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

Abstract

The pH-related change in morphology of vinblastine (VLB)-induced paracrystals formed in Chinese hamster ovary (CHO) cells was examined immunohistochemically in order to determine both the mechanism of tubulin crystallization and the influence of acidic pHs on cytoskeletal microtubules. Lowering the extracellular pH (pHe) rapidly reduced the intracellular pH (pHi) in CHO cells. Lowering the pHi to near the neutral range significantly accelerated the growth of VLB-induced paracrystals, compared to that of paracrystals formed at a physiological pHe. However, further cytoplasmic acidification caused by the addition of sodium azide into the culture medium induced the disappearance of typical paracrystals and the appearance of a highly organized meshwork of tubulin appearing as short, thick filaments at the light microscopic level. Treatments using different concentrations of VLB at different pHe's showed that low pHi's (6.7 and 6.3) suppressed paracrystal-formation at lower concentrations of VLB (5 x 10(-6) M and 10(-5) M). At higher concentrations of VLB (5 x 10(-5) M and 10(-4) M), only short filaments were formed at pHi 6.3. Electron microscopy revealed that the filaments had a ladder-like structure probably consisting of a stacked series of fused rings. This indicates that paracrystals may be modified by extremely low pH. These results show that paracrystals are unstable in living cells and that their formation is regulated by environmental pH.

Interaction of a new bis‐indol derivative, KAR‐2 with tubulin and its antimitotic activity

“…At higher concentrations (above 10 μg ml −1 ) KAR‐2 induced formation of tubulin paracrystals in CHO cells. These effects were qualitatively similar to those exerted by VBL (Dustin, 1984; Takanari et al , 1994; Dhamodharan et al , 1995). Therefore, we think that the mechanisms by which these drugs influence tubulin assembly/disassembly are not significantly different.…”

“…Both drugs inhibit the self‐assembly of tubulin into MTs at substoichiometric concentrations by the formation of a tubulin‐drug complex at the end of a growing MT and consequent blocking of self‐assembly (Wilson et al , 1976; Margolis et al , 1980) and suppression of dynamic instability (Dhamodharan et al , 1995). However at higher Vinca alkaloid concentrations, tubulin dimers are self‐associated irreversibly into linear polymers (Prakash & Timasheff, 1992) leading to the formation of paracrystals both in vivo and in vitro (Dustin, 1984; Na & Timasheff, 1986; Prakash & Timasheff, 1992; Takanari et al , 1994).…”

Time and cell cycle dependent formation of heterogeneous tubulin arrays induced by colchicine in Triticum aestivum root meristem

“…Our study demonstrated that tubulin arrays undergo time-dependent morphological changes in cells exposed to colchicine, starting out as a network of wavy cortical strands that mimics the distribution of interphase MTs, and then transformed into rod-like or needle-type bundles. The cortical strands were comparable to fusiform bodies (Takanari et al, 1994), and the rod-like or needle-type bundles were analogous to the needleshaped bundles induced in animal cells by Vinka alkaloids (Bensch and Malawista, 1969;Takanari et al, 1990).…”

“…In these experiments, cortical strands were detected during interphase, while another form of tubulin array, perinuclear reticulum, formed in mitotic cells. Some tubulincontaining arrays in higher plant cells have been called paracrystals, due to their morphological resemblance to the crystalloids induced by Vinca alkaloids (vinblastine and vincristine) in animal cells in vivo (Na and Timasheff, 1982;Starling, 1976;Takanari et al, 1990Takanari et al, , 1994. Paracrystals alter in shape and number per cell with increased drug concentration, starting out as long and needle-shaped, converting to numerous small squares/ barrels, and finally transforming to crystalloids, which are aggregates of tubulin molecules recognized only by tubulin immunofluorescence (Takanari et al, 1990).…”

Specific rearrangement of the microtubular network during the cell cycle phases and correlation with micronucleation and death induced by anticancer drugs in NIH/3T3 subcultures

“…Some authors have demonstrated that by reducing intracellular pH in proximity to neutrality, there was a significant acceleration of VBL-induced paracrystal growth. When the cytoplasmic pH value ranged from 6.3 to 6.7, paracrystals disappeared and short and thick tubulin bundles appeared (34). The variability of these cytoplasmic factors could be correlated, in the same culture, with the metabolic activity, in a specific phase of the cell cycle and with the cell kinetics in terms of proliferation and polyploidization.…”