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Additivity of Dilantin and Vinblastine Inhibitory Effects on Microtubule Assembly¹

School of Nursing [S. L.] and Department of Biochemistry [S. L., J. W. I., J. J. C.], University of Mississippi Medical Center, Jackson, Mississippi 39216

Dilantin (phenytoin) is a commonly used antiepileptic agent that is known to decrease conductance of sodium and calcium ions and delay outward potassium currents. Separate from its antiseizure activity, dilantin interferes with microtubule protein polymerization. It induces metaphase arrest and potentiates the effects of the antimitotics vincristine and vinblastine in cell culture. We show here by fluorescence binding studies that dilantin interacts directly with tubulin at a low affinity site [K_a = 3.5 (± 2.5) x 10³ M^-1; K_d = 286 µM]. We quantitatively examined the effect of dilantin on bulk microtubule formation and found that the drug raises the critical concentration for microtubule polymerization in 2 M glycerol identically in the presence or absence of vinblastine. The change in free energy for microtubule polymerization attributable to 400 µM dilantin [ G = 117 (± 28) cal/mol] is additive with vinblastine effects. Under the same conditions, mean microtubule lengths are 7.7 ± 4.3 µm (n = 558) and 7.4 ± 4.0 µm (n = 477) in the presence or absence of dilantin, respectively. Dilantin has no effect on vinblastine-induced tubulin spiral formation, as measured by sedimentation velocity. Our data suggest that the mechanism for the antimicrotubule effects of dilantin involves sequestration of tubulin heterodimers in 1:1 drug:tubulin complexes that do not participate in tubulin polymerization. The dilantin binding site is distinct from the Vinca binding site, and these independent binding modes account for the additive effects in vitro. The sequestration of tubulin heterodimers could explain the combined drug synergy in cell cultures if it disrupted interactions with proteins that regulate microtubule dynamics and/or cell cycle events.