Nitric oxide induces apoptosis in a human colonic epithelial cell line, T84

Chronic inflammation is associated with inducible nitric oxide synthase expression in infiltrating and resident cells (epithelia, neurons) and an exaggerated release of nitric oxide. NO can induce apoptosis in macrophages and tumour cell lines. We investigated whether NO induced cell death in an epithelial (T84) cell fine via apoptosis. Culture T84 cells were exposed to a bolus of NO (40 or 80 μM) dissolved in Hank's balanced salt solution (HBSS) supplemented with 10% fetal calf serum (FCS). After incubation for 4 h at 37°C in 5% CO2, cells were either stained for DNA fragmentation with the TdT-mediated dUTP–biotin nick end labelling (TUNEL) method, or cytosolic DNA fragments quantified by a cell death detection ELISA assay. Nitric oxide induced apoptosis in a dose-dependent manner which preceded frank cell death (failure to exclude Trypan blue). These data suggest that epithelial cell death may be NO dependent and via apoptosis, in states of gut inflammation.


Introduction Materials and Methods
Most animal cells display a physiological form Cell culture.. T84 cells were obtained from the of cell death, which is influenced by the extra-American Type Culture Collection (Rockville, MD, cellular microenvironment. The mode of this USA) at passage 52 on receipt. Cells were grown cell death is often associated with characteristic in a 1:1 mixture of Ham's F-12 medium and Dulmorphological, biochemical and molecular becco's modified Eagle's (high glucose) medium changes. Cells undergoing apoptosis demon-supplemented with 15mM Na-N-2-hydroxystrate typical nuclear and cytoplasmic condensaethylpiperazine-N-2-ethanesulfonic acid buffer tion followed by cell fragmentation. From the (pH 7.4), i mM -glutamine, 40t.tg/ml penicillin, molecular standpoint, apoptosis is associated 90btg/ml streptomycin, and 5% FCS. Medium with the activation of nucleases that degrade the NaHCO3 concentration was 1.2 g/l, and cell culchromosomal DNA into oligonucleosomal fragtures were maintained in a humidified 5% CO2 ments 180-200 bp in length. 2 While apoptosis incubator at 37C. Harvested cells were plated in is historically not linked to states of inflamma-6-well tissue culture plates and allowed to grow tion, it has become increasingly apparent that to confluence over 24h before use. inflammatory mediators, e.g., oxidants and cytokin s can in uc 34 e, d e apoptosis. Nitric oxide is a Preparation of NO solution: NO solution was major secretory product of mammalian cells, prepared as described previously. 2 Briefly, in a with critical functions in homeostasis and host chemical hood, HBSS (GIBCO BRL, Gaithersdefence. 5 Chronic gut inflammation is associated burg, MD, USA) supplemented with 10% FCS with enhanced production of NO while inhibiwas deoxygenated with 100% N2 for 30 min. 67 tion of NOS ameliorates gut inflammation.' NO Subsequently, a 10% NO-90% N2 gas mixture has been demonstrated to induce apoptosis in was bubbled into the flask for at least 30 min. macrophages and tumour cells. 8'9 As the epithe-The final concentration of this stock solution of lium is a site of iNOS expression and marked NO was 160t.tM, as previously determined by 12 cell death in gut inflammation, m' we hypothechemiluminescence. Media pH was not altered sized that NO may induce apoptosis in cultured by this procedure. epithelial cells.
Cell viability: Aliquots of cells from control and NO-treated samples were examined for viability

Results and Discussion
Detection of DNA fragmentation: Comparison of the cleavage of DNA into oligonucleosomal fragments revealed that DNA fragmentation increased in a dose-dependent manner in response to NO in the culture medium (Fig. 1).
These results were validated with the in situ apoptosis detection kit (TUNEL). Cell viability by Trypan blue dye exclusion was negative for cells exposed to NO at concentrations up to 801.tM, indicating that DNA fragmentation preceded cell death. In other experiments, we have observed that cells exposed to concentrations of NO of 1201.tM and higher did not survive the 4h incubation period of this protocol, based on the failure of these cells to exclude Trypan blue and/or their detachment from culture dish. NO has been shown to induce apoptosis in macrophages and tumor cells. 8'9 In chronic gut inflammation NO synthesis is augmented via iNOS expression in both experimental animals and humans. 6'm'4 Our data indicate that, during gut inflammation, the overproduction of NO by iNOS in epithelia and infiltrating leukocytes may result in epithelial cell death via apoptosis.
Exaggerated NO synthesis in enterocytes of an inflammed mucosa may be regarded as a mechanism to establish a chemical barrier which is not conducive for translocation by intestinal flora. In so doing, the enterocyte is at risk of DNA damage. Apoptosis may be a defence against the survival of transformed cells. These effects of nitric oxide on epithelial cell death were observed with a dose of nitric oxide which must be considered transient, as it is oxidized within minutes under normoxic conditions to nitrite/nitrate, which alone were ineffective on cell death. The cascade of mechanisms initiated by this brief exposure to nitric oxide which culminates in apoptosis, remains to be investigated. Inflammatory bowel disease is a predisposing risk factor for colon cancer and we speculate that this may result from a failure to initiate programmed cell death in cells transformed by oxidant stress, excess nitric oxide or related species.