No abstract

Taxol inhibited HeLa cell proliferation by inducing a sustained mitotic block at the metaphase/anaphase boundary. Half-maximal inhibition of cell proliferation occurred at 8 nM taxol, and mitosis was half-maximally blocked at 8 nM taxol. Inhibition of mitosis was associated with formation of an incomplete metaphase plate of chromosomes and an altered arrangement of spindle microtubules that strongly resembled the abnormal organization that occurs with low concentrations of vinblastine and other antimitotic compounds. No increase in microtubule polymer mass occurred below 10 nM taxol. The mass of microtubules increased half-maximally at 80 nM taxol and attained maximal levels (5 times normal) at 330 nM taxol. At submicromolar concentrations, taxol suppressed growing and shortening at the ends of microtubules reassembled in vitro from bovine brain tubulin in a manner that resembled suppression by vinblastine. Taxol was concentrated in HeLa cells several hundredfold to levels that were similar to those which suppressed dynamic instability in vitro. The results indicate that taxol shares a common antiproliferative mechanism with vinblastine. At its lowest effective concentrations, taxol appears to block mitosis by kinetically stabilizing spindle microtubules and not by changing the mass of polymerized microtubules.

No abstract

E7389, which is in phase I and II clinical trials, is a synthetic macrocyclic ketone analogue of the marine sponge natural product halichondrin B. Whereas its mechanism of action has not been fully elucidated, its main target seems to be tubulin and/or the microtubules responsible for the construction and proper function of the mitotic spindle. Like most microtubule-targeted antitumor drugs, it inhibits tumor cell proliferation in association with G 2 -M arrest. It binds to tubulin and inhibits microtubule polymerization. We examined the mechanism of action of E7389 with purified microtubules and in living cells and found that, unlike antimitotic drugs including vinblastine and paclitaxel that suppress both the shortening and growth phases of microtubule dynamic instability, E7389 seems to work by an end-poisoning mechanism that results predominantly in inhibition of microtubule growth, but not shortening, in association with sequestration of tubulin into aggregates. In living MCF7 cells at the concentration that half-maximally blocked cell proliferation and mitosis (1 nmol/L), E7389 did not affect the shortening events of microtubule dynamic instability nor the catastrophe or rescue frequencies, but it significantly suppressed the rate and extent of microtubule growth. Vinblastine, but not E7389, inhibited the dilution-induced microtubule disassembly rate. The results suggest that, at its lowest effective concentrations, E7389 may suppress mitosis by directly binding to microtubule ends as unliganded E7389 or by competition of E7389-induced tubulin aggregates with unliganded soluble tubulin for addition to growing microtubule ends. The result is formation of abnormal mitotic spindles that cannot pass the metaphase/ anaphase checkpoint. [Mol Cancer Ther 2005;4(7): 1086 -95]

The growing and shortening dynamics of individual bovine brain microtubules at their plus ends at steady state in vitro, assembled from isotypically pure a4u, abim, or acsv tubulin dimers, were determined by differential interference contrast video microscopy. Microtubules assembled from the purified afm isotype were considerably more dynamic than microtubules made from the apu or a4iv isotypes or from unfractionated phosphocellulose-purified tubulin. Furthermore, increasing the proportion of the aoil isotype in a mixture of the alI and atom isotypes suppressed microtubule dynamics, demonstrating that microtubule dynamics can be influenced by the tubulin isotype composition.The data support the hypothesis that cells might determine the dynamic properties and functions of its microtubules in part by altering the relative amounts of the different tubulin isotypes.Microtubules (1) are required for structural organization and for many kinds of movements within the eukaryotic cell cytoplasm. They are especially prominent in the central nervous system where they appear to be essential for the organization and function of axonal and dendritic processes of neurons. Although it is unclear how microtubule functions in cells are controlled, a large body of evidence indicates that the dynamic properties of the microtubules play an important role (for reviews, see refs. 2 and 3). For example, microtubule dynamics increase dramatically when cells progress from interphase to mitosis (4-6), and the rapid dynamics of spindle microtubules appear to be important for establishing spindle microtubule organization, for facilitating the linkage of chromosomes to the spindles, and for chromosome movement (2-7).Little is known about how microtubule dynamics are regulated in cells. In vitro and in cells, microtubule ends switch between states of growing and shortening, a process known as "dynamic instability" (8-13), apparently due to the gain and loss ofa stabilizing GTP-or GDP-P1-liganded tubulin cap at the microtubule ends. Also, in vitro and in cells, net growing of microtubules can occur at one microtubule end and net shortening can occur at the opposite end, a process termed "treadmilling" or "flux" (9, 14-16). Both dynamic instability and treadmilling are logical targets for control. Biochemical studies in vitro have indicated that microtubule dynamics in cells could be regulated at several levels. One possibility is that control of microtubule dynamics involves regulation of the gain and loss of the stabilizing cap. A second possibility is that control of microtubule dynamics occurs through interactions of microtubule-associated proteins (MAPs) with microtubule surfaces and ends.Another possible mechanism for control of microtubule polymerization dynamics could involve the isotypic composition of the tubulin itself. Tubulin is composed oftwo 50-kDa polypeptide subunits, a and (3, which exist in multiple forms called isotypes. For example, mammalian brain tubulin consists of at least five a-and five j-tubulin isotypes (17,...