Jordan M A, Thrower D, Wilson L
Department of Biological Sciences, University of California, Santa Barbara 93106.
J Cell Sci. 1992 Jul;102 ( Pt 3):401-16. doi: 10.1242/jcs.102.3.401.
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.
许多与微管蛋白结合的药物对有丝分裂的抑制作用被认为是由于微管解聚。然而，我们之前发现低浓度的长春碱和长春新碱可阻断HeLa细胞的有丝分裂，而纺锤体微管很少或没有解聚，纺锤体在形态上看起来正常或接近正常。在本研究中，我们在较宽的浓度范围内表征了长春碱、鬼臼毒素和诺考达唑对HeLa细胞有丝分裂纺锤体组织的影响。已知这三种药物在体外会影响微管聚合动力学，并使细胞中的微管解聚。我们想进一步探究这些药物对有丝分裂的抑制是否是通过对微管产生比微管净解聚更微妙的影响来实现的。我们比较了长春碱、鬼臼毒素和诺考达唑对纺锤体微管、染色体和中心体组织以及微管总质量的影响。通过免疫荧光显微镜检查纺锤体组织，并通过针对微管蛋白的定量酶联免疫吸附测定法在分离的细胞骨架上测定微管聚合物质量。随着药物浓度的增加，所有三种药物作用下有丝分裂纺锤体的组织变化方式相同。这些变化与有丝分裂停滞相关，但不一定伴随着微管净解聚。使用鬼臼毒素时，有丝分裂停滞伴随着微管解聚。相反，使用长春碱和诺考达唑时，在纺锤体微管完全存在的情况下发生有丝分裂停滞。所有三种药物都诱导了纺锤体微管几乎相同的重排、中期染色体越来越异常的组织以及中心体的碎片化。数据表明，这些抗有丝分裂药物主要通过抑制纺锤体微管的动力学而非简单地使微管解聚来阻断有丝分裂。
