Pulse treatment of interphasic HeLa cells with nanomolar doses of docetaxel affects centrosome organization and leads to catastrophic exit of mitosis.
In order to investigate the role of centrosome duplication in mitotic spindle morphogenesis, we designed a 1 hour pulse treatment protocol on synchronized HeLa cells with nanomolar doses of taxoids that might impair centrosome biogenesis but would allow the recovery of normal microtubule (Mt) dynamics before mitosis. We were prompted to use this approach as docetaxel (DOC; taxotereTM), a taxoid known to promote Mt polymerization, was shown to be more cytotoxic when applied during S phase. We show that pulse drug exposure is most efficient in late S and in G2 and results in a marked disorganization of the centrosome in G2, the pericentriolar material (PCM) being dissociated from centrioles. Separation of centrosomes at the G2-M transition is also impaired and mitotic spindle morphogenesis is grossly abnormal: although in most spindles chromosomes align in a metaphase plate, the two centrosomes stay most often unseparated at one pole and most of the NuMA protein accumulates at the other. Interestingly, we find that the centrosomes' ability to duplicate is not abolished as they are still able to trigger parthenogenetic development of frog eggs. Despite spindle asymmetry, the progression through mitosis is not blocked. This results in a catastrophic exit from mitosis, each mitotic cell generating several micronucleated cells linked together by multiple midbodies. Lack of mitotic block appears therefore as the prime cause of cell lethality. These experiments suggest that NuMA redistribution at the onset of mitosis depends upon the correct redistribution of PCM between centriole pairs. They also indicate that the presence of aberrant spindle poles does not alert the surveillance mechanism controlling the exit of mitosis.
Paoletti
A
A
Institut Curie, Section Recherche, UMR144 du CNRS, Paris, France.
Giocanti
N
N
Favaudon
V
V
Bornens
M
M
D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D002453:Cell Cycle / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D018385:Centrosome / Q000187:drug effects* / Q000378:metabolism; D000077143:Docetaxel; D004305:Dose-Response Relationship, Drug; D005260:Female; D016195:G2 Phase / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D007399:Interphase / Q000187:drug effects*; D008938:Mitosis / Q000187:drug effects*; D017239:Paclitaxel / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000633:toxicity; D010312:Parthenogenesis / Q000187:drug effects; D008941:Spindle Apparatus / Q000187:drug effects; D043823:Taxoids; D014982:Xenopus laevis
D000972:Antineoplastic Agents, Phytogenic; D043823:Taxoids; D000077143:Docetaxel; D017239:Paclitaxel