Initiation of human colon cancer cell proliferation by trypsin acting at protease-activated receptor-2

The protease-activated receptor-2 (PAR-2) is a G protein-coupled receptor that is cleaved and activated by trypsin. We investigated the expression of PAR-2 and the role of trypsin in cell proliferation in human colon cancer cell lines. A total of 10 cell lines were tested for expression of PAR-2 mRNA by Northern blot and RT-PCR. PAR-2 protein was detected by immunofluorescence. Trypsin and the peptide agonist SLIGKV (AP2) were tested for their ability to induce calcium mobilization and to promote cell proliferation on serum-deprived cells. PAR-2 mRNA was detected by Northern blot analysis in 6 out of 10 cell lines [HT-29, Cl.19A, Caco-2, SW480, HCT-8 and T84]. Other cell lines expressed low levels of transcripts, which were detected only by RT-PCR. Further results were obtained with HT-29 cells: (1) PAR-2 protein is expressed at the cell surface; (2) an increase in intracellular calcium concentration was observed upon trypsin (1–100 nM) or AP2 (10–100 μM) challenges; (3) cells grown in serum-deprived media supplemented with trypsin (0.1–1 nM) or AP2 (1–300 μM) exhibited important mitogenic responses (3-fold increase of cell number). Proliferative effects of trypsin or AP2 were also observed in other cell lines expressing PAR-2. These data show that subnanomolar concentrations of trypsin, acting at PAR-2, promoted the proliferation of human colon cancer cells. The results of this study indicate that trypsin could be considered as a growth factor and unravel a new mechanism whereby serine proteases control colon tumours. © 2001 Cancer Research Campaign http://www.bjcancer.co

Mast cells and biogenic amines in radiation-induced pulmonary fibrosis

Sprague-Dawley rats were exposed to a single X-ray dose of 30 Gy over the lungs and examined at 1-wk intervals during the following 3 to 8 wk. Mast cells were counted after specific staining with toluidine blue at a low pH and the mast-cell amines, histamine (Hi) and serotonin (5-HT), were measured using high-performance liquid chromatography. Irradiation induced pneumonitis followed by pulmonary mast-cell hyperplasia and progressive fibrosis 4 to 8 wk after irradiation. By week 4, immature-looking mast cells with a few granules started to appear, followed by a gradual increase in mast cells that reached very high levels after 8 wk, up to 40 to 200 times the normal. The pulmonary Hi and 5-HT content increased concomitantly from 6 and 1 micrograms/g to a maximum of 200 and 18 micrograms/g, respectively. These high levels of amine content and mast-cell densities greatly exceed those of any normal tissue. There was a strong correlation between the Hi and 5-HT content in both normal (r = 0.87) and irradiated (r = 0.93) lung tissue, as well as between the mast-cell density and amine content after irradiation (r = 0.86), thereby indicating that both amines derived from mast cells. The Hi/5-HT quotients were much lower in both normal and irradiated lung tissue (5 and 9, respectively) than in other tissues where these amines are stored in mast cells, or in isolated peritoneal mast cells (43). This relatively higher 5-HT content in pulmonary mast cells suggests that this amine performs a specific function in the lung