An alkaloid from opium, noscapine, is used as an antitussive drug and has low toxicity in humans and mice. We show that noscapine binds stoichiometrically to tubulin, alters its conformation, affects microtubule assembly, and arrests mammalian cells in mitosis. Furthermore, noscapine causes apoptosis in many cell types and has potent antitumor activity against solid murine lymphoid tumors (even when the drug was administered orally) and against human breast and bladder tumors implanted in nude mice. Because noscapine is water-soluble and absorbed after oral administration, its chemotherapeutic potential in human cancer merits thorough evaluation.

gamma-Tubulin is a newly identified member of the tubulin family whose sequence is highly conserved from yeast to man. This minor microtubule protein is localized to the microtubule organizing centres and a mutation in the gene encoding it produces a microtubuleless mitotic arrest in the filamentous fungus Aspergillus nidulans. Here we investigate the in vivo function of gamma-tubulin in mammalian cells using a synthetic peptide to generate a polyclonal antibody that binds to a highly conserved segment of gamma-tubulin. After microinjection into cultured mammalian cells, immunofluorescence localization revealed that this antibody binds to native centrosomes at all phases of the cell cycle. In the presence of the gamma-tubulin antibody, microtubules fail to regrow into cytoplasmic arrays after depolymerization induced by nocodazole or cold. Furthermore, cells injected immediately before or during mitosis fail to assemble a functional spindle. Thus in vivo gamma-tubulin is required for microtubule nucleation throughout the mammalian cell cycle.

Abstract. We assessed the mechanical properties of PC-12 neurites by applying a force with calibrated glass needles and measured resulting changes in neurite length and deflection of the needle. We observed a linear relationship between force and length change that was not affected by multiple distensions and were thus able to determine neurite spring constants and initial, nondistended, rest tensions. 81 out of 82 neurites showed positive rest tensions ranging over three orders of magnitude with most values clustering around 30-40 lxdynes. Treatment with cytochalasin D significantly reduced neurite rest tensions to an average compression equal to 14% of the former tension and spring constants to an average of 17 % of resting values. Treatment with nocodazole increased neurite rest tensions to an average of 282 % of resting values but produced no change in spring constant. These observations suggest a particular type of complementary force interaction underlying axonal shape; the neurite actin network under tension and neurite microtubules under compression. Thermodynamics suggests that microtubule (MT) assembly may be regulated by changes in compressive load. We tested this effect by releasing neurite attachment to a polylysine-coated surface with polyaspartate, thus shifting external compressive support onto internal elements, and measuring the relative change in MT polymerization using quantitative Western blotting. Neurons grown on polylysine or collagen without further treatment had a 1:2 ratio of soluble to polymerized tubulin. When neurites grown on polylysine were treated with 1% polyaspartate for 15-30 min, 80% of neurites retracted, shifting the soluble: polymerized tubulin ratio to 1:1. Polyaspartate treatment of cells grown on collagen, or grown on polylysine but treated with cytochalasin to reduce tension, caused neither retraction nor a change in the soluble:polymerized tubulin ratio. We suggest that the release of adhesion to the dish shifted the compressive load formerly borne by the dish onto MTs causing their partial depolymerization. Our observations are consistent with the possibility that alterations in MT compression during growth cone advance integrates MT assembly with the advance.INCE the pioneering studies of Yamada et al. (59), many investigators have found a clear cut "division of labor" in the role of the cytoskeleton in growth cone motility and axonal elongation. Anti-microtubule (MT) I drugs cause neurites to collapse but have no effect on growth cone motility functions, e.g. ruffling, microspike activity, etc. (8,17,24,30,31,49). Conversely, anti-actin drugs inhibit growth cone motility functions but neurites remain extended (8,24,31,49). Further, drugs that disrupt the actin network stabilize the neurite to retraction, while drugs that disrupt MTs cause retraction. Also, drugs that augment actin assembly cause retraction while drugs that augment MT assembly stabilize neurites to retraction and sometimes cause extension (15,31,35,49). Similarly, the networks move at different rate...

Noscapine, a microtubule-interfering agent, has been shown to arrest mitosis, to induce apoptosis, and to have potent antitumor activity. We report herein that two brominated derivatives of noscapine, 5-bromonoscapine (5-Br-nosc) and reduced 5-bromonoscapine (Rd 5-Br-nosc), have higher tubulin binding activity than noscapine and affect tubulin polymerization differently from noscapine. In addition, they are able to arrest cell cycle progression at mitosis at concentrations much lower than noscapine. Interestingly, whereas noscapine-arrested cells have nearly normal bipolar spindles, cells arrested by 5-Br-nosc and Rd 5-Br-nosc form multipolar spindles. Nevertheless, noscapine and the two derivatives all affect the attachment of chromosomes to spindle microtubules and they impair the tension across paired kinetochores to similar degrees. 5-Br-nosc and Rd 5-Br-nosc are also more active than noscapine in inhibiting the proliferation of various human cancer cells, including those that are resistant to paclitaxel and epothilone. Our results thus indicate a great potential for the use of 5-Br-nosc and Rd 5-Br-nosc both as biological tools for studying microtubule-mediated processes and as chemotherapeutic agents for the treatment of human cancers.

We have previously identified the opium alkaloid noscapine as a microtubule interacting agent that binds stoichiometrically to tubulin and alters its conformation. Here we show that, unlike many other microtubule inhibitors, noscapine does not significantly promote or inhibit microtubule polymerization. Instead, it alters the steady-state dynamics of microtubule assembly, primarily by increasing the amount of time that the microtubules spend in an attenuated (pause) state. Further studies reveal that even at high concentrations, noscapine does not alter the tubulin polymer/monomer ratio in HeLa cells. Cells treated with noscapine arrest at mitosis with nearly normal bipolar spindles. Strikingly, although most of the chromosomes in these cells are aligned at the metaphase plate, the rest remain near the spindle poles, both of which exhibit loss of tension across kinetochore pairs. Furthermore, levels of the spindle checkpoint proteins Mad2, Bub1, and BubR1 decrease by 138-, 3.7-, and 3.9-fold, respectively, at the kinetochore region upon chromosome alignment. Our results thus suggest that an exquisite control of microtubule dynamics is required for kinetochore tension generation and chromosome alignment during mitosis. Our data also support the idea that Mad2 and Bub1/BubR1 respond to kinetochore-microtubule attachment and/or tension to different degrees.

We report in this article that the retraction of PC 12 neurites, unlike that of other cultured neurons, is due to tension within the neurite. Retraction is rapid and independent of metabolic energy. Transection of one arm of a branched neurite immediately causes the remaining arm to take up a new equilibrium position between attachment points. Similarly, detachment of one growth cone of a cell causes the cell body to move to a new equilibrium position between the remaining neurites. These observations provide direct evidence for the suspension of the cell soma among a network of tensioned neurites. We used retraction as an assay for neurite tension to examine the role of actin filaments and microtubules in neurite support and elongation. Our data suggest that microtubules (MTs) within PC 12 neurites are under compression, supporting tension within the actin network. Treatment of cells with drugs that disrupt actin networks, cytochalasin D or erythro-9-[3-(2-hydroxynonyl)]adenosine eliminates retraction regardless of the absence of MTs, lack of adhesion to the substratum, or integrity of the neurite. Conversely, stimulation of actin polymerization by injection of phalloidin causes retraction of neurites. Treatments that depolymerize MTs, nocodazole or cold, cause retraction of neurites, which suggests that microtubules support this tension, i.e., are under compression. Stabilization of MTs with taxol stabilizes neurites to retraction and under appropriate circumstances can drive neurite extension. Taxol-stimulated neurite extension is augmented by combined treatment with anti-actin drugs. This is consistent with the actin network's normally exerting a force opposite that of MT assembly. Cytochalasin and erythro-9-[3-(2-hydroxynonyl)] adenosine were found to increase slightly the dose of nocodazole required for MT depolymerization. This is consistent with the postulated balance of forces and also suggests that alteration of the compression borne by the microtubules could serve as a local regulator for MT polymerization during neurite outgrowth.There is general agreement that axonal growth is intimately dependent upon the assembly and spatial organization of the neuronal cytoskeleton (3,16). Despite this agreement, the role of the cytoskeletal elements in axonal growth is poorly understood. The mechanism for integrating the polymerization of actin and microtubules (MTs) ~ is completely unknown. However, several different lines of evidence point to a local, i.e. controlled within the axon, mechanism for the integration of cytoskeletal assembly with axonal growth: axons and neurites severed from the cell body can regrow (18,24,28); new growth cones appear on the normally quiescent sides of neurites in response to MT depolymerization (4, 28); neurite outgrowth can be guided by local changes in substratum adhesiveness (17); and isolated neurites or growth cones can respond to a gradient of nerve growth factor (NGF) (6, 23).We recently reported that rate and extent of neurite outgrowth of PC 12 rat pheochromocy...

Abstract. fl-Tubulin is encoded in vertebrate genomesby a family of six to seven functional genes that produce six different polypeptide isotypes. We now document that although rat PC-12 cells express five of these isotypes, only two (classes II and III) accumulate significantly as a consequence of nerve growth factor-stimulated neurite outgrowth. In contrast to previous efforts that have failed to detect in vivo distinctions among different fl-tubulin isotypes, we demonstrate using immunoblotting with isotype-specific antibodies that three/3-tubulin polypeptides (classes I, II, and IV) are used preferentially for assembly of neurite microtubules (with ,x,70% of types I and II assembled but only ~50% of type III in polymer). Immunofluorescence localization shows that an additional isotype (V) is partially excluded from neurites. Distinctions in in vivo localization of the neuron-specific, class III isotype have also been directly observed using immunofluorescence and immunogold electron microscopy. The sum of these efforts documents that some in vivo functional differences between tubulin isotypes do exist.

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