cytochalasin-b and Adenocarcinoma

Aneuploidy and chromosomal instability (CIN) are common features of human malignancy that fuel genetic heterogeneity. Although tolerance to tetraploidization, an intermediate state that further exacerbates CIN, is frequently mediated by TP53 dysfunction, we find that some genome-doubled tumours retain wild-type TP53. We sought to understand how tetraploid cells with a functional p53/p21-axis tolerate genome-doubling events.. We performed quantitative proteomics in a diploid/tetraploid pair within a system of multiple independently derived TP53 wild-type tetraploid clones arising spontaneously from a diploid progenitor. We characterized adapted and acute tetraploidization in a variety of flow cytometry and biochemical assays and tested our findings against human tumours through bioinformatics analysis of the TCGA dataset.. Cyclin D1 was found to be specifically overexpressed in early but not late passage tetraploid clones, and this overexpression was sufficient to promote tolerance to spontaneous and pharmacologically induced tetraploidy. We provide evidence that this role extends to D-type cyclins and their overexpression confers specific proliferative advantage to tetraploid cells. We demonstrate that tetraploid clones exhibit elevated levels of functional p53 and p21 but override the p53/p21 checkpoint by elevated expression of cyclin D1, via a stoichiometry-dependent and CDK activity-independent mechanism. Tetraploid cells do not exhibit increased sensitivity to abemaciclib, suggesting that cyclin D-overexpressing tumours might not be specifically amenable to treatment with CDK4/6 inhibitors.. Our study suggests that D-type cyclin overexpression is an acute event, permissive for rapid adaptation to a genome-doubled state in TP53 wild-type tumours and that its overexpression is dispensable in later stages of tumour progression.

Topics: Adenocarcinoma; Aminopyridines; Benzimidazoles; Cell Line, Tumor; Colorectal Neoplasms; Cyclin C; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinase Inhibitor p21; Cytochalasin B; Diploidy; Flow Cytometry; Gene Knockdown Techniques; Genes, p53; HCT116 Cells; Humans; Protein Kinase Inhibitors; Tetraploidy; Tumor Suppressor Protein p53

Several studies have shown that curcumin can induce apoptosis and inhibit growth in human A549 lung adenocarcinoma cells. However, the mechanism is not completely understood yet. In the present study, we investigated the in vitro effect of curcumin on cell viability, apoptosis and disorganization of the actin cytoskeleton in A549 cells. Our results showed that curcumin significantly inhibited the viability of A549 cells in a dose- and time-dependent manner by induced apoptosis. The apoptotic process was associated with a disorganization of the architecture of actin microfilaments and a decrease in the levels of F-actin. DMSO-treated control cells exhibited a well-defined F-actin network that was mainly organized into stress fibers. The actin fibers in cells treated with curcumin or the positive control drug cytochalasin B were disorganized, disassembled, or disrupted, however, the disorganization of actin fibers and apoptosis could be prevented by phalloidin, an F-actin stabilizing compound. Thus, these results demonstrated that actin filament disorganization might play a central role in the curcumin-induced apoptosis of A549 cells.

Topics: Actin Cytoskeleton; Actins; Adenocarcinoma; Antineoplastic Agents, Phytogenic; Apoptosis; Cell Line, Tumor; Cell Survival; Curcuma; Curcumin; Cytochalasin B; Cytoskeleton; Dose-Response Relationship, Drug; Humans; Lung Neoplasms; Phalloidine; Phytotherapy; Plant Extracts; Poisons

Inorganic arsenic is a major environmental contaminant associated with an increased risk of human skin cancer. Arsenic modulates cellular signaling pathways that affect diverse processes such as cell proliferation, differentiation, and apoptosis, including genotoxic damage. The p53 protein plays a central role in mediating stress and DNA damage responses, leading to either growth arrest or apoptosis. Several signal transduction pathways activated under a plethora of stressing conditions increase p53 protein levels. To further understand the molecular mechanisms involved in the arsenic mode of action, we explored the effects of this metalloid on the activation of the phosphatidyl inositol 3-kinase (PI3K)/Ca2+/diacylglicerol dependent protein kinase/protein kinase B (PKB) signaling cascade and its repercussion in p53 activation in two epithelial cell types: primary normal human keratinocytes cultures (NHK) and the carcinoma-derived C33-A cell line. Although in both cell systems arsenic leads to an increase in p53 and its binding to DNA, the final outcome is different. In NHK, arsenic triggers a sustained activation of the PI3K/PKB/glycogen synthase kinase-3 beta pathway, driving the cell into a cell-differentiated stage in which the proliferation signals are turned down. In sharp contrast, in C33-A cells, arsenic leads to a transient increase in p53 followed by a drastic reduction in its nuclear levels and an increase in cell proliferation. These findings favor the notion that p53-stage and transcriptional abilities are important to understand modifications in the proliferation-differentiation balance, an equilibrium that is severely impaired by arsenic.

Topics: Adenocarcinoma; Arsenites; Cell Nucleus; Cell Proliferation; Cell Survival; Cytochalasin B; DNA Damage; DNA-Binding Proteins; Dose-Response Relationship, Drug; Drug Combinations; Environmental Pollutants; Enzyme Inhibitors; Epithelial Cells; Female; HeLa Cells; Humans; Infant, Newborn; Male; Micronuclei, Chromosome-Defective; Proto-Oncogene Proteins c-akt; Signal Transduction; Sodium Compounds; Tumor Suppressor Protein p53; Uterine Cervical Neoplasms

To study the interplay of drugs and energy metabolism of tumor cells, metabolic changes induced by chloroacetaldehyde and cytochalasin B were analyzed in colon carcinoma cells LS174T.. O(2)-consumption and extracellular acidification were recorded using a bioelectronic sensor-chip system, which monitors these parameters in a culture continuously for at least 24 h. In parallel cultures cell number, cellular ATP-content, mitochondrial transmembrane potential, and the content of reactive oxygen species (ROS) were determined.. When cell death was induced by chloroacetaldehyde (50 muM), the rate of acidification declined gradually for the next 15 h, while O(2)-consumption decreased rapidly within 30 min. This correlated with a loss in mitochondrial potential. However, cellular ATP-level showed a transient increase at 2 h; also ROS levels increased up to 6 h. In cells treated with cytochalasin B (2 muM), which inhibits glucose uptake, the rate of O(2)-consumption increased and the acidification activity dropped, even upon glutamine depletion. Mitochondrial membrane potential transiently increased after 1 h, while ATP-content decreased; there was no change in the level of ROS.. The pattern of changes in basic energy metabolism differs with the type of cell death and growth inhibition involved in the cytotoxic action of two different drugs.

Topics: Acetaldehyde; Adenocarcinoma; Biosensing Techniques; Cell Line, Tumor; Colorectal Neoplasms; Cytochalasin B; Energy Metabolism; Humans; Membrane Potentials; Mitochondria; Oxygen Consumption; Reactive Oxygen Species

We have previously established a vincristine resistant human lung cancer cell line (PC-9/VCR) by a stepwise exposure of parental line PC-9 to vincristine. In this study the resistant cells showed enhanced vincristine cytotoxicity in the presence of cytochalasin B and D. The increase in cytotoxicity was associated with an enhanced accumulation and a reduced efflux of vincristine. Colchicine and taxol had no effects on vincristine accumulation. Several cytoplasmic proteins were overexpressed in the resistant cells. The two major ones, with molecular weights of 58.8 kDa and 83.2 kDa, were shown by western blotting to be beta-tubulin and actin, respectively. The polymerized tubulin level in the resistant cells was significantly (p < 0.05) higher than that in the parental cells. These results suggest that the cellular cytoskeletons might play an important role in VCR resistance in the PC-9/VCR human lung cancer cell line.

Topics: Adenocarcinoma; Cell Survival; Colchicine; Cyclosporine; Cytochalasin B; Cytochalasin D; Cytoskeleton; Drug Resistance, Neoplasm; Drug Synergism; Humans; Lung Neoplasms; Neoplasm Proteins; Paclitaxel; Tubulin; Tumor Cells, Cultured; Verapamil; Vincristine

Hemidesmosomes (HDs) mediate adhesion of epithelial cells to the extracellular matrix and have morphological associations with intermediate-size filaments (IFs). Hemidesmosomal molecular components including HD1, the two bullous pemphigoid antigens, and the integrin alpha 6 beta 4 have been identified in HDs of stratified and complex epithelium. In this study, we report that HT29-Fu cells, a human colonic tumor cell line, express two hemidesmosomal components (HD1, alpha 6 beta 4) associated in an adhesion structure termed type II HDs. Immunofluorescence studies showed a colocalization of HD1 and alpha 6 beta 4 in basal patches between actin stress fibers. Using cytochalasin B or vinblastine, two drugs which disrupt the cytoskeleton, we demonstrate that the redistribution of HD1 was probably induced by the reorganization of the basal cytokeratin network. We also show that in vitro HD1 binds to polymerized cytokeratin intermediate filaments; this suggests that HD1 in intestinal epithelial cells functions as a linker protein connecting cytokeratin filaments to the basal plasma membrane, probably through the beta 4 subunit of the integrin alpha 6 beta 4.

Topics: Actins; Adenocarcinoma; Cell Adhesion; Cell Differentiation; Cell Polarity; Colonic Neoplasms; Cytochalasin B; Cytoskeleton; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Intermediate Filament Proteins; Intermediate Filaments; Keratins; Neoplasm Proteins; Organelles; Tumor Cells, Cultured; Vinblastine

We established an in vitro cytokinesis-block micronucleus assay of human tumours for estimation of the proportion of cells undergoing mitosis (the dividing fraction, DF), the time for the number of nuclei to double and the radiosensitivity in terms of the micronucleus frequency, based on a concept described previously. Under certain conditions, the nuclear number doubling time (NNDT) was considered to represent the potential doubling time. Tumour specimens obtained at surgery were disaggregated into single-cell suspensions and were directly cultured in the presence of cytochalasin B with or without irradiation. At various intervals, the percentage of multinucleate cells (the plateau value represented the DF), the average number of nuclei per cell and the number of micronuclei in binucleate cells were determined. DF and NNDT values were obtained in 58 of the 73 tumours investigated, and the micronucleus frequency was obtained in 54 of these 58 tumours. The DF ranged from 4.1% to 71% and the NNDT ranged from 3.1 to 83 days. A DF > or = 20% was associated with a higher recurrence rate in patients undergoing curative operation. A correlation was found between the NNDT and the time to relapse in patients with recurrent disease. The average number of micronuclei per binucleate cell at 2 Gy of irradiation (after subtraction of the value at 0 Gy) ranged from 0.052 to 0.35. Tumours which produced more micronuclei after irradiation showed a better response to radiotherapy. This assay can be readily performed on human tumours and appears to have promise as a predictive assay for radiation therapy.

Topics: Adenocarcinoma; Brain Neoplasms; Carcinoma; Carcinoma, Squamous Cell; Carcinoma, Transitional Cell; Cell Division; Cell Nucleus; Cell Survival; Cytochalasin B; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Humans; Micronucleus Tests; Neoplasms; Predictive Value of Tests; Prognosis; Radiation Tolerance; Remission Induction; Sarcoma; Treatment Outcome; Tumor Cells, Cultured

Human lung adenocarcinoma cells develop bipolar shape with prominent pseudopodia (greater than or equal to 200 microns) when cultured in the presence of autocrine motility factor (AMF)-like substance or on fibronectin-coated substrata. AMF was partially purified from a human lung adenocarcinoma cell line and has a peak biological activity at a molecular mass of 67 kDa. Using time-lapse photography, we observed that during AMF- or fibronectin-induced cell translocation, the nuclei of some bipolar cells are transported to the opposite end of the cell, while gross cell shape and position remain unchanged. Following this nuclear movement, which we call "nucleokinesis," the posterior pseudopodium is retracted behind the nucleus. Thus, extension of a pseudopodium followed by nucleokinesis in the same direction and retraction of the cell body behind the nucleus is a normal motile sequence in translocating bipolar cells. This suggests that nucleokinesis is a distinct step in whole-cell translocation of bipolar cells on biological substrata and that pseudopodia can be used as nuclear transport organs. In contrast, adenocarcinoma cells cultured on artificial substrata and in the absence of AMF display a fibroblast-like motility pattern with the nucleus centrally located within the migrating cell.

Topics: Adenocarcinoma; Cell Movement; Cell Nucleus; Colchicine; Culture Media; Cytochalasin B; Fibronectins; Glucose-6-Phosphate Isomerase; Growth Substances; Humans; In Vitro Techniques; Lung Neoplasms; Neoplasm Proteins; Tumor Cells, Cultured; Video Recording

The effects of insulin on glucose transport and metabolism were examined in cultured HT29 human colonic adenocarcinoma cells. The presence of glucose transporters was verified by D-glucose displaceable [3H]cytochalasin B binding. The Kd and Bmax values from cytochalasin B binding studies were 190 +/- 30 nM and 8.4 +/- 1.4 pmol/mg protein, respectively. Glucose transport determined with 3-O-methylglucose showed saturable kinetics with a Km of 5.8 +/- 0.4 mM and a Vmax of 0.047 +/- 0.003 mumol/mg protein per min at 25 degrees C. Moreover, in HT29 cells, two classes of insulin binding sites were detected in radioligand binding experiments. Although insulin failed to stimulate glucose transport, it was found to activate glycolysis in HT29 cells. Glucose consumption increased from 0.33 +/- 0.03 mumol/mg protein per h to 0.49 +/- 0.05 mumol/mg protein per h and lactate production was augmented from 0.67 +/- 0.04 mumol/mg protein per h to 0.87 +/- 0.06 mumol/mg protein per h in response to 10(-7) to 10(-5) M insulin. Insulin also enhanced mannose metabolism. Apart from these two hexoses, HT29 cells exhibited a surprisingly narrow substrate specificity. With the possible exception of glyceraldehyde, little lactate was produced from alternative substrates, including adenosine, inosine, ribose, deoxyribose, dihydroxyacetone, galactose and fructose either with or without insulin. Despite its limited utilization by the glycolytic pathway, adenosine was readily salvaged for de novo synthesis of adenine nucleotides. These findings suggest that insulin directly influences substrate utilization through the glycolytic pathway in HT29 cells without activating the glucose transport pathway.

Topics: 3-O-Methylglucose; Adenocarcinoma; Adenosine; Adenosine Triphosphate; Biological Transport; Colonic Neoplasms; Cytochalasin B; Epithelium; Glucose; Glycolysis; Hexoses; Insulin; Insulin-Like Growth Factor I; Methylglucosides; Monosaccharide Transport Proteins; Tumor Cells, Cultured

Alterations in mouse mammary tumor virus (MMTV) production and composition were induced by exposure of mammary tumor cells to cytodisruptive agents. Treatment with 2.1 microM cytochalasin D (CD) for 24 h reduced MMTV yield by 80% and electron microscopic examination of these cells did not reveal budding virions. Immune precipitation and quantitative immunofluorescence studies demonstrated that CD had no significant effect on MMTV polypeptide synthesis or surface expression suggesting that CD inhibited late steps in MMTV maturation. Decreases in MMTV production were also observed as a result of 24 h exposure of the cells to 2.1 microM cytochalasin B (CB). However, an initial 70% increase in the levels of extracellular virions within the first 18 h of treatment preceded diminution of virus production. In addition, CB was unable to abrogate maturation and release of MMTV particles as revealed by electron microscopic evaluation of thin sections of treated cells. Colcemid at 0.28 microM had no effect on virus production during the first 24 h of exposure although MMTV yield was reduced by 60-70% after 36 h of treatment. Polypeptide profiles of MMTV purified from cell cultures treated with any of the three cytodisruptive agents were altered and included 5-7 polypeptides not typically present in MMTV from untreated cells. These cytodisruptive agents did not significantly affect viability and protein metabolism of MJY-alpha cells; the data suggest that alterations in MMTV replication were due to disruption of the cellular cytoskeleton.

Topics: Adenocarcinoma; Animals; Antigens, Viral; Cytochalasin B; Cytochalasin D; Cytochalasins; Cytoskeleton; Demecolcine; Female; Mammary Neoplasms, Experimental; Mammary Tumor Virus, Mouse; Mice; Mice, Inbred BALB C; Viral Proteins; Virion; Virus Replication

Tissue culture monolayers of seven human intracranial tumours comprising 2 astrocytomas, 3 meningiomas, 1 secondary squamous cell carcinoma and 1 secondary adenocarcinoma were examined by a double immunofluorescent staining technique to demonstrate Concanavalin A (Con A) surface receptors and cytoplasmic actin in the same cell. Tumour cells, treated with fluoresceinisothiocyanate-labelled Con A (FITC-Con A) showed staining in cell margins or in a random distribution over the cell surface. Incubating the cells with FITC-Con A at 37 degrees for increasing periods of time resulted first in staining of clusters and later of perinuclear globules. Cells, pretreated with 4% paraformaldehyde at 4 degrees for 10 min or with cytochalasin B at 37 degrees for 30 min showed staining restricted to cell margins. In the cytochalasin B-treated cells, the peripheral staining was in the form of coarse clusters. Double fluorochrome studies showed that the anti-actin antibody (AAA) staining occurred in sites closely related to those stained by FITC-Con A both in untreated as well as in cytochalasin B-treated cells. The findings suggest that Con A receptors, as an example of a stable cell membrane determinant in human tumour cells, are associated with actin and that their mobility on the cell surface is dependent on an intact cytoplasmic actin system.

Topics: Actins; Adenocarcinoma; Astrocytoma; Brain Neoplasms; Carcinoma, Squamous Cell; Colchicine; Cytochalasin B; Cytoplasm; Fluorescent Antibody Technique; Humans; In Vitro Techniques; Meningioma; Receptors, Concanavalin A; Receptors, Drug

Topics: Adenocarcinoma; Cell Membrane; Cytochalasin B; Humans; Lung Neoplasms; Mesothelioma