Molecular characterization of salivary gland malignancy using the Smgb-Tag transgenic mouse model

The molecular mechanisms underlying salivary gland tumorigenesis remain unclear. In order to identify genetic changes that occur during the development of invasive adenocarcinoma from normal salivary gland, we used the Smgb-Tag transgenic mouse model. This transgene induces the progressive development of dysplasia to invasive adenocarcinoma in the submandibular salivary gland. Gene expression patterns from 20 submandibular glands (two normal, nine dysplasia and nine adenocarcinoma samples) were assessed using a mouse 15 K cDNA array. Unsupervised hierarchical clustering was used to group gene expression based on 157 differentially expressed genes distinguishing between dysplasias and adenocarcinomas. Further analysis identified 25 significantly overexpressed and 28 underexpressed cDNA sequences in adenocarcinoma as compared to dysplasia. Differential expression of five genes (Lcn2, Ptn, Cd24a, Mapk6 and Rnps1) was validated by quantitative real-time RT-PCR in a total of 48 mouse salivary gland tissues (seven histologically normal, 13 dysplasias and 28 adenocarcinomas), including the 20 samples analyzed by cDNA arrays. Immunohistochemical analysis was used to validate the expression of Ptn and Cd24a at the protein level in a subset of 16 mouse salivary glands (four normal, five dysplasia and seven adenocarcinoma samples), as well as in 23 human submandibular gland tumors (16 pleomorphic adenomas, three adenoid cystic carcinomas, one acinic cell carcinoma, one adenocarcinoma NOS, one myoepithelial and one mucoepidermoid carcinoma). We thus demonstrated that the Smgb-Tag transgenic mouse model is a useful tool for the identification of genes that are deregulated in salivary gland adenocarcinomas. Our data suggest that Ptn and Cd24a may be genetic markers associated with salivary gland tumorigenesis and/or progression.     

Ultrastructural study of acinar and intercalated duct organization of submandibular and parotid salivary gland

According to some current hypotheses, the morphology and organization of the intercalated duct/acinar interface of salivary gland have implications for the induction of tumors in this organ. However, this region has received limited detailed investigation. To study the organization of the terminal ductal segments of salivary gland, conventional transmission electron microscopy of human parotid and submandibular glands and canine submandibular gland was combined with 3-dimensional observations of polymer casts of the canine submandibular ductal system; the latter were prepared by retrograde injection of acrylic resin via the main excretory duct with subsequent digestion of the gland tissue. The division of intercalated ducts, into first- and second-order branches, and acinar arrangement is more complex than previously suggested. The entire surface of each elongated second-order intercalated duct is covered with acini projecting in all directions. In the human gland, some acini abut directly on the intercalated duct surface, whereas others are connected by a short stalk of intercalated duct cells; in comparison with canine submandibular gland, the latter may be a modification producing a third-order of the intercalated duct unit. All of these features combine to produce a highly efficient secretory apparatus with a large proportion of acinar cells to each intercalated duct.     

Dynamics of parenchymal cell division, differentiation, and apoptosis in the young adult female mouse submandibular gland

The submandibular salivary gland of the young adult female mouse has two secretory cell types, acinar and granular duct, which are separated by intercalated ducts. Based on the occurrence of autologous cell division in these cells, they have been traditionally classified as expanding populations. However, differentiation from stem or progenitor cells in the intercalated ducts, usually associated with renewing populations, has also been detected. The question of renewing or expanding populations is resolved by quantitating and integrating the rates of autologous cell division, differentiation, and apoptosis for each cell type. The integrated data shows that both acinar and granular duct cell populations exhibit a substantial positive growth index, whereas the growth index for the intercalated duct cells is moderately negative. On balance, it suggests that the submandibular gland of the young adult female mouse is still growing. Comparison of young female mice with older females suggests that, although overall parenchymal growth slows with age, there is no longer a net loss of intercalated duct cells. Comparison with young adult male submandibular glands indicates that gender differences exist in the rates and mechanisms used for maintaining the different cell populations. The acinar and granular duct cell populations in young adult female mouse submandibular glands are expanding at the expense of the intercalated duct cell population, which appears to be contracting.     

Expression and localization of cell growth factors in the salivary gland: a review

The salivary gland secretes not only digestive enzymes but also various cell growth factors. Especially in rodents, granular ducts which develop between striated and intercalated ducts, are known to secrete epidermal growth factor (EGF) and nerve growth factor (NGF). Out of newly discovered growth factors, we have examined hepatocyte growth factor (HGF), transforming growth factor-beta (TGF-beta), insulin-like growth factor I (IGF-I), and basic fibroblast growth factor (FGF-2) and have demonstrated their expression and localization in the rat submandibular gland. HGF and TGF-beta show a very similar distribution pattern to EGF, i.e., exclusive localization in the secretory granules of granular duct cells. These factors are suggested to function as i) exocrine salivary factors, or ii) endocrine factors after reabsorption. In contrast, FGF-2 shows a different pattern of cellular and subcellular localization from EGF and others. FGF-2 is localized in cytoplasm of striated and excretory duct cells and pillar cells in granular ducts, but not in secretory granules of granular duct cells. Since the receptor for FGF also present in the same cells as its ligand, FGF is suggested to function in the salivary gland as an autocrine/paracrine factor. The possible physiological roles of salivary growth factors in the digestive organs are discussed based on our own data and an extensive literature.     

Simple method to isolate the intact duct system of the rat submandibular gland

The excurrent duct system of the rat submandibular gland consists of a number of distinct segments. Using the direction of salivary flow as a reference point, these segments are, in order, intercalated duct, granular convoluted tubule, striated duct, excretory duct, main excretory duct (MED), and salivary bladder (which is an expanded portion of the MED). Because these ducts (with the exception of the MED and the salivary bladder) are encased in secretory endpieces, they are difficult to locate and to observe by scanning electron microscopy. A simple method has been devised to rid the gland of these obscuring endpieces so that the detailed architecture of the duct system can be examined. Rat submandibular glands were fixed initially by vascular perfusion with half‐strength Karnovsky's fixative. The connective tissue capsule was removed from extirpated glands and the glands remained in fixative for varying lengths of time. For our purposes, a 30‐minute immersion in the aldehyde mixture was optimum. After the sublingual gland was removed, the submandibular gland was softy struck with forceps having rounded tips, then shaken in fixative or buffer. The tissue that remained was postfixed in osmium tetroxide. This method results in the complete divestment of nonductular parenchyma from the rat submandibular gland, leaving the duct system clean and ready for microscopic examination. Anat Rec 254:74–75, 1999. © 1999 Wiley‐Liss, Inc.     

Immunohistochemical localization of carbonic anhydrases I, II, and VI in the developing rat sublingual and submandibular glands

Carbonic anhydrase has been localized to the acini and ducts of mature rat salivary glands. This enzyme has been associated with ion transport, a prominent function of striated and excretory ducts in salivary glands, suggesting that it might be used as a marker of ductal differentiation. The purpose of this study was to immunohistochemically document developmental changes in carbonic anhydrase in the ducts of the rat sublingual and submandibular glands. Immunohistochemistry was performed with antibodies to human carbonic anhydrase isoenzymes I, II and VI on sections of sublingual and submandibular glands from rats at representative postnatal developmental ages. Reactions were weak in the ducts of both glands at 1 day, then progressively increased. By 42 days, reactions had the adult pattern of virtually none in the mucous or seromucous acini, moderate to strong in the striated and excretory ducts, and none to weak in the intercalated ducts. Weak to moderate reactions were observed in the granular convoluted tubules of the submandibular gland as they became recognizable at age 42 days. Reactions to carbonic anhydrase I and II antibodies also increased from none (1 day) to modest (42 days) in the demilunes of the sublingual gland. The order of reaction intensity of the antibodies was II > I > VI. When localized via these anti-human antibodies, carbonic anhydrase is a useful marker of the functional differentiation of the striated and excretory ducts of the developing rat sublingual and submandibular glands.     

Localization of prostate-specific antigen-like immunoreactivity in human salivary gland and salivary gland tumors

Immunoreactivity of prostate-specific antigen (PSA), a kallikrein-like enzyme present in the seminal plasma, was demonstrated by indirect immunoperoxidase staining using a PSA antiserum in the apical cytoplasm along the luminal border of small-sized duct epithelial cells of the major salivary (parotid and submandibular) gland of both sexes (56/56, 100%). No PSA-like immunoreactivity was seen in large-sized duct epithelial cells and acinar cells. Minor salivary gland ducts were negative. When inflammatory and atrophic changes were observed, ductal expression of PSA-like immunoreactivity was decreased (12/37, 32%) and the site of intracellular localization often became diffusely cytoplasmic. The immunoreactivity was absorbed by human seminal plasma. Immunoreactivities of prostatic acid phosphatase and sex hormone receptors were undetectable in the salivary gland. Twenty-nine (34%) of 86 salivary gland tumors with ductal differentiation were immunoreactive for PSA mainly in the cytoplasm. A PSA monoclonal antibody ER-PR8 detected immunoreactivity in the prostate but not in the salivary glands or their tumors. Prostate-specific antigen-like immunoreactivity in small-sized (intercalated) duct epithelial cells of the major salivary gland and their tumors may be due to cross-reactivity of the antiserum with kallikrein-like substances.     

Immunolocalization of carbonic anhydrase isozymes in rat and mouse salivary and exorbital lacrimal glands

Carbonic anhydrase (CA) isozyme I and isozyme II have been localized with the immunoperoxidase bridge method in cells of mouse and rat salivary glands and exorbital lacrimal glands. Immunostaining proved optimal in Carnoy fixed specimens for some sites and in Bouin fixed glands for other sites. Staining in mouse largely resembled that in rat glands, but minor species differences were observed. Serous acinar cells in the submandibular gland stained uniformly and exclusively for CA I. From 50 to 100% of the serous acinar cells in the parotid glands evidenced content of both CA I and CA II. A minor population of serous acinar cells in the mouse exorbital lacrimal gland stained for CA I and CA II, but these glands in the rat failed to stain. Immunostaining was observed in ducts in Bouin fixed glands. Some cells in striated ducts of submandibular and sublingual glands stained for CA I and CA II and other cells in these ducts were negative. Such cellular heterogeneity was also observed in excretory ducts of submandibular and sublingual glands. These findings thus demonstrate the presence of CA in two morphologically and functionally diverse cell populations in rodent salivary glands. Immunolocalization of the CA isozymes in serous acinar cells and intercalated duct cells, presumably packaged in secretory granules, implies a role for this enzyme in salivary secretions whereas localization of CA in striated and excretory ducts suggests their traditional function in fluid and electrolyte transport.     

Histochemical localization of ouabain-sensitive, K+-dependent p-nitrophenylphosphatase (Na+-K+-ATPase) activity in the submandibular gland of the mouse: effect of androgen, thyroid hormone, or postnatal age

Ouabain-sensitive Na+-K+-ATPase activity was localized histochemically in the submandibular gland of the mouse under various conditions using p-nitrophenylphosphate as substrate at pH 9. In untreated adult males and females, intense staining was seen in the basally striated portions of the epithelial cells lining the excretory and striated ducts. The region of the lateral cell membranes, but not of the apical plasmalemma, also stained. In granular convoluted tubules (GCTs), strong staining was seen only in a narrow band of the basalmost region of the cells; in males this stained region was thinner than in females, and frequently was absent. The baso-lateral margins of acinar and intercalated duct cells gave a very weak reaction. In untreated males, or in females that were treated with dihydrotestosterone, overall staining for the enzyme was always less than in untreated females, due to the diminished reactivity of androgen-stimulated GCT cells and the decreased number of striated ducts. However, in females treated with triiodothyronine, enhanced activity of Na+-K+-ATPase was indicated by stronger staining in all cell types, including the hypertrophied GCT cells. Na+-K+-ATPase activity was undetected in the submandibular glands at birth, but moderate staining was seen in the larger excretory and striated ducts by 5 days of age. From 10 days of age onward, intense staining was seen in the excretory and striated portions of the ramifying duct system. Developing GCT cells could not be distinguished from their precursor cells in the striated ducts until 25 days of age. These data indicate that the salt-handling capacity of the submandibular gland of the mouse varies with both endocrine status and age.     

Tissue-specific expression of the tight junction proteins claudins and occludin in the rat salivary glands

Tight junctions (TJs) are essential features of endothelial barrier membranes and of fluid-secreting epithelial cells, such as in the salivary glands. Novel integral membrane proteins have been identified as components of TJs, namely claudins and occludin. The aim of the present study was to determine the distribution of occludin and claudins in the large salivary glands of the rat. The parotid, submandibular and sublingual salivary glands were harvested from adult Sprague-Dawley rats and cryostat sections were stained using immunoperoxidase and immunofluorescence methods. Claudin-1 was expressed in endothelial cells of microvessels and in short selected segments of the duct system. Claudin-3 was expressed principally in the acinar cells and intercalated ducts, while claudin-4 was principally expressed by the striated and interlobular ducts. Claudin-5 was specific to endothelial cells of microvessels. Occludin was ubiquitously detected in the duct system. Double labelling and confocal microscopy showed some co-localization of claudin-3 with claudin-4, and minimal co-localization of occludin with claudin-4, in the striated ducts. Claudin 2 was not detected in any of the salivary glands. The results indicate specificity of the chemical composition of tight junctions in the rat salivary glands, and may reflect different physiological roles for TJs in the glandular and duct epithelial cells, and in endothelial cells of salivary gland microvessels.     

Elastic fibers in the duct system of the rat submandibular salivary gland.

The submandibular salivary gland originates from the floor of the mouth whose mucosa contains elastic fibers. Therefore, such fibers were sought in the duct system of the derivative organ. In adult rats, light microscopy has indeed revealed fine, circumferential, elastic fibers near the basement membrane of the duct epithelium. In the larger extralobular ducts, they were separated from several layers of longitudinal elastic fibers by a capillary-rich zone sparse in elastic fibers except for fine angular ones. More peripherally, larger angular-appearing fibers were frequently present near the submandibular parasympathetic ganglia in the duct wall. As duct diameter decreased, elastic fiber size and number diminished. Intralobularly, the smaller striated ducts, granular and intercalated ducts, and acini generally lacked such fibers. Electron microscopy of the extraglandular portion of the main duct revealed fibrils extending from both fibroblasts and elastic fibers that were close to the epithelium. Microfibrils coursed from them toward the lamina densa. Anchoring filaments joined the lamina densa to the basal plasma membrane of the epithelium. Elastic fibers also appeared to connect to both capillaries and collagen via finer intermediate structures. These associations might permit dynamic interactions of fibroblasts, fibers, smaller fibrillar components, vasa, and the duct epithelium. This interplay could occur during feeding and grooming when tongue protrusion and neck extension stretch the submandibular duct and the gland itself. As a result, the tensile forces engendered could modify cell geometry and the calibers of the larger ducts' lumens and intercellular spaces, thus affecting the flow and composition of salivary secretion.     

Characterization of cytoskeletal proteins in basal cells of human parotid salivary gland ducts

Summary From previous immunofluorescent, immunohistochemical and ultrastructural studies, myoepithelial cells have been reported to be absent from the striated and excretory ducts of human salivary gland. Yet recently, certain anti-cytokeratin monoclonal antibodies which specifically label the myoepithelium of salivary gland acini and intercalated ducts have also been found to stain basally situated cells in both striated and excretory ducts. In this study, we have used eight samples of normal human parotid gland (methacarn-fixed and frozen sections) to establish if basal cells of striated and excretory ducts have the cytoskeletal protein complement (actin and cytokeratins) of myoepithelial cells. Using a muscle-specific actin, HHF35, not only is the myoepithelium of acini and intercalated ducts stained in all cases, but stellate and spindle shaped cells are also detected all along the inter- and intralobular striated ducts in four of the eight examples. With double-labeled frozen sections and fluorescent microscopy, the actin-specific probe, phalloidin, and the myoepithelial-selective anti-cytokeratin 14 antibody, 312C8-1, confirm that the striated duct does have a population of basal cells with the cytoskeletal protein make-up of myoepithelium. The monoclonal antibody 8.12 (specific for cytokeratin 13 and 16) also stains some basal cells of striated and excretory ducts, as well as luminal cells of ducts at all levels, but does not label the myoepithelium of acini and intercalated ducts. Both the anti-cytokeratin antibodies and the actin-detecting mechanisms reveal that the basal cell population of striated and excretory ducts is more heterogeneous, and likely functionally more complex, than has been realized previously. Such findings are not in agreement with certain aspects of current theories of the histogenesis of salivary gland tumours.The authors appreciate the funding provided by the Moe Levin Family Foundation, Montreal, Canada     

Ultrastructure of the cat sublingual gland

The sublingual gland of the cat consists primarily of branched secretory tubules that open into an abbreviated duct system. The simple epithelium that composes the secretory tubules consists of an admixture of mucous and serous cells, with the former predominating. Some secretory tubules are capped by a serous demilune. Regardless of position, almost all serous cells have prominent basal folds and border on at least one intercellular canaliculus as well as on the tubule lumen. Serous cells possess an extensive array of irregular, distended cisternae of rough-surfaced endoplasmic reticulum that frequently contain dense intracisternal granules. Serous granules are relatively few in number and rarely show evidence of substructure. Mucous cells, which lack basal folds, contain an apical mass of secretory material in the form of partially fused droplets. The duct system is somewhat less ordered than in most major salivary glands; secretory tubules empty into structures resembling intercalated ducts or may be in direct continuity with ducts intermediate in morphology between intercalated and excretory ducts. The absence of striated ducts noted in this study may be correlated with the high sodium content of cat sublingual saliva. The main excretory duct of the sublingual gland closely resembles that of the cat submandibular gland in terms of morphology, but exhibits little of the transport functions reported in the latter duct.     

生后小鼠颌下腺神经生长因子基因表达的发育变化

为了探讨在发育过程中小鼠颌下腺神经生长因子基因表达的变化，应用原位杂交组织化学技术和图像分析方法对新生至２月龄ＩＣＲ雄性小鼠颌下ＮＧＦｍＥＮＡ基因表达的变化，应用原位杂交研究。结果显示：新生小儿颌下腺未见ＮＧＦｍＲＮＡ杂交信号，８－１０ｄ龄小鼠颌下腺ＮＧＦ原位杂交信号呈阴性至弱阳性。     

Cellular localization of kallikreins in rat submandibular and sublingual salivary glands: immunofluorescence tracing related to histological characteristics.

Immunohistochemical localization of rat salivary gland kallikrein was related to glandular structures in tissue processed and stained by various methods. In the submandibular gland, most of the kallikrein was located to cytoplasmic granules of the granular tubules. Cells of the striated ducts showed a faint cytoplasmic staining with a bright luminal rim that occasionally was seen also in the excretory ducts. Minor amounts of kallikrein was found in the interstitial tissue. In the sublingual gland, kallikrein was found in the cytoplasm of the striated duct cells and as a luminal rim in the main ducts. Acini were negative in both glands. Fixation in Helly's fluid preserved cytoplasmic granules and was thus superior for intracellular localization of kallikrein, whereas ethanol fixation, due to absence of non-specific background staining, afforded the most sensitive method for detection of small amounts of antigen. In the submandibular gland, best identification of granular tubules and striated ducts was achieved with DMAB-nitrite staining for tryptophan and counterstaining with Mayer's haemalum on sections of tissue fixed in Helly's fluid. In the sublingual gland, the duct system was best demonstrated by haematoxylin-eosin staining.     

Three-dimensional reconstruction of adult female mouse submandibular gland secretory structures

Computer-assisted reconstructions of adult female mouse submandibular gland have been used to positionally characterize within the three-dimensional structure likely intermediates in secretory cell replacement. The locations of striated granular duct cells and granular intercalated duct cells are consistent with a role as intermediates between intercalated duct cells and granular duct cells or acinar cells, respectively. Average volumes of the two putative intermediate cell types are also consistent with this role. The reconstructions suggest that, in addition to a "streaming" mechanism for secretory cell replacement, formation of new secretory structures composed of multiple acini and second-order intercalated ducts may also contribute to the cell replacement process.     

三叶因子2在大鼠下颌下腺的表达及性差

目的:探讨三叶因子2(TFF2)在大鼠下颌下腺的定位及其在不同性别中的表达差异.方法:对大鼠下颌下腺进行TFF2免疫组织化学显色和RT-PCR检测TFF2mRNA表达.结果:TFF2免疫反应阳性物质主要位于闰管、纹状管和小叶间导管上皮细胞胞质,特别是近管腔面更多,纹状管和小叶间导管管腔内亦有阳性物质分布;颗粒区管的少颗粒暗细胞或无颗粒细胞强阳性;多颗粒细胞弱阳性或阴性.雄性大鼠的下颌下腺表达TFF2mRNA明显多于雌性.结论:TFF2主要分布在下颌下腺导管的上皮细胞,以雄性丰富,且主要以腔分泌方式发挥作用.     

MUC1 and MUC2 expression in salivary gland tumors and in non-neoplastic salivary gland tissue

The expression of MUC1 and MUC2 was studied in salivary gland tumors and non-neoplastic salivary gland tissue. Formalin-fixed paraffin-embedded specimens from 101 patients (21 pleomorphic adenomas (PA), 22 Warthin's tumors (WT), 26 adenoid cystic carcinomas (ACC), 13 acinic cell adenocarcinomas (ACA), 9 mucoepidermoid carcinomas (MC), and 10 specimens of non-neoplastic parotid and submandibular gland tissue) were immunostained. All salivary gland tumors expressed MUC1. A strong immunoreactivity was noted in WT and MC, a moderate in ACC and ACA, and a weak in PA. Strong expression of MUC2 was noted in all WT, moderate expression in MC, and weak expression in PA and ACA. All cases of ACC except for two were negative for MUC2. In general, MUC1 expression was stronger than that of MUC2. Non-neoplastic salivary gland tissue revealed a moderate MUC1 and MUC2 expression in excretory ducts and a strong expression in striated ducts. The apical plasma membrane of some serous acini expressed MUC1. Mucous acini were negative for both antigens. No change in immunoreactivity was noted in cases of chronic sclerosing sialadenitis. In conclusion, the different expression pattern of MUC1 and MUC2 in salivary gland neoplasia may be of additional value for the classification of salivary gland tumors.