In situ localization of amylase mRNA and protein. An investigation of amylase gene activity in normal human parotid gland

The distribution of human salivary amylase mRNA was studied by in situ hybridization to a [32P]-labeled amylase cDNA probe. Amylase mRNA was localized to the apical portion of acinar cells in frozen sections of human parotid salivary gland. No hybridization was noted in ductal cells, skeletal muscle, or in connective tissue. These results were consistent with immunohistochemical localization of amylase. The technique of in situ hybridization was modified to permit localization of amylase mRNA in variously fixed, paraffin-embedded parotid glands. Although the hybridization signal decreased with all fixatives, the pattern of localization paralleled that obtained with frozen sections. No advantage was noted in fixation with ethanol-acetic acid or Bouin solution over routine fixation with formalin. These results have important implications for researchers interested in studies of gene expression. We have demonstrated that routinely fixed paraffin blocks of human tissue can be used for cellular localization of specific mRNA. In coordination with immunocytochemistry, in situ hybridization offers a powerful tool for studies of mRNA and protein expression in individual cells.     

Cellular characteristics of long-term cultured rat parotid acinar cells

Summary We have successfully maintained and biochemically characterized differentiated rat parotid acinar cells cultured for long periods (6 mo.). The cells were cultured on a reconstituted basement membrane matrix in a medium containing a variety of agents that promote cellular proliferation and differentiation. The cultured cells retain the characteristics of the parental parotid acinar cells. They exhibit both secretory granules and abundant cellular organelles required for protein synthesis and secretion. In situ hybridization and immunocytochemistry demonstrate high levels of proline-rich protein mRNA and protein, and lower levels of amylase mRNA and protein, in their cytoplasm. These findings suggest that rat parotid acinar cells can be maintained in a differentiated state in vitro for long periods, and can serve as a useful model system for studying the regulation of exocrine secretory processes.     

P63 is expressed in basal and myoepithelial cells of human normal and tumor salivary gland tissues.

p63 is essential for epithelial cell survival and may function as an oncogene. We examined by immunohistochemistry p63 expression in human normal and tumor salivary gland tissues. In normal salivary glands, p63 was expressed in the nuclei of myoepithelial and basal duct cells. Among 68 representative salivary gland tumors, 63 displayed p63 reactivity. In all tumor types differentiated towards luminal and myoepithelial lineages (pleomorphic adenomas, basal cell adenomas, adenoid cystic carcinomas, and epithelial-myoepithelial carcinomas), p63 was expressed in myoepithelial cells, whereas luminal cells were always negative. Similarly, in mucoepidermoid carcinomas, basal, intermediate, and squamous cells expressed p63, in contrast to luminal mucous cells. p63 reactivity was also restricted to basal cells in Warthin tumors and oncocytomas. Myoepitheliomas and myoepithelial carcinomas all expressed p63. The only five negative tumors were three of four acinar cell carcinomas and two of three adenocarcinomas. In conclusion, p63 is expressed in the nuclei of normal human salivary gland myoepithelial and basal duct cells. p63 expression is retained in the modified myoepithelial and basal cells of human salivary gland tumors, which suggests a role for p63 in oncogenesis of these complex tumors.     

Characterization of lacrimal gland carbonic anhydrase VI.

We have previously demonstrated by immunohistochemistry the presence of secreted carbonic anhydrase (CA VI) in the acinar cells of the rat lacrimal glands. In this study we purified the sheep lacrimal gland CA VI to homogeneity and demonstrated by Western analysis that it has the same apparent subunit molecular weight (45 kD) as the enzyme isolated from saliva. RT-PCR analysis showed that CA VI mRNA from the lacrimal gland was identical to that of the parotid gland CA VI mRNA. An RIA specific for sheep CA VI showed the lacrimal gland tissue concentration of the enzyme to be 4.20 +/- 2.60 ng/mg protein, or about 1/7000 of the level found in the parotid gland. Immunohistochemistry (IHC) and in situ hybridization (ISH) showed that lacrimal acinar cells expressed both immunoreactivity and mRNA for CA VI. Moreover, CA VI immunoreactivity was occasionally observed in the lumen of the ducts. Unlike the parotid gland, in which all acinar cells expressed CA VI immunoreactivity and mRNA, only some of the acinar cells of the lacrimal gland showed expression. These results indicate that the lacrimal gland synthesizes and secretes a very small amount of salivary CA VI. In tear fluid, CA VI is presumed to have a role in the maintenance of acid/base balance on the surface of the eye, akin to its role in the oral cavity.     

Parafibromin expression in lung normal tissue and carcinoma: its comparison with clinicopathological parameters of carcinoma

Parafibromin is a protein encoded by the hyperparathyroidism 2 oncosuppressor gene and its down-regulated expression is involved in the pathogenesis of parathyroid, gastric and colorectal carcinomas. To clarify the roles of parafibromin expression in lung carcinomas, it was examined by immunohistochemistry and in situ hybridization on tissue microarray containing lung carcinomas (n=144) and normal lung tissue (n=20), with a comparison to clinicopathological parameters of carcinomas. Lung carcinoma cell lines and tissues were studied for parafibromin expression by Western blot and RT-PCR. Down-regulated expression of parafibromin mRNA was found in lung carcinoma in comparison with matched normal tissue (p<0.05). Parafibromin protein was found in the cilia and nuclei of pseudo-stratified columnar epithelium, and the nuclei of lung carcinoma. According to immunostaining and in situ hybridization, there was no difference in parafibromin expression between histological subtypes of lung carcinoma (p>0.05). The Kaplan-Meier method indicated that nuclear parafibromin expression was positively correlated with adenocarcinoma patients (p<0.05). Down-regulated parafibromin mRNA expression might play an important role in lung carcinogenesis, but not in its histogenesis. Strong parafibromin expression in cilia of the pseudo-stratified columnar epithelium indicated its possible involvement in cell mobility. Parafibromin expression could be employed to indicate the favorable prognosis of patients with adenocarcinoma.     

Immunocytochemical localization of amylase in gerbil salivary gland acinar cells processed by rapid freezing and freeze-substitution fixation

We examined the immunocytochemical localization of amylase in cryofixed serous acinar cells of gerbil major salivary glands by indirect immunostaining, using anti-gerbil parotid amylase antibody and protein A-gold complex. Fresh tissue blocks were quickly frozen by the metal-contact method, using liquid helium, and were freeze-substituted with either osmium-acetone solution or glutaraldehyde-containing acetone. They were then embedded in an epoxy resin mixture which was polymerized at 60 degrees C. Some tissue blocks substituted with aldehyde-acetone solution were embedded in Lowicryl K4M, polymerized at -30 degrees C. Thin sections of epoxy resin-embedded materials were treated with an oxidizing agent before immunostaining. The labeling density on the materials processed by various protocols for preparatory procedures was quantitatively compared to examine the usefulness of application of cryofixation to immunocytochemistry. The central dense core of heterogeneous secretory granules in the serous acinar cells of the parotid and sublingual glands was heavily labeled with immunogold, regardless of substitution media and embedding resins employed. The immunolabeling pattern clearly distinguished between the dense core and the surrounding matrix. Labeling density in the cryofixed materials was about 1.5 times greater than in those processed by conventional chemical fixation. Seromucous secretory granules in the submandibular gland acinar cells were only faintly labeled. The results obtained indicate that application of immunostaining to quick-frozen, substitution-fixed tissues is useful for high-resolution immunocytochemistry.     

Human kallikrein 13 expression in salivary gland tumors

The human kallikrein 13 protein (hK13) is expressed in many normal tissues. Petraki et al have previously described presence of hK13 in salivary gland tissue, localized to duct epithelia and some acinar cells. The aim of this study was to determine whether hK13 is expressed in salivary gland tissues and salivary gland tumors (both benign and malignant), in order to compare normal with tumor tissues. Pleomorphic adenomas (PA), adenoid cystic carcinomas (ACC), polymorphous low grade adenocarcinomas (PLGA), acinic cell carcinomas (ACI), mucoepidermoid carcinomas (MEC) and adenocarcinomas not otherwise specified (ANOS) of both minor and major salivary glands were examined. The results of this study indicate that most salivary gland tumors show high levels of expression of hK13. Overall, staining in PA was significantly less than that seen in normal salivary gland tissue. PLGA, ACC and ANOS each stained significantly more than normal salivary gland tissue while MEC and ACI did not. Ductal cells and cells lining duct-like structures showed a higher intensity of staining than non-ductal cells in most tumors. Tumors which exhibited only non-ductal cells also exhibited cytoplasmic staining. In conclusion, we demonstrate the high expression of hK13 in several common salivary gland tumors.     

Increased expression of the enzyme beta 1-4-galactosyltransferase is associated with human parotid neoplasms

Human biopsy samples of parotid gland neoplasms were examined for the level of enzyme activity of the glycosyltransferase, beta 1-4-galactosyltransferase. An analysis of an adenoid cystic carcinoma, Warthin's tumor, mucoepidermoid carcinoma, and five pleomorphic adenomas all revealed elevated levels of enzyme activity. Evidence for plasma membrane beta 1-4-galactosyltransferase activity was provided by membrane fractionation as well as intact cell enzyme assays. On the other hand, the major protein of human saliva, salivary alpha-amylase, was substantially reduced in the same tissue compared with adjacent normal parotid gland tissue. The trichloroacetic acid-soluble proteins isolated from gland homogenates were also reduced in two of the carcinoma samples but increased in the pleomorphic adenomas. Additionally, the proliferation of these cells, in vitro, could be retarded by culturing in media containing the galactosyltransferase specific modifier protein, alpha-lactalbumin, or the nucleotide sugar, UDP-galactose.     

Glial fibrillary acidic protein and desmin in salivary neoplasms

The presence of intermediate filament proteins (IFP) in normal salivary gland tissue and in a number of salivary gland neoplasms has been investigated by immunohistochemical techniques on frozen sections. Cytokeratins (CKs) were seen in almost all normal epithelial cells. In the parotid gland and in palatal gland tissue, a co-expression of cytokeratin and glial fibrillary acidic protein (GFAP) was seen in some myoepithelial cells, but this was not apparent in the submandibular gland. In some pleomorphic adenomas, carcinomas in pleomorphic adenomas, one mucoepidermoid carcinoma, one mucus-producing adenopapillary carcinoma and one adenoid cystic carcinoma, cells expressing three different IFP classes were found (CKs, vimentin, GFAP). These cells were most often situated peripherally in the tumour cords or ducts. The cytokeratin pattern in these cells, as revealed by mAbs PKK1-3, was similar to that in normal myoepithelial cells. Furthermore, reactivity for a fourth class of IFP, desmin, could be seen in this cell type in two carcinomas in pleomorphic adenomas, and also in a few cells in a pleomorphic adenoma and an adenoid cystic carcinoma. Thus the pattern of IFP expression in salivary gland neoplasms, is very complex, and cannot always be related to the normal tissue.     

Coordination of murine parotid secretory protein and salivary amylase expression.

PSP, parotid secretory protein, and salivary amylase are the major secretory proteins of mouse parotid gland where they appear in a constant ratio. Here we describe the isolation of the PSP gene and show through expression analysis on this and the salivary amylase gene that the two genes are transcribed in a coordinate fashion in adult animals, whereas the activation profiles are different during postnatal development. An explanation is put forward that involves activation of the genes at different stages of the acinar cell differentiation, leading in adults to the maximal and thus proportionate expression.     

Expression and induction by beta-adrenergic agonists of the cystatin S gene in submandibular glands of developing rats.

Repeated administration of the beta-adrenergic agonist isoprenaline (isoproterenol, IPR), which produces hypertrophic/hyperplastic enlargements of rat submandibular and parotid glands, induces synthesis of a secretory protein shown to be a cysteine proteinase inhibitor, rat cystatin S. In the current study, Northern blot and hybridizations in situ were carried out to establish the developmental and beta-adrenergic regulation of the expression of the cystatin S gene. Cystatin S mRNA was not detected in submandibular glands of 20-day-old fetuses, nor in the glands of newborn or 10-day-old rats. However, steady-state levels of cystatin S mRNA increased between 21 and 28 days, reaching a conspicuously high concentration at 28 days; cystatin S mRNA then declined rapidly to a barely detectable level in glands of 32-day-old rats. IPR administration for 4 days induced high levels of cystatin S mRNA in submandibular glands of developing and adult rats. In both prepubertal and mature animals, induction of cystatin S mRNA in submandibular glands was more pronounced in female than in male animals. Hybridizations in situ revealed cystatin S mRNA only in acinar but not in duct cells of the submandibular gland. Developmentally, expression of the cystatin S gene coincided with acinar cell differentiation. These data suggest a complex neural, hormonal and developmental regulation of salivary cystatin genes.     

In situ hybridization of albumin mRNA in normal liver and liver tumors: Identification of hepatocellular origin

In situ hybridization was performed to detect albumin mRNA in normal liver, liver cirrhosis, primary liver tumors and secondary liver neoplasms. In areas of normal liver, and liver cirrhosis, signals for albumin mRNA were present in hepatocytes, whereas no signals were seen in other cells such as endothelial and Küpffer cells, bile duct epithelium and smooth muscle cells. In 53 of 56 hepatocellular carcinomas signals were present in tumor cells but in eight cholangiocarcinomas and 14 metastatic adenocarcinomas from large bowel or pancreas, carcinoma cells were negative for albumin mRNA. In three metastatic tumors (from two neuroendocrine carcinomas and one gastric leiomyosarcoma), tumor cells contained no signals, while the surrounding hepatocytes showed diffuse grains. In 15 of the 84 specimens examined in situ hybridization was applied to routine formalin-fixed and paraffin-embedded blocks and strong signals were obtained for albumin mRNA. We conclude that in situ hybridization of human albumin is a valid tool in the differential diagnosis of hepatocellular carcinoma from cholangiocarcinomas and tumors metastatic to the liver.     

Cloning and characterization of a novel animal lectin expressed in the rat sublingual gland.

We cloned a rat gene that is expressed primarily in the sublingual gland and named the predicted 503 amino-acid protein SLAMP (sublingual acinar membrane protein). SLAMP has 63% homology with human ERGIC-53-like protein, a member of the family of animal L-type lectins. Using a cDNA probe for SLAMP mRNA and rabbit antisera against SLAMP, we examined the expression and localization of SLAMP in major rat organs and tissues. With both Northern and Western blot analyses, abundant expression of SLAMP was demonstrated predominantly in the sublingual gland, with single sizes of the mRNA and protein 1.8 kb and 50 kDa, respectively, but not in other organs or tissues, including the parotid and submandibular glands. With immunohistochemistry, SLAMP was localized to the mucous acinar cells, but not to the serous demilunes or the duct system. With immunoelectron microscopy, SLAMP was localized predominantly to regions corresponding to the ER-Golgi intermediate compartment. Besides the sublingual gland, SLAMP immunoreactivity was also demonstrated in mucous cells of the minor salivary glands in oral cavity and of Brunner's glands in the duodenum. These results suggested that rat SLAMP plays a specific role in the early secretory pathway of glycoproteins in specific types of mucous cells.     

Molecular cloning of mouse PSP mRNA.

PSP is the most abundant translation product of mouse parotid glands where its production is co-ordinated with that of salivary amylase. The synthesis of these two proteins apparently is restricted to this tissue. In order to enable us to study common regulatory elements in the genes of the two proteins, double stranded cDNA, synthesized for parotid gland poly (A)+ RNA, was cloned. DNA sequencing of three clones complementary to the most abundant messenger indicated overlap and resulted in a total sequence of 867 nucleotides. Translation of this sequence revealed that at one end the amino acid sequence was the same as the N-terminal sequence of PSP. The sequence contains 60 nucleotides coding for part of or the complete signal peptide, 645 nucleotides coding for the PSP protein, and 162 nucleotides that apparently are not translated. Southern blot analysis suggests a simple structure for the PSP gene in mouse and man.