EFFECTS OF STEGANACIN AND TAXOL IN MICROTUBULES

Abstract

The experiments reported in this thesis are concerned with the molecular and cellular basis of action of two experimental antitumor drugs, steganacin and taxol.;Steganacin is a potent inhibitor of microtubule polymerization in vitro. Turbidity measurements and electron microscopy were used to monitor the assembly reaction. Steganacin inhibits the yield of the polymerization reaction (1 mg ml('-1) tubulin) by 50% at a concentration of 1.5 (mu)M. The drug is a competitive inhibitor of {lcub}('3)H{rcub}colchicine binding to purified tubulin (K(,I) is 3.1 (mu)M). The apparent affinity of {lcub}('3)H{rcub}colchicine for tubulin was calculated to be 2.1 (mu)M.;Steganacin blocks the replication of HeLa cells in mitosis, in a manner similar to that of colchicine. The mitotic index of a 10 (mu)M steganacin-treated suspension of HeLa cells (2 x 10('5) cells ml('-1)) increased from 3 to 94% in 24 hrs, but the cell number remained constant during the time course of the experiment. BALB/c fibroblast cells that were incubated with 10 (mu)M steganacin for 2 hrs at 37(DEGREES)C did not display their microtubules, as visualized by indirect immunofluorescence microscopy.;Steganacin, like colchicine and podophyllotoxin, inhibits the uptake of nucleosides by HeLa cells. The drug (50 (mu)M) inhibits uptake of {lcub}('14)C{rcub}uridine by 65% at 4(DEGREES)C, as early as 2 min after addition of the drug.;In contrast to steganacin, taxol enhances the rate, yield, and number of nucleations in the microtubule polymerization reaction in vitro. In addition, taxol renders microtubules resistent to depolymerization by cold (4(DEGREES)C) and CaCl(,2) (4 mM). Microtubule polymerization has been monitored in the presence of taxol by (1) an increase in turbidity at 350 nm as a function of time, (2) sedimentation, and (3) electron microscopy. Taxol decreases the characteristic lag time for assembly (1 mg ml('-1) tubulin) in a dose-dependent manner. The drug increases the yield of the polymerization 1.5-fold and causes a decrease in the critical concentration of tubulin required for assembly. Microtubules polymerized in the presence of taxol are approximately one third as long as control microtubules at apparent equilibrium. Therefore, there is an approximate 3.8-fold increase in the number of microtubules (or nucleations) in the reaction mixture. The optimal effects of the drug are observed at stoichiometric equivalence with tubulin dimers.;Experiments with taxol-treated HeLa and BALB/c fibroblast cells correlate well with the in vitro results. Taxol inhibits the replication of Hela cells by 50% at 10 nM. Flow microfluorometry experiments have demonstrated that cells treated with a concentration of taxol that inhibits replication 100% accumulate in the G(,2) and M phases of the cell cycle. Up to 90% of taxol-treated cells are in mitosis at 20 hrs as determined by electron microscopy.;Indirect immunofluorescence microscopy was used to demonstrate that taxol stabilizes cytoplasmic microtubules in cells. BALB/c fibroblast cells that have been treated for 22 hrs with 10 (mu)M taxol display, in addition to their cytoplasmic microtubules, bundles of microtubules that appear to radiate from a common site or sites. These taxol-treated microtubule cytoskeletons are resistant to depolymerization by cold (4(DEGREES)C) and steganacin. The structure and the distribution of the microtubules in the taxol-treated cells have been confirmed by electron microscopy.;Taxol, like other spindle poisons, inhibits 3T3 fibroblast cell migration. However, these cells are still able to produce mobile lamellipodia and filopodia. Taxol does not inhibit the uptake of nucleosides by HeLa cells, in contrast to steganacin, colchicine, and podophyllotoxin.;The effects of steganacin and taxol on microtubules may explain the observed antitumor activities of the two drugs.