Instability of pleomorphic tubulin paracrystals artificially induced by Vinca alkaloids in tissue‐cultured cells

Abstract

The processes of tubulin paracrystal induction in Chinese hamster ovary cells treated with a Vinca alkaloid, ie, vinblastine or vincristine, and treated simultaneously with one of the Vinca alkaloids and colcemid or colchicine were followed by four different microscopic techniques, in particular by tubulin-immunofluorescence. Vinca alkaloid alone, in lower concentrations, induced basically tactoid or needle-shaped (N-shaped) paracrystals. However, the formation of crystalloid was greatly enhanced by increasing the concentration of Vinca alkaloid. Square or barrel-shaped (S-shaped) and hexagonal paracrystals were also commonly induced by simultaneous treatment with a Vinca alkaloid and colcemid or colchicine. Large rectangular paracrystals often displayed fibrillar or lamellar fine structures which ran perpendicular to the long axis but tended to cleave into fragments by spontaneous splitting. Electron micrographs revealed the fine structure of crystalloids to be aggregates of numerous filaments. The growth of paracrystals, particularly N-shaped crystals, was markedly inhibited when cells were exposed to drug(s) at a low temperature (4 degrees C). We confirmed that both N- and S-shaped paracrystals dissociated rapidly after the culture medium was replaced with fresh, drug-free medium. Glutaraldehyde-fixed paracrystals treated with RNase solution were stained with acridine orange, showing a weak orange color. Possible factors involved in the assembly and disassembly of tubulin paracrystals are discussed.

In vivo modulation of vincristine-induced neurotoxicity in Lymnaea stagnalis, by the ACTH(4?9)analogue Org 2766

“…It can be assumed that a spontaneous dissociation of paracrystals had taken place with decreasing VCR concentrations. Similar observations indicated unstability of VCR-induced crystals in vivo in Chinese Hamster Ovary-cells [29]. Moreover, no crystalline material was observed in autophagic bodies in our material, supporting the idea of spontaneous dissociation of the crystals.…”

Microtubule dynamics as a target in oncology

“…In contrast, the gross effect of microtubule destabilization is observed when sufficient drug is present to bind and disrupt tubulin interactions along the surface of the microtubule (Figure 2). The vincas also have affinity to free tubulin heterodimers and can give rise to tubulin paracrystals at high concentrations 11. While tubulin binding and suppression of microtubule dynamics are credited for the antineoplastic properties of the vinca alkaloids, these properties also lead to many of the observed side effects of these agents.…”

Effects of acidic pH on the formation of vinblastine‐induced paracrystals in Chinese hamster ovary cells

“…It is well known that some alkaloids are microtubule poisons and interfere with assembly by blocking the growing end of microtubule, thus preventing elongation and assembly of tubulin subunits [14]. However, in the case of Vinca alkaloids, it is characteristic that at higher concentrations they restructure free tubulin dimers in living cells into paracrystals which consist of tubular structures [4,17,27,40,45]. To explain this phenomenon, Owellen et al [37] proposed that tubulin paracrystals are formed when the molecular ratio between tubulin dimers and vinblastind (VLB) becomes 1:1.…”

“…Furthermore, tan, a microtubule-associated protein, has been shown to be involved in polymerization [30]. Nev-ertheless these molecular mechanisms are unable to explain completely the polymorphism of VLB-induced paracrystals [45]: the paracrystals change in shape and number per cell with increasing VLB-concentration, starting out as long needles/tactoids (N-shaped), converting to numerous, small squares/barrels (S-shaped) and finally converting to crystalloids which are aggregates of tubulin molecules recognized only by tubulin immunofluorescence.…”