RESUMEN
The transcription factor nuclear factor (NF)-kappaB regulates the expression of genes that can influence cell proliferation and death. Here we analyze the contribution of NF-kappaB to the regulation of epithelial cell turnover in the colon. Immunohistochemical, immunoblot, and DNA binding analyses indicate that NF-kappaB complexes change as colonocytes mature: p65-p50 complexes predominate in proliferating epithelial cells of the colon, whereas the p50-p50 dimer is prevalent in mature epithelial cells. NF-kappaB1 (p50) knockout mice were used to study the role of NF-kappaB in regulating epithelial cell turnover. Knockout animals lacked detectable NF-kappaB DNA binding activity in isolated epithelial cells and had significantly longer crypts with a more extensive proliferative zone than their wild-type counterparts (as determined by proliferating cell nuclear antigen staining and in vivo bromodeoxyuridine labeling). Gene expression profiling reveals that the NF-kappaB1 knockout mice express the potentially growth-enhancing tumor necrosis factor (TNF)-alpha and nerve growth factor-alpha genes at elevated levels, with in situ hybridization localizing some of the TNF-alpha expression to epithelial cells. TNF-alpha is NF-kappaB regulated, and its upregulation in NF-kappaB1 knockouts may result from an alleviation of p50-p50 repression. NF-kappaB complexes may therefore influence cell proliferation in the colon through their ability to selectively activate and/or repress gene expression.
Asunto(s)
Colon/anatomía & histología , Células Epiteliales/fisiología , Mucosa Intestinal/fisiología , FN-kappa B/fisiología , Fosfatasa Alcalina/metabolismo , Animales , Electroforesis en Gel de Poliacrilamida , Expresión Génica , Etiquetado Corte-Fin in Situ , Ratones , Ratones Noqueados , ARN Mensajero/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
The transcription factor nuclear factor-kappaB (NF-kappaB) regulates genes that can influence cell proliferation, apoptosis, and inflammatory responses. Since these events can contribute to carcinogenesis, we examined the expression of NF-kappaB inhibitory proteins (IkappaBs) in normal and transformed colonic epithelial cells. Immunohistochemical analysis of the mouse colon revealed a high level of IkappaBbeta expression in epithelial cells relative to the rest of the tissue, whereas IkappaBalpha was found primarily in cells of the lamina propria. Mouse colon tumors showed a similar cell-specific staining pattern. Immunoblot analysis of IkappaBbeta from mouse colonocytes and the human HT-29 colon cancer cell line indicated that most of the IkappaBbeta in these cells was similar to the C-terminal-truncated IkappaBbeta2 isoform. Cell fractionation studies were consistent with IkappaBbeta being a major regulator of p65-p50 NF-kappaB complexes in HT-29 cells. Interestingly, two larger proteins specifically recognized by IkappaBbeta antibodies (p106 and p112) were found in HT-29 cells and in colon tissue of carcinogen-exposed mice. The p106 and p112 proteins bound to NF-kappaB, and their levels changed during the transient interleukin-1beta activation of NF-kappaB in HT-29 cells. Evidence was obtained indicating that p106 and p112 are stably ubiquitinated forms of IkappaBbeta. We propose that deficiencies in the proteasomal degradation of IkappaBbeta lead to p106 and p112 accumulation, which in turn alter NF-kappaB regulation in colon cancer cells.
Asunto(s)
Línea Celular Transformada , Colon/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas I-kappa B , Animales , Células Epiteliales/metabolismo , Células HT29 , Humanos , RatonesRESUMEN
A toxic dose of acetaminophen (APAP) reduces the activity of NF-kappaB in mouse liver. NF-kappaB inactivation may be important for APAP toxicity, as this transcription factor can play a central role in maintaining hepatic viability. We recently reported that APAP likewise inhibits serum growth factor activation of NF-kappaB in a mouse hepatoma cell line (Hepa 1-6 cells). Here we present evidence that APAP's antioxidant activity may be involved in this NF-kappaB inhibition in Hepa 1-6 cells. Like the antioxidants N-acetylcysteine (NAC) and pyrrolidinedithiocarbamate (PDTC), APAP was found to suppress the H(2)O(2)-induced oxidation of an intracellular reactive oxygen species probe (dihydrodichlorofluorescein) in Hepa 1-6 cells. Treatment of Hepa 1-6 cells with H(2)O(2) was sufficient for NF-kappaB activation and IkappaBalpha degradation, and APAP was able to block both of these events. The APAP inhibition of NF-kappaB activation by serum growth factors may also be due to APAP's antioxidant activity, as the antioxidants NAC and PDTC likewise inhibit this activation. The potential role of NF-kappaB and oxidant-based growth factor signal transduction in APAP toxicity is discussed.
Asunto(s)
Acetaminofén/farmacología , Analgésicos no Narcóticos/farmacología , Proteínas I-kappa B , FN-kappa B/metabolismo , Acetilcisteína/farmacología , Animales , Antioxidantes/farmacología , Western Blotting , Carcinoma Hepatocelular/metabolismo , Sondas de ADN/química , Proteínas de Unión al ADN/metabolismo , Fluoresceínas/farmacología , Peróxido de Hidrógeno/farmacología , Ratones , Inhibidor NF-kappaB alfa , FN-kappa B/antagonistas & inhibidores , Oxidación-Reducción/efectos de los fármacos , Pirrolidinas/farmacología , Transducción de Señal/efectos de los fármacos , Tiocarbamatos/farmacología , Células Tumorales CultivadasRESUMEN
BACKGROUND & AIMS: The transcription factor nuclear factor-kappaB (NF-kappaB) plays a central role in regulating immune and inflammatory responses. Because butyrate deficiency has been associated with inflammatory bowel disease, we examined the effect of butyrate on NF-kappaB activity in the human HT-29 colonic cell line. METHODS: The influence of butyrate (4 mmol/L) on NF-kappaB activity was determined using the gel mobility shift assay. The effect of butyrate on the expression of NF-kappaB subunits and inhibitory proteins was determined by immunoblotting. NF-kappaB-regulated gene expression was assayed by primer extension of intercellular adhesion molecule 1 and Mn superoxide dismutase messenger RNA, and by analysis of a transfected luciferase reporter. RESULTS: Exposure of HT-29 cells to butyrate eliminated their constitutive NF-kappaB, p50 dimer activity. This inhibition corresponded with a reduction in p50 nuclear localization, without a reduction in expression. Butyrate also selectively modulated activation of NF-kappaB, suppressing its activation by tumor necrosis factor alpha and phorbol ester more than 10-fold, without affecting the activity induced by interleukin (IL)-1beta. Butyrate did, however, enhance formation of the stronger p65-p50 transcriptional activator in IL-1beta-stimulated cells. The changes in NF-kappaB activation did not correlate with changes in IkappaBalpha levels. Gene expression reflected DNA binding. The influence of butyrate on NF-kappaB may result in part from its ability to inhibit deacetylases because the specific deacetylase inhibitor trichostatin A has a similar effect. CONCLUSIONS: These findings suggest that the influences of butyrate on colonic inflammatory responses may result in part from its influence on NF-kappaB activation. This activity of butyrate apparently involves its ability to inhibit deacetylases.
Asunto(s)
Butiratos/farmacología , Colon/metabolismo , Mucosa Intestinal/metabolismo , FN-kappa B/metabolismo , Acetilación , Línea Celular , Colon/citología , Citocinas/fisiología , Dimerización , Regulación de la Expresión Génica/fisiología , Humanos , Mucosa Intestinal/citología , Mucosa Intestinal/efectos de los fármacosRESUMEN
The nonsteroidal antiinflammatory drugs (NSAIDs) indomethacin and salicylic acid and the short chain fatty acid butyrate are effective colon cancer chemopreventive agents that increase reactive oxygen species (ROS) generation in colon cancer cells. Here we demonstrate that these agents sensitize the normally resistant human HT-29 colon cancer cell line to apoptosis induced by TNF-alpha or a Fas ligating antibody. The role of ROS in this sensitization is supported by the finding that direct exposure of the cells to H2O2 is sufficient for sensitization. Neither TNF-alpha nor Fas ligation alter basal or chemopreventive agent-activated ROS generation, suggesting that the death ligands and chemopreventive agents act in a complementary fashion. The dual chemopreventive agent/death ligand treatments do not increase Fas, TNF receptor 1, Bak or c-myc expression (although salicylic acid moderately induces of Fas expression). Cell death does correlate with alterations in NF-kappa B activity: the NSAIDs, butyrate and H2O2 enhance c-Rel complex formation by TNF-alpha and provide an overall enhancement of NF-kappa B activation by Fas. The antioxidant N-acetylcysteine (NAC) blocks cell death and NF-kappa B activation induced by Fas ligation, suggesting a potential role for NF-kappa B in Fas-induced apoptosis in these cells. The effects of NAC on TNF-alpha-induced cell death are more complex, with NAC being marginally protective and itself enhancing the formation of c-Rel containing complexes at higher concentrations (25 mM). The influence of NSAIDs and butyrate on ROS generation and death ligand sensitivity may be relevant to their ability to suppress colon carcinogenesis.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Butiratos/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/metabolismo , Glicoproteínas de Membrana/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Acetilcisteína/farmacología , Anticuerpos/farmacología , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Neoplasias Colorrectales/prevención & control , Proteína Ligando Fas , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Peróxido de Hidrógeno/farmacología , Indometacina/farmacología , Glicoproteínas de Membrana/antagonistas & inhibidores , FN-kappa B/metabolismo , Ácido Salicílico/farmacología , Células Tumorales CultivadasRESUMEN
Nonsteroidal anti-inflammatory drugs (NSAIDs) and short-chain fatty acids are effective suppressors of colorectal cancer that may work in part by accentuating apoptosis of transformed cells. Since reactive oxygen species (ROS) can play an important role in regulating cell growth and cell death, we determined the effect of the NSAIDs indomethacin and salicylic acid, and the short-chain fatty acids butyrate and propionate on ROS metabolism in the HT-29 human colorectal carcinoma cell line. We find that all of these agents increase cellular peroxide generation, as determined by two independent assays. Arachidonic acid was also found to increase ROS generation, and could synergize with indomethacin in this reaction. The NSAIDs and short-chain fatty acids under study all possess a carboxyl group, and this carboxyl group is essential for salicylic acid's ability to increase ROS production. Although the two NSAIDs examined increase peroxide production, they were both found to suppress superoxide generation by vitamin K3 (menadione), a redox cycling compound similar to those found in the colon. The short-chain fatty acids did not have this activity. The ability of these NSAIDs and short-chain fatty acids to alter cellular ROS metabolism may contribute to their chemopreventive activity.