Tubulin alterations in taxol-induced apoptosis parallel those observed with other drugs.
We have described previously that during apoptosis cellular tubulin is reorganized into visible tubulin structures that correlate with apoptotic morphology. Such changes have been observed in human leukaemic cells treated with a variety of cytotoxic agents. These structures are unlike those seen in untreated non-mitotic or mitotic cells. As taxol is known to act by enhancing the polymerization of tubulin in the initiation and extension of microtubules (MTs), and has been shown to induce and stabilize the formation of tubulin structures in a variety of cells, we examined the involvement of tubulin in apoptosis induced by taxol. Apoptosis was induced in a human T-cell leukaemic line, CCRF-CEM, following treatment with 10 nM taxol. The morphological features typical of apoptosis were apparent in taxol-treated cells after drug addition. Immunocytochemical analysis using a monoclonal antibody to beta-tubulin indicated that taxol induced visible tubulin polymerization. DNA fragmentation was detected at 10 hr post-treatment. Flow cytometric analysis of taxol-treated cells showed a time-dependent accumulation of cells in G2/M phase with the appearance of a hypodiploid peak coincident with the detection of DNA fragmentation. Microtubule structures observed following taxol treatment were of three types. At the time of DNA fragmentation, 50% of the cells displayed tubulin structures associated with apoptotic morphology similar to those seen in apoptosis induced by treatment with methotrexate (10(-8) M) or etoposide (17 microM). Twenty percent of the cells were arrested in mitosis at this time. These cells contained either multiple asters or disordered mitotic spindles, and did not display apoptotic morphology. The remaining cells, while normal in morphology, had extensive tubulin polymerization in the cytoplasm and around the nucleus. We examined the time-course of tubulin mRNA expression in apoptosis induced by taxol, methotrexate and etoposide. The level of tubulin mRNA displayed a transient increase after treatment, and prior to the onset of DNA fragmentation with each of the three drugs. These results suggest that during apoptosis taxol induces tubulin changes that display characteristics similar to those observed during apoptosis following treatment with drugs that do not interact directly with tubulin.
Ireland
C M
CM
Children's Leukaemia and Cancer Research Centre, Prince of Wales Children's Hospital, Sydney, Australia.
Pittman
S M
SM
D017209:Apoptosis; D002460:Cell Line / Q000187:drug effects; D018385:Centrosome / Q000187:drug effects; D005047:Etoposide / Q000633:toxicity; D008727:Methotrexate / Q000633:toxicity; D017239:Paclitaxel / Q000633:toxicity*; D012313:RNA / Q000032:analysis; D014404:Tubulin / Q000187:drug effects* / Q000235:genetics / Q000648:ultrastructure
D014404:Tubulin; D012313:RNA; D005047:Etoposide; D017239:Paclitaxel; D008727:Methotrexate