Differential Expression of Peroxisomal Proteins in Distinct Types of Parotid Gland Tumors.

Salivary gland cancers are rare but aggressive tumors that have poor prognosis and lack effective cure. Of those, parotid tumors constitute the majority. Functioning as metabolic machinery contributing to cellular redox balance, peroxisomes have emerged as crucial players in tumorigenesis. Studies on murine and human cells have examined the role of peroxisomes in carcinogenesis with conflicting results. These studies either examined the consequences of altered peroxisomal proliferators or compared their expression in healthy and neoplastic tissues. None, however, examined such differences exclusively in human parotid tissue or extended comparison to peroxisomal proteins and their associated gene expressions. Therefore, we examined differences in peroxisomal dynamics in parotid tumors of different morphologies. Using immunofluorescence and quantitative PCR, we compared the expression levels of key peroxisomal enzymes and proliferators in healthy and neoplastic parotid tissue samples. Three parotid tumor subtypes were examined: pleomorphic adenoma, mucoepidermoid carcinoma and acinic cell carcinoma. We observed higher expression of peroxisomal matrix proteins in neoplastic samples with exceptional down regulation of certain enzymes; however, the degree of expression varied between tumor subtypes. Our findings confirm previous experimental results on other organ tissues and suggest peroxisomes as possible therapeutic targets or markers in all or certain subtypes of parotid neoplasms.

Poly-L-Arginine Induces Apoptosis of NCI-H292 Cells via ERK1/2 Signaling Pathway.

Cationic protein is a cytotoxic protein secreted by eosinophils and takes part in the damage of airway epithelium in asthma. Poly-L-arginine (PLA), a synthetic cationic protein, is widely used to mimic the biological function of the natural cationic protein in vitro. Previous studies demonstrated the damage of the airway epithelial cells by cationic protein, but the molecular mechanism is unclear. The purpose of this study aimed at exploring whether PLA could induce apoptosis of human airway epithelial cells (NCI-H292) and the underlying mechanism. Methods. The morphology of apoptotic cells was observed by transmission electron microscopy. The rate of apoptosis was analyzed by flow cytometry (FCM). The expressions of the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), Bcl-2/Bax, and cleaved caspase-3 were assessed by western blot. Results. PLA can induce apoptosis in NCI-H292 cells in a concentration-dependent manner. Moreover, the phosphorylation of the ERK1/2 and the unbalance of Bcl2/Bax, as well as the activation of caspase-3, were involved in the PLA-induced apoptosis. Conclusions. PLA can induce the apoptosis in NCI-H292 cells, and this process at least involved the ERK1/2 and mitochondrial pathway. The results could have some indications in revealing the apoptotic damage of the airway epithelial cells. Besides, inhibition of cationic protein-induced apoptotic death in airway epithelial cells could be considered as a potential target of anti-injury or antiremodeling in asthmatics.

Protumoral effect of macrophage through Axl activation on mucoepidermoid carcinoma.

OBJECTIVE: This study aims to test the potential involvement of Axl signaling in the protumoral effect of tumor-associated macrophages (TAMs) in mucoepidermoid carcinoma (MEC). MATERIALS AND METHODS: We carried out cocultured experiments by incubation of MEC cells (UTMUC-1) and macrophages (THP-1) and examined Axl activation status. The expression of MMPs and behavior change were examined in UT-MUC-1 cells. The effect of Axl signaling on co-cultured cancer cells was further investigated by knockdown Axl expression and suppression by Axl-specific inhibitor R428. RESULTS: Activation of Axl signaling and increased expression and activity of MMP-2 and MMP-9 along with increased invasion/migration ability in MEC cells were observed when co-cultured with TAMs. Upon knockdown of Axl in MEC or addition of R428 in the co-cultured system, these co-cultured effects were diminished. CONCLUSION: TAMs play a protumoral role in MEC via activation of the Axl signaling pathway, up-regulating MMPs expression, and increasing invasion/migration ability.

Aldehyde dehydrogenases in early stage lung cancer: nuclear expression.

PURPOSE: Aldehyde dehydrogenase enzymes are a family of intracellular enzymes that participate in cellular detoxification, differentiation and drug resistance through the oxidation of cellular aldehydes. The isoform 1 (ALDH1) has been proved useful for the identification of cancer stem cells. The ALDH1 cytoplasmatic expression has been associated with poor prognostis in several tumours, such as non-small cell lung cancer. The role of the ALDH1 nuclear expression remains unknown. METHODS: We conducted a historical cohort study in 89 patients diagnosed of stage I non-small cell lung cancer treated with surgery between 2009 and 2004 in the Thoracic Surgery Department in the Universitary Hospital Puerta de Hierro. We selected from this sample those cases with nuclear expression of the ALDH1. RESULTS: Three of the 89 (3.3 %) patients showed a nuclear expression of the ALDH1. The three of them are still alive with a median time of follow up of 73 months (more than 6 years). CONCLUSION: We have identified ALDH1 as a nuclear protein in early stage non-small cell lung cancer. It might have a function in cell cycle control, associating a better prognosis to these patients. More studies are necessary to clarify the role of nuclear expression of ALDH1.

Abundant expression of mTOR kinase in salivary gland tumors - potentials as therapy target?

BACKGROUND: Salivary gland tumors constitute 3-6% of all head and neck neoplasms in adults. Because of limited advances made in the treatment of metastatic disease, the more important is the role of new therapeutic strategies, including molecular therapy. The mammalian target of rapamycin (mTOR) has recently been established as a therapeutic target for several drugs. MATERIAL: Evaluation of phospho-mTOR as a possible therapy target by patients with salivary gland tumors. Immunohistochemical semi-quantitative analyses of the expression of phospho-mTOR(Ser2448) were processed on a tissue microarray containing samples from more than 900 patients. For statistical analysis, contingency table and chi-squared test (likelihood) were used. RESULTS: We observed at least weak phospho-mTOR expression in 25.6-41.2% of all 4 histological adenoma and in 36.8-61.6% of all 11 histological carcinoma subtypes analyzed. No association was seen between phospho-mTOR expression and tumor grade in mucoepidermoid carcinomas. CONCLUSION: In conjunction with literature data providing the evidence for a functional role of mTOR in salivary gland tumors, we conclude that treatment with mTOR-antagonists might potentially also be efficient in wide variety of salivary gland carcinomas.

Kallikrein-related peptidase 10 expression in salivary gland tissues and tumours.

OBJECTIVES: Kallikrein-related peptidase 10 (KLK10) has been implicated in the development of several types of cancer. The purpose of this study was to analyze the expression of KLK10 in 3 types of salivary gland tumour and normal salivary glands. MATERIALS AND METHODS: A standard immunoperoxidase staining technique was used to assess the immunoexpression profile of KLK10 in normal salivary glands and 3 types of salivary gland tumour: pleomorphic adenoma, adenoid cystic carcinoma and mucoepidermoid carcinoma. RESULTS: Pleomorphic adenomas showed significantly lower KLK10 levels than control tissues. Neither of the malignant tumours (adenoid cystic carcinoma and mucoepidermoid carcinoma) showed a significant alteration in the immunoreactive scores of KLK10 in comparison with the normal salivary gland tissues. KLK10 immunoreactive scores were comparable in adenoid cystic carcinoma and mucoepidermoid carcinoma. Pleomorphic adenoma had significantly lower levels of KLK10 than mucoepidermoid carcinoma. CONCLUSIONS: The finding of lower KLK10 levels in pleomorphic adenoma suggests aberrant expression in a tumour that develops primarily from myoepithelial cells. A kallikrein cascade may play a role in the development and/or outcome of some salivary gland tumours.

Immuno-histochemical evaluation of Cathepsin D in malignant salivary gland carcinomas.

OBJECTIVE: Cathepsin D is a lysosomal acid protease secreted in increased levels in several malignancies. However, its role in salivary gland tumors has not been studied extensively. The present study aims to assess the expression of Cathepsin D in malignant salivary gland tumors and to compare its expression in these tumors. STUDY DESIGN: A total of 30 cases of malignant salivary gland carcinomas which included 16 cases of adenoid cystic carcinoma (ACC), 9 cases of mucoepidermoid carcinoma (MEC), and 5 cases of polymorphous low grade adenocarcinoma (PLGA) were evaluated immunohistochemically using anti-Cathepsin D antibody. RESULT: All the cases showed positivity (100%) for Cathepsin D with intense expression noted in ACC and MEC as compared to PLGA. Comparison of these tumors revealed statistical significant difference in expression between ACC and PLGA. CONCLUSION: Intense expression of Cathepsin D in high grade carcinomas may be a marker for invasive potential and aggressive behavior.

Human kallikrein 14 (KLK14) expression in salivary gland tumors.

OBJECTIVE: To analyze the expression of human kallikrein 14 (KLK14) in salivary gland tumors. METHODS: A standard immunoperoxidase staining technique was used to assess the expression profile of KLK14 in normal salivary glands and tumors including pleomorphic adenoma (PA; n=17), adenoid cystic carcinoma (ACC; n=13) and mucoepidermoid carcinoma (MEC; n=9). Tumor stage, grade, patient age and gender, and site of occurrence were recorded. These clinical parameters were correlated with KLK14 levels in malignant tumors. The expression profiles for KLK3, 5, 6, 8 and 13 were also retrieved. RESULTS: Normal salivary glands, PA, ACC and MEC showed strong expression of KLK14 in ductal and non-ductal cells. Both PA and ACC showed higher KLK14 levels than normal glands and MEC tissues. There were no statistically significant associations between levels of KLK14 and clinical parameters. CONCLUSIONS: The differences in the levels of KLK14 suggest that KLKs may aid in the differential diagnosis of salivary gland tumors. The coexpression of KLKs suggests their possible involvement in an enzymatic pathway activated in salivary gland. KLK14 may be a promising new biomarker in salivary gland tumors.

The expression and prognostic significance of Cks1 in salivary cancer.

Cks1 is an essential factor in facilitating Skp2-dependent degradation of p27, but its role in salivary malignancies is unknown. Expression of cyclin-dependent kinase subunit 1 (Cks1) was examined in 64 salivary malignancies, compared with p27, S-phase kinase protein 2 (Skp2), Ki-67, p53, and TDT-mediated dutp-biotin nick end labeling (TUNEL) expression, and with THE patient's clinical and pathological parameters. Cks1 expression was markedly increased in 30 patients (47%) and strongly correlated with increased expression of Skp2, Ki-67, p53, and TUNEL, but inversely with p27 levels. High expression of Cks1 WAS strongly associated with lymph node metastases and poor prognosis and survival. Cks1 alterations may have a significant biological role in the pathogenesis of salivary cancer.

Peroxiredoxin I is overexpressed in oncocytic lesions of salivary glands.

BACKGROUND: Oncocytic lesions, particularly frequent in the salivary glands, are characterized by cells with an atypical accumulation of mitochondria. This accumulation has been recognized as a compensatory mechanism to intrinsic functional defects of these organelles, resulting in energy production impairment and increased generation of reactive oxygen species (ROS), including hydrogen peroxide (H(2)O(2)). Peroxiredoxin I (Prx I) is a H(2)O(2) scavenging protein and the expression of its yeast homolog was reported to be influenced by mitochondrial function. METHODS: In this study, we evaluated Prx I expression in oncocytic lesions of salivary glands by immunohistochemistry. RESULTS: Our results showed that Prx I is overexpressed in oncocytes regardless of the salivary gland lesion where they appear. CONCLUSIONS: These results suggest that Prx I expression in oncocytes is related to its ability to decompose mitochondrial-derived H(2)O(2) and that it could provide to the cells a protective role in an environment that, by continuously producing potential DNA-damaging ROS, predisposes to genome instability and cellular transformation.

Epidermal growth factor receptor and human epidermal growth receptor 2 expression in parotid mucoepidermoid carcinoma: possible implications for targeted therapy.

The expression of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) was analyzed in immunohistochemical preparations from 46 primary parotid mucoepidermoid carcinomas (MEC). For the cases with lymph node metastases, the receptor expressions were investigated in parallel samples, primary tumour and metastasis, from each patient (n=11). The goal was to evaluate whether any of these receptors are suitable as a target for radionuclide-based imaging and therapy. The HercepTest scoring was used for the analysis of both HER2 and EGFR expression (0, 1+, 2+ or 3+). EGFR overexpression (2+/3+) was found in 67.4% (31/46) of the primary tumours. Out of the 11 cases with evaluated paired samples, EGFR overexpression was observed in 81.8% (9/11) of the primary tumours and 72.7% (8/11) of the corresponding lymph node metastases. There was only one patient who had EGFR overexpression in the primary tumours which changed to negative in the lymph node metastases but no changes occurred reciprocally. The HER2 overexpression was only found in 4.3% (2/46) of the primary mucoepidermoid carcinoma and none of the lymph node metastases (0/11). EGFR and HER2 stainings were mainly found in the cell membranes. It was concluded that the majority of parotid mucoepidermoid carcinomas express EGFR strongly in their cell membranes and that lymph node metastases generally express EGFR to approximately the same extent as in the primary tumours. The stability in the EGFR expression is encouraging in the effort to develop radionuclide-based EGFR imaging agents. It is also possible that EGFR targeting agents (e.g. Iressa, Tarceva, Erbitux or radiolabelled antibodies) can be applied for the therapy of mucoepidermoid carcinoma.

Overexpression of cyclooxygenase-2 correlates with cytoplasmic HuR expression in salivary mucoepidermoid carcinoma but not in pleomorphic adenoma.

BACKGROUND: The overexpression of cyclooxygenase (COX)-2 in several human carcinomas suggests that COX-2 is related to carcinogenesis. Although COX-2 expression has been shown to be up-regulated in carcinomas of the salivary gland, its mechanisms are not completely understood. HuR is an mRNA-binding protein that controls the stability of certain transcripts including COX-2. METHODS: The expression of COX-2 and HuR was determined by immunohistochemistry in 28 cases of salivary pleomorphic adenoma and 18 cases of salivary mucoepidermoid carcinoma. RESULTS: 28.6% and 72.2% of the pleomorphic adenomas and mucoepidermoid carcinomas showed high COX-2 expression respectively. 35.7% of pleomorphic adenomas and 72.2% of mucoepidermoid carcinomas were tested positive for HuR in the cytoplasm of tumor cells. There was a correlation between a high COX-2 immunoreactivity and cytoplasmic HuR expression in mucoepidermoid carcinomas but not in pleomorphic adenomas. CONCLUSION: This study suggests that cytoplasmic HuR is correlated with COX-2 expression in salivary mucoepidermoid carcinomas. In addition, the immunoreactivity of COX-2 and cytoplasmic HuR might be used to evaluate the nature of a borderline malignancy in the salivary glands.

DNA methyltransferase 1 expression and promoter methylation of E-cadherin in mucoepidermoid carcinoma.

BACKGROUND: Loss of E-cadherin expression is found frequently in many types of human malignancies, including mucoepidermoid carcinoma (MEC). CpG methylation in the promoter region has proven important in the regulation of gene expression implicated in malignant transformation. DNA methyltransferases (DNMTs) are the major enzymes involved in establishing genomic methylation patterns. The current study was designed to test the hypothesis that CpG methylation of the promoter region of the E-cadherin gene may inactivate its expression and to examine DNMT 1 (DNMT1) protein expression in MEC. METHODS: Genomic DNA was obtained from paraffin embedded sections by laser microdissection in 46 MEC specimens. Methylation status of the E-cadherin promoter was examined by utilizing the methylation-specific polymerase chain reaction assay. To examine E-cadherin and DNMT1 proteins expression levels, the MEC specimens and adjacent epithelial tissues were studied immunohistochemically. Chi-square analysis was used to evaluate the correlation of protein expression and E-cadherin methylation status with clinicopathologic parameters. Comparisons of the survival rate between patients with DNMT1-positive and DNMT1-negative patients were made using Kaplan-Meier analysis. RESULTS: The data showed that all normal tissues expressed E-cadherin, and no promoter methylation was detected. Of the MEC samples analyzed, methylation allele was found in 33 of 46 samples (72%), and reduced E-cadherin expression was found in 21 of 46 samples (45%). DNMT1-positive expression was observed in 29 of 46 MEC samples (63%). A significant correlation was found between E-cadherin expression and the methylation status of E-cadherin promoter (P = 0.021). In addition, increased DNMT1 expression was correlated with histologic grade, clinical stage, and a poor prognosis in patients with MEC. CONCLUSIONS: Hypermethylation of CpG sites at the 5' promoter of E-cadherin was a common event associated with E-cadherin expression levels in MEC, suggesting an epigenetically mediated loss of E-cadherin function in these tumors. Increased DNMT1 protein expression may play a critical role in the carcinogenesis and disease progression of MEC.

Extra-cellular signal-regulated ERK-1/ERK-2 pathway activation in human salivary gland mucoepidermoid carcinoma: association to aggressive tumor behavior and tumor cell proliferation.

Information on oncogenetic events accompanying salivary gland mucoepidermoid carcinoma is so far limited. Activation of extracellular signal-regulated kinases ERK-1 and ERK-2 is strongly correlated to cancer. Using an antibody specific for phosphorylated (active) ERK-1/ERK-2, we examined human salivary gland mucoepidermoid carcinoma samples by immunohistochemistry. The comparison in paired tumor and normal tissue samples showed that phosphorylated ERK-1/ERK-2 immunoreactivity was higher in tumor cells as compared to surrounding normal salivary parenchyma. ERK-1/ERK-2 phosphorylation was observed in approximately 39% of mucoepidermoid carcinomas. Those tumors where the ERK-1/ERK-2 pathway was activated had a more aggressive tumor behavior as compared to the group where this pathway was inactive. The association of ERK-1/ERK-2 phosphorylation to a worse prognosis was independent of histological grade. ERK-1/ERK-2 phosphorylation was associated with increased Ki67 and cyclin A indexes, which indicated that ERK-1/ERK-2 pathway activation increased tumor cell proliferation. There was no relationship between ERK-1/ERK-2 phosphorylation and HER-2/neu or p16/INK4a protein expression. In conclusion, ERK-1/ERK-2 pathway is active in salivary gland mucoepidermoid carcinoma and this activation is associated to a more aggressive tumor behavior and a higher proliferative activity. These data suggest that deregulation of ERK-1/ERK-2 pathway contributes to mucoepidermoid carcinoma phenotype and, possibly, represents a target for new anticancer drugs.

[Expression of PTEN protein in oromaxillofacial tumor cell lines].

AIM: To investigate expression of PTEN protein in oromaxillofacial tumor cell lines and to compare the difference of PTEN protein expression between highly metastatic cancer cell lines and their parent cell lines. METHODS: ABC immunohistochemical staining was applied for detecting expression of PTEN protein in 8 kinds of cancer cell lines, including tongue cancer cell lines Tca8113 and HSC-3,oral bottom cancer cell line HSC-2, buccal cancer cell line BcaCD885, mucoepidermoid carcinoma cell line MEC-1, adenoid cystic carcinoma cell line SACC83, highly metastatic tongue cancer cell line Tb-TLP and highly metastatic mucoepidermoid carcinoma cell line M3SP2. RESULTS: Of the 8 kinds of cancer cell lines, 5 kinds of cancer cell lines were positive for PTEN protein expression. PTEN protein was located in cytoplasm around nucleus. 3 kinds of cancer cell lines, BcaCD885, Tb-TLP and M3SP2, lacked expression of PTEN protein. CONCLUSION: Deficiency of PTEN protein expression may play a certain role in cancer cell metastasis.

Immunohistochemical study of the distribution of endogenous biotin and biotin-binding enzymes in ductal structures of salivary gland tumours.

To clarify the pathologic value of endogenous biotin in the salivary gland, we examined in a series of neoplasms of the salivary gland by immunohistochemical staining the distribution of endogenous biotin and of biotin-binding enzymes, namely, acetyl CoA carboxylase (AC), which is a cytosolic enzyme, and pyruvate carboxylase (PC), which is a mitochondrial enzyme. In pleomorphic adenoma, we found biotin and PC in ductal epithelial elements, while AC was found mainly in myoepithelial elements. Carcinoma ex pleomorphic adenoma, adenocarcinoma and mucoepidermoid carcinoma were frequently immunopositive for biotin, PC and AC, while adenoid cystic carcinoma was rarely immunopositive for biotin, PC or AC. These results indicate that endogenous biotin might be associated with the mitochondrial enzyme, which is present at high levels in ductal cells of the salivary gland. However, the neoplastic cells in adenoid cystic carcinoma seemed to have an unusual expression of biotin and related enzymes.

Detection of telomerase activity in oral lesions.

Telomerase is a ribonucleoprotein complex intimately associated with cell immortalization and neoplastic transformation. In almost all types of cancer this enzyme is reactivated and stabilizes telomere length. It may be necessary for continuous cell proliferation. In this study we used a non-radioactive polymerase chain reaction assay to analyze telomerase activity in various tissue specimens taken from the oral cavity. Four of 4 (100%) squamous cell carcinoma cell lines, 28 of 29 (96%) malignant tumors, 10 of 28 (36%) benign lesions, and none of the 14 (0%) oral control tissues possessed telomerase activity. Moreover, 4 of 15 (27%) oral rinses and 3 of 3 (100%) samples of ascites and pleural effusion taken from patients with oral malignancy were telomerase positive. These findings indicate that the evaluation of telomerase activity in tissue and body fluid specimens may provide information useful in the diagnosis of oral malignancy.

Immunohistochemical evaluation of transglutaminase C in tumours of salivary glands.

Transglutaminase C (TGase C), a family of Ca(2+)-dependent enzymes and an essential component in the cross-linking of peptide bonds, has been found to be a marker of epithelial differentiation with a possible role in cellular apoptosis, extracellular matrix stabilisation and Ca2+ binding, thereby having a potential role in tumour growth, differentiation and invasive behaviour. The expression of TGase C was evaluated in normal human salivary glands and their neoplastic lesions which included pleomorphic adenoma (n = 30), Warthin's tumour (n = 5), adenoid cystic carcinoma (n = 10), acinic cell carcinoma (n = 5), mucoepidermoid carcinoma (n = 5) and control tissue specimens of normal oral mucosa and squamous cell carcinoma, using polyclonal antibody, the specificity of which was determined by Western blotting, generated by immunising rabbits with purified transglutaminase. The TGase C was observed in the epithelial cells in the control tissue specimens examined. Pleiomorphic adenoma revealed reaction products in luminal tumour cells, the non-luminal or modified myoepithelial cells and their plasmacytoid variants, squamous metaplastic cells and chondroid cells. Adenoid cystic carcinomas had tumour cells in the luminal cells of tubular and cribriform structures and the acinic cell carcinoma had from low to moderate immunoreactivity in the tumour cell component and a diffuse immunoreactivity in the stroma for TGase C. Mucoepidermoid carcinoma showed no reaction products in the mucous-producing cells, while intermediate and epidermoid cells had immunoreactivity in the cell cytoplasm. As the presence of TGase C in salivary gland tumours was confined to those tumour cells which form the predominant histomorphology in each tumour subtype, it may be suggested that these enzymes may have a potential role in the regulation of cellular function in neoplastic salivary tissues affecting tumour growth, differentiation and neoplastic behaviour.

Over-expression of glutathione S-transferases, DT-diaphorase and an apparently tumour-specific cytosolic class-3 aldehyde dehydrogenase by Warthin tumours and mucoepidermoid carcinomas of the human parotid gland.

Cytosolic class-3 aldehyde dehydrogenase (ALDH-3) may help to protect organisms from certain environmental aldehydes by catalysing their detoxification. Consistent with this notion are the reports that relatively high levels of this enzyme are present in tissues, e.g. stomach mucosa and lung, that are so-called ports of entry for such agents. Further, it is found in human saliva. The present investigation revealed that small amounts of this enzyme are also present in human salivary glands; mean values for ALDH-3 activities (NADP-dependent enzyme-catalysed oxidation of benzaldehyde) in cytosolic fractions prepared from submandibular and parotid glands were 52 (range: 29-92) and 44 (range: 13-73) mIU/g tissue, respectively. Essentially identical or slightly lower levels of this enzyme activity were found in pleomorphic adenomas, an undifferentiated carcinoma, and an adenocystic carcinomas, of the parotid gland. On the other hand, Warthin tumours, and mucoepidermoid carcinomas of the parotid gland exhibited relatively elevated levels of ALDH-3 activity; mean values were 1200 (range: 780-1880) and 810 (range: 580-1200) mIU/g tissue, respectively. The ALDH-3 found in normal salivary glands was, as judged by physical, immunological and kinetic criteria, identical to human stomach mucosa ALDH-3 whereas the ALDH-3 present in Warthin tumours, and mucoepidermoid carcinomas, of the parotid gland appeared to be a subtle variant thereof. Qualitatively paralleling the relatively elevated ALDH-3 levels in mucoepidermoid carcinomas and Warthin tumours were relatively elevated levels of glutathione S-transferase (alpha and pi) and DT-diaphorase. As was the case with ALDH-3 levels, glutathione S-transferase (alpha and pi) and DT-diaphorase levels were not elevated in pleomorphic adenomas. Glutathione S-transferase mu was not detected in the two normal parotid gland samples, or in the single pleomorphic adenoma sample, tested. It was found in the single mucoepidermoid carcinoma sample, and in one of the two Warthin tumour samples tested. Cellular levels of ALDH-3, glutathione S-transferases and/or DT-diaphorase could be useful criteria when the decision to be made is whether a salivary gland tumour is a mucoepidermoid carcinoma. ALDH-3 and glutathione S-transferases are known to catalyse the detoxification of two agents that are used to treat salivary gland tumours, viz. cyclophosphamide and cisplatin, respectively. Thus, elevated levels of these enzymes in the mucoepidermoid carcinomas must account for, or at least contribute to, the relative ineffectiveness of these agents when used to treat this tumour.

Mast cell tryptase is a mitogen for epithelial cells. Stimulation of IL-8 production and intercellular adhesion molecule-1 expression.

Tryptase, a protease unique to the mast cell secretory granule, is released in substantial quantities into the respiratory tract of patients with inflammatory disease of the airways. We have investigated the potential of tryptase to act as a mitogen for bronchial epithelial cells and to stimulate release of IL-8 and expression of ICAM-1. Tryptase was isolated from extracts of human lung tissue using ammonium sulphate precipitation, octyl agarose, and heparin agarose chromatography. Purified tryptase stimulated DNA synthesis in the human epithelial cell line H292, as measured by [3H] thymidine incorporation. Maximal growth was observed after 24 h using 25 mU/ml of tryptase (where 1 micron is defined as that which can hydrolyze 1 mumol of the peptide substrate N-alpha-benzoyl-DL-arginine p-nitroanilide hydrochloride per minute at 25 degrees C), a concentration that is likely to be achieved in vivo. Inhibitors of tryptase activity, including leupeptin and benzamidine hydrochloride, significantly decreased tryptase-induced stimulation of DNA synthesis, indicating the requirement for an active catalytic site. Tryptase stimulated a catalytic site-dependent release of IL-8 from epithelial cells after 24 h, and this was associated with up-regulation of ICAM-1 expression, as revealed by FACS analysis. Tryptase may play a critical role in epithelial repair and in the recruitment of granulocytes following mast cell activation.