amanitins and Adenocarcinoma

Pretreatment of HT29-D4 epithelial adenocarcinoma colic cells with des-IGF-1 upregulated TNF alpha-mediated activation of IL-8 expression at different levels (protein, mRNA, and hnRNA). RNA transcription but not RNA stabilization was found to be involved. In this cell line, cooperation of NF-kappa B with other factors appeared essential for IL-8 expression. Indeed, TNF alpha-induced NF-kappa B translocation was not sufficient to support enhancement of the transcription and des-IGF-1 did not promote but partly inhibited both the TNF alpha-induced NF-kappa B activation and I kappa B alpha degradation through a PI-3K-dependent pathway. A CCAAT/enhancer binding protein (C/EBP) site located on the IL-8 gene enhancer cooperated with a NF-kappa B binding site and led to the upregulation of IL-8 expression. Binding of C/EBP alpha to this sequence disappeared in IGF-1 treated cells. This event may be important for the cross-talk between IGF-1- and TNF alpha-mediated pathways leading to the control of inflammatory processes and the decision concerning apoptosis or cell survival.

Topics: Adenocarcinoma; Amanitins; Binding Sites; CCAAT-Enhancer-Binding Protein-alpha; CCAAT-Enhancer-Binding Proteins; Colonic Neoplasms; Down-Regulation; Humans; I-kappa B Proteins; Insulin-Like Growth Factor I; Interleukin-8; Kinetics; NF-kappa B; NF-KappaB Inhibitor alpha; Peptide Fragments; Phosphatidylinositol 3-Kinases; RNA, Messenger; Transcription Factor AP-1; Transcriptional Activation; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Up-Regulation

The cystic fibrosis transmembrane conductance regulator (CFTR) was studied in HT-29 human colonic carcinoma cells with the aim of assessing possible mechanisms of up-regulation of its expression. CFTR was identified and quantified in total cell extracts by Western immunoblots using a monoclonal anti-CFTR antibody and was functionally assessed by tracer Cl-efflux from intact cells. It was found that various stimuli that lead to a sustained (greater than or equal to 8 h) elevation of intracellular cyclic AMP elicited a marked and specific increase in CFTR expression in cell membranes and concomitant activation of Cl- secretion. Further activation of Cl- secretion was obtained by additional short term activation by cyclic AMP analogues or cyclic AMP-inducing agents. Blockers of transcription or translation largely depressed the cAMP-mediated induction of CFTR levels and associated function, indicating that the inductive phenomenon was at the transcriptional level. The results imply the involvement of putative cyclic AMP responsive (and related) elements that are present in the CFTR gene promoter and that are known to modulate eukaryotic gene expression. Activation of these elements by various stimuli might provide pharmacological tools for up-regulation of CFTR expression at both biochemical and physiological levels.

Topics: Adenocarcinoma; Amanitins; Blotting, Western; Chlorides; Colforsin; Cyclic AMP; Cycloheximide; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Dactinomycin; DNA; Humans; Membrane Proteins; Promoter Regions, Genetic; Protein Biosynthesis; Tetradecanoylphorbol Acetate; Transcription, Genetic; Tumor Cells, Cultured; Up-Regulation

The C-24 oxidation pathway plays a major role in the degradation of vitamin D metabolites in kidney and other target tissues. The aim of the present study was to establish an intestinal cell culture system to study the mechanisms regulating the vitamin D catabolic pathway. 25-Hydroxyvitamin D3-24-hydroxylase (24-hydroxylase), the first enzyme in the catabolic sequence, was examined in Caco-2 cells, a human colon adenocarcinoma cell line which exhibits differentiated functions of absorbing intestinal epithelial cells. While untreated Caco-2 cells did not exhibit 24-hydroxylase activity, significant catabolic activity was induced by prior treatment of cell monolayers with 1,25-dihydroxyvitamin D3(1,25-(OH)2D3). Induced 24-hydroxylase D3 (25OHD3) and 1,25-(OH)2D3 was detected 6 h after treatment of cells with 10(-8)M 1,25-(OH)2D3, peaked at 16 h, and decreased thereafter. Treatment of cells with 10(-7) M 1,25-(OH)2D3 elicited a maximal 24-hydroxylase response. Comparable time courses of induction by 1,25-(OH)2D3 and 1,25-(OH)2D3-dose response curves were observed in cultured human skin fibroblasts and Caco-2 cells. 25OHD3 was not as good an inducer of the vitamin D catabolic pathway in Caco-2 cells as 1,25-(OH)2D3. Induction of 24-hydroxylase activity by 1,25-(OH)2D3 was inhibited by pretreatment of Caco-2 cells with either actinomycin D, alpha-amanitin, or cycloheximide suggesting that mRNA and protein synthesis are required for induction. The present study demonstrates that 1,25-(OH)2D3-treated Caco-2 cells express the vitamin D catabolic pathway and, therefore, constitute a useful in vitro model to study the mechanism of induction by 1,25-(OH)2D3.

Topics: Adenocarcinoma; Amanitins; Calcifediol; Calcitriol; Colonic Neoplasms; Cycloheximide; Cytochrome P-450 Enzyme System; Dactinomycin; Enzyme Induction; Fibroblasts; Humans; Hydroxylation; Intestines; Kinetics; Steroid Hydroxylases; Tumor Cells, Cultured; Vitamin D; Vitamin D3 24-Hydroxylase

Nuclear extract from Morris hepatoma 3924A was fractionated by DEAE-Sephadex chromatography. The fraction eluting with 300 mM (NH4)2SO4 (DE-C) was used for transcribing cloned mouse metallothionein-I (MT-I) gene in a run-off assay. This fraction contained the majority of RNA polymerase II as well as the transcription factor(s). Accuracy of MT-I DNA transcription was confirmed by S1 nuclease mapping. Low concentrations (1 microgram/ml) of alpha-amanitin inhibited the reaction, indicating that RNA polymerase II directed the transcription. Unfractionated nuclear extracts from the hepatoma or a rat mammary adenocarcinoma as well as whole cell extract obtained from the mammary tumor also transcribed MT-I gene. The extent of transcriptional activity was in the following order: hepatoma nuclear fraction DE-C greater than whole cell extract derived from rat mammary adenocarcinoma cells greater than nuclear extract derived from rat hepatoma or rat mammary adenocarcinoma cells. These studies have demonstrated that a fractionated nuclear extract obtained from a tissue supports efficient and accurate RNA polymerase II-mediated transcription of MT-I DNA.

Topics: Adenocarcinoma; Amanitins; Animals; Cell Extracts; Cell Fractionation; Cell Nucleus; Cell-Free System; Chromatography, Ion Exchange; DNA; Liver Neoplasms, Experimental; Mammary Neoplasms, Experimental; Metallothionein; Mice; Rats; RNA Polymerase II; Transcription, Genetic

DNA-dependent RNA polymerase was solubilized from nuclei of Ehrlich ascites carcinoma (EAC) cells by sonic disruption in the presence of 0.3 M (NH4)2 SO4, and the multiple RNA polymerases were separated by chromatography on DEAE-Sephadex A-25. Elution with a nine-step gradient of (NH4)2 SO4 yielded five peaks of activity designated RNA polymerases Ia, Ib, IIa, IIb, and III, of which IIb was the most prominent. Linear-gradient elusion also yielded five peaks of the same designation, but Ia and Ib, as well as IIa and IIb, were not well separated. IIa and IIb were inhibited completely by 0.1 mug alpha-amanitin/ml, whereas the other forms were not. EAC RNA polymerases Ia, Ib, IIa, and IIb possessed Mg2+ ion, Mn2+ ion, and (NH4)2 SO4 optima, molecular weights, and thermal sensitivities similar to those reported for other mammalian DNA-dependent RNA polymerases. As measured by relative ribonucleoside monophosphate incorporation, with native calf thymus DNA template, EAC RNA polymerases Ia and Ib synthesized ribosomal RNA-like products, whereas forms IIa, IIb, and the parent enzyme mixture synthesized compounds that were more similar to DNA. No species specificity was found for DNA templates, and denatured DNA was consistently preferred to the native template by RNA polymerases IIa and IIb; the two kinds of template were about equally efficient for RNA polymerases Ia and Ib. Although EAC RNA polymerases Ia, IIa, and IIb were inhibited by daunomycin, form IIa was preferentially affected. 3',5'-Cyclic AMP, 3',5'-Cyclic GMP, and gibberellic acid, implicated as RNA polymerase regulators in other systems, were generally ineffective. The levels of nuclear RNA polymerase activities, per mg DNA, of 3 mouse ascites tumors (EAC, 6C3HED lymphosarcoma, and TA3 adenocarcinoma) were compared with those from 3 normal mouse tissues (kidney, liver, and spleen). On the average, the tumor cell nuclei contained (per mg of DNA) 8.9, 1.5, 2.7, 20.0, and 3.8 times the activities of RNA polymerases Ia, Ib, IIa, IIb, and III, respectively, as the normal cells, but the difference was significantly only for IIb.

Topics: Adenocarcinoma; Amanitins; Animals; Carcinoma, Ehrlich Tumor; Cell Nucleus; Chromatography, Ion Exchange; Daunorubicin; DNA-Directed RNA Polymerases; DNA, Neoplasm; Kidney; Liver; Lymphoma, Non-Hodgkin; Magnesium; Male; Manganese; Mice; Molecular Weight; Neoplasms, Experimental; RNA, Ribosomal; Species Specificity; Spleen; Temperature; Templates, Genetic