cytochalasin-d and Adenocarcinoma

Therapeutic delivery of small interfering RNA (siRNA) is a major challenge that limits its potential clinical application. Four fatty acids derivatives of octa-arginine (R8) were synthesized and evaluated for the delivery of siRNA into hepatocellular carcinoma Hep G2 and human lung adenocarcinoma A549 cells. The results showed that the long chain acid oleic acid or stearic acid derivatives of R8, OA-R8 and StA-R8, were more efficient in siRNA complexation and form nanoparticles with greater stability compared to the native R8. Cellular uptake of fluorescence-labeled siRNA delivered by OA-R8 and StA-R8 in Hep G2 and A549 cells was substantially 40-50 times higher than unmodified R8. A significant reduction in siRNA cellular uptake was observed in the presence of sucrose and cytochalasin D, indicating endocytosis as a primary mechanism of cellular entry. A survivin siRNA was used to prepare nanoparticles with OA-R8 or StA-R8 and evaluated for silencing of survivin mRNA and protein in A549 cells, and the inhibition efficiencies of survivin protein reached to 50.3% and 54.6%, respectively. The results showed greater effectiveness with the derivatized R8. Taken together, these findings showed that long chain fatty acid derivatives of R8 are efficient delivery agents for siRNA and may facilitate its therapeutic application.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Arginine; Cell Line, Tumor; Cell-Penetrating Peptides; Cytochalasin D; Dose-Response Relationship, Drug; Endocytosis; Fatty Acids; Hep G2 Cells; Humans; Lung Neoplasms; Nanoparticles; RNA, Small Interfering; Sucrose

The stem cell marker nestin is an intermediate filament protein that plays an important role in cell integrity, migration, and differentiation. Nestin expression occurs in approximately one third of pancreatic ductal adenocarcinoma (PDAC), and its expression strongly correlates with tumor staging and metastasis. Little is known about the mechanisms by which nestin influences PDAC progression. Here, nestin overexpression in PDAC cells increased cell motility and drove phenotypic changes associated with the epithelial-mesenchymal transition (EMT) in vitro; conversely, knockdown of endogenous nestin expression reduced the migration rate and reverted cells to a more epithelial phenotype. Mouse xenograft studies showed that knockdown of nestin significantly reduced tumor incidence and volume. Nestin protein expression was associated with Smad4 status in PDAC cells; hence, nestin expression might be regulated by the TGF-β1/Smad4 pathway in PDAC. We examined nestin expression after TGF-β1 treatment in human pancreatic cancer PANC-1 and PANC-1 shSmad4 cells. The TGF-β1/Smad4 pathway induced nestin protein expression in PDAC cells in a Smad4-dependent manner. Moreover, increased nestin expression caused a positive feedback regulator of the TGF-β1 signaling system. In addition, hypoxia was shown to induce nestin expression in PDAC cells, and the hypoxia-induced expression of nestin is mediated by the TGF-β1/Smad4 pathway. Finally, the antimicrotubule inhibitors, cytochalasin D and withaferin A, exhibited anti-nestin activity; these inhibitors might be potential antimetastatic drugs. Our findings uncovered a novel role of nestin in regulating TGF-β1-induced EMT. Anti-nestin therapeutics may serve as a potential treatment for PDAC metastasis.

Topics: Adenocarcinoma; Animals; Biomarkers, Tumor; Carcinogenesis; Carcinoma, Pancreatic Ductal; Cell Movement; Cytochalasin D; Disease Progression; Epithelial-Mesenchymal Transition; Gene Knockdown Techniques; Humans; Male; Mice; Mice, SCID; Microtubules; Neoplasm Metastasis; Nestin; Pancreas; Pancreatic Neoplasms; Signal Transduction; Smad4 Protein; Stem Cells; Transforming Growth Factor beta1; Up-Regulation; Withanolides; Xenograft Model Antitumor Assays

The tumor suppressor p21WAF/CIP1 mediates the proliferation arrest via p53-dependent or -independent gene transactivation following distinct environmental stresses. In this study, we show that direct destabilization of the actin cytoskeleton by actin-targeting reagents leads to a p53-independent up-regulation of p21WAF/CIP1. The actin-targeting agent cytochalasin B (10 microM) quickly disrupted the actin cytoskeleton of p53 wild-type and p53-null cells accompanied by up-regulation of p21WAF/CIP1. Nevertheless, both total p53 and ser-15 phosphorylated p53 were not accumulated concomitantly, compared to the effect caused by ionizing irradiation. P53-independent up-regulation of p21WAF/CIP1 was also observed by two other actin-targeting agents cytochalasin D and latrunculin B, but not by the microtubule inhibitor colcemid. Furthermore, we showed that p21WAF/CIP1 mRNA level was not increased, whereas the protein degradation was delayed. A reduction of ubiquitination for p21WAF/CIP1 protein was detected using immunoprecipitation/immunoblot analysis. Up-regulation of p21WAF/CIP1 was not associated with cytotoxicity induced by cytochalasin B that influenced DNA integrity and plating efficiency only after 24 h of treatment. In addition, up-regulated p21WAF/CIP1 was accompanied by reduction of phosphorylation on retinoblastoma (Rb) protein in p53-null cells, implying that p21WAF/CIP1 might in part account for the molecular regulation of cytochalasin B induced G1 phase arrest. Together, current results suggest that p21WAF/CIP1 level can be mediated by actin organization in the absence of p53 via a post-transcriptional machinery, and it may contribute to the growth ablation by agents targeting the actin cytoskeleton.

Topics: Actins; Adenocarcinoma; Bone Neoplasms; Bridged Bicyclo Compounds, Heterocyclic; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p21; Cytochalasin D; Cytoskeleton; DNA, Neoplasm; Genes, p53; Humans; Lung Neoplasms; Osteosarcoma; RNA Processing, Post-Transcriptional; RNA, Messenger; RNA, Neoplasm; Thiazolidines; Ubiquitin

Metastases kill 90% of cancer patients. It is thus a major challenge in cancer therapy to inhibit the spreading of tumor cells from primary tumor sites to those particular organs where metastases are likely to occur. Whereas the actin cytoskeleton is a key component involved in cell migration, agents targeting actin dynamics have been relatively poorly investigated. Consequently, valuable in vitro pharmacological tools are needed to selectively identify this type of agent. In response to the absence of any standardized process, the present work aims to develop a multi-assay strategy for screening actin-affecting drugs with anti-migratory potentials. To validate our approach, we used two cancer cell lines (MCF7 and A549) and three actin-affecting drugs (cytochalasin D, latrunculin A, and jasplakinolide). We quantified the effects of these drugs on the kinetics of actin polymerization in tubes (by means of spectrofluorimetry) and on the dynamics of actin cytoskeletons within whole cells (by means of fluorescence microscopy). Using quantitative videomicroscopy, we investigated the actual effects of the drugs on cell motility. Finally, the combined drug effects on cell motility and cell growth were evaluated by means of a scratch-wound assay. While our results showed concordant drug-induced effects on actin polymerization occurring in vitro in test tubes and within whole cells, the whole cell assay appeared more sensitive than the tube assay. The inhibition of actin polymerization induced by cytochalasin D was paralleled by a decrease in cell motility for both cell types. In the case of jasplakinolide, which induces actin polymerization, while it significantly enhanced the locomotion of the A549 cells, it significantly inhibited that of the MCF-7 ones. All these effects were confirmed by means of the scratch-wound assay except of the jasplakinolide-induced effects on MCF-7 cell motility. These later seemed compensated by an additional effect occurring during wound recolonization (possibly acting on the cell growth features). In conclusion, the use of multi-assays with different levels of sophistication and biological relevance is recommended in the screening of new actin-affecting drugs with potentially anti-migratory effects.

Topics: Actins; Adenocarcinoma; Antineoplastic Agents; Breast Neoplasms; Bridged Bicyclo Compounds, Heterocyclic; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Cytochalasin D; Cytoskeleton; Depsipeptides; Humans; Inhibitory Concentration 50; Lung Neoplasms; Neoplasm Invasiveness; Statistics, Nonparametric; Thiazoles; Thiazolidines

Recent studies have identified novel actions for 2-aminoethoxydiphenyl borate (2-APB) in triggering calcium release and enhancing calcium influx induced by the depletion of intracellular calcium stores. In this study, we have examined the effects of 2-APB on the human lung adenocarcinoma A549 cell line, which we have previously shown displays a unique calcium influx response, when ER calcium stores are depleted by thapsigargin (TG) treatment. Here, we show that low concentrations of 2-APB failed to induce the rapid augmentation of TG-activated calcium influx previously reported for other cell types. We observed that store-operated calcium (SOC) channels in the A549 cell line exhibited short-term sensitivity to low doses of 2-APB, perhaps reflecting a delayed augmentation of SOC channel activity or the recruitment of 2-APB-insensitive SOC channels. In both intact and permeabilized cells, 2-APB effectively discharged a subset of A549 calcium pools corresponding to the hormone-sensitive intracellular calcium stores. The 2-APB-induced calcium release produced a long-lasting perturbation of the adenosine triphosphate (ATP)-releasable calcium pools, effectively uncoupling ATP-activated calcium release even, when stores are replenished with calcium. In contrast to previous reports, we found that disruption of either the actin or microtubule-based cytoskeleton failed to block the 2-APB-induced effects on calcium signaling in A549 cells. Our study describes novel cytoskeletal-independent effects of 2-APB on Ca2+-signaling pathways, revealing differentially sensitive Ca2+-influx pathways and long-term perturbation of hormone-sensitive Ca2+ stores.

Topics: Actins; Adenocarcinoma; Adenosine Triphosphate; Antineoplastic Agents, Phytogenic; Boron Compounds; Calcium; Calcium Channels; Calcium Signaling; Cell Line, Tumor; Cytochalasin D; Cytoskeleton; Digitonin; Dose-Response Relationship, Drug; Enzyme Inhibitors; Green Fluorescent Proteins; Humans; Indicators and Reagents; Inositol 1,4,5-Trisphosphate; Ionomycin; Lung Neoplasms; Microscopy, Confocal; Nucleic Acid Synthesis Inhibitors; Paclitaxel; Thapsigargin; Time Factors

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion-selective channel whose dysfunction leads to the onset of cystic fibrosis. CFTR activation is normally elicited by stimulation of the cAMP pathway, which effects protein kinase A activation. However, previous studies from our laboratory indicate that the actin cytoskeleton is also required for a proper CFTR function. In this report, the regulatory role of actin filament organization in the activation of CFTR was explored. Maneuvers to modify the steady-state organization of actin filaments elicit the activation of CFTR in the absence of a functional cAMP pathway. Partial disruption of the actin cytoskeleton of CFTR-expressing cells with cytochalasin D (CD) induced CFTR activation in the absence of an activated PKA. Similar findings were obtained by intracellular dialysis with the actin-severing protein gelsolin. However, extended treatment with CD leading to the collapse of the actin cytoskeleton rendered CFTR completely insensitive to direct PKA activation. cAMP activation of CFTR was also found to be dysfunctional in cells lacking the actin-crosslinking protein ABP-280, which was recovered after dialysis of the cells with filamin, a homologue of ABP-280. The present data indicate that an organized actin network is required for the proper cAMP-dependent activation of CFTR. The possibility is also explored that actin must be directly associated with CFTR to elicit its activation, further suggesting that this channel protein may bind actin as well.

Topics: Actin Cytoskeleton; Adenocarcinoma; Amino Acid Sequence; Animals; Contractile Proteins; Cyclic AMP-Dependent Protein Kinases; Cystic Fibrosis Transmembrane Conductance Regulator; Cytochalasin D; Female; Filamins; Gelsolin; Humans; Mammary Neoplasms, Animal; Melanoma; Mice; Microfilament Proteins; Molecular Sequence Data; Nucleic Acid Synthesis Inhibitors; Protozoan Proteins; Tumor Cells, Cultured

Lipocortin 1 (annexin I) is a calcium- and phospholipid-binding annexin protein which can be externalised from cells despite the lack of a signal sequence. To determine its cellular distribution lipocortin 1 in A549 human lung adenocarcinoma cells was localised by light- and electron-microscopic immunocytochemistry and by cell fractionation and western blotting. Lipocortin 1 immunoreactivity is concentrated in prominent patches associated with the plasma membrane. The intensity of these patches varied with the confluence and duration of the culture and was not detectably diminished by an EDTA wash before fixation. Tubulin and cytokeratin 8 were colocalized with lipocortin 1 in the patches. Within the cells lipocortin 1 was distributed throughout the cytoplasm. Electron microscopy revealed prominent immunoreactivity along the plasma membrane with occasional large clusters of gold particles in contact with the membrane surface of the cells; within the cytoplasm the membrane of some vesicle/vacuole structures and some small electron-dense bodies was immunoreactive, but no immunogold particles were associated with the multilamellar bodies. Subcellular fractionation, extraction and western blotting showed that lipocortin 1 in the membrane pellet was present as two distinct fractions; one, intimately associated with the lipid bilayer, which behaved like an integral membrane protein and one loosely attached which behaved like a peripheral membrane protein. The results show that a substantial amounts of lipocortin 1 is concentrated in focal structures associated with and immediately beneath the plasma membrane. These might form part of the mechanism by which lipocortin 1 is released from the cells.

Topics: Adenocarcinoma; Annexin A1; Biological Transport; Blotting, Western; Cell Compartmentation; Cell Membrane; Cytochalasin D; Cytoplasm; Cytoskeleton; Fluorescent Antibody Technique; Humans; Keratins; Lung Neoplasms; Membrane Proteins; Microscopy, Immunoelectron; Microtubules; Nucleic Acid Synthesis Inhibitors; Tubulin; Tumor Cells, Cultured

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

Changes in lamellipod extension and chemotaxis in response to EGF were analysed for MTLn3 cells (a metastatic cell line derived from the 13762NF rat mammary adenocarcinoma). Addition of EGF produced a cessation of ruffling followed by extension of hyaline lamellipods containing increased amounts of F-actin at the growing edge. A non-metastatic cell line (MTC) derived from the same tumor did not show such responses. Lamellipod extension was maximal within 5 min, followed by retraction and resumption of ruffling. Maximal area increases due to lamellipod extension occurred at about 5 nM EGF. Chemotactic and chemokinetic responses, measured using a microchemotaxis chamber, were also greatest at 5 nM. Cytochalasin D inhibited EGF-stimulated responses including lamellipod extension, increases in F-actin in lamellipods, and chemotaxis. Nocodazole affected chemotaxis at higher concentrations but not EGF-induced lamellipod extension. We conclude that polymerization of F-actin at the leading edges of lamellipods is necessary for extension of lamellipods and chemotaxis of MTLn3 cells in response to EGF. The motility and chemotaxis responses of this metastatic cell line have strong similarities to those seen in well-characterized chemotactic cells such as Dictyostelium and neutrophils.

Topics: Actins; Adenocarcinoma; Animals; Cell Movement; Chemotaxis; Cytochalasin D; Dose-Response Relationship, Drug; Epidermal Growth Factor; Mammary Neoplasms, Experimental; Microtubules; Nocodazole; Pseudopodia; Rats; 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

Protein kinase A (PKA)-activation of epithelial Na+ channels requires actin filaments. Mouse mammary adenocarcinoma cells expressing the human cystic fibrosis transmembrane conductance regulator (CFTR) or mock transfectants were used to determine whether CFTR is also modulated by the actin cytoskeleton. The actin filament disrupter cytochalasin D (CD; approximately 5 micrograms/ml) readily activated whole cell currents in CFTR but not in mock-transfected (MOCK) cells. Addition of actin to the cytosolic side of quiescent excised inside-out patches of CFTR but not MOCK cells also activated CFTR. The actin-activated Cl- channels (symmetrical Cl-) had a linear conductance of 9.3 pS and were inhibited by diphenylamine-2-carboxylate and monoclonal antibodies raised against CFTR. Channel activity was also blocked by addition of the actin-binding proteins deoxyribonuclease I and filamin. Incubation of CFTR cells with CD (approximately 15 micrograms/ml) for > 6 h prevented CFTR activation by the addition of either 8-bromoadenosine 3',5'-cyclic monophosphate plus forskolin under whole cell conditions or PKA under excised inside-out conditions. However, CFTR activation was restored by subsequent addition of actin. The data indicate that CFTR is regulated by actin filaments whose effect may, in turn, be associated with the PKA-dependent pathway.

Topics: Actins; Adenocarcinoma; Amino Acid Sequence; Animals; Carrier Proteins; Cell Line; Chloride Channels; Contractile Proteins; Cyclic AMP; Cystic Fibrosis Transmembrane Conductance Regulator; Cytochalasin D; Cytoskeleton; Filamins; Humans; Kinetics; Mammary Neoplasms, Experimental; Membrane Potentials; Membrane Proteins; Mice; Microfilament Proteins; Molecular Sequence Data; Patch-Clamp Techniques; Recombinant Proteins; Sequence Homology, Amino Acid; Time Factors; Transfection; 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

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