Centrosome and spindle pole microtubules are main targets of a fluorescenttaxoid inducing cell death

Microtubules offer a very large local concentration of binding sites for cy totoxic taxoids or for hypothetical endogenous regulators. Several compound s from diverse sources stabilize microtubules and arrest cell division simi larly to the antitumour drug Taxol. We have investigated the subcellular lo cation of the Taxol binding sites, employing a fluorescent taxoid (FLUTAX) that reversibly interacts with the Taxol binding sites of microtubules and induces cellular effects similar to Taxol. The specific binding of FLUTAX t o a fraction of the available cellular binding sites effectively inhibits d ivision of cultured human tumour cells at G(2)/M, and triggers apoptotic de ath. The loci of reversible binding, directly imaged in intact U937 cells t reated with cytotoxic doses of fluorescent taxoid are the centrosomes, with a few associated microtubules in interphase cells, and the spindle pole mi crotubules in mitotic cells, instead of uniformly labelling the microtubule cytoskeleton. Cytoskeletal lesions induced and visualized with FLUTAX cons ist of microtubule bundles and abnormal mitoses, including monopolar spindl es and highly fluorescent multipolar spindles. The multiple asters and mono polar spindles mark arrested U937 leukaemia and OVCAR-3 ovarian carcinoma c ells on their path to apoptosis (as well as K562, HeLa, and MCF-7 cells). D epending on the FLUTAX treatment, OVCAR-3 cells died from abnormal mitosis or from a subsequent interphase with double chromatin content and lobulated nuclei (micronuclei), indicating impairment of centrosome separation. Frag mented centrosomes could be observed in FLUTAX-treated non-transformed 3T3. A31 cells, which developed micronuclei but were resistant to apoptosis. The se results strongly suggest that centrosomal impairment by taxoid binding d uring interphase, in addition to the suppression of the kinetochore microtu bule dynamics in the mitotic spindle, is a primary cause of cell cycle de-r egulation and cell death. Cell Motil. Cytoskeleton 49:1-15, 2001. (C) 2001 Wiley-Liss, Inc.