demecolcine and Cell-Transformation--Neoplastic

Drosophila neuroblasts are a model system for studying stem cell self-renewal and the establishment of cortical polarity. Larval neuroblasts generate a large apical self-renewing neuroblast, and a small basal cell that differentiates. We performed a genetic screen to identify regulators of neuroblast self-renewal, and identified a mutation in sgt1 (suppressor-of-G2-allele-of-skp1) that had fewer neuroblasts. We found that sgt1 neuroblasts have two polarity phenotypes: failure to establish apical cortical polarity at prophase, and lack of cortical Scribble localization throughout the cell cycle. Apical cortical polarity was partially restored at metaphase by a microtubule-induced cortical polarity pathway. Double mutants lacking Sgt1 and Pins (a microtubule-induced polarity pathway component) resulted in neuroblasts without detectable cortical polarity and formation of "neuroblast tumors." Mutants in hsp83 (encoding the predicted Sgt1-binding protein Hsp90), LKB1, or AMPKα all show similar prophase apical cortical polarity defects (but no Scribble phenotype), and activated AMPKα rescued the sgt1 mutant phenotype. We propose that an Sgt1/Hsp90-LKB1-AMPK pathway acts redundantly with a microtubule-induced polarity pathway to generate neuroblast cortical polarity, and the absence of neuroblast cortical polarity can produce neuroblast tumors.

Topics: AMP-Activated Protein Kinase Kinases; AMP-Activated Protein Kinases; Animals; Animals, Genetically Modified; Brain; Cell Cycle Proteins; Cell Polarity; Cell Transformation, Neoplastic; Demecolcine; Drosophila melanogaster; Drosophila Proteins; Guanine Nucleotide Dissociation Inhibitors; Heat-Shock Proteins; Larva; Microscopy, Confocal; Microtubules; Molecular Chaperones; Mutation; Neural Stem Cells; Protein Kinases; Signal Transduction; Tubulin Modulators

Tetraploid cells with unstable chromosomes frequently arise as an early step in tumorigenesis and lead to the formation of aneuploid cells. The mechanisms responsible for the chromosome instability of polyploid cells are not fully understood, although the supernumerary centrosomes in polyploid cells have been considered the major cause of chromosomal instability. The aim of this study was to examine the integrity of mitotic spindles and centrosomes in proliferative polyploid cells established from normal human fibroblasts. TIG-1 human fibroblasts were treated with demecolcine (DC) for 4 days to induce polyploidy, and the change in DNA content was monitored. Localization of centrosomes and mitotic spindles in polyploid mitotic cells was examined by immunohistochemistry and laser scanning cytometry. TIG-1 cells treated with DC became almost completely tetraploid at 2 weeks after treatment and grew at the same rate as untreated diploid cells. Most mitotic cells with 8C DNA content had only two centrosomes with bipolar spindles in established tetraploid cells, although they had four or more centrosomes with multipolar spindles at 3 days after DC treatment. The frequency of aneuploid cells increased as established tetraploid cells were propagated. These results indicate that tetraploid cells that form bipolar spindles with two centrosomes in mitosis can proliferate as diploid cells. These cells may serve as a useful model for studying the chromosome instability of polyploid cells.

Topics: Aneuploidy; Cell Culture Techniques; Cell Proliferation; Cell Transformation, Neoplastic; Cells, Cultured; Centrosome; Chromosomal Instability; Demecolcine; DNA; Fibroblasts; Humans; Spindle Apparatus; Tetraploidy

Pentaploid H1 (ES) cells (5H1 cells) were accidentally obtained through one-cell cloning of octaploid H1 (ES) cells (8H1 cells) that were established from tetraploid H1 (ES) cells (4H1 cells) polyploidized using demecolcine. The number of chromosomes of 5H1 cells was 100, unlike the 40 of diploid H1 (ES) cells (2H1 cells), 80 of 4H1, and 160 of 8H1 cells. The durations of G(1), S, and G(2)/M phases of 5H1 cells were 3, 7, and 6 h, respectively, almost the same as those of 2H1, 4H1, and 8H1 cells. The cell volume of 5H1 cells was half of that of 8H1 cells, suggesting that 5H1 cells were created through abnormal cell divisions of 8H1 cells. The morphology of growing 5H1 cells was a spherical cluster similar to that of 2H1 cells and differing from the flagstone-like shape of 4H1 and 8H1 cells. Pentaploid solid tumors were formed from 5H1 cells after interperitoneal injection into the mouse abdomen, and they contained endodermal, mesodermal, and ectodermal cells as well as undifferentiated cells, suggesting both that the DNA content of 5H1 cells was retained during tumor formation and that the 5H1 cells were pluripotent. The DNA content of 5H1 cells was stable in long-term culturing as 2H1 cells, meaning that 5H1 and 2H1 cells shared similarities in DNA structure. The excellent stability of the DNA content of 5H1 cells was explained using a hypothesis for the DNA structure of polyploid cells because the pairing of homologous chromosomes in 5H1 cells is spatially forbidden.

Topics: Animals; Antineoplastic Agents, Phytogenic; Cell Culture Techniques; Cell Differentiation; Cell Division; Cell Lineage; Cell Transformation, Neoplastic; Cells, Cultured; Chromosomal Instability; Chromosome Aberrations; Demecolcine; DNA; Gene Expression Regulation, Neoplastic; Mice; Neoplasms; Pluripotent Stem Cells; Polyploidy; Stem Cell Transplantation

The behaviour of epitheliocytes, their transformed analogues, and fibroblasts was studied on special culture substrates--lattices with large square openings (the area of an opening was 2000 microm2). It was shown that normal epithelocytes and fibroblasts initially attached to and spread on the lattice bars, were soon displaced into the lattice openings and appeared to be "sagged" in the substrate-free spaces. The cells remained attached to the bars only by their edges (epitheliocytes) or lateral processes (fibroblasts), whereas basal surfaces of the cells had no contacts with the substrate. Displacement of the cells from the bars into the lattice openings was observed only if during spreading the cell body was located on two perpendicular bars. In this position the cell body underwent bending which presumably induced stretching of the cell and its displacement into the opening. Unlike epitheliocytes, which gradually "covered" the lattice openings completely, the fibroblasts were retracted and elongated upon their displacement, "crossing" the openings by their bodies and processes. The epitheliocytes transformed by the ras oncogene and displaying a fibroblast-like shape, most often remained on the bars and were not displaced into the lattice openings. Induction of the epithelioid phenotype in fibroblasts by the agents, depolymerizing (colcemid) or disintegrating (taxol) the cytoskeletal system of microtubuli, was accompanied by a change in the behaviour of the cells: the treated fibroblasts, like epitheliocytes, acquired the ability to "cover" the lattice openings. Possible mechanisms of the cell reactions to the substrate having discontinuous configuration are discussed. It is supposed that these distinctions in reactions of epitheliocytes and fibroblast-like cells may result from different bending ability of the cells and/or differences between forces responsible for the cell adhesion to the lattice bars and forces stretching the cells over the lattice openings.

Topics: Animals; Cell Adhesion; Cell Culture Techniques; Cell Line, Transformed; Cell Movement; Cell Size; Cell Surface Extensions; Cell Transformation, Neoplastic; Cells, Cultured; Culture Media; Cytoskeleton; Demecolcine; Epithelial Cells; Extracellular Matrix; Fibroblasts; Fibronectins; Focal Adhesions; Genes, ras; Humans; Mice; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Microscopy, Video; Microtubules; Paclitaxel; Phenotype; Rats

To understand better the role of the microtubular system in the development and maintenance of morphological organization of nonpolarized and polarized cells of the same origin we examined the effects of two microtubule-specific drugs, colcemid and taxol, on discoid cultured epithelial rat cells of the IAR-2 line and on polarized cells obtained from this line by transfection of mutated N-ras oncogene; morphometric, immunomorphologic, and videomicroscopic methods were used. Depolymerization of microtubules by colcemid did not cause major changes in the discoid shape of IAR cells but altered organization of actin cortex; in particular, it led to disappearance of circumferential bundle of actin microfilaments. Taxol reorganized the normal network of microtubules radiating from the perinuclear centers into numerous arrays of short microtubules not associated with any centers. Taxol-treated cells had wider circumferential bundles of microfilaments than control cells and morphometric analysis showed that their contours were closer to geometric circle than those of control or of colcemid-treated cells. These data show that function of the microtubular system is essential for maintenance of the characteristic morphological organization of discoid cells; we propose to name this function "contra-polarization." Contra-polarization is not prevented and is even promoted by taxol; this result suggests that a decentralized system of microtubules is sufficient for this function. In contrast, maintenance of polarized morphology of IAR-2 cells transfected by the N-ras oncogene is inhibited not only by colcemid but also by taxol and thus requires the presence of a normal centralized microtubular system.

Topics: Animals; Cell Polarity; Cell Transformation, Neoplastic; Cells, Cultured; Cytoskeleton; Demecolcine; Epithelium; Genes, ras; In Vitro Techniques; Microtubules; Paclitaxel; Rats; Transfection; Video Recording

Cytogenetic changes were investigated during the spontaneous progression of CHEF18 Chinese hamster cells towards tumorigenicity. We further report the chromosomal characterization of a series of spontaneous anchorage-independent clones, as well as of a series of tumor-derived cell lines resulting from injection of late passage cells in nude mice. The high karyotypic homogeneity (presence of four marker chromosomes strictly associated in all the metaphases analyzed) in all clones and tumor-derived cell lines prompted us to alter the specific pattern of chromosomal aberrations in order to identify which if any of the aberrations were more strictly related to transformation. For this purpose we treated a tumor-derived cell line with Colcemid and analyzed the reversion of anchorage-independent phenotype in the subclones showing an altered association of the four marker chromosomes. We conclude that two of four marker chromosomes contribute to anchorage independence.

Topics: Animals; Cell Transformation, Neoplastic; Chromosome Aberrations; Cricetinae; Cricetulus; Demecolcine; Karyotyping; Tumor Cells, Cultured; Tumor Stem Cell Assay

How important are the changes of microtubule control for the realization of actin cortex changes during neoplastic transformation? To answer this question we studied the actin cytoskeleton and intermediate filaments condition after colcemid destruction or taxol disintegration of microtubule system in non-transformed cells BALB/c 3T3 and in the same cells transformed by Ha-ras gene. We have come to a conclusion that the differences between non-transformed and transformed cells in the actin cytoskeleton organization remain the same after specific inhibitor action on the microtubules; after the microtubules are destroyed the differences between the two cell types appear in the intermediate filament organization; there are reasons to assume that changes in the actin cortex structure may play the central role in morphological transformation expression.

Topics: Actins; Animals; Cell Transformation, Neoplastic; Clone Cells; Cytoskeleton; Demecolcine; Fibroblasts; Genes, ras; Intermediate Filaments; Mice; Mice, Inbred BALB C; Microtubules; Paclitaxel; Transfection

This study shows that artificial increase in cell site leads to morphological normalization of transformed fibroblasts. Mouse L cells (clone 171/5) were used. As most transformed cells, they were poorly spread on the substratum, made only dot-like focal contacts with it, rounded quickly at room temperature and did not contain prominent actin cables. Giant cells were obtained by incubation of these cells in the medium supplemented with mitomycin C (0.15-0.20 mcg/ml). DNA synthesis and mitosis were blocked by this treatment, while protein synthesis was changing very slightly. As a consequence, the cell size increased dramatically from 3 to 11 days of the cell incubation in the mitomycin containing medium. The degree of cell spreading per mcg of protein increased significantly in the giant cells. These cells do not round after moderate cooling, and well developed system of actin cables and matured streak-like focal contacts associated with these cables are formed in them. These results, along with our previous data on the restoration of cell spreading and cytoskeleton structure in giant multinucleated cells, provide strong evidences that the increase in cell size per se can induce qualitative changes in cell morphology. It can be suggested that there are some scaling-dependent factors regulating the processes of cytoskeleton assembly and formation of cell-substrate contacts.

Topics: Animals; Cell Line, Transformed; Cell Transformation, Neoplastic; Cytoskeleton; Demecolcine; DNA, Neoplasm; L Cells; Mice; Mitomycin; Temperature; Time Factors; Tumor Cells, Cultured

The effects of doxorubicin (adriamycin, ADR) and daunorubicin (daunomycin, DAU), two anthracyclinic antibiotics, on a human breast carcinoma cell line (CG5) were studied by cytochemical and morphological methods. Both ADR and DAU were capable of inducing the multinucleation and spreading phenomena, associated with a decrease of the cell growth rate. DAU appeared to be more effective than ADR at the tested concentrations (10(-5), 5 x 10(-5) mM), in affecting the cell growth as well as in inducing multinucleation. As revealed by scanning electron microscopy, spreading and multinucleation were accompanied by a remarkable redistribution of surface structures. Moreover, a dose- and time-dependent rearrangement of the underlying cytoskeletal components was clearly detected. In addition, both ADR and DAU at 5 x 10(-5) mM seemed to favor the rebuilding of microtubules after treatment with colcemid, while a higher dose (10(-4) mM) exerted the opposite effect. Furthermore, both anthracyclines prevented the action of the antimicrotubular agent. When recovered after treatment with cytochalasin B, in presence of ADR (or DAU) (5 x 10(-5), 10(-4) mM), cells showed a microfilament pattern rearranged differently as compared to that of cells recovered in anthracycline-free medium. The results reported here strongly suggest the involvement of actin and tubulin in CG5 cell response to ADR and DAU treatments. Thus, the cytoskeletal apparatus is confirmed as another target involved in the mechanism of action of anthracyclines.

Topics: Actins; Antibiotics, Antineoplastic; Breast Neoplasms; Cell Transformation, Neoplastic; Cytochalasin B; Cytoskeleton; Daunorubicin; Demecolcine; Doxorubicin; Humans; Microscopy, Electron; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Tubulin; Tumor Cells, Cultured

A pentapeptide, pyroGlu-Glu-Asp-Ser-GlyOH, was previously isolated from mouse skin and shown to inhibit reversibly proliferation of murine keratinocytes, both in intact skin and in culture. In the present report we have shown that proliferation of normal human keratinocytes in culture is also inhibited by the pentapeptide, whether the cells are grown under standard conditions or prevented from stratifying in medium containing a low concentration of calcium ions. An SV40-transformed line of human keratinocytes, SVK14, was completely insensitive to the pentapeptide and a line derived from a squamous cell carcinoma of the oral cavity, SCC-9, was less sensitive than the normal cells. The effect of the pentapeptide on terminal differentiation was determined by measuring the proportion of cells expressing involucrin after a single dose or repeated doses of the pentapeptide: no consistent change in the number of terminally differentiating cells was observed. Cell extracts and conditioned medium from all three cell types contained the epidermal pentapeptide, suggesting a role for the peptide in both autocrine (normal keratinocytes) and paracine (SVK14, SCC-9) growth control. A proportion of the pentapeptide isolated from conditioned medium was phosphorylated; since it was as active as the non-phosphorylated form, when assayed on mouse epidermis, the role of phosphorylation remains to be determined.

Topics: Amino Acid Sequence; Cell Differentiation; Cell Transformation, Neoplastic; Cells, Cultured; Demecolcine; DNA Replication; Growth Inhibitors; Humans; Infant, Newborn; Keratinocytes; Kinetics; Male; Mitosis; Mitotic Index; Molecular Sequence Data; Oligopeptides; Protein Precursors; Pyrrolidonecarboxylic Acid; Thymidine

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

The role of the cytoskeleton in morphological normalization of transformed cells was studied. Mouse cells of the L197/6 clonal line were fused by polyethylene glycol and replated. The multinuclear cells were more spread than control ones: the ratio of the cell-occupied area to the number of the nuclei increased 2-3 times as a result of multinucleation. Instead of the spindle-like morphology typical for control cells they became star-like with larger lamellar regions located between radially oriented cell processes. According to the immunofluorescent data these processes contained thick bundles of microtubules and intermediate filaments. Destruction of these bundles with colcemide led to a decrease in the area occupied by multinuclear cells but did not change significantly the area occupied by control cells. The role of microtubules and intermediate filaments in cell spreading is discussed.

Topics: Animals; Cell Fusion; Cell Nucleus; Cell Transformation, Neoplastic; Clone Cells; Cytoskeleton; Demecolcine; Fluorescent Antibody Technique; L Cells; Mice; Microtubules

Multinucleation of various cultured cells was produced by polyethylene glycol-induced fusion or by cytochalasin-induced block of mitosis. It was found that multinucleation induced by both methods considerably improved deficient spreading of all the tested transformed fibroblastic lines; average substratum area occupied by one cell and divided per number of nuclei was 2.0-2.5 times larger for multinucleated cells than for mononucleated ones. Improved spreading was accompanied by increased area of lamellar cytoplasm, increased number of focal contacts, and, in certain lines, by the appearance of actin bundles; numerous microtubules and intermediate filaments radiated from perinuclear zones into the lamellas of multinucleated cells. The number of cell-associated fibronectin fibrils was not increased by multinucleation. Cycloheximide did not prevent the improvement of spreading, suggesting that this effect was not due to any alterations of protein synthesis. Colcemid considerably decreased the effect of multinucleation but did not abolish it completely. It is suggested that increase of spreading is due to multinucleation-associated alterations of quantitative interrelationships between various cell components. One of these alterations is probably increased density of microtubules per unit length of outer cell edge.

Topics: Animals; Avian Sarcoma Viruses; Cell Line; Cell Nucleus; Cell Transformation, Neoplastic; Cells, Cultured; Cricetinae; Culture Media; Cycloheximide; Demecolcine; L Cells; Mice; Quail

Treatment of Syrian hamster embryo cells in culture with 0.01-0.1 micrograms/ml of colcemid for 48 h resulted in morphological and neoplastic transformation of the cells. Cell transformation was observed with doses which were non-cytotoxic and did not cause mitotic inhibition of the cells. Higher dose of colcemid (greater than 0.1 microgram/ml) resulted in mitotic inhibition of the cells and a significant loss of colony forming ability, but no increase in the frequency of morphological transformation. Treatment of the cells with transforming doses of colcemid did not result in any measurable induction of gene mutations or structural chromosome aberrations; however, numerical chromosome changes in the treated cells were observed. A 14-fold increase in the number of aneuploid cells with a near diploid chromosome complement was found in cultures treated with 0.1 micrograms/ml colcemid and both chromosome loss and gain were induced. The dose response curves for colcemid induced morphological transformation and aneuploidy were similar. These results are consistent with a role of carcinogen-induced chromosome non-disjunction in carcinogenesis.

Topics: Aneuploidy; Animals; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Cricetinae; Demecolcine; Dose-Response Relationship, Drug; Spindle Apparatus

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

Topics: Animals; Cell Transformation, Neoplastic; Chromosome Aberrations; Clone Cells; Demecolcine; Fibroblasts; Ploidies; Rats

When primary baby mouse skin (BMS) cultures were subcultured for 48 hours into media containing 10(-6) to 10(-7) M colchicine or demecolcine, the number of altered cell foci appearing after 3-4 weeks' maintenance at 36 degrees C was substantially enhanced over drug-free controls. This applied whether or not the primary cultures had been irradiated with white fluorescent light. The additional presence of cytochalasin D and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) sometimes improved and sometimes partly suppressed the enhancing effect of the antitubulin drugs, and these drugs were omitted for reproducible focus enhancement. The enhancement depended on passage through DNA synthesis in presence of colchicine, which did not prevent concurrent or subsequent DNA synthesis but induced a substantial proportion (greater than 33%) to replicate in the tetraploid (4n to 8n) chromosome configuration. Another effect was to induce widespread asymmetric nuclear division, allowing the potential for chromosome loss. All these effects occurred within the first one or few cell cycles after removal of the antitubulin drugs. The results suggest that the generation of tetraploidy perhaps followed by chromosome loss may be an important factor in the rapid induction of altered cell foci. Pre-existing DNA damage is another important factor.

Topics: Animals; Cell Transformation, Neoplastic; Cells, Cultured; Colchicine; Cytochalasin D; Cytochalasins; Demecolcine; DNA; Light; Mice; Oxygen; Polyploidy; Skin; Tetradecanoylphorbol Acetate

The Chinese hamster ovary (CHO) cell, like other transformed cells, has lost the fibronectin deposit around its membrane. Treatment with cyclic AMP derivatives restores the typical fibroblastic deposit of fibronectin. Thus, the reverse transformation process induced by cyclic AMP (cAMP) in the CHO cell restores this important property as well as other morphological, biochemical, and growth behavioral characteristics of the normal fibroblastic state. The fibronectin deposit occurs significantly later in time than do other characteristics of the reverse transformation reaction and may therefore reflect a metabolic action that requires other cAMP effect to precede it. The restoration of fibronectin deposition in response to cAMP derivatives is also exhibited by vole cells transformed by avian sarcoma virus, but it is not by HeLa cell. Addition of Colcemid, which disrupts microtubules, to CHO cells containing a fibronectin deposit induced by cAMP derivatives causes little or no erosion of the deposit, but cytochalasin B, which disrupts 5-nm microfilaments, eliminates it completely. Thus, various features of the action of cAMP derivatives on CHO and related cells require integrity of the cellular microfibrils--in some cases microtubules only, in some cases 5-nm microfilaments only, and in some cases both classes of fibrils.

Topics: Animals; Bucladesine; Cell Line; Cell Transformation, Neoplastic; Cricetinae; Cyclic AMP; Cytochalasin B; Cytoskeleton; Demecolcine; Extracellular Space; Female; Fibronectins; Fluorescent Antibody Technique; Microtubules

The statmokinetic reaction of malignant-transformed cells of a hamster tumor (CHT-1) was induced by colcemid. The reversibility of the reaction is primarily related to the repolymerization of spindle microtubules from the tubuline pool of the precursors. The character of the statmokinetic reaction reversibility in the test cells evidences the activity of polymerization processes in them. The course of these processes in the presence of copper ions did not differ from that in nonmalignant-transformed cells.

Topics: Animals; Animals, Newborn; Cell Line; Cell Transformation, Neoplastic; Copper; Cricetinae; Demecolcine; Kinetics; Microtubules; Mitosis; Sarcoma, Experimental; Time Factors

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Demecolcine; Fibroblasts; Mice; Microtubules; Pseudopodia

Seven different transformation stigmata of the transformed CHO cell line, including morphological characteristics, growth behavior, cell membrane biochemical properties, and failure of fibronectin deposition, are reversed by addition of cAMP derivatives to the medium. Simultaneously the microtubular pattern changes from a sparse, relatively random set to an orderly arrangement of tubules largely parallel to each other and to the long axis of the resulting fibroblastic cell. Agents like colcemid and cytochalasin B, respectively disorganizing microtubular and particular microfilamentous structures, prevent at least certain aspects of the reverse transformation reaction induced by cAMP in interphase cells. It is proposed that malignant transformation can be effected by damage to the microtubular and microfilamentous structures which changes cell constitution and behavior in two ways: (1) chromosomal instability is introduced which promotes continuous selection for variants better able to resist environmental signals to limit reproduction and (2) a variety of metabolic defects in biochemical processes such as specific membrane functions are introduced which may alter the growth responses of the cell. This picture offers a reasonable explanation for a number of aspects of normal and malignant cell behavior.

Topics: Animals; Cell Line; Cell Membrane; Cell Transformation, Neoplastic; Cricetinae; Cricetulus; Cyclic AMP; Cytochalasin B; Demecolcine; Female; Fibronectins; Microtubules; Ovary

Transformed cells often display knobs (or blebs) distributed over their surface throughout most of interphase. Scanning electron microscopy (SEM) and time-lapse cinematography on CHO-K1 cells reveal roughly spherical knobs of 0.5-4 micron in diameter distributed densely around the cell periphery but sparsely over the central, nuclear hillock and oscillating in and out of the membrane with a period of 15-60 sec. Cyclic AMP derivatives cause the phenomenon of reverse transformation, in which the cell is converted to a fibroblastic morphology with disappearance of the knobs. A model was proposed attributing knob formation to the disorganization of the jointly operating microtubular and microfilamentous structure of the normal fibroblast. Evidence for this model includes the following: 1) Either colcemid or cytochalasin B (CB) prevents the knob disappearance normally produced by cAMP, and can elicit similar knobs from smooth-surfaced cells; 2) knob removal by cAMP is specific, with little effect on microvilli and lamellipodia; 3) immunofluorescence with antiactin sera reveals condensed, amorphous masses directly beneath the membrane of CB-treated cells instead of smooth, parallel fibrous patterns of reverse-transformed cells or normal fibroblasts; 4) transmission electron microscopy (TEM) of sections show dense, elongated microfilament bundles and microtubules parallel to the long axis of the reverse-transformed CHO cell, but sparse, random microtubules throughout the transformed cell and an apparent disordered network of 6-nm microfilaments beneath the knobs; 5) cell membranes at the end of telophase, when the spindle disappears and cleavage is complete, display typical knob activity as expected by this picture.

Topics: Animals; Bucladesine; Cell Cycle; Cell Line; Cell Membrane; Cell Transformation, Neoplastic; Cricetinae; Cricetulus; Cytochalasin B; Cytoskeleton; Demecolcine; Female; Lung; Microscopy, Electron, Scanning; Microtubules; Ovary

Previous studies have shown that the cell surface expression of thymus leukemia antigen (TL) on ASL-1w leukemia cells varies with the progression of the cells through the growth cycle. Expression of TL is maximal in S phase, and its quantitative expression varies directly with DNA synthesis. In the present study, the effect of anti-TL serum on the growth of ASL-1w cells was examined. The antiserum, tested in the absence of complement, affected the growth of these cells in biphasic manner. When the antiserum concentration was 0.1% or greater, there was a rapid decline in the rate of DNA synthesis, and after 5 to 7 hr, cell death. When the antiserum concentration was 0.067% or less, the decline in the rate of synthesis of DNA did not become apparent until 5 to 6 hr after treatment. Under these conditions, there was approximately a 20% increase in cell number after 24 hr of culture. The hypothesis that treatment of ASL-1w cells with the lesser concentration of anti-TL serum blocks the cells in G2 phase of the cell cycle is discussed.

Topics: Animals; Antigens, Neoplasm; Cell Transformation, Neoplastic; Complement System Proteins; Demecolcine; DNA; Guinea Pigs; Immune Sera; Leukemia, Experimental; Mice; Mitosis; Thymus Gland

By a further development of the technique for time-lapse filming of organ cultures, it has become possible to observe the internal structure of the living tissues and to study the dynamics of intercellular contacts within the tissue. This method has been applied to a study of the interaction between the tissues of the chick chorioallantoic membrane and non-malignant fibroblasts, polyoma virus-transformed fibroblasts, and Harding-Passey melanoma cells. The behaviour of these cells in the absence and in the presence of colcemid has been investigated. From these studies it is concluded that disturbances in cell shape brought about by colcemid do not affect the invasive properties whereas the surface polypodial activity shown by the malignant cells is a major factor in tumour invasion.

Topics: Cell Membrane; Cell Movement; Cell Physiological Phenomena; Cell Transformation, Neoplastic; Demecolcine; Neoplasm Invasiveness; Organ Culture Techniques; Photography; Pseudopodia; Time Factors

The frequency of colcemid-induced genome mutations (aneuploidy and polyploidy) in normal and SV40-transformed cultures of Djungarian hamster embryonic cells was studied. Genome mutations were easily induced by the drug in transformed but not in normal cultures. Elevation of colcemid concentration and prolongation of the incubation period did not substantially increase the frequency of genome mutations in normal cells. An attempt was made to study the causes of the differences in sensitivity to colcemid-mutagenicity of normal and transformed cells. Transformed cells did not include more 3H-colchicine than normal cells, and binding of the drug to cell homogenates was similar in both kinds of cultures. According to these data the higher sensitivity of transformed cells to colcemid is not connected either with increased permeability of the cell to the drug or with changes in its binding to tubulin.

Topics: Aneuploidy; Animals; Cell Transformation, Neoplastic; Cells, Cultured; Colchicine; Cricetinae; Demecolcine; Fibroblasts; In Vitro Techniques; Mutation; Polyploidy; Simian virus 40; Stimulation, Chemical

The two-electrode method of input resistance measuring was shown to give lower values of resistance compared with the one-electrode balance method. Gradual growth of estimates of input resistance and membrane potential in the cause of experiment is associated with decrease in the shunting effect of the electrode puncture. The mean values of steady Rin of sparse and confluent culture did not differ when the cells were electrically disconnected (38 plus or minus 1.2 MO and 41 plus or minus 1.8 MO respectively) and differed in electrically coupled fibroblasts (54 plus or minus 4.9 MO --sparse; 29 plus or minus 2.2 MO -- confluent culture). This difference is thought to be associated with shunting effect of electrical connections. The specific resistance of membrane was estimated in normal and transformed fibroblasts.

Topics: Animals; Cell Transformation, Neoplastic; Culture Techniques; Cytological Techniques; Demecolcine; Electric Conductivity; Electrophysiology; Fibroblasts; L Cells; Membrane Potentials; Mice; Microelectrodes; Oscillometry

The effect of the non-ionic detergent tween 80 on one colcemid-resistant and one sensitive subline of mouse L cells has been studied. The colcemid-resistant subline was also resistant to colchicine and Vinca alkaloids. Tween 80 at concentrations of 0.01% (v/v) or higher increased the sensitivity of drug-resistant cells to the antimitotic effect of colcemid, colchicine and vinblastine. Tween 80 also potentiated the initial rate and the maximal level of the (3-H)-cholchicine uptake by both sensitive and resistant cells. However, the detergent at concentrations of up to 1% had no effect on (3-H)-colchicine binding by cell homogenates. Thus it appears that the effects of tween 80 were due to an increase in cell membrane permeability to the drugs. The effect was completely reversible. The cells did not become adapted to the sensitizing action of tween 80 even after prolonged incubation in medium containing the detergent. A considerable increase in the permeability to the drug was obtained with doses of tween 80 which were non-toxic and which had no effect on cell proliferation, morphology and locomotion. The increase in membrane permeability caused by tween 80 at these concentrations was selective, the membrane permeability to ions and to (3-H)-2-deowy-D-glucose being, in fact, unchanged.

Topics: Animals; Cell Membrane Permeability; Cell Transformation, Neoplastic; Colchicine; Demecolcine; Deoxyglucose; Drug Interactions; L Cells; Membrane Potentials; Mice; Mitosis; Polyethylene Glycols; Polysorbates; Stimulation, Chemical; Temperature; Time Factors; Vinca Alkaloids

Stationary cultures of the mouse transformed cells L and S-40 sensitive to topoinhibition were found to be insensitive to the action of hyaluronidase, RNAase, and colcemid in doses known to stimulate multiplication of normal mouse fibroblasts. These cultures were still insensitive to the action of medium change and removal of a part of the monolayer.

Topics: Animals; Cell Transformation, Neoplastic; Demecolcine; DNA; DNA, Neoplasm; Fibroblasts; Hyaluronoglucosaminidase; L Cells; Mice; Ribonucleases

Topics: Animals; Cell Division; Cell Line; Cell Transformation, Neoplastic; Chromatin; Demecolcine; Deoxycytidine; DNA-Directed RNA Polymerases; DNA, Viral; Epithelium; Escherichia coli; Isotope Labeling; Liver; Mitosis; Nucleic Acid Hybridization; Rats; RNA, Viral; Simian virus 40; Thymidine; Time Factors; Transcription, Genetic; Tritium; Uridine

Topics: Adenocarcinoma; Aneuploidy; Biopsy; Carcinoma, Squamous Cell; Cell Division; Cell Movement; Cell Transformation, Neoplastic; Demecolcine; Diploidy; Ear Neoplasms; Humans; Karyotyping; Lymphoma; Mitosis; Nose Neoplasms; Pharyngeal Neoplasms

Mitotic, nonmalignant Balb/c 3T3 cells exhibit endogenous, surface galactosyltransferase activity that does not require intercellular contact throughout the assay period. In this respect, mitotic 3T3 cells resemble malignant Balb/c 3T12 cells which similarly show no contact requirement for optimum transferase activity in any phase of their cell cycle. Previously, it was shown that randomly growing populations of 3T3 cells have lower galactosyltransferase activity when assayed under conditions which decreased cell contact. This led to the conclusion that these normal (3T3) and malignant (3T12) cells differed in that intercellular contact is required for optimum activity of surface galactosyltransferases on the normal cell type. The present data indicate that mitotic 3T3 cells may be capable of expressing enzyme activities exhibited at all times by malignant cells. That is, mitotic 3T3 cells and randomly growing 3T12 cells may readily catalyze galactosyltransferase reactions between enzymes and acceptors on the same cell. Interphase 3T3 cells, on the other hand, might require that enzymes glycosylate acceptors on adjacent cells. A model is proposed that suggests that changes in the spatial arrangement of surface enzymes and acceptors or variations in the fluidity of the cell membrane can account for this contact-related glycosylation.

Topics: Animals; Autoradiography; Carbon Radioisotopes; Cell Division; Cell Line; Cell Membrane; Cell Transformation, Neoplastic; Contact Inhibition; Demecolcine; Galactose; Glucosamine; Hexosyltransferases; Mice; Mice, Inbred BALB C; Mice, Inbred Strains; Mitosis; Time Factors; Tritium; Trypsin; Uridine Diphosphate Sugars

Topics: Acetates; Animals; Cell Adhesion; Cell Movement; Cell Transformation, Neoplastic; Cells, Cultured; Chelating Agents; Cytochalasins; Demecolcine; Fibroblasts; Glycols; Kinetics; Mathematics; Mice; Potassium; Procaine; Simian virus 40; Sodium; Time Factors

Topics: Animals; Antineoplastic Agents; Cell Line; Cell Membrane Permeability; Cell Transformation, Neoplastic; Cricetinae; Demecolcine; Embryo, Mammalian; Fibroblasts; HeLa Cells; Humans; Male; Mice