cytochalasin-d and Colonic-Neoplasms

Higher levels of focal adhesion kinase (FAK) are expressed in colon metastatic carcinomas. However, the signaling pathways and their mechanisms that control cell adhesion and motility, important components of cancer metastasis, are not well understood. We sought to identify the integrin-mediated mechanism of FAK cleavage and downstream signaling as well as its role in motility in human colon cancer GEO cells. Our results demonstrate that phosphorylated FAK (tyrosine 397) is cleaved at distinct sites by integrin signaling when cells attach to collagen IV. Specific blocking antibodies (clone P1E6) to integrin alpha2 inhibited FAK activation and cell motility (micromotion). Ectopic expression of the FAK C-terminal domain FRNK attenuated FAK and ERK phosphorylation and micromotion. Calpain inhibitor N-acetyl-leucyl-leucyl-norleucinal blocked FAK cleavage, cell adhesion, and micromotion. Antisense approaches established an important role for mu-calpain in cell motility. Expression of wild type mu-calpain increased cell micromotion, whereas its point mutant reversed the effect. Further, cytochalasin D inhibited FAK phosphorylation and cleavage, cell adhesion, locomotion, and ERK phosphorylation, thus showing FAK activation downstream of actin assembly. We also found a pivotal role for FAK Tyr(861) phosphorylation in cell motility and ERK activation. Our results reveal a novel functional connection between integrin alpha2 engagement, FAK, ERK, and mu-calpain activation in cell motility and a direct link between FAK cleavage and enhanced cell motility. The data suggest that blocking the integrin alpha2/FAK/ERK/mu-calpain pathway may be an important strategy to reduce cancer progression.

Topics: Biotinylation; Butadienes; Calpain; Cell Adhesion; Cell Line, Tumor; Cell Movement; Colonic Neoplasms; Cytochalasin D; Electric Impedance; Enzyme Activation; Enzyme Inhibitors; Focal Adhesion Protein-Tyrosine Kinases; Humans; Integrin alpha2; Mitogen-Activated Protein Kinases; Nitriles; Nucleic Acid Synthesis Inhibitors; Oligonucleotides, Antisense; Phosphorylation; Point Mutation; Precipitin Tests; Protein Structure, Tertiary; Signal Transduction; Substrate Specificity; Tyrosine

Increased extracellular pressure stimulates colon cancer cell adhesion by activating focal adhesion kinase (FAK) and Src. We investigated the role of the cytoskeleton in pressure-induced inside-out FAK and Src phosphorylation and pressure-stimulated adhesion. We perturbed actin polymerization with phalloidin, cytochalasin D and latrunculin B, and microtubule organization with colchicine and paclitaxol. We compared the effects of these agents on pressure-induced SW620 and human primary colon cancer cell adhesion and inside-out FAK/Src activation with outside-in adhesion-dependent FAK/Src activation. Cells pretreated with cytoskeletal inhibitors were subjected to 15 mmHg increased pressure and allowed to adhere to collagen I coated plates or prevented from adhesion to pacificated plates for 30 min. Phalloidin, cytochalasin D, latrunculin B and colchicine pretreatment completely prevented pressure-stimulated and significantly inhibited basal SW620 cell adhesion. Taxol did not inhibit pressure-induced colon cancer cell adhesion, but significantly lowered basal adhesion. Cytochalasin D and colchicine had similar effects in pressure-stimulated primary human malignant colonocytes. Phalloidin, cytochalasin D, latrunculin B and colchicine prevented pressure-induced SW620 FAK phosphorylation but not Src phosphorylation. FAK phosphorylation in response to collagen I adhesion was significantly attenuated but not completely prevented by these inhibitors. Although Src phosphorylation was not increased on adhesion, the cytoskeleton disrupting agents significantly lowered basal Src phosphorylation in adherent cells. These results suggest that both cytoskeleton-dependent FAK activation and cytoskeleton-independent Src activation may be required for extracellular pressure to stimulate colon cancer cell adhesion. Furthermore, the cytoskeleton plays a different role in pressure-activated FAK and Src signaling than in FAK and Src activation in adherent cells. We, therefore, hypothesize that cytoskeletal interactions with focal adhesion signals mediate the effects of extracellular pressure on colon cancer cell adhesion.

Topics: Cell Adhesion; Cell Line, Tumor; Cells, Cultured; Colchicine; Colon; Colonic Neoplasms; Cytochalasin D; Cytoskeleton; Dimethyl Sulfoxide; Humans; Integrins; Models, Biological; Paclitaxel; Phalloidine; Pressure; Signal Transduction

The establishment and maintenance of epithelial polarity are crucial for tissue organization and function in mammals. Epithelial cadherin (E-cadherin) is expressed in epithelial cell membrane and is important for cell-cell adhesion, intercellular junctions formation, as well as epithelial cell polarization. We report herein that CAS (CAS/CSE 1), the human cellular apoptosis susceptibility protein, interacts with E-cadherin and stimulates polarization of HT-29 human colon epithelial cells. CAS binds with E-cadherin but not with beta-catenin in the immunoprecipitation assays. Interaction of CAS with E-cadherin enhances the formation of E-cadherin/beta-catenin cell-cell adhesive complex. Electron microscopic study demonstrated that CAS overexpression in cells stimulates intercellular junction complex formation. The disorganization of cellular cytoskeleton by cytochalasin D, colchicine, or acrylamide treatment disrupts CAS-stimulated HT-29 cell polarization. CAS-mediated HT-29 cell polarity is also inhibited by antisense E-cadherin DNA expression. Our results indicate that CAS cooperates with E-cadherin and plays a role in the establishment of epithelial cell polarity.

Topics: Acrylamide; Apoptosis; Blotting, Western; Cadherins; Cell Polarity; Cellular Apoptosis Susceptibility Protein; Colchicine; Colonic Neoplasms; Cytochalasin D; Cytoskeleton; DNA; Epithelial Cells; Humans; Oligonucleotides, Antisense; Precipitin Tests; Protein Binding; Reverse Transcriptase Polymerase Chain Reaction; Tumor Cells, Cultured

The delivery of cells to specific regions of the vasculature is a critical step in many therapeutic strategies. These include the packaging of DNA or RNA in cell "vehicles" for delivery to tissues, the reconstitution of differentiated cells to an organ using embryonic stem cells, and the enhancement of the immune response using effector lymphocytes. In most cases, these cells must be injected systemically. Unfortunately, ex vivo manipulation or activation can affect cell visco-elastic properties, making it difficult for the injected cells to traverse capillary beds. Compounding the problem is the fact that common agents used in the laboratory for increasing cell deformability generally have adverse side effects on the therapeutic potential of the cells. Using micropipet aspiration techniques, cytotoxicity assays and in vivo trafficking studies we show that: (1) the rigidity of injected effector cells directly affects resistance to passage through tissue; (2) modulation of cytoskeletal organization can be used to decrease cell rigidity, but can also compromise therapeutic efficacy; and (3) thioglycollate, an agent which does not influence effector lymphocyte cytotoxic activity, reduces cell rigidity and entrapment in the lungs.

Topics: Animals; Cell Size; Colonic Neoplasms; Cytochalasin D; Cytoskeleton; Cytotoxicity, Immunologic; Elasticity; Female; Hemorheology; Humans; Killer Cells, Natural; Lung; Lymphocyte Activation; Mice; Mice, Nude; Neoplasm Transplantation; Neovascularization, Pathologic; Thioglycolates

Although infection of the intestinal and biliary tracts by Cryptosporidium parvum is a major problem in patients with the acquired immunodeficiency syndrome, the specific microbial and host molecules involved in C. parvum infection are unknown. We tested the hypothesis that lectin-carbohydrate interactions and cytoskeleton reorganization are involved in the infection of biliary and intestinal epithelia by C. parvum.. In vitro models of cryptosporidial infection using human biliary and intestinal epithelial cell lines were used to assay C. parvum attachment and invasion.. Exposure of C. parvum sporozoites to the sugar, galactose-N-acetylgalactosamine (Gal/GalNAc), and to bovine mucin reduced C. parvum attachment to biliary and intestinal epithelia up to 70%. Preincubation of cell monolayers with either lectins specific to Gal/GalNAc, or glycosidases that specifically release Gal/GalNAc oligosaccharides from glycoproteins, decreased attachment up to 80%. Cytochalasin B and cytochalasin D, but not nocodazole, decreased invasion of cells by C. parvum up to 70% without affecting attachment. During cell invasion (but not attachment), confocal microscopy showed recruitment of actin (but not tubulin) in biliary and intestinal epithelia directly adjacent to C. parvum.. Gal/GalNAc epitopes of glycoproteins on the epithelial apical membrane and Gal/GalNAc-specific sporozoite surface lectins are involved in the mechanism(s) of C. parvum attachment to intestinal and biliary epithelial cells, and actin remodeling in host cells is required for C. parvum invasion.

Topics: Animals; Antigens, Tumor-Associated, Carbohydrate; Bile Ducts; Cattle; Cell Adhesion; Cell Line; Colonic Neoplasms; Cryptosporidiosis; Cryptosporidium parvum; Cytochalasin B; Cytochalasin D; Epithelial Cells; Glycoside Hydrolases; Humans; Intestinal Mucosa; Lectins; Mucins; Nocodazole; Tumor Cells, Cultured

Integrin-mediated tumour cell adhesion to extracellular matrix (ECM) components is an important step in the development of metastatic lesions. Thus, integrin expression and integrin-mediated adhesion of colon carcinoma cells to various ECM components was examined. Poorly (HT-29P) and highly (HT-29LMM) liver-metastatic colon carcinoma cells were used to study the rates of adhesion to collagen I (C I), collagen IV (C IV), laminin (LN), fibronectin (FN), or vitronectin (VN) in a static adhesion assay (10-120 min). Cells were untreated or treated with oligopeptides (RGD, GRGDS, YIGSR, RGES), anti-integrin antibodies, or colchicine, nocodazole, cycloheximide, acrylamide or cytochalasin D (to disrupt cytoskeletal structures). Both cell lines expressed similar patterns of integrin expression (alpha2, alpha3, ,alpha6, alphav, beta1, beta4, and beta5) by immunocytochemistry and immunoprecipitation. HT-29LMM cells showed significantly higher rates of adhesion to LN (P < 0.001) and FN (P < 0.001), but significantly poorer rates of adhesion to C I (P < 0.05) and C IV (P < 0.001) than HT-29P cells, respectively, adhesion to VN was insignificant. RGD and GRGDS inhibited HT-29LMM cell adhesion to FN only. Pretreatment with anti-beta, or anti-alpha2 integrin subunits suppressed adhesion to C I and C IV, and adhesion to LN was inhibited with anti-beta1 or anti-alpha6 integrin. Anti-beta1 or anti-alphav blocked adhesion to FN. Pretreatment of cells with cytochalasin D, cycloheximide or acrylamide inhibited adhesive interactions of both cell lines to the ECM components. In contrast, colchicine and nocodazole had no effect. The results demonstrate that adhesion of HT-29 cells to ECM is mediated, in part, by different integrins, depending on the substrate. Poorly and highly metastatic HT-29 cells possessed different patterns of adhesion to the various ECM substrates, but these differences were not due to different expression of integrin subunits. The results also suggested that the initial adhesion of poorly or highly metastatic HT-29 cells to ECM components requires, in part, the presence of native action and intermediate filaments, but not of microtubules. Thus the adhesion of tumour cells to ECM components may be dependent on signal transduction and assembly of microfilaments.

Topics: Acrylamide; Antibodies; Cell Adhesion; Colchicine; Colonic Neoplasms; Cycloheximide; Cytochalasin D; Cytoskeleton; Extracellular Matrix; Humans; Integrins; Neoplasm Metastasis; Nocodazole; Tumor Cells, Cultured

The organization of the endoplasmic reticulum (ER)-intermediate compartment (IC)-Golgi system was studied in tumoral HT-29 cells. Depending on the culture conditions, these cells are either undifferentiated or exhibit enterocytic differentiation after reaching confluence. In differentiated HT-29 cells, these organelles were organized as in most cell types. They displayed a classical structure and appeared associated with microtubules, as nocodazole altered both their structure and intracellular localization. Likewise, membrane dynamics of the Golgi appeared normal: as in many other cells, brefeldin A (BFA) induced retrograde transport from the Golgi to the ER, demonstrated by tubulation of the Golgi elements and shift of the galactosyltransferase activity from the Golgi- to the RER-enriched fraction, isolated by subcellular fractionation. In contrast, atypical features were observed in undifferentiated HT-29 cells: the Golgi structure exhibited abnormal swellings; the IC elements were very rare. Only cytochalasin D altered the structure and intracellular localization of the three organelles, suggesting that they were associated with microfilaments instead of microtubules. The membrane dynamics were unusual: brefeldin A led to a vesiculation of the Golgi elements with a slowed-down retrograde transport of galactosyltransferase. HT-29 cells engaged in the differentiation process, but which were still undifferentiated, showed mainly the features of undifferentiated cells, with a few characteristics of differentiated cells. These results indicate that the structure of the Golgi apparatus, IC and ER, their relationships to cytoskeletal elements and membrane dynamics depend on the state of differentiation of HT-29 cells. Although they are tumoral, differentiated HT-29 cells exhibit features observed in non-tumoral polarized epithelial cells. On the contrary, undifferentiated HT-29 cells display important abnormalities that may be related to their metastatic properties.

Topics: Antineoplastic Agents; Brefeldin A; Cell Differentiation; Cell Division; Cell Membrane; Colonic Neoplasms; Cyclopentanes; Cytochalasin D; Cytoskeleton; Endoplasmic Reticulum; Golgi Apparatus; Humans; Microscopy, Electron; Microscopy, Immunoelectron; Nocodazole; Protein Synthesis Inhibitors; Tumor Cells, Cultured

During cancer progression, tumor cells interact with stromal cells. As a consequence, matrix metalloproteinases are produced that contribute to the degradation of the extracellular matrix. This study used coculture systems to investigate fibroblast interaction with three colon cancer cell lines isolated from a single patient. Cells from primary colorectal carcinoma, but not from corresponding liver or lymph node metastases, induced gelatinase B expression by fibroblasts of different tissue origin. Remarkably, direct cell-cell contact was required for this induction, which occurred at the pretranslational level (as revealed by Northern blot analysis) and was completely blocked by anti-beta1 integrin monoclonal antibody, but only partially blocked by anti-alpha5 or anti-alpha(v). Induction was also inhibited by cytochalasin D, staurosporine, or dexamethasone, suggesting the need, respectively, for an organized actin cytoskeleton, protein kinase C, and AP-1-driven gene transcription. Our data suggest that direct tumor-stromal cell contact is one inductive event involved in matrix metalloproteinase expression by stromal cells.

Topics: Animals; Antibodies, Monoclonal; Carcinoma; Cell Communication; Cells, Cultured; Coculture Techniques; Collagenases; Colonic Neoplasms; Connective Tissue; Connective Tissue Cells; Cytochalasin D; Cytoskeleton; Dexamethasone; Enzyme Induction; Enzyme Inhibitors; Extracellular Matrix Proteins; Fibroblasts; Genistein; Humans; Integrin beta1; Isoflavones; Keratinocytes; Liver Neoplasms; Lung Neoplasms; Matrix Metalloproteinase 9; Mice; Mice, Nude; Neoplasm Metastasis; Protein Kinase C; Staurosporine; Transcription Factor AP-1; Tumor Cells, Cultured

We have shown previously [McCool, Forstner and Forstner (1994) Biochem. J. 302, 111-118] using pulse-chase labelling of mucin with [3H]threonine that LS180 colonic tumour cells synthesize and secrete MUC2 without the addition of secretagogues. Treatment of the LS180 cells with monensin to disrupt Golgi function was also found to inhibit baseline secretion almost completely. In this paper we show that addition of nocodazole to inhibit microtubule assembly reduced baseline secretion by 53% over a 6 h chase period. In contrast, cytochalasin D did not affect the rate of unstimulated mucin synthesis or secretion, suggesting that baseline secretion is not influenced by disruption of actin microfilaments. In addition, regulated mucin secretion by LS180 cells was studied in response to carbachol, phorbol 12-myristate 13-acetate and A23187. Mucin released in response to secretagogues behaved identically on SDS/PAGE to that secreted under baseline conditions. T84 cells and the B6 subclone of the HT29 cell line responded in a similar manner to LS180 cells and secreted high-molecular-mass mucin which included MUC2 and behaved like LS180 mucin on SDS/PAGE. Neither monensin nor nocodazole significantly affected secretagogue-stimulated mucin secretion. Since these compounds inhibited secretion of labelled mucin under baseline conditions, mucin released by secretagogues must have come from a separate, unlabelled mucin pool in stored granules. Cytochalasin D, on the other hand, caused the release of small amounts of stored mucin, suggesting that actin microfilaments participate in regulated exocytosis. Thus two kinds of mucin secretion occur in LS180 cells. Unregulated secretion depends upon continuous transport of mucin granules from Golgi vesicles to the cell surface and does not utilize stored mucin, whereas regulated secretion involves the release of mucin from storage granules and is not affected by microtubule or Golgi disruption.

Topics: Adenocarcinoma; Biomarkers, Tumor; Calcimycin; Carbachol; Cell Size; Colchicine; Colonic Neoplasms; Cytochalasin D; Cytoplasmic Granules; Golgi Apparatus; Humans; Microscopy, Electron; Monensin; Mucin-2; Mucins; Neoplasm Proteins; Nocodazole; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Intestinal alkaline phosphatase (AP) is secreted by Caco-2 cells bound to surfactant-like particles (SLP), which can be localized by electron microscopy to the basolateral space and the intestinal lumen, especially over tight junctions. To investigate the hypothesis that SLP are secreted basolaterally and enter the lumen through the tight junction, Caco-2 cells were incubated with agents known to increase permeability at tight junctions. Cytochalasin D and phorbol 12-myristate 13-acetate increased Caco-2 cell monolayer permeability and the appearance of particles in apical medium two- to threefold, as monitored by mannitol movement and AP activity, respectively. Blocking the apical secretory pathway by nocodazole or colchicine had no effect on either parameter. Estimation of SLP content demonstrated an increase in apical media particles similar to that determined by AP activity. Quantitative image analysis established that apical SLP content increased 4-10 times, whereas total cell particle content remained unchanged. These data indicate that SLP may be secreted initially into the basolateral space and then transported to the intestinal lumen through the tight junctions.

Topics: Adenocarcinoma; Alkaline Phosphatase; Animals; Antibodies; Cell Line; Cell Membrane; Cell Membrane Permeability; Colonic Neoplasms; Cytochalasin D; Humans; Hypertonic Solutions; Intercellular Junctions; Intestine, Small; Microscopy, Electron; Organelles; Rabbits; Raffinose; Sucrose; Surface-Active Agents; Tetradecanoylphorbol Acetate; Time Factors; Tumor Cells, Cultured

Apical and basolateral endocytic pathways in polarised Caco-2 cells were investigated by following the uptake, recycling and transcytosis of the galactose-binding protein toxin ricin, as a membrane marker. Differences in the extent and kinetics of lectin uptake, recycling and transcytosis were observed at the apical and basolateral domains and altered with the age of the cell monolayer. Treatment of polarised Caco-2 cells with cytochalasin D showed a domain-specific, concentration-dependent inhibition of apical endocytosis of ricin. Inhibition of apical endocytosis by cytochalasin D was not due to a gross change in brush border morphology, although actin stress fibres within the cell body were disrupted. It is not clear whether inhibition of apical endocytosis in polarized epithelial cells by cytochalasin D is caused simply by disruption of a mechanochemical motor involving microvillar actin filaments. The cytochalasin D effect was also observed when measuring uptake of folate, suggesting apical domain-specific inhibition of caveolar, as well as clathrin-mediated, endocytic routes.

Topics: Actin Cytoskeleton; Adenocarcinoma; Animals; Cell Line; Cell Polarity; Colonic Neoplasms; Cytochalasin D; Depression, Chemical; Dogs; Endocytosis; Folic Acid; Humans; Kidney; Microvilli; Neoplasm Proteins; Ricin; Tumor Cells, Cultured

During the course of the present biochemical and ultrastructural studies, we found that the expression of either the undifferentiated or the differentiated HT-29 cell phenotype determined the intracellular fate of ricin. Although the recognition of ricin at the cell surface required interaction with the galactose-binding site on both cell populations, the lag time before ricin started to inhibit protein synthesis was longer in the differentiated than the undifferentiated cells. Dose-response studies and "time-addition" experiments performed with NH4Cl, which raises the pH of acidic vesicles and organelles, showed that ricin uptake as well as the movement of the toxin to the translocation site were affected in the differentiated cells. In contrast, NH4Cl acted on only post-internalization events in the undifferentiated cells. When the addition of cytochalasin D, an actin-depolymerizing drug, was staggered, the differentiated cells were found to be protected against ricin only during the very early stage of the internalization process. In contrast, the undifferentiated cells were protected during both the early and late stages of endocytosis. Moreover, electron microscopic examination showed that cytochalasin D altered the structure of the Golgi apparatus only in the undifferentiated cells. 3-Methyladenine, a specific inhibitor of the autophagic pathway, protected the undifferentiated and differentiated cells against ricin to about the same extent. We concluded that to enter the differentiated cells, ricin followed the classical endosome-Golgi pathway. In contrast, in the undifferentiated cells, ricin reaches the cytosol by two distinct routes: the minor one involves the endosome-Golgi pathway; the major one involves a cytochalasin D-sensitive pathway.

Topics: Actin Cytoskeleton; Adenocarcinoma; Ammonium Chloride; Cell Differentiation; Colon; Colonic Neoplasms; Cytochalasin D; Endocytosis; Humans; Hydrogen-Ion Concentration; Microtubules; Ricin; Tumor Cells, Cultured

The effect of phorbol 12-myristate 13-acetate (PMA), a phorbol ester known to stimulate protein kinase C, on taurine transport was studied in the human colon carcinoma cell line HT-29. PMA (1 microM) was found to inhibit taurine uptake in confluent monolayers of this cell line by approximately 70% after pretreatment of the cells with the compound for 1 h (IC50 = 42.7 +/- 2.6 nM). The inhibitory effect of PMA on the taurine transporter could be confirmed by using beta-alanine, another substrate for the transporter. Kinetic analysis of taurine uptake indicated that the PMA effect was associated with a decrease in the maximal velocity (954 +/- 26 vs. 676 +/- 28 pmol.10 min-1.mg of protein-1) and an increase in the Michaelis-Menten constant (9.8 +/- 0.5 vs. 13.3 +/- 1.0 microM). The inhibition of taurine uptake could be blocked by cotreatment of the cultures with staurosporine, an inhibitor of protein kinase C. The inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate had no effect. Treatment of the cells with PMA did not alter the uptake of leucine and lysine, stimulated the uptake of aspartic acid, and inhibited the uptake of proline. The PMA effect on taurine uptake was not prevented by cycloheximide, actinomycin D, colchicine, or cytochalasin D. Comparison of the taurine transport activity in HT-29 cells with that in Caco-2, another human colon carcinoma cell line, revealed that the latter cell line also expressed the taurine transporter but at a much reduced level (about one-fifth compared with HT-29).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Biological Transport; Colchicine; Colonic Neoplasms; Cycloheximide; Cytochalasin D; Dactinomycin; Humans; Kinetics; Protein Kinase C; Taurine; Tetradecanoylphorbol Acetate; Time Factors; Tumor Cells, Cultured