Antiproliferative mechanism of action of cryptophycin-52: kinetic stabilization of microtubule dynamics by high-affinity binding to microtubule ends

Abstract

Cryptophycin-52 (LY355703) is a new synthetic member of the cryptophycin family of antimitotic antitumor agents that is currently undergoing clinical evaluation. At high concentrations (>/=10 times the IC50), cryptophycin-52 blocked HeLa cell proliferation at mitosis by depolymerizing spindle microtubules and disrupting chromosome organization. However, low concentrations of cryptophycin-52 inhibited cell proliferation at mitosis (IC50 = 11 pM) without significantly altering spindle microtubule mass or organization. Cryptophycin-52 appears to be the most potent suppressor of microtubule dynamics found thus far. It suppressed the dynamic instability behavior of individual microtubules in vitro (IC50 = 20 nM), reducing the rate and extent of shortening and growing without significantly reducing polymer mass or mean microtubule length. Using [3H]cryptophycin-52, we found that the compound bound to microtubule ends in vitro with high affinity (Kd, 47 nM, maximum of approximately 19.5 cryptophycin-52 molecules per microtubule). By analyzing the effects of cryptophycin-52 on dynamics in relation to its binding to microtubules, we determined that approximately 5-6 molecules of cryptophycin-52 bound to a microtubule were sufficient to decrease dynamicity by 50%. Cryptophycin-52 became concentrated in cells 730-fold, and the resulting intracellular cryptophycin-52 concentration was similar to that required to stabilize microtubule dynamics in vitro. The data suggest that cryptophycin-52 potently perturbs kinetic events at microtubule ends that are required for microtubule function during mitosis and that it acts by forming a reversible cryptophycin-52-tubulin stabilizing cap at microtubule ends.

Figure 1

Structure of cryptophycin-52. R = H, cryptophycin-1; R = CH₃, cryptophycin-52.

Figure 2

The effects of cryptophycin-52 on Hela cell mitotic spindles. Microtubules (A, C, E, and G) and chromosomes (B, D, F, and H). (A and B) A control cell spindle with a well defined compact metaphase plate of chromosomes (arrowhead). Spindles of cells treated either with 10 or 20 pM cryptophycin-52 were similar. Shown in C and D are cells treated with 10 pM cyptophycin-52 (20 h); one spindle is normal and bipolar whereas the other spindle has chromosomes nearer one spindle pole (arrow in D). Shown in E and F are two cells treated for 20 h at 20 pM cryptophycin-52. Both spindles are relatively normal in organization. (G and H) At 300 pM cryptophycin-52 (20 h), most microtubules are depolymerized and chromosomes are in a ball-shaped mass. (Bar = 2 μm; ×5,500.)

Figure 3

Effects of cryptophycin-52 on microtubule assembly. Tubulin (13 μM) was mixed with S. purpuratus flagellar seeds in PMME buffer containing 2 mM GTP and incubated at 37°C in the absence or presence of different concentrations of cryptophycin-52 for 35 min to polymerize the microtubules to steady state. (A) The effects of cryptophycin-52 on polymer mass and on mean microtubule length were determined as described in the Materials and Methods. (B) The stoichiometries of cryptophycin-52 binding per microtubule and per mole of tubulin in microtubules were determined as described under Materials and Methods. Mean length of control microtubules was 6.06 ± 2.3 μm. Inset shows a double-reciprocal plot of cryptophycin-52 binding to microtubules. Error bars = SEM.

Figure 4

Length changes of individual microtubules at their plus ends at steady state in the absence (A) and presence (B) of 50 nM cryptophycin-52. The lengths of individual microtubules were measured from real-time video tape recordings as described under Materials and Methods.

Figure 5

Effects of bound cryptophycin-52 on rates of growing and shortening (A), mean length grown or shortened (B), and dynamicity (C). The average lengths that microtubules grew during growing events were determined by dividing the summed growing lengths for all microtubules for a particular condition by the total number of growing events measured for that condition. The mean shortening length per shortening excursion was determined similarly. Error bars = SEM.