J Dent Res 83(7): 567-571, 2004
© 2004 International and American Associations for Dental Research
Salivary Agglutinin/DMBT1SAG Expression is Up-regulated in the Presence of Salivary Gland Tumors
F.J. Bikker1,
J.E. van der Wal2,
A.J.M. Ligtenberg1,*,
J. Mollenhauer3,
J.M.A. de Blieck-Hogervorst1,
I. van der Waal4,
A. Poustka3, and
A.V. Nieuw Amerongen1
1 Department of Dental Basic Sciences, Academic Centre for Dentistry Amsterdam (ACTA), Van der Boechorststraat 7, 1081 BT Amsterdam, the Netherlands;
2 Department of Pathology and Laboratory Medicine, University Hospital Groningen, Groningen, The Netherlands;
3 Department of Molecular Genome Analysis, Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, Germany; and
4 Department of Oral & Maxillofacial Surgery and Oral Pathology, Free University medical centre (VUmc)/ACTA, Amsterdam, the Netherlands;
* corresponding author, ajm.ligtenberg{at}vumc.nl
 |
ABSTRACT
|
|---|
Salivary agglutinin (SAG) is encoded by the gene Deleted in Malignant Brain Tumors 1 (DMBT1) and represents the salivary variant of DMBT1 (DMBT1SAG). While SAG is a bona fide anti-caries factor, DMBT1 was proposed as a candidate tumor-suppressor for brain, digestive tract, and lung cancer. Though DMBT1SAG is expressed in the salivary glands, its expression in salivary gland tumors is unknown. Here we analyzed DMBT1SAG expression in 20 salivary gland tumors and 14 tumor-flanking tissues by immunohistochemistry. DMBT1SAG in salivary gland tumors resembles the changes of expression levels known from DMBT1 in tumors in other cancer types. Particularly, DMBT1SAG was up-regulated in 10/14 tumor-flanking tissues, and a strong staining of the luminal content in the tumor and/or the tumor-flanking tissue was observed in 14/20 cases. This suggests that, in addition to its role in caries defense, SAG may serve as a potential tumor indicator and/or tumor suppressor in salivary gland tissue.
KEY WORDS: DMBT1 • gp-340 • salivary agglutinin • SRCR • salivary gland tumor
|
INTRODUCTION
|
|---|
Salivary agglutinin (SAG) is a 300- to 400-kDa glycoprotein that was originally identified as the protein responsible for the calcium-dependent Streptococcus mutans-aggregating properties of parotid saliva (Rundegren and Ericson, 1981; Rundegren and Arnold, 1987). For this reason, it has been implicated in the protection against caries (Carlen et al., 1998).
SAG is identical to the respiratory tract glycoprotein gp-340. Both proteins are encoded by the gene Deleted in Malignant Brain Tumors 1 (DMBT1) at chromosome 10q25.3-q26.1 and thus represent the DMBT1-isoforms secreted to the saliva and the lung surfactant, respectively (Mollenhauer et al., 1997; Holmskov et al., 1999; Prakobphol et al., 2000; Ligtenberg et al., 2001). DMBT1GP340 is putatively involved in respiratory tract protection, because it interacts with the lung defense collectins—surfactant proteins D (SP-D) and A (SP-A)—and is able to stimulate alveolar macrophage migration (Holmskov et al., 1999; Tino and Wright, 1999).
While analysis of these data suggests a certain functional overlap between DMBT1SAG and DMBT1GP340, i.e., in pathogen defense, further, quite distinct, functions have been attached to DMBT1 as well. In vitro studies demonstrated that the rabbit homologue of DMBT1 (and therefore SAG) triggers epithelial differentiation, when interacting with galectin-3 in the extracellular matrix (ECM; Vijayakumar et al., 1999; Hikita et al., 2000; Mollenhauer et al., 2000 , 2003). Furthermore, different tumors were shown to display loss of DMBT1 expression, apparently depending on the time point of DMBT1 localization in the ECM (Mollenhauer et al., 2001 HREF="#MOLLENHAUER-ETAL-2002A">, 2002a, 2003). Unifying concepts have postulated that both its protective functions, especially its role in pathogen defense, and its putative functions in cellular differentiation are of importance to counteract tumorigenesis (Holmskov et al., 1999; Takeshita et al., 1999; Wu et al., 1999; Mollenhauer et al., 2000, 2001, 2002a, Mollenhauer et al., b, 2003; Bikker et al., 2002a; Kang and Reid, 2003).
Based on these concepts, it appeared tempting to investigate whether SAG, which previously was exclusively linked to caries defense, is subjected to the same principles in salivary gland tumors as DMBT1 in tumors at various other sites in the human body. Here we report on the DMBT1SAG expression pattern in salivary gland tumors and tumor-flanking tissue.
|
MATERIALS & METHODS
|
|---|
Abbreviations
ACC, adenoid cystic carcinoma; ACT, acinic cell carcinoma; ECM, extracellular matrix; DMBT1, deleted in malignant brain tumors 1; gp-340, glycoprotein-340; Lab, labial; mAb, monoclonal antibody; MEC, mucoepidermoid carcinoma; PA, pleomorphic adenoma; Pal, palatal; Par, parotid; SAG, salivary agglutinin, SM, submandibular; SP-A, surfactant protein A; SP-D, surfactant protein-D; and SRCR, scavenger receptor cysteine-rich domain.
Tumor and Normal Samples
Sections of normal, labial glandular tissues (n = 3), parotid glandular tissues (n = 4), and normal submandibular glandular tissues (n = 2), and sections of adenoid cystic carcinomas (n = 5), mucoepidermoid carcinomas (n = 5), acinic cell carcinomas (n = 5) (malignant carcinomas), and pleomorphic adenomas (n = 5) (benign carcinomas) were used for immunostaining with DMBT1SAG (Table 1
). Fourteen of the 20 tissues studied (parotid and submandibular) contained tumor-flanking normal tissue, i.e., tissue immediately adjacent to the tumor (Table 1; examples, Fig., C; Mollenhauer et al., 2002a). The sections were obtained from human salivary gland tissues that had been removed for therapeutic or diagnostic purposes by the Department of Oral & Maxillofacial Surgery and Oral Pathology. Because of the small size of the biopsies taken for diagnostic purposes, 6 biopsies did not contain tumor-flanking tissue. The study was approved by the Institutional Ethical Board of the VUmc at Amsterdam, and informed consent was obtained from all tissue donors.
View larger version (165K):
[in this window]
[in a new window]
|
Figure. Immunohistochemical localization of DMBT1SAG in human healthy salivary gland tissue, tumor-surrounding tissue, and salivary gland tumors. The sections were counterstained with hematoxylin/eosin. (A) Healthy parotid tissue: DMBT1SAG is localized in the ducts. (B) Parotid tumor-surrounding tissue (ACC). DMBT1SAG is localized in the intercalated ducts (icd) and serous acini (a). (C) ACC surrounding tissue (upper right side) showing strong staining of luminal content of striated ducts next to ACC (lower left side). (D) Healthy submandibular gland tissue. Serous acini (a) and demilune cells (d) were DMBT1SAG-positive. (E) Submandibular tumor-surrounding tissue (PA). DMBT1SAG is localized in the intercalated ducts and strongly present in the luminal content (lc). (F) Healthy labial tissue. DMBT1SAG is localized in the ducts, demilune cells (d), and serous acini (a). (G) ACC. No DMBT1SAG was found in ACCs. (H,I) MEC, the mucus-producing cells, were DMBT1SAG-positive (m). The epidermoid-cell and intermediate-cell components were totally negative. Moreover, the luminal content (lc) stained strongly positive (H). (J) ATC, showing no DMBT1SAG expression. (K) ACT, a minor subset of tumor cells showing DMBT1SAG expression. (L), PA. DMBT1SAG staining strongly positive in the secretory product and focally some tumor cells surrounding the secretory product.
|
|
Antibodies
For immunohistochemical detection of DMBT1SAG, tissue specimens were probed with monoclonal antibody (mAb) 213-6 that recognizes a peptide epitope of DMBT1 (Holmskov et al., 1999; Bikker et al., 2002b). For healthy parotid tissue, we used mAb 213-6 and mAb 213-1 (Holmskov et al., 1999; Bikker et al., 2002b). The mAbs were kindly provided by Dr. Uffe Holmskov (University of Southern Denmark, Odense, Denmark).
Immunohistochemistry on Human Tissue Specimens
Sections were cut from neutral-buffered formaldehyde-fixed paraffin-embedded tissue blocks and mounted on ChemMate Capillary Gap Slides (DAKO, Glostrup, Denmark), dried at 60°C, deparaffinized, and hydrated. Antigen retrieval was performed by means of microwave heating in Target Retrieval Solution (DAKO) for 11 min at full power (900 W), and 15 min at 400 W. After being heated, slides remained in the buffer for 15 min. Antigen retrieval was followed by blocking of endogenous biotin, with the Dako Biotin-Blocking System (DAKO). Incubation with mAb 213-6 and 213-1 (17 µg/mL) was done for 25 min at room temperature. Immunostaining was automated by means of the ChemMate HRP/DAB detection kit (K5001, DAKO, Glostrup, Denmark) on the TechMate 1000 instrument (DAKO, Glostrup, Denmark). Immunostaining was followed by brief nuclear counter-staining in Mayer’s hematoxylin/eosin. Finally, coverslips were mounted with AquaTex (Merck, Darmstadt, Germany). We generated controls by replacing the primary monoclonal antibody with an unrelated monoclonal antibody of the same subclass as mAb 213-6 (IgG1).
|
RESULTS
|
|---|
DMBT1SAG Expression in Normal and Tumor-flanking Tissue
Recent studies suggested that DMBT1 is up-regulated in tumor-flanking tissues in liver, lung, and breast cancer (Mollenhauer et al., 2001, 2002a). To test whether this might also be the case for DMBT1SAG in the salivary gland, we compared its expression in tumor-flanking tissues with data previously obtained for the normal salivary gland (Table 2) (Bikker et al., 2002b).
The tumor-flanking parotid gland tissue showed staining of the luminal aspects of intercalated duct cells in 11/11 cases, similar to the pattern observed in the healthy tissue (Figs. A, B
). Seven cases displayed up-regulation in the serous acini (Fig., B), and 7 cases revealed substantial staining of the luminal content (Fig., C), which was not observed in healthy tissues (Fig., A). In contrast to healthy submandibular gland tissue (Fig., D), 3/3 tumor-flanking submandibular gland tissues showed up-regulation of DMBT1SAG in the intercalated ducts, and 2 of these displayed a corresponding strong staining of the luminal content as well (Fig., E). Remarkably, DMBT1SAG was completely down-regulated in the demilune cells in the tumor-flanking tissue of the submandibular gland (3/3 cases; Fig., E). In healthy labial tissue, staining was observed in ducts, demilune cells, and serous acini (Fig., F). Due to the lack of either tumor-flanking tissue or corresponding normal tissue from healthy glands, no comparisons could be made for tongue and labial/palatal gland tumors.
Taken together, the analyses revealed up-regulation of DMBT1SAG by secretory cells in 10/14 (71%) tumor-flanking tissues (Table 2). Compared with normal tissues, staining for DMBT1SAG in the luminal content of tumor-flanking normal tissues was observed (9/14 tumor-flanking tissues vs. 0/9 normal tissues; p = 0.003 according to the two-tailed Fisher’s exact test). Moreover, a switch of the cell types that express DMBT1SAG appeared to take place. In the parotid and submandibular gland tissue, expression was induced in serous acini and in the intercalated ducts, respectively, while it was silenced in demilune cells. Consistently, expression and/or secretion of DMBT1SAG was enhanced in the presence of a tumor. This can be at least partly traced back to the up-regulation of DMBT1SAG expression in the serous acini and intercalated ducts.
DMBT1SAG Expression in Salivary Gland Tumors
Studies of other tumor types arising from monolayered epithelia or exocrine glands indicated a down-regulation of DMBT1 in the tumor cells compared with the tumor-flanking tissues (Mollenhauer et al., 2001 , 2002a). All 20 salivary gland tumors displayed decreased DMBT1SAG expression (Table 3) compared with positive structures in either the tumor-flanking tissues or healthy tissues (Table 2): 11/12 tumor-flanking and 9/9 normal tissues with 60–90% positive cells, respectively, vs. 0/20 tumors; p < 0.00001 for both comparisons according to the two-tailed Fisher’s exact test). The 5 ACCs were negative for DMBT1SAG (Fig., G). Four of 5 MECs showed strong staining of the luminal content of the ducts (Fig., H). In these tumors, the epidermoid- and intermediate-cell components consistently were negative, while a variable fraction (< 5% to 60%) of the mucus-producing cells was strongly positive in 3/5 cases (Figs. H, I). One of the 5 MECs totally lacked DMBT1SAG expression. Likewise, 3/5 ATCs were negative (Fig., J). In the remaining 2 cases, focal staining of parts that maintained a duct-like structure was observed (Fig., K). One case showed reactivity in the luminal content. Four of the 5 PAs displayed strong staining of the secretory product with focal-positive surrounding tumor cells (Fig., L). Moreover, secretory cells that preserved a luminal context were mostly DMBT1SAG-positive, while the solid areas did not show reactivity (examples in Figs. H, I, K, L). Accordingly, 9 out of 20 tumors showed strong DMBT1SAG staining of the luminal content (Table 3), while this was not the case in any of the 9 normal tissues (Table 2; p = 0.03 according to the two-tailed Fisher’s exact test).
|
DISCUSSION
|
|---|
For about two decades, SAG has been intensely investigated with regard to its role in binding and aggregation of cariogenic bacteria in the oral cavity. Recent discoveries have greatly expanded both the available data and the view on this molecule. We and others reported that SAG is identical to DMBT1 (Prakobphol et al., 2000; Ligtenberg et al., 2001). DMBT1 was detected in the salivary glands with an expression pattern virtually indistinguishable from that of SAG (Mollenhauer et al., 2001).
These findings had reciprocal impacts. On the one hand, the role of DMBT1 in the prevention of infection-induced cancer has come into focus, because we demonstrated direct interaction with the gastric-cancer-causing Helicobacter pylori and traced it back to the polymorphic scavenger receptor cysteine-rich (SRCR) domains of DMBT1 (Prakobphol et al., 2000; Bikker et al., 2002a). On the other hand, the relationship to DMBT1 considerably broadened the view on SAG, because one now must anticipate that it plays a role in the defense against various epidemiologically relevant pathogens in various organs, as well as in the oral cavity (Holmskov et al., 1999; Mollenhauer et al., 2000, 2001; Prakobphol et al., 2000). In particular, however, this relationship also means that SAG is potentially linked to tumorigenesis (Mori et al., 1999; Takeshita et al., 1999; Wu et al., 1999; Mollenhauer et al., 2000 , 2001, 2002a, Mollenhauer et al., b, 2003). Studies on DMBT1 indicated that SAG represents a highly unusual molecule for a potential tumor suppressor (Mollenhauer et al., 2000, 2001 HREF="#MOLLENHAUER-ETAL-2002A">, 2002a,Mollenhauer et al., b, 2003). Because several reports pointed to a lack of inactivating mutations, but frequent loss of expression in cancer, we focused on expression studies of DMBT1SAG in salivary gland tumors.
With few exceptions, DMBT1 is secreted luminally by monolayered epithelia and glands, and luminal secretion is commonly assumed to be associated with protective functions (Holmskov et al., 1999; Mollenhauer et al., 2000, 2001, 2002a; Bikker et al., 2002a). In tissues with constitutive DMBT1 expression, loss of expression may take place directly after resolution of the monolayered structure and secretion of DMBT1 to the ECM. In contrast, tissues without or with low DMBT1 expression may experience induction of DMBT1 expression at early stages of tumorigenesis, which is then followed by a resolution of the monolayered structure, translocation of DMBT1 to the ECM, and finally, again, by a loss of its expression (Mollenhauer et al., 2001, 2002a). Analysis of these and further data suggested that DMBT1 translocation to the ECM may be unfavorable for tumor growth, which is strongly supported by the fact that its rabbit homologue is able to trigger cell differentiation when locating to the ECM (Hikita et al., 2000). Analysis of our present data may provide support for these models and might extend them to DMBT1SAG.
We can confirm that an induction of DMBT1SAG also takes place in the salivary gland in the presence of tumors. Ten of 14 tumor-flanking tissues showed de novo expression of DMBT1SAG in structures that are negative in the respective healthy tissues. Remarkably, other cell types, i.e., demilune cells, show an accompanying silencing of DMBT1SAG, which raises the questions whether different cell types might produce different variants and whether these might exert different functions.
In agreement with the hypothesis that DMBT1 might be less compatible with tumor growth when locating to the ECM, only tumor parts that maintained a luminal context showed expression, while solid areas without luminal context were devoid of DMBT1SAG. Also, in MEC, the better-differentiated mucus-producing cells retained DMBT1SAG expression, while the less-differentiated epidermoid and intermediate cells were negative. Most remarkably, however, 14/20 salivary gland tumors displayed a strong staining for DMBT1SAG in the luminal content in either the tumor-flanking tissue and/or the tumor itself, both of which most likely contribute to this effect. As a consequence, this could mean that the anti-caries factor SAG might advance to a potential tumor marker in the oral cavity. Increased DMBT1SAG levels in saliva or saliva collected from a particular salivary gland could represent an indicator for the onset or the presence of a salivary gland tumor. This defines a clear need for further studies that aim at quantification of DMBT1SAG in the saliva of cancer patients and at comparison with other diseases, such as oral infection and inflammation.
|
ACKNOWLEDGMENTS
|
|---|
We thank Wim Vos, Thea Tadema, and Elisabeth Bloemena from the Department of Pathology, VUmc, for their practical assistance. This study was financially supported by The Netherlands Interuniversity Research School of Dentistry (IOT).
Received September 4, 2003;
Last revision April 28, 2004;
Accepted May 5, 2004
|
REFERENCES
|
|---|
Bikker FJ, Ligtenberg AJ, Nazmi K, Veerman EC, van’t Hof W, Bolscher JG, et al. (2002a). Identification of the bacteria-binding peptide domain on salivary agglutinin (gp-340/DMBT1), a member of the scavenger receptor-cysteine rich superfamily. J Biol Chem 277:32109–32115.[Abstract/Free Full Text]
Bikker FJ, Ligtenberg AJ, van der Wal JE, van den Keijbus PA, Holmskov U, Veerman EC, et al. (2002b). Immunohistochemical detection of salivary agglutinin/gp-340 in human parotid, submandibular, and labial salivary glands. J Dent Res 81:134–139.[Abstract/Free Full Text]
Carlen A, Bratt P, Stenudd C, Olsson J, Stromberg N (1998). Agglutinin and acidic proline-rich protein receptor patterns may modulate bacterial adherence and colonization on tooth surfaces. J Dent Res 77:81–90.[Abstract/Free Full Text]
Hikita C, Vijayakumar S, Takito J, Erdjument-Bromage H, Tempst P, Al Awqati Q (2000). Induction of terminal differentiation in epithelial cells requires polymerization of hensin by galectin 3. J Cell Biol 151:1235–1246.[Abstract/Free Full Text]
Holmskov U, Mollenhauer J, Madsen J, Vitved L, Gronlund J, Tornoe I, et al. (1999). Cloning of gp-340, a putative opsonin receptor for lung surfactant protein D. Proc Natl Acad Sci USA 96:10794–10799.[Abstract/Free Full Text]
Kang W, Reid KB (2003). DMBT1, a regulator of mucosal homeostasis through the linking of mucosal defense and regeneration? FEBS Lett 540:21–25.[ISI][Medline]
Ligtenberg TJ, Bikker FJ, Groenink J, Tornoe I, Leth-Larsen R, Veerman EC, et al. (2001). Human salivary agglutinin binds to lung surfactant protein-D and is identical with scavenger receptor protein gp-340. Biochem J 359:243–248.[ISI][Medline]
Mollenhauer J, Wiemann S, Scheurlen W, Korn B, Hayashi Y, Wilgenbus KK, et al. (1997). DMBT1, a new member of the SRCR superfamily, on chromosome 10q25.3–26.1 is deleted in malignant brain tumours. Nat Genet 17:32–39.[ISI][Medline]
Mollenhauer J, Herbertz S, Holmskov U, Tolnay M, Krebs I, Merlo A, et al. (2000). DMBT1 encodes a protein involved in the immune defense and in epithelial differentiation and is highly unstable in cancer. Cancer Res 60:1704–1710.[Abstract/Free Full Text]
Mollenhauer J, Herbertz S, Helmke B, Kollender G, Krebs I, Madsen J, et al. (2001). Deleted in Malignant Brain Tumors 1 is a versatile mucin-like molecule likely to play a differential role in digestive tract cancer. Cancer Res 61:8880–8886.[Abstract/Free Full Text]
Mollenhauer J, Helmke B, Muller H, Kollender G, Lyer S, Diedrichs L, et al. (2002a). Sequential changes of the DMBT1 expression and location in normal lung tissue and lung carcinomas. Genes Chromosomes Cancer 35:164–169.[ISI][Medline]
Mollenhauer J, Muller H, Kollender G, Lyer S, Diedrichs L, Helmke B, et al. (2002b). The SRCR/SID region of DMBT1 defines a complex multi-allele system representing the major basis for its variability in cancer. Genes Chromosomes Cancer 35:242–255.[ISI][Medline]
Mollenhauer J, Deichmann M, Helmke B, Muller H, Kollender G, Holmskov U, et al. (2003). Frequent downregulation of DMBT1 and galectin-3 in epithelial skin cancer. Int J Cancer 105:149–157.[ISI][Medline]
Mori M, Shiraishi T, Tanaka S, Yamagata M, Mafune K, Tanaka Y, et al. (1999). Lack of DMBT1 expression in oesophageal, gastric and colon cancers. Br J Cancer 79:211–213.[ISI][Medline]
Prakobphol A, Xu F, Hoang VM, Larsson T, Bergström J, Johansson I, et al. (2000). Salivary agglutinin, which binds Streptococcus mutans and Helicobacter pylori, is the lung scavenger receptor cysteine-rich protein gp-340. J Biol Chem 275:39860–39866.[Abstract/Free Full Text]
Rundegren J, Arnold RR (1987). Differentiation and interaction of secretory immunoglobulin A and a calcium-dependent parotid agglutinin for several bacterial strains. Infect Immun 55:288–292.[Abstract/Free Full Text]
Rundegren J, Ericson T (1981). Effect of calcium on reactions between a salivary agglutinin and a serotype c strain of Streptococcus mutans. J Oral Pathol 10:269–275.[ISI][Medline]
Takeshita H, Sato M, Shiwaku HO, Semba S, Sakurada A, Hoshi M, et al. (1999). Expression of the DMBT1 gene is frequently suppressed in human lung cancer. Jpn J Cancer Res 90:903–908.[ISI][Medline]
Tino MJ, Wright JR (1999). Glycoprotein-340 binds surfactant protein-A (SP-A) and stimulates alveolar macrophage migration in an SP-A-independent manner. Am J Respir Cell Mol Biol 20:759–768.[Abstract/Free Full Text]
Vijayakumar S, Takito J, Hikita C, Al Awqati Q (1999). Hensin remodels the apical cytoskeleton and induces columnarization of intercalated epithelial cells: processes that resemble terminal differentiation. J Cell Biol 144:1057–1067.[Abstract/Free Full Text]
Wu W, Kemp BL, Proctor ML, Gazdar AF, Minna JD, Hong WK, et al. (1999). Expression of DMBT1, a candidate tumor suppressor gene, is frequently lost in lung cancer. Cancer Res 59:1846–1851.[Abstract/Free Full Text]
This article has been cited by other articles:
|
|
|
|
 
E. Stoddard, G. Cannon, H. Ni, K. Kariko, J. Capodici, D. Malamud, and D. Weissman
gp340 Expressed on Human Genital Epithelia Binds HIV-1 Envelope Protein and Facilitates Viral Transmission
J. Immunol.,
September 1, 2007;
179(5):
3126 - 3132.
[Abstract]
[Full Text]
[PDF]
|
|
|
TOP
ABSTRACT
INTRODUCTION
