Paclitaxel Inhibits Progression of Mitotic Cells to G₁ Phase by Interference with Spindle Formation without Affecting Other Microtubule Functions during Anaphase and Telephase

Abstract

Very low concentrations of paclitaxel, a clinically active anticancer agent isolated from the bark of the Pacific yew tree, were found to produce micronuclei in human colon carcinoma cells, suggesting inhibition of mitotic spindle assembly or function. The possibility that paclitaxel acts at the level of the mitotic spindle was investigated by evaluating its ability to inhibit the progression of mitotic cells to G₁ phase. Paclitaxel inhibited mitotic progression with a median inhibitory concentration of 4 nm, a concentration equivalent to the median cytotoxic concentration, without arresting cells in mitosis. A direct correlation was shown to exist between the cytotoxic potency and ability to inhibit mitotic progression for analogues of paclitaxel and antimicrotubule agents but not for the topoisomerase II-active agents etoposide and teniposide. After release from the nocodazole block, cells synchronized in mitosis remained sensitive to very low concentrations of paclitaxel for <30 min, the time required for spindle formation, yet remained sensitive to vinblastine for >90 min. This result indicates that very low concentrations of paclitaxel inhibit formation of mitotic spindles in cells without affecting function of preformed spindles and without arresting cells in mitosis. Continuous exposure to low nanomolar concentrations of paclitaxel for more than one cell cycle resulted in cells with DNA contents >4C and as much as 8C. These results support a hypothesis that, by not being capable of segregating sister chromatids, paclitaxel-treated cells eventually reform nuclear membranes around individual or clusters of chromosomes, revert to G₁ phase cells containing 4C DNA, and enter S phase, resulting in cells with as much as 8C DNA content. It is proposed that this is the primary cytotoxic mechanism of paclitaxel.