Differential in vitro association of vinca alkaloid-induced tubulin spiral filaments into aggregated spirals

Abstract

Vinblastine, vincristine, vindesine, and vinorelbine, the four vinca alkaloids used in cancer therapy, differ in their antitumoral spectra and toxicities, but not in their inhibitory effects on microtubule assembly in vitro. At higher drug concentrations, vinca alkaloids induce the assembly of spiral filaments of tubulin, which, in turn, can interact laterally and form paracrystals. Using methods that distinguish spiral filaments and paracrystals (aggregated spirals), we found that spiral filament formation was largely independent of the incubation temperature, of the alkaloid used, and of the presence or absence of microtubule-associated proteins (MAPs). In contrast, the formation of aggregated spirals was markedly dependent on the alkaloid used, on the incubation temperature, and on the absence or presence of MAPs. Aggregated spirals failed to assemble in the presence of high concentrations of MAP-1A or MAP-1B, whereas they assembled readily with tau and MAP-2. Differences in patterns of turbidity development using pure tubulin allowed the classification of thirteen cytotoxic vinca alkaloids into five distinct groups, with centrifugal recovery of aggregated spirals in close agreement with the various turbidity patterns. With microtubule protein, i.e. tubulin preparations containing MAPs, only four groups were defined by turbidity patterns, and centrifugal protein recovery was more divergent. Vinblastine, vincristine, vindesine, and vinorelbine fell into distinct groups under both reaction conditions, and thus they appear to have qualitatively distinguishable in vitro interactions with tubulin. These differential effects on spiral filament and aggregated spiral assembly revealed that the four drugs induce different constraints on the tubulin molecule.