herbimycin and Colonic-Neoplasms

The extravasation of metastatic cells is regulated by molecular events involving the initial adhesion of tumor cells to the endothelium and subsequently the migration of cells in the host connective tissue. E-selectin on endothelial cells and sialyl Lewis X carbohydrate component on tumor cells are mainly involved in the adhesion of colon carcinoma cells to the endothelium of target organ. Interaction of T84 colon cancer cells to purified E-selectin in vitro caused an increase in the tyrosine phosphorylation of a number of proteins as well as the modulation of cellular properties correlated to the metastatic phenotype. Specifically, E-selectin-stimulated actin reorganization, increased collagenase secretion, and induced cell migration. Treatment of T84 cells with herbimycin A inhibited cell adhesion as well as selectin-induced increase of cell migration, and cytoskeleton assembly. Our data demonstrate that binding of cancer cells to E-selectin starts signal transduction pathways which may affect the tumor metastatic abilities.

Topics: Benzoquinones; Carcinoma; Cell Adhesion; Cell Movement; Cells, Cultured; Coculture Techniques; Colonic Neoplasms; E-Selectin; Endothelium, Vascular; Enzyme Inhibitors; Humans; Lactams, Macrocyclic; Matrix Metalloproteinase 2; Neoplasm Metastasis; Oligosaccharides; Phosphoproteins; Phosphorylation; Phosphotyrosine; Protein-Tyrosine Kinases; Quinones; Rifabutin; Sialyl Lewis X Antigen; Tumor Cells, Cultured

Fifteen different homeobox genes were identified from normal colon mucosa, untreated COLO 205 and herbimycin A treated COLO 205 cells in a degenerate primer RT-PCR screen. Several of the homeobox genes, including Cdx-1, Cdx-2, Pdx-1 and Hox A5, showed a trend toward differential expression in normal colon mucosa, and undifferentiated COLO 205 cells. Hox A5 was recently shown to suppress growth and induce p53-dependent apoptosis. To determine if Hox A5 was differentially expressed in differentiation of colon epithelial cells, we quantified Hox A5 expression by real-time quantitative RT-PCR. Expression of Hox A5 was 5.3- and 4.8-fold higher in normal colon mucosa compared to COLO 205 and HT-29 cells, respectively, suggesting that Hox A5 expression was higher in differentiated compared to undifferentiated colon epithelial cells. To avoid the complexity of tissue specimens and the influence of individual variation in Hox A5 expression, the effect of differentiation on Hox A5 expression was studied in COLO 205 cells treated with herbimycin A. The quantitative study showed that Hox A5 expression was increased when COLO 205 cells were induced to differentiate. The expression of Hox A5 was about 2-fold higher in the cells treated for 48 h compared to the untreated poorly-differentiated cells. The present study shows that Hox A5 may be involved in intestinal cell differentiation, in addition to its role in apoptosis.

Topics: Benzoquinones; Cell Differentiation; Colonic Neoplasms; DNA Primers; Electrophoresis, Agar Gel; Enzyme Inhibitors; Gene Expression; Genes, Homeobox; Homeodomain Proteins; Humans; Intestinal Mucosa; Lactams, Macrocyclic; Phosphoproteins; Quinones; Reverse Transcriptase Polymerase Chain Reaction; Rifabutin; RNA, Messenger; RNA, Neoplasm; Tumor Cells, Cultured

Cystic fibrosis is an autosomal recessive genetic disease, produced by a mutation in the CFTR gene that impairs its function as a chloride channel. In this work, we have examined the effects of interleukin-1beta (IL-1beta) on the expression of CFTR in human colonic T84 cells. Treatment of T84 cells with IL-1beta (0.25 ng/ml) for 4 h resulted in an increased CFTR expression (mRNA and protein). However, higher doses of IL-1beta (1 ng/ml and over) produced inhibition of CFTR mRNA and protein expression. The protein kinase C (PKC) inhibitors H7 (50 microM) and GF109203X (1 microM) inhibited the stimulatory effect of IL-1beta. Similar effects were seen in the presence of the protein tyrosine kinase (PTK) inhibitors genistein (60 microM) and herbymicin A (2 microM). These results suggest that some PKC isoform(s) and at least a PTK might be involved in the CFTR up-regulation induced by IL-1beta. The repression of CFTR up-regulation by cycloheximide (35.5 microM) suggests the participation of a de novo synthesized protein. Results obtained by using the RNA polymerase II inhibitor DRB (78 microM), suggest that the increased mRNA levels seen after IL-1beta treatment are not due to an increased stability of the message. We conclude that the CFTR mRNA and protein levels are modulated by IL-1beta, this cytokine being the first extracellular protein known to up-regulate CFTR gene expression.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adenocarcinoma; Benzoquinones; Cell Line; Colon; Colonic Neoplasms; Cycloheximide; Cystic Fibrosis Transmembrane Conductance Regulator; Enzyme Inhibitors; Genistein; Humans; Indoles; Interleukin-1; Lactams, Macrocyclic; Maleimides; Neoplasm Proteins; Phosphorylation; Protein Kinase C; Protein Processing, Post-Translational; Protein Synthesis Inhibitors; Quinones; Rifabutin; RNA, Messenger; RNA, Neoplasm; Signal Transduction; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Up-Regulation

The ansamycin antibiotics, herbimycin A (HA) and geldanamycin (GM), bind to a conserved pocket in heat shock protein 90 (Hsp90) and alter the function of this chaperone protein. Occupancy of this pocket results in the degradation of a subset of signaling molecules. These include proteins known to associate with Hsp90, e.g., the steroid receptors and Raf, as well as certain transmembrane tyrosine kinases, such as the ErbB receptor family. In a variety of tumor cell lines, treatment with HA potently inhibited cellular proliferation by inducing G1 arrest. This arrest was accompanied by hypophosphorylation of the retinoblastoma gene product (RB) and rapid down-regulation of cyclin D- and E-associated kinase activities. Inhibition of kinase activity was found to result from loss in expression of cyclins D1, D3, and E, as well as the associated cyclin-dependent kinases, cyclin-dependent kinase 4 and cyclin-dependent kinase 6. In addition, HA treatment also caused a late induction of p27(Kip1) protein. The loss of cyclin D preceded the other effects of HA, suggesting that it might be the primary cause of G1 arrest. To determine whether the effects of HA are mediated by selective inhibition of the cyclin D-RB pathway, HA was added to tumor cell lines lacking functional RB. HA treatment of Rb-negative tumor cell lines failed to elicit a G1 arrest. In addition, after release from synchronization with nocodazole, Rb-negative but not Rb-positive cell lines were able to progress through G1 into S phase in the presence of HA. Together, these findings suggest that induction of G1 arrest by HA results from down-regulation of cyclin D expression and its associated kinase activity. Furthermore, these findings imply that Hsp90 selectively regulates signaling pathways upstream of RB.

Topics: Antibiotics, Antineoplastic; Benzoquinones; Blotting, Western; Breast Neoplasms; Cell Cycle Proteins; Cell Division; Colonic Neoplasms; Cyclin A; Cyclin D1; Cyclin D3; Cyclin E; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Cyclins; Down-Regulation; Flow Cytometry; G1 Phase; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Microtubule-Associated Proteins; Mutation; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Quinones; Retinoblastoma Protein; Rifabutin; Signal Transduction; Time Factors; Tumor Cells, Cultured; Tumor Suppressor Proteins

Herbimycin A, a tyrosine kinase inhibitor, induces cellular differentiation and delayed apoptosis in Colo-205 cells, a poorly differentiated human colon carcinoma cell line. Cell cycle analysis in conjunction with end labeling of DNA fragments revealed that G2 arrest preceded apoptotic cell death. Ultrastructural examination of herbimycin-treated cells demonstrated morphologic features of epithelial differentiation, including formation of a microvillar apical membrane and lateral desmosome adhesions. A marked accumulation of mitochondria was also observed. Fluorometric analysis using the mitochondrial probes nonyl-acridine orange and JC-1 confirmed a progressive increase in mitochondrial mass. However these cells also demonstrated a progressive decline in unit mitochondrial transmembrane potential (DeltaPsim) as determined by the DeltaPsim-sensitive fluorescent probes rhodamine 123 and JC-1 analyzed for red fluorescence. In concert with these mitochondrial changes, Colo-205 cells treated with herbimycin A produced increased levels of reactive oxygen species as evidenced by oxidation of both dichlorodihydrofluorescein diacetate and dihydroethidium. Cell-free assays for apoptosis using rat-liver nuclei and extracts of Colo-205 cells at 24 h showed that apoptotic activity of Colo-205 lysates requires the early action of mitochondria. Morphological and functional mitochondrial changes were observed at early time points, preceding cleavage of poly (ADP-ribose) polymerase. These results suggest that apoptosis in differentiated Colo-205 cells involves unrestrained mitochondrial proliferation and progressive membrane dysfunction, a novel mechanism in apoptosis.

Topics: Adenocarcinoma; Animals; Apoptosis; Benzoquinones; Cell Cycle; Cell Differentiation; Cell Division; Cell Survival; Cell-Free System; Colon; Colonic Neoplasms; Enzyme Inhibitors; Fluorescent Dyes; Humans; Intracellular Membranes; Lactams, Macrocyclic; Liver; Membrane Potentials; Mitochondria; Oxidation-Reduction; Poly(ADP-ribose) Polymerases; Protein-Tyrosine Kinases; Quinones; Rats; Reactive Oxygen Species; Rifabutin; Tumor Cells, Cultured

The effect of herbimycin A, an ansamycin antibiotic which inhibits cellular transformation by retroviral tyrosine kinases, on the monolayer growth of seven colon tumor cell lines and one cell line established from normal colonic mucosa, CCL239, was examined. Each colon tumor cell line tested showed dose-dependent growth inhibition in response to herbimycin A. A 125ng ml-1 dose of the antibiotic caused greater than 40% growth inhibition in all colon tumor cell lines after two cell doublings. In contrast, at similar herbimycin A concentrations only 12% inhibition was observed in 'normal' CCL239 cells. No major morphologic changes were observed at the light microscopic level in any of the tumor cell lines or CCL239 cells in response to treatment with herbimycin A. Studies using the HT29 colon adenocarcinoma cell line showed dose-dependent inactivation of pp60c-src by herbimycin A, resulting in decreased autophosphorylation, enolase phosphorylation and steady-state levels, which correlated with cellular growth inhibition. Herbimycin A-induced reductions in pp60c-src kinase activity preceded changes in pp60c-src steady-state levels. Growth and pp60c-src inhibition were reversible following removal of herbimycin A from cell culture media. Our results suggest that regulation of pp60c-src tyrosine kinase activity may be important in growth control of colon tumor cells.

Topics: Adenocarcinoma; Antibiotics, Antineoplastic; Benzoquinones; Cell Division; Cell Line; Colonic Neoplasms; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Humans; Immunoblotting; Lactams, Macrocyclic; Precipitin Tests; Proto-Oncogene Proteins pp60(c-src); Quinones; Rifabutin; Tumor Cells, Cultured