Key Event Title
|Level of Biological Organization|
Key Event Components
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP|
|Tubulin binding and aneuploidy||KeyEvent|
|Homo sapiens||Homo sapiens||Moderate||NCBI|
|Xenopus laevis||Xenopus laevis||Moderate||NCBI|
|All life stages||High|
Key Event Description
Microtubules are polar structures, and in each filament, subunits are added to one extremity (the plus end) and removed from the other one (the minus end) [reviewed in Marchetti et al. 2016]. Microtubules are dynamic structures characterized by features such as dynamic instability and treadmilling. Dynamic instability defines the ability of microtubules to grow or shorten [Mitchison & Kirschner, 1984; Wade and Hyman, 1997]; the process is based on a multitude of events regulating the assembly/disassembly of the subunits. Treadmilling is the process by which, in the presence of an active loss of subunits (at the minus end) and acquisition of subunits (at the plus end), a steady-state is maintained, and the length of the microtubule remains unchanged [Waterman-Sloter and Salmon, 1997]. Microtubule dynamics can be affected as a result of microtubule depolymerization or microtubule stabilization.
How It Is Measured or Detected
Microtubule depolymerization is generally assessed by an acellular tubulin polymerization assay [Salmon et al., 1984; Wilson et al., 1984; Wallin and Hartley-Asp, 1993; Ibanez et al., 2003; Liu et al., 2010]. A reaction mixture containing tubulin and a test agent, after preincubation, is chilled on ice. GTP is added, and turbidity development is followed at 350 nm in a temperature-controlled recording spectrophotometer. The extent of the reaction is then measured and the area under the curve is used to determine the concentration that inhibited tubulin polymerization by 50% (IC50) [Hamel, 2003]. A concentration of 2.5 μM of colchicine is needed to inhibit microtubule polymerization by 50% [Zavala et al., 1980] and the ability of new chemicals to induce this effect is benchmarked against this value (e.g., combretastatin A-4 IC50 is 1.2 μM [Pettit et al., 1998]).
Domain of Applicability
Depolymerization of microtubules has been measured in many somatic cell types, in addition to frog and mouse eggs, and in human cells, including eggs, in culture [Salmon et al. 1984; Wilson et al., 1984; Ibanez et al., 2003; Liu et al., 2010]. Quantitative cell-based assays for assessing microtubule activities of compounds are achieved by measuring the indirect effects on cell cycle which result from a disruption of microtubule networks. These methods utilize either fluorescent microscopy or cell cycle analysis. In fluorescent microscopic studies, either the α- or β-tubulin can be labeled directly with a tubulin antibody-conjugated fluorescent probe, or indirectly via a secondary antibody [Zhou et al., 2009]. Tubulin stabilizers and destabilizers cause cell cycle arrest at the G2/M phase [Bhalla, 2003], and therefore measurement of the percentage of cells arrested in G2/M phase is used as a surrogate endpoint for microtubule activity.
Evidence for Perturbation by Stressor
Colchicine interferes with microtubule dynamics at lower concentrations while it induces a net depolymerization at higher concentrations which is a consequence of the inability of further extending the microtubules [Stanton et al., 2011]. This dual action is in common with other spindle poisons (e.g. vinca derivatives) [Panda et al., 1996]. All microtubule-binding agents alter microtubule dynamics, engaging cell cycle surveillance mechanisms that arrest cell division in metaphase. This mitotic stall may then lead to various irremediable effects such as mitotic catastrophe, apoptosis or aneuploidy [Kops et al., 2005].
After addition of colchicine at concentrations of 0.1-3.0 mM, microtubule polymerization decreased rapidly and simultaneously thoughout the central spindle and aster (Salmon et al, 1984)
Bhalla KN. 2003. Microtubule-targeted anticancer agents and apoptosis. Oncogene 22:9075-9086.
Hamel E. 2003. Evaluation of antimitotic agents by quantitative comparisons of their effects on the polymerization of purified tubulin. Cell Biochem Biophys 38:1-22.
Ibanez E, Albertini DF, Overstrom EW. 2003. Demecolcine-induced oocyte enucleation for somatic cell cloning: coordination between cell-cycle egress, kinetics of cortical cytoskeletal interactions, and second polar body extrusion. Biol Reprod 68:1249-1258.
Kops GJ, Weaver BA, Cleveland DW. 2005. On the road to cancer: aneuploidy and the mitotic checkpoint. Nat Rev Cancer 5:773-785.
Lambrus BG, Holland AJ. 2017. A new mode of mitotic surveillance. Trends Cell Biol 27:314-321.
Liu S, Li Y, Feng HL, Yan JH, Li M, Ma SY, Chen ZJ. 2010. Dynamic modulation of cytoskeleton during in vitro maturation in human oocytes. Am J Obstet Gynecol 203:151.e151-157.
Mailhes JB, Carabatsos MJ, Young D, London SN, Bell M, Albertini DF. 1999. Taxol-induced meiotic maturation delay, spindle defects, and aneuploidy in mouse oocytes and zygotes. Mutat Res 423:79-90.
Marchetti F, Massarotti A, Yauk CL, Pacchierotti F, Russo A. 2016. The adverse outcome pathway (AOP) for chemical binding to tubulin in oocytes leading to aneuploid offspring. Environ Mol Mutagen 57:87-113.
Mitchison T, Kirschner M. 1984. Dynamic instability of microtubule growth. Nature. 312:237-242.
Panda D, Jordan MA, Chu KC, Wilson L. 1996. Differential effects of vinblastine on polymerization and dynamics at opposite microtubule ends. J Biol Chem 271:29807-29812.
Pettit GR, Toki B, Herald DL, Verdier-Pinard P, Boyd MR, Hamel E, Pettit RK. 1998. Antineoplastic agents. 379. Synthesis of phenstatin phosphate. J Med Chem 41:1688-1695.
Salmon ED, McKeel M, Hays T. 1984. Rapid rate of tubulin dissociation from microtubules in the mitotic spindle in vivo measured by blocking polymerization with colchicine. J Cell Biol 99:1066-1075.
Stanton RA, Gernert KM, Nettles JH, Aneja R. 2011. Drugs that target dynamic microtubules: a new molecular perspective. Med Res Rev 31:443-481.
Wade RH, Hyman AA. 1997. Microtubule structure and dynamics. Curr Opin Cell Biol 9:12-17.
Wallin M, Hartley-Asp B. 1993. Effects of potential aneuploidy inducing agents on microtubule assembly in vitro. Mutat Res 287:17-22.
Waterman-Sloter CM, Salmon ED. 1997. Microtubule dynamics: treadmilling comes around again. Curr Biol 7:R369-R372.
Wilson L, Miller HP, Pfeffer TA, Sullivan KF, Detrich HW, 3rd. 1984. Colchicine-binding activity distinguishes sea urchin egg and outer doublet tubulins. J Cell Biol 99:37-41.
Zavala F, Guenard D, Robin JP, Brown E. 1980. Structure--antitubulin activity relationship in steganacin congeners and analogues. Inhibition of tubulin polymerization in vitro by (+/-)-isodeoxypodophyllotoxin. J Med Chem 23:546-549.
Zhou YB, Feng X, Wang LN, Du JQ, Zhou YY, Yu HP, Zang Y, Li YJ, Li J. 2009. LGH00031, a novel ortho-quinonoid inhibitor of cell division cycle 25B, inhibits human cancer cells via ROS generation. Acta Pharmacol Sin 30;1359-1368.