demecolcine and Cell-Transformation--Viral

Adenovirus-transformed cells were tested for their ability to synthesize DNA in the presence of cell cycle inhibitory drugs. We show that transformed cells are completely resistant to the mitotic inhibitor colcemid, partly resistant to lovastatin, mimosine, aphidicolin and genistein but not to hydroxyurea or thymidine. When treated with colcemid, AdE1-transformed cells continue to synthesize DNA but do not divide and, therefore, become highly polyploid. This effect is dependent on the presence of both E1A and E1B.

Topics: Adenovirus E1 Proteins; Animals; Cell Cycle; Cell Line, Transformed; Cell Transformation, Viral; Demecolcine; Mitosis; Polyploidy; Rats

Bovine papillomavirus (BPV) previously has been reported to exist in transformed rodent cell lines as both chromosomally integrated and extrachromosomal forms. In the BPV-transformed mouse cell line ID13, extrachromosomal BPV molecules replicate throughout S phase of the cell cycle in a random choice mode. We report here that these replication properties were altered for chromosomally integrated BPV DNA in five independent ID13 subclones. In all of the subclones, the integrated BPV sequences, which had no detectable deletions or mutations, existed in head-to-tail tandem arrays that replicated once per cell cycle, predominantly late in S phase. In contrast, extrachromosomal BPV molecules present in other subclones of the same cell line replicated in the random choice mode observed previously for non-integrated BPV. Our results indicate that the replication origin of integrated BPV either is inactivated as a consequence of chromosomal insertion, leading to the replication of BPV from origins in the flanking chromosomal DNA, or alternatively is reprogrammed to function in a once-per-cell cycle mode predominantly late in S phase.

Topics: Animals; Blotting, Southern; Bovine papillomavirus 1; Cattle; Cell Cycle; Cell Line, Transformed; Cell Transformation, Viral; Centrifugation, Density Gradient; Chromosomes; Clone Cells; Demecolcine; DNA Probes; DNA Replication; Electrophoresis, Gel, Pulsed-Field; Flow Cytometry; In Situ Hybridization, Fluorescence; Mice; Virus Integration; Virus Replication

The growth characteristics of B lymphocytes infected with Epstein-Barr virus (lymphoblastoid cells) have been investigated by flow cytometric analysis of DNA content and by estimation of cell culture doubling times. It was found that the manipulative procedures involved in the cell cycle analysis resulted in a slowing of the growth rate. This slowing of growth was brought about by the prolongation of cell cycle transit times and by the entry of cells into a short-lived non-cycling pool. The entry of a proportion of the cells into the non-cycling pool may be the normal response of lymphoblastoid cells to non-optimal conditions. The non-cycling cells survived in culture with a T 1/2 of approximately 30-60 hr and continued to secrete immunoglobulin. Their surface transferrin receptors were considerably reduced, which suggests that the failure to divide may have resulted from a failure of growth factor receptors to reach a threshold value following mitosis.

Topics: B-Lymphocytes; Cell Cycle; Cell Division; Cell Transformation, Viral; Cells, Cultured; Demecolcine; DNA; Flow Cytometry; Herpesvirus 4, Human; Humans; Interphase; Kinetics; Receptors, Transferrin

Cultured human fibroblasts showed a typical fibrillar organization of microtubules in immunofluorescence, including the vimentin type of intermediate filament as well as actin-containing microfilaments. During infection with herpes simplex virus type 1 (HSV-1), the vimentin organization was maintained whereas actin, myosin and tubulin showed a progressive association with the viral glycoproteins within juxtanuclear structures. These structures could also be revealed with fluorochrome-coupled wheat germ agglutinin. Disruption of the microtubules by demecolcine treatment or their stabilization by taxol treatment did not prevent the aggregation of viral proteins in the cytoplasm. Taxol stabilization of the microtubules allowed the juxtanuclear accumulation of the glycoproteins in HSV-infected cells whereas treatment with demecolcine led to an accumulation of the glycoproteins either in small vesicles in the cytoplasm or in the focal adhesion areas of the cells. Production of infectious intracellular virus particles was reduced in cells treated with demecolcine or with taxol before and during infection. The results of this study indicate that the normal intracellular transport and distribution of the HSV glycoproteins and the formation of infectious virus are dependent on the presence of intact microtubules.

Topics: Alkaloids; Antibodies, Monoclonal; Cell Transformation, Viral; Cells, Cultured; Cytoskeleton; Demecolcine; Fibroblasts; Fluorescent Antibody Technique; Humans; Microtubules; Paclitaxel; Simplexvirus; Tubulin; Vimentin

Mitogenically stimulated human and mouse lymphocytes enter the cell cycle (G0, G1A, G1B, S, G2+M) via a newly recognized subphase, G1'. This subphase precedes G1A and is distinct from G0. The G1' subphase is absent in immortalized and tumorigenic lymphoblastoid cell lines (LCLs) by cytofluorimetric criteria. Furthermore, colcemid inhibits transition through the G0/G1' as well as G2 phases in mitogen-stimulated lymphocytes and in LCLs. Tumorigenic LCLs are not sensitive to growth inhibition by colcemid during early G1. These observations suggest that a progressive series of changes have occurred during G0/G1' which lead to deregulation of growth control.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Demecolcine; Humans; Interphase; Lymphocyte Activation; Lymphocytes; Mice

We have used a multi-parameter flow-cytometric technique to analyse changes in cell-cycle phase distribution (early and late G1, S and G2+M phases) for normal and X-ray-sensitive (ataxia-telangiectasia, A-T) lymphoblastoid cells exposed to X-irradiation and sodium butyrate (either alone or in combination). Sodium butyrate, an inhibitor of histone deacetylase, is a useful pharmacological tool for determining the proposed role of a histone acetylation-based chromatin surveillance system in controlling cell-cycle responses to DNA damage. We report that X-irradiated A-T cells (acute doses up to 1.5 Gy) demonstrate deficiencies in the capacity to traverse G1 and G2+M phases, although we can find no evidence of the specific involvement of a sodium butyrate-sensitive process in normal cells or abnormalities in the responses of A-T cells to the drug. We conclude that abnormal cellular control of G1 transition in A-T may be the basis of disturbed cellular differentiation in vivo, particularly in non-proliferating tissues under conditions of accumulated environmental or spontaneous DNA damage.

Topics: Ataxia Telangiectasia; Butyrates; Butyric Acid; Cell Cycle; Cell Division; Cell Transformation, Viral; Chromatin; Demecolcine; Deoxyribonucleases; DNA; Flow Cytometry; Herpesvirus 4, Human; Histone Deacetylase Inhibitors; Humans; Lymphocytes; Ribonucleases; RNA; Time Factors

We transformed synchronized mouse L-thymidine kinase deficient cells with a cloned thymidine kinase gene and carrier DNA at different times in the cell cycle. The frequency of resistant colonies was determined after growth in hypoxanthine-aminopterin-thymidine medium for two weeks (late expression) and the frequency of thymidine kinase positive cells was determined 72 hours after transformation (early expression) by incorporation of 3H-thymidine and autoradiography. The frequency of late expression was several-fold higher when cells were exposed to DNA during S-phase, whereas the frequency of early expression did not depend on the phase of the cell cycle.

Topics: Animals; Cell Cycle; Cell Transformation, Viral; Cloning, Molecular; Demecolcine; Genes; Interphase; Kinetics; L Cells; Mice; Simplexvirus; Thymidine Kinase

Topics: Animals; Cell Adhesion; Cell Cycle; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cytochalasin B; Demecolcine; DNA; DNA Replication; Mice; Peptide Hydrolases; Protein Biosynthesis; RNA; RNA, Heterogeneous Nuclear

The drug cytochalasin B (CB), which disrupts the cellular microfilament network, allows the identification of as yet unclassified structural differences between normal and Rous sarcoma virus-transformed chicken embryo fibroblasts. When exposed to CB, normal chick fibroblasts attain an arborized or dendritic morphology. This results as the cytoplasm collapses upon the remaining structural and adhesive components of the cell. Rous sarcoma virus-transformed cells did not form or maintain these dendritic-like processes in the presence of CB and, as a result, rounded up but still remained attached to the substrate. With a temperature-sensitive mutant of Rous sarcoma virus, LA24A, it was possible to show that these effects are completely reversible and dependent on the expression of pp60src. The cytoskeleton in these CB-treated cells was examined by both immunofluorescence and electron microscopy. After exposure to CB, the microfilaments were found to be disrupted similarly throughout both the transformed and the nontransformed cells. In the nontransformed cells arborized by exposure to CB, the extended processes were found to contain intermediate filaments in an unusually high concentration and degree of organization. The distribution of these filaments in the central body of the arborized cells was random. This lower concentration and random distribution was similar to that seen throughout the transformed cells rounded up by exposure to CB. The failure of these transformed cells to arborize in CB indicates that the structural component(s) which is necessary for the formation or maintenance or both of the arborized state is altered by the expression of pp60src.

Topics: Animals; Avian Sarcoma Viruses; Cell Transformation, Viral; Cells, Cultured; Chick Embryo; Cycloheximide; Cytochalasin B; Cytoskeleton; Demecolcine; Fibroblasts; Mutation; Oncogene Protein pp60(v-src); Rats; Skin; Temperature; Viral Proteins

The effects of rabies virus on host cells were studied and compared to those obtained with another rhabdovirus, vesicular stomatitis virus [J. Virol. 34, 777-781 (1980)]. We show here: (1) that rabies infection has no effect on cell morphology, while infection with vesicular stomatitis virus caused cell retraction. Thus, only vesicular stomatitis virus induced a depolymerization of the microfilaments; and (2) that microtubules and microfilaments do not play a major role in rabies virus production, as it is suggested by results obtained with several effectors (colcemid, colchicine and cytochalasin-B) which directly or indirectly affect cytoskeleton organization. The same properties were observed with directly or indirectly affect cytoskeleton organization. The same properties were observed with vesicular stomatitis virus. Furthermore, the use of cytochalasin-B shows that an inhibition of glycosylation of the virion spike protein occurs only in rabies infected cells. As vesicular stomatitis viral glycosylation is normal in cytochalasin-B treated cells, results obtained indicate that two types of interactions can occur between a virion and the host-cell depending on the rhabdovirus type.

Topics: Animals; Cell Line; Cell Transformation, Viral; Chick Embryo; Colchicine; Cricetinae; Cytochalasin B; Cytochalasin D; Cytochalasins; Demecolcine; Kidney; Kinetics; Rabies virus; Vesicular stomatitis Indiana virus; Viral Proteins; Virion; Virus Replication

The rate of appearance, in a newly formed heterokaryon population, of cells bearing completely intermixed mouse and human surface antigens may be used to estimate diffusion constants for antigens on individual cells. From this estimate, it appears that the surface antigens in most cells do not diffuse at the rate expected, but rather move more slowly, by a factor of ten or more, than expected from either measured or calculated diffusion constants for proteins freely mobile in the plane of a lipid membrane. Differences in diffusion rates between cells are not due to effects of Sendai virus, or of trypsin. Restrictions on diffusion are apparently not due to cytochalasin B- or Colcemid-sensitive elements.

Topics: Animals; Antigens, Surface; Cell Line; Cell Membrane; Cell Transformation, Viral; Cytochalasin B; Demecolcine; Diffusion; Humans; Hybrid Cells; Mice; Parainfluenza Virus 1, Human