[PubMed] [Google Scholar] 28. butyric acid-induced apoptosis at any dose examined. Although cytotoxic anti-Fas antibody affected butyric acid-induced apoptosis, a synergistic effect was not seen. Time-dependent activation of caspase-8 and Tetrodotoxin -9 was acknowledged in butyric acid- as well as Fas-mediated apoptosis. IETD-CHO and LEHD-CHO, specific inhibitors of caspase-8 and -9, respectively, completely clogged Fas-mediated apoptosis and partially prevented butyric acid-induced apoptosis. These results Tetrodotoxin suggest that the Fas-FasL connection is not involved in butyric acid-induced apoptosis and that caspase-8 and -9-dependent apoptosis plays an important part in butyric acid-induced apoptosis, as well as Fas-induced apoptosis. Butyric acid, one of the short chain fatty acids, suppresses the proliferation of a variety of malignancy cell lines in vitro (14, 20). Our earlier study (16) shown that short-chain fatty acids, especially volatile fatty acids present in the tradition filtrates of for 5 min, and washed twice with ice-cold PBS. The cells were resuspended in 400 l of hypotonic lysis buffer (0.2% Triton X-100, 10 mM Tris, 1 mM EDTA [pH 8.0]) and Tetrodotoxin centrifuged for 15 min at 13,800 (26). Half the supernatants, which contained small DNA fragments, as well as the pellet comprising large pieces of DNA and cell debris, were utilized for the diphenylamine (DPA) assay (observe below). DNA fragmentation assay. The DPA reaction was performed by the method of Paradones et al. (29). Perchloric acid (0.5 M) was added to the other half of the DNA (resuspended with 200 l of hypotonic lysis buffer) and to the pellets containing uncut the supernatants containing DNA fragments, and then 2 quantities of a solution containing 0.088 M DPA, 98% (vol/vol) glacial acetic acid, 1.5% (vol/vol) sulfuric acid, and a 0.5% (vol/vol) concentration of 1 1.6% acetaldehyde answer were added. The samples were stored at 4C for 48 h. The colorimetric reaction was quantified spectrophotometrically at 575 nm having a model UV-160A UV spectrophotometer (Shimazu Co. Ltd., Tokyo, Japan). The percentage of fragmentation was determined as the percentage of DNA in the supernatants to the total DNA. Circulation cytometry analysis. PBMC (4 106) and Jurkat cells (1 106) in 1 ml of medium were cultured for the indicated occasions with or without 5 mM butyric acid. To measure Fas manifestation, cells (106) were then harvested and stained with fluorescein isothiocyanate-labeled anti-human Fas MAb (clone DX2) or with an isotype control (mouse IgG1) (Becton Dickinson) for 30 min at 4C. After washing in PBS, the samples were analyzed having a FACScan apparatus within 1 h. Data from 106 cells were analyzed for each sample. Western blotting. Cells were lysed in lysis buffer (10 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, 1 mM EGTA, 0.1 mM phenylmethylsulfonyl fluoride, 8 g of aprotinin per ml, 2 g of leupeptin per ml) and centrifuged at 14,000 for 10 min at 4C. The supernatant was collected and the amount of protein was measured using the Bio-Rad (Hercules, Calif.) protein assay. Equal amounts (25 g) of protein from each sample were separated by sodium dodecyl sulfateC12.5% polyacrylamide gel electrophoresis and transferred to a polyvinylfluoride membrane (Millipore, Bedford, Mass.). Western blots were probed with mouse anti-human Fas or FasL MAbs, or with their isotype settings (mouse IgG1) from Transduction Laboratories (Lexington, Ky.). Main antibodies were detected using a goat-anti mouse horseradish peroxidase-conjugated secondary antibody (Amersham, Little Chalfont, United Kingdom). Detection of chemiluminescence was performed with an ECL Western blot detection kit (Amersham), according to the supplier’s recommendations. Measurement of caspase protease activity. After incubation of cells (106 per well) in 24-well cells tradition plates for the indicated occasions with 5 mM butyric acid or 10 ng of Tetrodotoxin cytotoxic anti-Fas MAb (CH-11) per ml, all the cells were collected, washed as explained above, and the caspase-8 and -9 activities were measured using a caspase fluorometric protease assay kit (MBL Co.). Levels of released 7-amino-4-trifluoromethylcoumarin (AFC) were measured Tetrodotoxin having a BioLumin 960 spectrofluorometer (Molecular Dynamics Japan, Tokyo, Japan) with excitation at 400 nm and emission at 505 nm. The results are indicated as the mean SEM of three different experiments with triplicate cultures. Ideals significantly different from the related bad control without stimulants, or the related inhibitor-free anti-Fas antibody or butyric acid ideals at 0.05 are indicated. Inhibition of caspase-8 with IETD-cleaving activity and of caspase-9 with LEHD-cleaving activity was accomplished using caspase-8 inhibitor Ac-IETD-CHO and caspase-9 inhibitor Ac-LEHD-CHO (Peptide Institute, Inc., Mouse monoclonal to PRKDC Osaka, Japan), respectively, given 1 h before the addition of butyric acid or anti-Fas antibody. Statistics. Multiple-group comparisons were made using a one-way analysis of variance followed by post hoc intergroup assessment from the Bonferroni-Dunn test. Where appropriate, Student’s test was used to compare two groups. RESULTS Manifestation of Fas in Jurkat and PBMC-T cells after butyric acid treatment. To test whether the Fas-FasL.