Enhanced interaction between tubulin and microtubule‐associated protein 2 via inhibition of MAP kinase and CDC2 kinase by paclitaxel

Abstract

Paclitaxel, an anti-mitotic anti-cancer agent, is active against solid tumors. The inhibition of depolymerization and promotion of microtubular assembly are essential for the anti-tumor activity of paclitaxel. Microtubule-associated proteins (MAPs) co-polymerize with tubulin and play some roles in microtubular dynamics. We examined the effect of paclitaxel on the interaction between tubulin and MAPs. Human lung-cancer cells, PC-14, were synchronized to G1/S border by the thymidine-double-block technique. After release from exposure to thymidine, the cells were treated briefly with 2 nM paclitaxel and the levels of alpha and beta tubulins and MAPs were examined after various times. Immunoblot analysis of paclitaxel-treated cells showed no changes in the overall expression of alpha and beta tubulins, microtubule-associated protein 2 (MAP2) or MAPs in comparison with controls. The samples were immunoprecipitated with anti-alpha- and anti-beta-tubulin antibodies and reblotted with an anti-MAP2 antibody, which showed that the amount of co-immuno-precipitated MAP2 in the synchronized cells, were increased by the brief paclitaxel treatment. These results suggest that paclitaxel treatment enhances the interaction between alpha and beta tubulins and MAP2. Since the phosphorylation state of MAP2 regulates the affinity of MAP2 for tubulins, and mitogen-activated protein (MAP) kinase is considered to be one of the kinases responsible for MAP2 phosphorylation, the effect of paclitaxel treatment on the MAP-kinase activity of synchronized PC-14 cells was examined. Two bands with molecular masses of 42 and 44 kDa were detected by an "intra-gel" MAP-kinase assay using myelin basic protein as the substrate. Paclitaxel treatment inhibited the MAP-kinase activity of PC-14 cells and inhibition was maximal at the G2/M phase of the cell cycle. Similar, concentration-dependent inhibition by paclitaxel of cellular MAP kinase of human synchronized small-cell lung carcinoma, H69, was observed. No inhibition of the MAP kinase of the paclitaxel-resistant sub-line H69/Txl by paclitaxel was observed, suggesting that some change of the MAP-kinase cascade had occurred in these cells. No direct inhibition of MAP-kinase activity by paclitaxel was observed in the cell-free assay (in vitro), suggesting that paclitaxel did not inhibit MAP kinase directly. Since it has been speculated that p34cdc2 kinase is also a kinase that phosphorylates MAP2, the effect of paclitaxel treatment on the p34cdc2-kinase activity of synchronized PC-14 and PC-9 cells was examined. Paclitaxel inhibited p34cdc2-kinase activation at the G2/M phase. These results suggest that paclitaxel inhibited MAP kinase and p34cdc2 kinase in vivo indirectly. These actions of paclitaxel may be responsible for the increased affinity between MAP2 and tubulins that it induces.

Induction of apoptosis by taxol and cisplatin and effect on cell cycle-related proteins in cisplatin-sensitive and -resistant human ovarian cells

“…It is therefore possible that the structure of the cyclin B1-cdc2 kinase complex could be modified by the binding of an unknown inactivating factor. However, an inhibition of p34cdc2 kinase activation at the G2-M phase by TX has been recently observed by Nishio et al (1995) in human lung cancer cell lines. A decrease in p34cdc2 catalytic activity was also transiently observed after CDDP-TX sequential exposure.…”

Anticancer effects of ZD6474, a VEGF receptor tyrosine kinase inhibitor, in gefitinib (“Iressa”)‐sensitive and resistant xenograft models

“…Human NSCLC cell lines PC-9 and PC-14 were used. 9,10) In addition, a gefitinib-resistant subline, PC-9/ZD, was derived from PC-9 cells by short-term exposure to the mutagen N-methyl-N′-nitro-N-nitrosoguanidine, continuous exposure to 0.2-0.5 µM gefitinib for 28 days, and subcloning. The resistant phenotype has been stable for at least 6 months under drug-free conditions.…”

Differential effect of vinorelbine versus paclitaxel on ERK2 kinase activity during apoptosis in MCF-7 cells

“…Kinase activity was calculated from the amount of phosphorylated substrate formed using liquid scintillation counting as described previously (Sommercorn et al, 1987;Wang et al, 1995). Alternatively, an aliquot of the substrate was subjected to 4%/10% stack SDS-PAGE (Nishio et al, 1995), and the phosphorylated substrate was analyzed by autoradiography, and quantitated by an imaging densitometer. …”