Effects of vinblastine, podophyllotoxin and nocodazole on mitotic spindles. Implications for the role of microtubule dynamics in mitosis.

Inhibition of mitosis by many drugs that bind to tubulin has been attributed to depolymerization of microtubules. However, we found previously that low concentrations of vinblastine and vincristine blocked mitosis in HeLa cells with little or no depolymerization of spindle microtubules, and spindles appeared morphologically normal or nearly normal. In the present study, we characterized the effects of vinblastine, podophyllotoxin and nocodazole over broad concentration ranges on mitotic spindle organization in HeLa cells. These three drugs are known to affect the dynamics of microtubule polymerization in vitro and to depolymerize microtubules in cells. We wanted to probe further whether mitotic inhibition by these drugs is brought about by a more subtle effect on the microtubules than net microtubule depolymerization. We compared the effects of vinblastine, podophyllotoxin and nocodazole on the organization of spindle microtubules, chromosomes and centrosomes, and on the total mass of microtubules. Spindle organization was examined by immunofluorescence microscopy, and microtubule polymer mass was assayed on isolated cytoskeletons by a quantitative enzyme-linked immunoadsorbence assay for tubulin. As the drug concentration was increased, the organization of mitotic spindles changed in the same way with all three drugs. The changes were associated with mitotic arrest, but were not necessarily accompanied by net microtubule depolymerization. With podophyllotoxin, mitotic arrest was accompanied by microtubule depolymerization. In contrast, with vinblastine and nocodazole, mitotic arrest occurred in the presence of a full complement of spindle microtubules. All three drugs induced a nearly identical rearrangement of spindle microtubules, an increasingly aberrant organization of metaphase chromosomes, and fragmentation of centrosomes. The data suggest that these anti-mitotic drugs block mitosis primarily by inhibiting the dynamics of spindle microtubules rather than by simply depolymerizing the microtubules.

[1]  A. Hyman,et al.  Two different microtubule-based motor activities with opposite polarities in kinetochores , 1991, Nature.

[2]  M. Jordan,et al.  Mechanism of inhibition of cell proliferation by Vinca alkaloids. , 1991, Cancer research.

[3]  T. Mitchison,et al.  Poleward Microtubule Flux in Mitotic Spindles Assembled in Vitro , 2002 .

[4]  T. Mitchison,et al.  Mitotic spindle assembly by two different pathways in vitro , 1991, The Journal of cell biology.

[5]  M. Jordan,et al.  Quantitation of cellular tubulin in microtubules and tubulin pools by a competitive ELISA. , 1991, Journal of immunological methods.

[6]  R. Schimke,et al.  Cell line-specific differences in the control of cell cycle progression in the absence of mitosis. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[7]  D. Beach,et al.  Distinct nuclear and spindle pole body populations of cyclin–cdc2 in fission yeast , 1990, Nature.

[8]  C. Rieder,et al.  Kinetochores capture astral microtubules during chromosome attachment to the mitotic spindle: direct visualization in live newt lung cells , 1990, The Journal of cell biology.

[9]  M. Sheetz,et al.  Localization of cytoplasmic dynein to mitotic spindles and kinetochores , 1990, Nature.

[10]  J. McIntosh,et al.  Cytoplasmic dynein is localized to kinetochores during mitosis , 1990, Nature.

[11]  M. Jordan,et al.  Kinetic analysis of tubulin exchange at microtubule ends at low vinblastine concentrations. , 1990, Biochemistry.

[12]  C. Rieder,et al.  Kinetochores are transported poleward along a single astral microtubule during chromosome attachment to the spindle in newt lung cells , 1990, The Journal of cell biology.

[13]  M. Dorée,et al.  p34cdc2 is located in both nucleus and cytoplasm; part is centrosomally associated at G2/M and enters vesicles at anaphase. , 1989, The EMBO journal.

[14]  A. Murray,et al.  Dominoes and clocks: the union of two views of the cell cycle. , 1989, Science.

[15]  G. Borisy,et al.  Anaphase onset and dephosphorylation of mitotic phosphoproteins occur concomitantly. , 1989, Journal of cell science.

[16]  T. Mitchison,et al.  Polewards microtubule flux in the mitotic spindle: evidence from photoactivation of fluorescence , 1989, The Journal of cell biology.

[17]  M. Schilstra,et al.  The effect of podophyllotoxin on microtubule dynamics. , 1989, The Journal of biological chemistry.

[18]  C. Sellitto,et al.  Distribution of pericentriolar material in multipolar spindles induced by colcemid treatment in Chinese hamster ovary cells. , 1988, Journal of cell science.

[19]  R. Nicklas,et al.  The forces that move chromosomes in mitosis. , 1988, Annual review of biophysics and biophysical chemistry.

[20]  M. Jordan,et al.  Phase dynamics at microtubule ends: the coexistence of microtubule length changes and treadmilling , 1987, The Journal of cell biology.

[21]  H. Sakai,et al.  Redistribution of fluorescently labeled tubulin in the mitotic apparatus of sand dollar eggs and the effects of taxol. , 1987, Cell structure and function.

[22]  M. Gottesman,et al.  Chinese hamster ovary cells. , 1987, Methods in enzymology.

[23]  T. Albrecht,et al.  A radiolabeled monoclonal antibody binding assay for cytoskeletal tubulin in cultured cells , 1986, The Journal of cell biology.

[24]  W. J. Wheeler,et al.  Mitotic inhibition and aneuploidy induction by naturally occurring and synthetic estrogens in Chinese hamster cells in vitro. , 1986, Mutation research.

[25]  L. Wilson,et al.  Kinetics and Steady State Dynamics of Tubulin Addition and Loss at Opposite Microtubule Ends: The Mechanism of Action of Colchicine a , 1986, Annals of the New York Academy of Sciences.

[26]  M. Kirschner,et al.  Sites of microtubule assembly and disassembly in the mitotic spindle , 1986, Cell.

[27]  J. Parry,et al.  Diethylstilboestrol: the binding and effects of diethylstilboestrol upon the polymerisation and depolymerisation of purified microtubule protein in vitro. , 1985, Carcinogenesis.

[28]  R. Nicklas.  Mitosis in eukaryotic cells: an overview of chromosome distribution. , 1985, Basic life sciences.

[29]  E D Salmon,et al.  Tubulin dynamics in cultured mammalian cells , 1984, The Journal of cell biology.

[30]  J. McIntosh,et al.  Spindle microtubule dynamics in sea urchin embryos: analysis using a fluorescein-labeled tubulin and measurements of fluorescence redistribution after laser photobleaching , 1984, The Journal of cell biology.

[31]  M. Kirschner,et al.  Centrosome development in early mouse embryos as defined by an autoantibody against pericentriolar material , 1983, Cell.

[32]  M. Jordan,et al.  Differential radiolabeling of opposite microtubule ends: methodology, equilibrium exchange-flux analysis, and drug poisoning. , 1983, Analytical biochemistry.

[33]  R. Margolis,et al.  Interaction of vinblastine with steady-state microtubules in vitro. , 1982, Journal of molecular biology.

[34]  G. Borisy,et al.  Microtubule-nucleating activity of centrosomes in Chinese hamster ovary cells is independent of the centriole cycle but coupled to the mitotic cycle , 1981, The Journal of cell biology.

[35]  J. Mullins,et al.  Production of large numbers of mitotic mammalian cells by use of the reversible microtubule inhibitor nocodazole. Nocodazole accumulated mitotic cells. , 1980, Experimental cell research.

[36]  P. Schiff,et al.  Taxol stabilizes microtubules in mouse fibroblast cells. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[37]  G. Borisy,et al.  Test of four possible mechanisms for the temporal control of spindle and cytoplasmic microtubule assembly in HeLa cells. , 1980, The Journal of biological chemistry.

[38]  G. Sluder.  Role of spindle microtubules in the control of cell cycle timing , 1979, The Journal of cell biology.

[39]  P. Schiff,et al.  Promotion of microtubule assembly in vitro by taxol , 1979, Nature.

[40]  P. Woollard,et al.  MICROTUBULES , 1978, The Lancet.

[41]  M. De Brabander,et al.  Interaction of oncodazole (R 17934), a new antitumoral drug, with rat brain tubulin. , 1976, Biochemical and biophysical research communications.

[42]  M. De Brabander,et al.  The effects of methyl (5-(2-thienylcarbonyl)-1H-benzimidazol-2-yl) carbamate, (R 17934; NSC 238159), a new synthetic antitumoral drug interfering with microtubules, on mammalian cells cultured in vitro. , 1976, Cancer research.

[43]  H Meiners,et al.  [Effects of temperature]. , 1973, ZWR.

[44]  H. Kato,et al.  Nondisjunction of chromosomes in a synchronized cell population initiated by reversal of Colcemid inhibition. , 1970, Experimental cell research.

[45]  S. Malawista,et al.  MICROTUBULAR CRYSTALS IN MAMMALIAN CELLS , 1969, The Journal of cell biology.

[46]  S. Malawista,et al.  Vinblastine and Griseofulvin Reversibly Disrupt the Living Mitotic Spindle , 1968, Science.

[47]  E. Robbins,et al.  THE CENTRIOLE CYCLE IN SYNCHRONIZED HELA CELLS , 1968, The Journal of cell biology.

[48]  P. Rao,et al.  CHAPTER 17 – Effects of Temperature on the Mitotic Cycle of Normal and Synchronized Mammalian Cells , 1966 .

[49]  E. Robbins,et al.  CHAPTER 18 – Some Macromolecular Characteristics of Synchronized HeLa Cells , 1966 .