Cleavage of the urokinase receptor (uPAR) on oral cancer cells: regulation by transforming growth factor - ß1 (TGF-ß1) and potential effects on migration and invasion.

BACKGROUND: Urokinase plasminogen activator (uPA) receptor (uPAR) is up-regulated at the invasive tumour front of human oral squamous cell carcinoma (OSCC), indicating a role for uPAR in tumour progression. We previously observed elevated expression of uPAR at the tumour-stroma interface in a mouse model for OSCC, which was associated with increased proteolytic activity. The tumour microenvironment regulated uPAR expression, as well as its glycosylation and cleavage. Both full-length- and cleaved uPAR (uPAR (II-III)) are involved in highly regulated processes such as cell signalling, proliferation, migration, stem cell mobilization and invasion. The aim of the current study was to analyse tumour associated factors and their effect on uPAR cleavage, and the potential implications for cell proliferation, migration and invasion. METHODS: Mouse uPAR was stably overexpressed in the mouse OSCC cell line AT84. The ratio of full-length versus cleaved uPAR as analysed by Western blotting and its regulation was assessed by addition of different protease inhibitors and transforming growth factor - ß1 (TGF-ß1). The role of uPAR cleavage in cell proliferation and migration was analysed using real-time cell analysis and invasion was assessed using the myoma invasion model. RESULTS: We found that when uPAR was overexpressed a proportion of the receptor was cleaved, thus the cells presented both full-length uPAR and uPAR (II-III). Cleavage was mainly performed by serine proteases and urokinase plasminogen activator (uPA) in particular. When the OSCC cells were stimulated with TGF-ß1, the production of the uPA inhibitor PAI-1 was increased, resulting in a reduction of uPAR cleavage. By inhibiting cleavage of uPAR, cell migration was reduced, and by inhibiting uPA activity, invasion was reduced. We could also show that medium containing soluble uPAR (suPAR), and cleaved soluble uPAR (suPAR (II-III)), induced migration in OSCC cells with low endogenous levels of uPAR. CONCLUSIONS: These results show that soluble factors in the tumour microenvironment, such as TGF-ß1, PAI-1 and uPA, can influence the ratio of full length and uPAR (II-III) and thereby potentially effect cell migration and invasion. Resolving how uPAR cleavage is controlled is therefore vital for understanding how OSCC progresses and potentially provides new targets for therapy.

Preserved flow-mediated dilation in adults with cyanotic congenital heart disease.

Adults with cyanotic congenital heart disease (CCHD) have been shown to have endothelial dysfunction in the forearm resistance vessels as assessed with venous occlusion plethysmography. Whether these abnormalities are confined to the microvasculature or reflect generalized endothelial dysfunction remain unknown. We used high-resolution ultrasound to compare flow responses and endothelial-dependent flow-mediated dilation (FMD) in the brachial artery of 13 adult patients with CCHD and 14 healthy controls. High-dose vitamin C was infused to evaluate the possible role of reactive oxygen species on endothelial vasomotor function. FMD was measured both prior to and after vitamin C infusion. Sublingual glyceryl nitrate was given to assess endothelium-independent responses. FMD did not differ among patients with CCHD and controls either before (6.2 +/- 4.1, 5.1 +/- 2.6%, p = 0.44) or after (5.1 +/- 2.8, 5.2 +/- 3.1%, p = 0.90) vitamin C infusion. Endothelium-independent vasodilatation was similar in both groups (14.3 +/- 3.7, 13.2 +/- 4.4%). There were no differences in baseline flow or in measures of reactive hyperemia. Adults with CCHD appear to have preserved endothelial function in their conduit arteries. This suggests that these patients are not at an increased risk of premature atherosclerotic cardiovascular events.

Increased resistance towards oxidative stress accompanies enhancement of metastatic potential obtained by repeated in vivo passage of colon carcinoma cells in syngeneic rats.

The colon carcinoma cell line CC531 is metastatic to liver after splenic injection in syngeneic rats. After repeated in vivo passages, a subline was selected that produced liver metastases at a considerably higher rate than the original cell line. These cells were characterized by increased intracellular glutathione, proliferation and ability to restore glutathione after exposure to oxidative stress, thus indicating an elevated resistance to oxidative stress. Furthermore, the increased metastatic ability was also accompanied by increased proliferation rate, adhesion to extracellular matrix proteins and endothelial cells, and secretion of a 60 kD matrix metalloproteinase. When cultured in vitro for a prolonged time (more than 30 trypsinizations), the cells showed a reduced in vivo metastatic ability, reduced secretion of three metalloproteinases including the 60 kD proteinase, and reduced intracellular glutathione. These results indicate that metastatic ability can be influenced through several adaptive mechanisms, and that the cell's ability to resist oxidative stress and maintain intracellular glutathione are of central importance.

Butyrate reduces liver metastasis of rat colon carcinoma cells in vivo and resistance to oxidative stress in vitro.

Injection of the rat colon carcinoma cell line CC531 into spleen of syngeneic rats results in considerable amounts of liver metastases within 14 days. We investigated whether preincubation of the cells with butyrate reduced their metastatic ability in vivo and whether this was accompanied by reduction in related properties such as secretion of metalloproteinases and their ability to withstand oxidative stress. Butyrate incubation reduced cell growth rate and initiated apoptosis in a dose- and time-related manner, but proliferation was retrieved when cultivation was continued in medium without butyrate. Splenic injection of butyrate treated, proliferating cells resulted in significantly reduced amounts of tumor mass compared to untreated cells. The butyrate treated cells were more susceptible to oxidative stress than control cells, as demonstrated by increased number of apoptotic cells and reduced cell growth after exposure to menadione. A reduction in cellular glutathione was found after prolonged incubation with butyrate. Butyrate appeared not to alter the secretion of active metalloproteinases from the cells although an apparent increase in proforms was demonstrated. Neither did butyrate alter the synthesis of metalloproteinase inhibitors. Lastly, a reduced adhesion of the tumor cells to collagen coated matrix was found after butyrate treatment. Thus, the inhibitory effects of butyrate on tumor malignancy are caused by a diversity of mechanisms.

Nitric oxide exposure of CC531 rat colon carcinoma cells induces gamma-glutamyltransferase which may counteract glutathione depletion and cell death.

Gamma-glutamyltransferase (GGT) has a central role in glutathione homeostasis by initiating the breakdown of extracellular GSH. We investigated in the present study whether nitric oxide exposure of CC531 rat colon carcinoma cells modulates GGT and how the activity of the enzyme affects the level of intracellular GSH. The data show that GGT activity was induced in a dose-related manner by two NO-donors (spermineNONOate and nitrosoglutathione) and that antioxidants partly inhibited the induction. SpermineNONOate lowered intracellular GSH and induced apoptosis. Cultivating the cells in cystine-depleted medium also resulted in a 50% lowering of GSH, but this was avoided when GSH was added to the medium. This effect was mediated by the activity of GGT and shown after inhibiting GGT activity with acivicin and cyst(e)ine transporters with alanine and homocysteic acid. This shows that the cells benefit from GGT in maintaining the intracellular GSH level. Cells with induced GGT activity obtained after NO incubation showed a higher uptake rate of cysteine (2-fold), measured by incubating the cells with 5S-radiolabeled GSH. The enzyme was also induced by interferon-gamma and tumor necrosis factor-alpha, but this induction was not connected to activation of the endogenous nitric oxide synthase, as the addition of aminoguanidine, a NO-synthase inhibitor, did not affect the induction. The present study shows that the activity of GGT is upregulated by NO-donors and that the colon carcinoma cells, when cultivated in cystine-depleted medium, benefit from the enzyme in maintaining the intracellular level of GSH. Thus, the enzyme will add to the protective measures of the tumor cells during nitrosative stress.

The expression of gamma-glutamyltransferase in rat colon carcinoma cells is distinctly regulated during differentiation and oxidative stress.

Gamma glutamyltransferase (GGT) is a plasma membrane bound enzyme that initiates the degradation of glutathione. The presence of several promoters in the rat GGT gene indicates strict control and regulation of its expression. The aim of this study was to investigate whether the GGT gene was regulated differently after butyrate-induced differentiation and oxidative stress exposure of rat colon carcinoma cells and whether the regulation was related to the glutathione level. The activity of GGT was upregulated in a time-and-dose dependent manner after both butyrate and menadione incubations. The presence of antioxidants blocked the menadione but not the butyrate mediated induction of the enzyme. The level of intracellular glutathione was reduced after menadione, but not after butyrate incubations. Depletion of glutathione alone did not alter GGT activity. Reactive oxygen species (ROS) were not produced after incubations with butyrate, while menadione incubations produced ROS. The multiple GGT mRNA transcripts (mRNA I-V) that originate from the five distinct promoters were all present in the cell line. Incubations with butyrate enhanced mRNA II and IV transcripts whereas a reduction in mRNA IV-1 was noted during menadione incubations. The level of total GGT mRNA (I-V) was not altered when related to the amount of total beta-actin mRNA. We conclude that GGT activity can be upregulated by at least two distinct mechanisms during differentiation and oxidative stress. Apparently, the regulation of the enzyme is not directly linked to the intracellular level of glutathione.