Centrosome and spindle pole microtubules are main targets of a fluorescent taxoid inducing cell death.
Microtubules offer a very large local concentration of binding sites for cytotoxic taxoids or for hypothetical endogenous regulators. Several compounds from diverse sources stabilize microtubules and arrest cell division similarly to the antitumour drug Taxol. We have investigated the subcellular location 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 to a fraction of the available cellular binding sites effectively inhibits division of cultured human tumour cells at G(2)/M, and triggers apoptotic death. The loci of reversible binding, directly imaged in intact U937 cells treated with cytotoxic doses of fluorescent taxoid are the centrosomes, with a few associated microtubules in interphase cells, and the spindle pole microtubules in mitotic cells, instead of uniformly labelling the microtubule cytoskeleton. Cytoskeletal lesions induced and visualized with FLUTAX consist of microtubule bundles and abnormal mitoses, including monopolar spindles and highly fluorescent multipolar spindles. The multiple asters and monopolar spindles mark arrested U937 leukaemia and OVCAR-3 ovarian carcinoma cells on their path to apoptosis (as well as K562, HeLa, and MCF-7 cells). Depending 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. Fragmented centrosomes could be observed in FLUTAX-treated non-transformed 3T3.A31 cells, which developed micronuclei but were resistant to apoptosis. These results strongly suggest that centrosomal impairment by taxoid binding during interphase, in addition to the suppression of the kinetochore microtubule dynamics in the mitotic spindle, is a primary cause of cell cycle de-regulation and cell death.
Abal
M
M
Centro de Investigaciones Biológicas, CSIC, Madrid, Spain.
Souto
A A
AA
Amat-Guerri
F
F
Acuña
A U
AU
Andreu
J M
JM
Barasoain
I
I
D001665:Binding Sites; D002453:Cell Cycle / Q000187:drug effects; D016923:Cell Death / Q000187:drug effects*; D018385:Centrosome / Q000187:drug effects* / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000187:drug effects* / Q000378:metabolism; D017239:Paclitaxel / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology*; D043823:Taxoids; D020298:U937 Cells / Q000187:drug effects
C116523:7-O-(N-(4'-fluoresceincarbonyl)alanyl)taxol; D043823:Taxoids; D017239:Paclitaxel