivity in ELISA with purified recombinant enzymes; (2) immunocytology on Sf9 cells 2 days after infection with Baculo-virus containing GalNAc-transferases, GalNAc-T1, -T2, -T3, or the histo-blood group A enzyme (Bennett et al., 1995, 1996); and (3) differential immunoprecipitation of active recombinant enzymes. Details of assays are described in corresponding figure captions. Western blot analysis with purified recombinant enzymes was also performed with all antibodies, but it proved impossible to select antibodies reactive with both native and denatured enzymes.

Cell culture and immunocytology

Cell lines WI38 (human fibroblast), HL60 (human leukocyte), and various human cancer cell lines (A431 (epidermoid carcinoma), Colo205 (colon carcinoma), HeLa (cervix carcinoma), and MKN45 (gastric carcinoma)) were grown to subconfluency in the appropriate media as recommended by American Type Culture Collection. Cells were fixed in ice-cold acetone and then kept at -70°C before staining. In addition, cell lines (WI38, A431) were fixed in 3% paraformaldehyde, quenched with 50 mM ammonium chloride in PBS and permeabilized in 0.1% Triton X-100 prior to antibody staining. At this stage, 0.2% fish skin gelatin (Sigma) was added as a blocking agent.

Patients and tissues

Nonkeratinized labial mucosa was obtained from 10 young healthy volunteers. Spermatozoa were obtained from five young healthy volunteers. Samples of exocervical epithelium were obtained from seven hysterectomies where the exocervical epithelium was normal, and five samples of skin were obtained from unaffected areas of breast resections. Malignant tissue analyzed included 29 samples of oral squamous cell carcinomas each including adjacent normal appearing tissue originating from various regions of the mouth (Table II). The patients were operated on at the University Hospital (Rigshospitalet), Copenhagen, Denmark, in 1990-97. All tumors were histopathologically graded and 21/29 tumors were staged clinically according to the UICC classification (Hermanek et al., 1987); 2/29 were recurrent tumors, and 15/29 patients had received radio therapy prior to surgery. The experiments were approved by the local Human Investigations Committee (J#KF 03-004/95), and the use of mice was authorized by the Danish Animal Inspectorate.

All tissue samples were divided in two: one part was processed for conventional histopathologic diagnosis, and the other part was immediately embedded in Tissue-Tek and frozen in isopentane, precooled on dry ice, and stored at -70°C until use.

Immunohistology procedures

Sections were cut from frozen blocks at a thickness of 5 µm and mounted on gelatin-coated slides. Every fifth section was stained by hematoxylin-eosin and used as reference during evaluation. Antigens were detected in the tissue by a double-layer immunofluorescence technique. Sections were used unfixed, fixed in cold 10% buffered neutral formalin for 15 min, or fixed in cold acetone for 10 min, and incubated with undiluted hybridoma supernatants for 12-24 h at 4°C. Counterstaining for the cell nucleus was performed with propidium iodide (Sigma). Bound MAbs were detected with FITC-conjugated rabbit anti-mouse immunoglobulin absorbed with human serum (Code F-261, Dako, Denmark). For double staining with anti-keratin antibody the first antibody layer was mixed with an anti-keratin polyclonal antibody (1:1000) (code Z622, Dako, Denmark), and after blocking with goat serum (1:10) the second layer was replaced with a mixture of FITC-conjugated donkey anti-mouse immunoglobulin (1:100) and Texas red-conjugated affinity pure goat anti-rabbit immunoglobulin (1:200; Jackson Immunoresearch Lab, Baltimore, MD). Slides were mounted in glycerol containing p-phenylene-diamine and examined in a Zeiss fluorescence microscope using epiillumination. The microscope was equipped with FITC and Texas red interference filters and a 75W xenon lamp (FITC) and a 50W HBO lamp (Texas red).

Different methods of fixation of cell lines and tissues were tested in preliminary studies. The staining patterns for all three selected MAbs were similar in cell lines (WI38, A431) using acetone or 3% formaldehyde solution fixation followed by Triton X-100 permeabilization. Similar findings with the three MAbs were observed with fresh frozen tissue sections that were either unfixed, fixed in acetone, or fixed in 10% buffered formaldehyde. However, formalin fixed tissue sections embedded in paraffin by routine histological procedures clearly abolished the staining reactions for all three MAbs. Antigen retrieval in formalin-fixed, paraffin-embedded tissues using microwave oven with and without pretreatment with enzymes (Protease type XXIV (Sigma) 0.05% for 5 min) and a biotin-streptavidin method (Duet Kit, DAKO, Denmark) was not possible. Identification of immunoreactive cells in the connective tissue was confirmed by the staining of consecutive sections of five tumors with an indirect immuno-alkaline phosphatase technique in addition to our chosen method, the indirect FITC labeling technique. The epithelial component in sections of tumors was verified by anti-keratin staining in double immunofluorescence. Controls included omission of primary antibodies and replacement of primary antibodies with MAbs of other specificity but with same isotype, WKH1 (White et al., 1990), and 5C10 (Mandel et al., 1992a).

Confocal laser scanning microscopy

Frozen, acetone/paraformaldehyde fixed sections of 4 blocks (cases: 6, 8, 17, 27) were prepared for confocal microscopy by the same methods except that tissue sections were 12 µm thick. x-y sections were generated and the images obtained were analyzed using a CLSM 3/0 confocal microscope (Zeiss, Oberkochen, Germany) equipped with an argon ion laser omitting at 488 nm and a two channel detector system for green (FITC) and red (Texas red) allowing simultaneous confocal imaging of double-labeled material. Scans were performed as overlays or consecutive single frame scans, using a helium-neon laser 543 nm for excitation for Texas red for the latter.

Acknowledgments

We thank Drs. M. A. Hollingsworth and S. B. Levery for many helpful suggestions and critical reading of the manuscript. This work was supported by the Danish Cancer Society, the Mizutani Foundation for Glycoscience, the Ingeborg Roikjer Foundation, the Danish Medical Research Council, the Novo Nordisk Foundation, NIH 1 RO1 CA66234, and funds from the EU Biotech 4th Framework.

Abbreviations

GalNAc-transferase, UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase; GalNAc, N-acetylgalactosamine; MAb, monoclonal antibody; Sf9, Spodoptera frugiperda.

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