rhazinilam mimics the cellular effects of taxol by different mechanisms of action

Cell Motility and the Cytoskeleton 28:317-326 (1994)

Rhazinilam Mimics the Cellular Effects of Taxol by Different Mechanisms of Action

Bruno David, Thierry Sevenet, Michel Morgat, Daniel Guenard, Andre Moisand, Yvette Tollon, Odile Thoison, and Michel Wright

CNRS, Laboratoire de Pharmacologie et de Toxicologie Fondamentales, Toulouse Cedex (B.D., A.M., Y. T., M. W.), CNRS, lnstitut de Chimie des

substances Naturelles, (6. D., T.S., D.G., 0. T.), and CEN-Saclay, Service de Biochimie, Gif sur Yvette (M. M,), France

We have investigated the effects of the microtubule poison rhazinilam on microtubule assembly in vivo and in vitro. In mammalian cells, rhazinilam mimics the effects of taxol and leads to microtubule bundles, multiple asters, and microtubule cold stability. In vitro, rhazinilam protected preassembled microtubules from cold-induced disassembly, but not from calcium ion-induced disassembly. More- over, both at 0°C and at 37"C, rhazinilam induced the formation of anomalous tubulin assemblies (spirals). This process was prevented by maytansine and vinblastine, but not by colchicine. Preferential saturable and stoichiometric binding of radioactive rhazinilam to tubulin in spirals was observed with a dissociation constant of 5 bM. This binding was abolished in the presence of vinblastine and maytansine. In contrast, specific binding of radioactive rhazinilam to tubulin assembled in microtubules was undetectable. These results demonstrate that rhazinilam alters microtubule stability differently than taxol, and that the overall similar effects of rhazinilam and taxol on the cellular cytoskeleton are the con- sequence of two distinct mechanisms of action at the molecular level. Q 1994 Wiley-Liss, Inc.

Key words: maytansine, vinblastine, diphenylpyridazone, colchicine, taxol, tubulin, microtubule

INTRODUCTION

The microtubule cytoskeleton, which plays a cen- tral role in the spatial organization of eukaryotic cells and in mitosis, is the target of numerous xenobiotic agents. In vitro most of these substances interact directly with tubulin, prevent tubulin assembly and can lead to the formation of various types of anomalous tubulin assemblies [Fujiwara and Tilney, 1975; Roobol et al., 1977; Batra et al., 1986; Luduena et al., 19861. Taxol is an exception to this rule. It stabilizes and protects microtubules against cold- and calcium-induced disassembly and decreases the apparent critical concentration of tubulin necessary for microtubule assembly [Schiff et al., 19791. In agreement with these in vitro studies, colchicine-like microtubule poisons disassemble the interphase microtubule cytoskeleton and prevent the formation of the mitotic spindle, while taxol stabilizes microtubules against cold disassembly [Schiff and Horwitz, 19801 and induces

0 1994 Wiley-Liss, Inc.

the formation of microtubule bundles in interphase cells [De Brabander et al., 1981; Herman et al., 1983; Ma- surovsky et al., 1981; Forry-Schaudies et al., 19861. Moreover, taxol blocks the mitotic microtubule cytoskeleton in complex aster-like structures [De Brabander et al., 19811. We have previously observed that rhazinilam, a recently recognized microtubule poison that prevents microtubule assembly in vitro [Thoison et al., 19871, had effects on mammalian cells similar to taxol. It induces both microtubule bundling in interphase and blocks mitotic cells in multiple aster-like structures. However, despite the apparent similarity between the effects of taxol

Received November 20, 1993; accepted February 22, 1994

Address reprint requests to Dr. Michel Wright, CNRS, Laboratoire de Pharmacologie et de Toxicologie Fondamentales, 205 route de Nar- bonne, 31078 Toulouse Cedex, France.

318 David et al.

and rhazinilam on the microtubule cytoskeleton in vivo, we show that these two drugs exhibit a distinct mechanism of action on microtubule proteins.

MATERIALS AND METHODS Chemicals

Rhazinilam (Fig. 3 , inset) was extracted from the bark of Kopsia singapurensis, an arborescent Apocyn- aceae from south peninsular Malaysia [Teo et al., 19901. Dry ground bark (5.2 kg), soaked for 15 h in 8% am- monia (7 liter), was extracted four times for 2 h at 35°C with CH,CI, (30 liter). Crude rhazinilam (1.45 g) was obtained by chromatography (Silica gel Merk 7734, in hexane/ethyl acetate l i l ) . Pure rhazinilam (0.8 g) obtained after recrystallisation in ethyl ether was identified [Abraham and Rosenstein, 1972; Ratcliffe et al., 1973; Thoison et a]. , 19871 by UV, IR spectra, optical rotation, X-ray structure, NMR (proton NMR, I3C NMR and two- dimensional NMR ['H/'H and 'H/"C] with a Brucker WB-400) and mass spectrometric analyses (molecular ion peak at 294 mass units and a peak corresponding to M-CH,CH, at 265 mass units; electron impact AEI MS- 50). Tritiated rhazinilam (5 mCi/mole on C5) was syn- thesized (Fig. 3, inset) from monobrominated rhazinilam and purified by HPLC (C, micro-Bondapack column, methanol/water 4/1). Taxol and maytansine have been obtained from the National Cancer Institute (Bethesda, MD), vinblastine from P. Fabre Mkdicaments (Castres, France) and diphenylpyridazone from Dr. Hamel. All other chemicals were bought from Sigma Chemicals (St. Louis, MO), Boehringer (Mannheim, Germany) and Merk (Darmstadt, Germany).

Microscopic Observation of Mammalian Cells

PtK2 cells (Potorous triductylis kidney cells), washed in 100 mM PIPES (piperazine-N,N'-bis [2- ethanesulfonic acid]) pH 6.9, 1 mM EGTA (ethylene glycol-bis (P-aminoethyl ether) N,N,N',N'-tetraacetic acid), 1 mM MgCI, containing 4% w/w polyethylene glycol 6000, were permeabilized at 22"C, for 1 min in the same buffer with 0.5% Triton X-100 and washed in the same medium without detergent. Permeabilized cells, fixed for 0.5 h by 3% v/v formaldehyde and processed for immunolabeling (microtubule staining with anti-tubulin antibody YL 112 (specific for tyrosinated a-tubulin) [Kilmartin et al., 19821, and chromatin staining with 4' ,6-diamidino-2-phenylindole 0.2 pg/ml) were observed by epifluorescence (Zeiss Axiophot microscope, Nocticon camera from Lhesa Electronique (Cergy-Pontoise, France). The microtubule cytoskeleton and the nuclear images have been used to calculate a composite image showing both features (Sapphire image treatment from Quantel (Newbury, England), difference

and histogram functions). On these composite pictures the immunofluorescent image of the microtubule network is shown in white and the fluorescent image of the nucleus is shown in black (Figs. 1 and 2). For electron microscopy, lysed cells were fixed for 3-14 h at 22°C with 2.5% glutaraldehyde, with or without 4% tannic acid, processed for electron microscopy and observed with a Phillips electron microscope (EM 301) at 80 kV.

Microtubule Proteins

Sheep brain microtubule proteins were purified by two or three cycles of assemblyldisassembly at 37"CWC [Shelanski et al., 19731. Pure tubulin was obtained by an additional phosphocellulose chromatography [Weingar- ten et al., 19751. Tubulin-colchicine complexes unable to assemble at 37°C (5 mg/ml) were obtained by incubation of two-cycle-purified microtubule proteins with an excess of colchicine and separated from free colchicine on a Sephadex G-25 Pharmacia column.

Tubulin Assembly

Tubulin assembly and disassembly were monitored by the variations of absorbance at 350 nm with a spec- trophotometer (Beckman DU 64 equipped with a PC computer). The decrease of temperature from 37°C to 4°C occurred in less than 1 min, while 0°C was reached after 2.5 min. The increase of temperature from 0°C to 33°C occurred in less than 1 min, while 37°C was reached after 1.5 min. Unless otherwise stated, all experiments have been made in the presence of 1 mM GTP. The amount of sedimentable microtubule proteins was determined by ultracentrifuging 100 pI aliquots of microtubule proteins on 50 p1 cushions 15% (v/v) sucrose using a Beckman airfuge (rotor type 30", 27 Ib/inch2 for 15 min at 0°C or 37°C). Then the amount of protein was determined in the pellet [Lowry et al., 19511. In all experiments, tubulin assemblies were characterized by electron microscopy after negative staining with 2% (w/v) uranyl acetate.

Equilibrium Dialysis

Microdialysis chambers (0.2 ml compartments) [Takoudju et al., 1988b] were filled with 0.1 M PIPES, pH 6.9, 0.5 mM MgCl,, 1 mM EGTA, 1 mM GTP and ['HI-rhazinilam (12 to 300 pM in 4% (v/v) dimethyl sulfoxide, 2.4 to 4.5 mCi/mole) in one compartment and three-cycle-purified microtubule proteins ( 10 mg/ml) in the other compartment. Equilibrium was reached after 6 h with constant shaking at 0°C (crushed ice) and 37°C both in the absence or in the presence of microtubule proteins. These conditions affected neither the ability of tubulin kept at 0°C to form microtubules, nor the main- tenance, at 37"C, of tubulin assembly [Takoudju et al., 1988bl. In the presence of 200 pM ['HI-rhazinilam, the

Rhazinilam, A New Microtubule Poison 319

Fig. 1. Effects of rhazinilam on mammalian cells. PtK2 cells were treated in the presence of 125 FM rhazinilam. A and B: Immunoflu- orescence labelling (microtubules are shown in white and chromatin in black) of a cell blocked in early mitosis (A), and of an interphase cell

(B). C-F: Electron microscopy. The extraction procedure allows the observation of the microtubule structure in E and F. Some microtubule bundles (35%) seem to be laterally attached to an unknown structure (E arrows).

concentration of bound rhazinilam was a sigmoidal func- tion of the amount of microtubule proteins (0, 8, 32, and 45 pM [3H]-rhazinilam were bound in the presence of 1 , 3, 5 and 10 mg/ml three-cycle-purified microtubule proteins, respectively, at 0°C). Thus, a concentration of 10 mg/ml of three-cycle-purified microtubule proteins was used in all experiments. HPLC measurements (C,8 micro-Bondapack column; methanol/water 4/ l ) revealed that neither nonradioactive, nor ['H]-rhazinilam were modified during dialysis in the absence or in the presence of microtubule proteins (10 mglml). Adsorption of ['HI- rhazinilam to the dialysis chambers accounted for less

than 29% of the initial amount in the absence of microtubule proteins both at 0°C and 37°C. This effect was overcome by the determination of the concentration of [3H]-rhazinilam in each compartment [Takoudju et al . , 1988bl. The adsorption of microtubule proteins to the dialysis chambers was negligible both at 0°C and 37°C [Takoudju et al., 1988bl. Binding constants were determined by Scatchard analysis.

Gradient Centrifugation Separation of tubulin assemblies from unassembled

proteins was performed by sedimenting for 1 h at 40,000

320 David et al.

Fig. 2. Stabilization of microtubules against cold disassembly in the presence of rhazinilam. Immunofluorescence labelling (microtubules are shown in white and chromatin in black). Cells have been preincubated at 37°C for 3 h without (A: typical microtubule network) or

with 100 pM rhazinilam (C: large microtubule bundles). Then, cells were further incubated at 0°C for 4 h without (B: diffuse background of fluorescence around the nucleus) and with rhazinilam (D: microtubule bundles).

x g (rotor SW50 Beckman) a 0.3 ml aliquot in a 5 ml 5-20% sucrose linear gradient containing 2% (v/v) dimethyl sulfoxide, 100 mM PIPES, pH 6.9, 1 mM EGTA, 0.5 mM GTP and 0.5 mM MgCI,. Protein con- tent [Lowry et al., 195 I ] and radioactivity (20 pl in 3 ml of Ready gel, Beckman Instruments, Palo Alto, CA) of each fraction were determined.

RESULTS Rhazinilam Mimics the Action of Taxol on Mammalian Cells

Rhazinilam inhibited the growth of various mammalian cells with an ID,, of 0.6-1.5 p,M calculated over a 48 hours incubation in the presence of the drug (L1210, P388 murine leukemia, and PtK2 cells). PtK2 cells were further used in order to study the effect of rhazinilam on the microtubule cytoskeleton. Higher doses of rhazinilam were used since we looked for a rapid and clear effect on the microtubule cytoskeleton. When PtK2 cells were treated for 6 h by 6-125 pM rhazinilam, the immunofluorescence observation of the microtubule cy-

toskeleton showed that all mitotic figures were abnormal. Rhazinilam did not prevent the disappearance of the nuclear envelope. Chromosomes were assembled in one or several masses and 2-10 asters were present in the cytoplasm (Fig. 1A). In a few cases, postmitotic figures characterized by several nuclei and several microtubule asters were observed. The number of these abnormal mitotic figures (6 h incubation) increased with rhazinilam concentration and reached 17% of the cells in the presence of 125 pM rhazinilam (control mitotic index: 5.5%). Electron microscopic observations showed that these asters were made of typical microtubules. Most microtubules (64%; 28 microtubule sections recorded) were composed of 13 protofilaments, while 28% and 7% exhibited 12 and 14 protofilaments, respectively. In the presence of the highest concentrations of rhazinilam which have been used (63-125 pM), the immunofluorescent labelling of the interphase microtubule cytoskeleton revealed numerous cytoplasmic bundles (Fig. 1 B). Electron microscopy demonstrated that the bundles were composed of microtubules (Fig. 1C-E). After 6-24 h of treatment, 73% of microtubules were composed of 13


protofilaments, while 7% and 20% possessed 12 and 14 protofilaments (64 microtubule sections recorded). After 48 h, the frequency of 13 protofilament microtubules decreased to 54%, while 20% and 23% exhibited 12 and 14 protofilaments (52 microtubule sections recorded). In bipolarized interphase cells, microtubule bundles were generally located in two groups around the nucleus (Fig. 1B). Despite the presence of these large microtubule bundles, other microtubules were also present in the cytoplasm. Rhazinilam did not prevent microtubule assembly but rather, disturbed the microtubule cytoskeleton. We investigated whether rhazinilam could stabilize microtubules. Most cytoplasmic microtubules disappeared in control PtK2 cells submitted to cold treatment (0°C for 0.25-4 h; Fig. 2A,B). In contrast, in interphase cells preincubated for 6 h with 125 pM rhazinilam at 37"C, cytoplasmic microtubule bundles were not disassembled (Fig. 2C,D). However, these cold-resistant microtubule bundles did not prevent the cells from rounding up. Con- comitantly, with the loss of cell bipolarity (2-4 h cold treatment), the orientation of the microtubule bundles became randomized. Thus, it is likely that these microtubules did not play a role in the spatial organization of the cell. Cold stability of mitotic microtubules was iden- tical in cells preincubated with or without rhazinilam, since all but kinetochore microtubules were disassembled. In contrast with these observations, our preliminary observations have suggested that rhazinilam could prevent in vitro microtubule assembly [Thoison et al., 19871. Thus, we investigated further the effects of rhazinilam on mammalian tubulin in vitro.

In Vitro Rhazinilam Induces Anomalous Assemblies of Microtubule Proteins

Rhazinilam was added at 0°C to mammalian microtubule proteins, consisting of tubulin and microtubule-associated proteins (MAPS), capable of self-assembly at 37°C (Fig. 3C control). The absorbance increased with time (Fig. 3A), but was lower than when microtubules were assembled (Fig. 3C). No microtubules were detected by electron microscopy, but numerous anomalous longitudinal tubulin assemblies were observed. Among them, spirals (about 0.25 pm), formed by the longitudinal association of 2-6 protofilaments, were fre- quent (Fig. 4A,B). These anomalous tubulin assemblies (55% of the initial amount of microtubule proteins present in the reaction mixture according to the ultracentrifugation assay) were the only assemblies observed by electron microscopy. They could account for turbidity increase. Despite the variations that we observed in the shape and length of tubulin spirals formed in the presence of rhazinilam (Fig. 4A), there was a linear relationship (Fig. 3B) between the increment of absorbance at

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Fig. 3. Formation of tubulin assemblies after addition of rhazinilam on microtubule proteins at 0" C. Inset: Chemical formula of rhazinilam. A: Rhazinilam (100 +M, 1.5% dimethyl sulfoxide) was added to a cold solution of microtubule proteins ( 5 mg/ml) and the increase of absorbance was recorded [Gaskin et al., 19741. Anomalous tubulin assemblies formed at O'C, remained stable at 37°C (Sedimentation assay: 2.75 mg/ml at 0°C and 37°C). No microtubules could be detected by electron microscopy at both temperatures. B: Relation between the amount of sedimentable tubulin [Takoudju et al., 1988b] and absorbance. C: Formation of tubulin assemblies at 37°C with and without rhazinilam (control). Electron microscopic observations showed microtubules in the absence of rhazinilam, while only anomalous tubnlin assemblies were observed in the presence of rhazinilam.

350 nm and the amount of anomalous assemblies sedi- mented by ultracentrifugation [Takoudju et al., 1988133. The initial increase of absorbance per unit of time and the amount of anomalous assemblies (measured at the pla- teau of absorbance by the ultracentrifugation assay), increased with rhazinilam concentration (ID+ 10 and 25 pM, respectively). Anomalous assembly (80% of the initial amount of microtubule proteins present in the reaction mixture) was also observed at 0°C with pure tubulin [Weingarten et al., 19751 as judged by electron microscopy, absorbance increase and sedimentation assay. For microtubule proteins and pure tubulin, the pla- teau of absorbance did not decrease after a shift up from 0°C to 37°C (Fig. 3A) and electron microscopy showed the presence of anomalous assemblies only. Thus, longitudinal tubulin assemblies and spirals formed at 0"C, were stable at 37°C.

322 David et al.

Fig. 4. Anomalous tubulin assembly in the presence of rhazinilam (150 pM). A and B: Microtubule proteins were incubated at 0°C with 150 pM rhazinilam (Fig. 3). A: Overall view of the anomalous longitudinal tubulin assemblies. B: Detail of a spiral formed by the lon-

gitudinal association of 4 protofilaments (arrowheads). C-E: Presence of spirals at the two extremities of a microtubule preincubated at 37°C in the presence of rhazinilam (125 pM) and further submitted to a 30 min treatment at 0°C.

Rhazinilam was added to cold microtubule proteins or pure tubulin and the temperature was immediately raised to 37°C (Fig. 3C). The formation of microtubule was prevented as shown by electron microscopy. More- over, numerous anomalous tubulin assemblies containing spirals were observed. Ca2+ ions increased this effect. For example, anomalous tubulin assemblies were prepared by the addition of rhazinilam to a preparation of microtubule proteins ( 5 mglml). These tubulin assem-

blies corresponded to 60% of the initial amount of microtubule proteins present in the reaction mixture (sedimentation assay) and were characterized by electron microscopy. Addition of 4 mM Ca2+ led to a further increase of sedimentable proteins (88% of the initial amount of microtubule proteins present in the reaction mixture) and only spirals were observed by electron microscopy. This concentration of Ca2+ did not modify the absorbance of unassembled microtubule proteins (see for


tubulin assembly induced by rhazinilam (200 pM): 46% and 100% with 5 and 25 pM maytansine; 52%, 81% and 90% inhibition with 2, 10 and 25 pM vinblastine as judged by the sedimentation assay. Tubulin-colchicine complexes (5 mg/ml) assembled into spirals either with vincristine (100 pM) or rhazinilam (200 kM), in agreement with the interaction of colchicine to a binding site different from those of vinblastine and maytansine [Bryan, 1972; Owellen et al., 19721.

Rhazinilam Protects Microtubules Against Cold Disassembly

Addition of rhazinilam (100-150 pM) to preassembled microtubules prepared from microtubule proteins and pure tubulin did not significantly modify their assembly as determined by electron microscopy and absorbance monitoring (Fig. SA). In the presence of 150 pM rhazinilam, lowering the temperature to 0°C did not change the absorbance for more than 2 h. Microtubules were still present, although few spirals could also be observed by electron microscopy. The inhibition of microtubule cold disassembly occurred in proportion to the amount of drug added (Fig. SA, inset). Moreover, after an incubation of 15 to 30 min at o"C, spirals could be observed at one or both ends of microtubules in continuity with microtubule protofilaments (Fig. 4C). In contrast with the increased stability of microtubules to cold, rhazinilam did not interfere with Ca2+ -induced disassembly at 37°C (Fig. SB): at low rhazinilam concentrations (10 pM), the addition of Ca2+ led to a biphasic variation of absorbance; the initial decrease of absorbance was fol- lowed by an increase corresponding to the formation of spirals in the absence of microtubules, as shown by electron microscopy. Thus, it is possible that microtubules could disassemble into tubulin dimers which then reas- semble into anomalous tubulin assemblies. At higher rhazinilam concentrations (50-1 00 pM), electron microscopy revealed that the disappearance of microtubules did not lead to a decrease of absorbance due to the large and simultaneous formation of anomalous tubulin assemblies, in agreement with the highest spiralization potential of tubulin in the presence of both rhazinilam and calcium ions. At higher rhazinilam concentrations, it is not possible to determine whether microtubules were disassembled into tubulin dimers which then reassembled con- comitantly into spirals, or disassemble in forming directly anomalous tubulin assemblies.

Radioactive Rhazinilam Binds Stoichiometrically to Tubulin Spirals but Not to Microtubules

Rhazinilam showed two effects: formation of anomalous tubulin assemblies and microtubule protection from cold disassembly. Thus, it seemed likely that rhazinilam could bind to tubulin in both cases. In order to test this hypothesis, the binding of [3H]-rhazinilam to

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Fig. 5 . Rhazinilam protection of microtubules from cold (A) but not from calcium ion disasseinbly (B). Microtubules (tubulin concentration 5 mgiml) were incubated in the presence of various concentrations of rhazinilam (arrows). In A, the temperature was lowered to 0°C (vertical dashed line). The inset shows the increase cold protection of microtubules (decrease in the rate of disassembly) with increasing concentrations of rhazinilam. In B, 4 mM Ca2+ were added 2 min after rhazinilam addition.

example the addition of Ca2+ in the absence of rhazinilam in Fig. 5).

GTP was necessary for tubulin spiralisation. A microtubule protein preparation ( 10 mg/ml) was depleted from its GTP by 9 successive assembly/disassembly cycles at 37"C/OoC. Then it showed no increase of absorbance when the temperature was raised to 37°C. But addition of 1 mM GTP allowed the recovery of 92% of the initial amplitude of absorbance at 37°C. As shown by electron microscopy and the sedimentation assay, when this GTP-depleted microtubule protein preparation was incubated at 0°C with 150 pM rhazinilam, limited anomalous tubulin assembly occurred (29% of the maximum amount obtained by adding back 1 mM GTP).

Preincubation of microtubule proteins (5 mg/ml) with maytansine and vinblastine inhibited the anomalous

324 David et al.

tubulin (10 mg/ml) was studied by equilibrium microdialysis. A saturable binding of ['HI-rhazinilam to tubulin spirals was observed at 0°C with increasing concentrations of free rhazinilam (Fig. 6A, closed circles), but its low solubility made it impossible to determine whether [3H]-rhazinilam could be displaced by an excess of un- labelled drug. Tubulin being 90% pure, 0.5 mole of rhazinilam were bound per mole of tubulin (dissociation constant: 4.9 p,M; Hill coefficient: 0.87). The amount of spirals determined by ultracentrifugation increased lin- early with bound ['HI-rhazinilam (Fig. 6A, inset), suggesting that rhazinilam binds preferentially to tubulin spirals rather than to free tubulin. GTP-depleted tubulin failed to assemble into spirals as shown by electron microscopy and rhazinilam showed an unsaturable binding (Fig. 6A, squares) similar to those observed with serum albumin (10 mg/ml) at 0°C and 37°C. Thus, rhazinilam does not bind strongly to free tubulin. Since 55% of tubulin was assembled into spirals, we suggest that one mole of rhazinilam binds per mole of tubulin assembled in spirals. Direct binding of [3H]-rhazinilam to tubulin spirals was demonstrated in nonequilibrium conditions by sedimenting tubulin spirals onto a 5-20% linear sucrose gradient; most of [3H]-rhazinilam remained in the top fractions of the gradient and less than 3% was observed with the sedimenting spirals (apparent stoichiom- etry: 0.56 moles of rhazinilam per mole of tubulin). This peak of radioactivity was absent when tubulin was omit- ted or was replaced by serum albumin.

Although rhazinilam protected microtubules against cold disassembly, ['HI-rhazinilam did not show a saturable binding to microtubules assembled with or without taxol or to tubulin ribbons assembled in 30% dimethyl sulfoxide (Fig. 6B). In addition, no significant binding to these tubulin assemblies was observed after centrifugation in a sucrose gradient. Thus, the percentage of tubulin able to bind ['HI-rhazinilam in microtubules was probably very low. This observation was unexpected since we have shown that rhazinilam could prevent cold microtubule disassembly both in vivo and in vitro.

Nonsaturable binding curves were observed when microtubule proteins were preincubated (0.5 h at 0°C) with 50 pM maytansine or 400 pM vinblastine before equilibrium dialysis. Similarly, no [3H]-rhazinilam sed- imented in a sucrose gradient with tubulin spirals induced by the presence of 400 pM vinblastine and further incubated with [3H]-rhazinilam. These observations are in agreement with the inability of rhazinilam to interact with tubulin complexed with vinblastine.

DISCUSSION

We have shown that rhazinilam and taxol induced the same overall effects on mammalian cells. First, cells

I I I I I I ' 0 ' - 0

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0 10 20 30 40 50 60 70 80 90 FREE RHAZlNlLAM IpM)

Fig. 6. Binding of [3H]-rhazinilam to microtubule proteins by micro- equilibrium dialysis. Equilibrium dialysis of [3H]-rhazinilam was made against three-cycle-purified microtubule proteins and the concentrations of free and bound rhazinilam were calculated (see Mate- rials and Methods). A: Tubulin with (closed circles) or without GTP (squares) at 0°C. Inset: Relationship between bound [3H]-rhazinilam and the amount of sedimentable proteins (results obtained in another similar experiment). B: Microtubules without (82% of the initial amount of microtubule proteins present in the reaction mixture, closed circles) or with 20 pM taxol (98% of the initial amount of microtubule proteins present in the reaction mixture, open circles) and tubulin ribbons (79% of the initial amount of microtubule proteins present in the reaction mixture, squares) assembled in 30% dimethyl-sulfoxide at 37°C.

were blocked in mitosis and numerous microtubule asters were formed. Second, increasing drug concentrations induced both mitotic arrest and the presence of microtubule bundles in the cytoplasm of interphase cells. Third, the number of microtubule protofilaments was abnormal. Fourth, cytoplasmic microtubules were protected from cold disassembly. Thus, the action of rhazinilam on mammalian cells seemed to mimic the effects of taxol [Schiff and Horwitz, 1980; De Brabander et al., 1981; Masurovsky et al., 1981; Herman et al., 1983; Forry- Schaudies et al., 19861 although rhazinilam concentrations were higher than those of taxol.

In agreement with these observations, both rhazinilam and taxol stabilized microtubules from cold disassembly in vitro [Schiff et al., 19791. However, in vitro, they presented numerous differences including stabilization of microtubules in the presence of Ca2+ ions [Schiff et al., 19791, promotion of microtubule assembly [Schiff et al., 19791, spiralization, sensitivity to GTP [Schiff and Horwitz, 19811, and binding to microtubules [Parness

Rhazinilam. A New Microtubule Poison 325

and Horwitz, 1981; Manfredi et al., 1982; Takoudju et al., 1988bl. Moreover, [3H]-acetyl taxol binds to tubulin spirals induced by rhazinilam [Takoudju et al., 1988al suggesting that taxol and rhazinilam do dot interact at the same binding site on the tubulin molecule. Surprisingly, protection of microtubules from cold disassembly occurs in the absence of a significant binding of rhazinilam to microtubules. This observation contrasts with the stoi- chioinetric binding of taxol to microtubules [Parness and Horwitz, 198 1 ; Takoudju et al., 1988b], These observations show that rhazinilam and taxol stabilize microtubules by different mechanisms. Thus, the apparent similarity of effects of these two drugs in mammalian cells are secondary consequences of the overall stabilization of microtubules.

We investigated whether rhazinilam could share some common properties with other microtubule poisons, Rhazinilam induced the formation of anomalous tubulin assemblies, mostly composed by short spirals both at 0°C and 37”C, while no microtubules were assembled at 37°C. These spirals were assembled either from pure tubulin or from tubulin/colchicine complexes, suggesting that rhazinilam does not interact with the colchicine binding site. Although spirals induced by rhazinilam were morphologically similar to those formed at 0°C by diphenylpyridazone derivatives, a chemically unrelated microtubule poison [Batra et al., 19861, these two microtubule poisons exhibited different effects. In contrast to rhazinilam, diphenylpyridazone derivatives were unable to induce tubulin spirals at 37°C and to prevent rapid cold microtubule disassembly (unpublished observations). Spirals assembled by rhazinilam and vinblastine [Fujiwara and Tilney, 19751 were morphologically different: anomalous tubulin assemblies formed in the presence of rhazinilam were rather short and made by the association of a variable number of longitudinal filaments, while those formed in the presence of vinblastine were homogeneous and constituted long helices. However, they exhibit some similarities: they were stable at 0°C and 37°C; they could be observed at microtubule ends [Warfield and Bouck, 19741; their assembly required GTP, was stimulated by the presence of Ca2+ ions [Ventilla et al., 1975; Na and Timasheff, 19821, and was prevented by maytansine [Luduena et al., 19861. The saturable binding of [3H]-rhazinilam was abolished when tubulin was complexed with vinblastine or maytansine. Conversely, the binding of [‘HI-vinblastine to tubulin was strongly inhibited when tubulin was preincubated with rhazinilam (unpublished results). But we have not been able to determine whether these inhibitions were competitive. Thus, rhazinilam may interact, at least partly, with the common tubulin binding site of maytansine and vinblastine [Mandelbaum-Shavit et al., 1976; Bhattacharya and Wolff, 1977; York et al., 19811, al-

though more complex mechanisms could account for these observations. Comparison of the planar chemical formula of rhazinilam and vinblastine might suggest a similarity between rhazinilam and the vindoline portion of vinblastine. However, the three-dimensional configu- rations of these molecules exhibit large differences. Vin- doline lacks the pyrrolic aromatic ring and the nine- membered peptidic ring present in rhazinilam, and the stereochemistry of the side chain at C20 is different. Finally, vindoline is unable to perturb the assembly and disassembly of microtubules like the dextrarotary enan- tiomer of rhazinilam (unpublished results).

The action of rhazinilam, i.e., tubulin spiralization and protection of microtubules from cold disassembly, is dependent on the assembly state of tubulin. This effect has never been observed with other microtubule poisons. The protection of microtubules from cold disassembly is a very unexpected observation for a microtubule poison preventing microtubule assembly. The cold stability of microtubules in the presence of rhazinilam could be due to the binding of rhazinilam to a few tubulin molecules at the ends of microtubules as suggested by the formation of tubulin spirals at both microtubule ends (Fig. 3C). This hypothesis could account for our inability to ob- serve any binding of radioactive rhazinilam to microtubule over the background. It might also explain the un- folding of the microtubule ends in the presence of rhazinilam. Thus, the differential stability of microtubule towards cold and calcium disassembly in the presence of rhazinilam suggests that these two processes are rnedi- ated by a different mechanism on microtubules. Rhazi- nilam cannot be considered as a promising pharmacolog- ical agent. First, a high concentration is needed to induce cytotoxic effects. Second, its biotransformation in hu- man serum leads to inactive derivatives (unpublished results). However, its novel mode of action demonstrates that the search for new microtubule poisons remains a worthwhile challenge.

ACKNOWLEDGMENTS

The interest of Prof. P. Potier, the help of Prof. J . Cros, Prof. B. Roques, Dr. H. Hamid and Dr. K.C. Chan have been appreciated. We are indebted for the financial support of “L’ Association pour la Recherche contre le Cancer” and the help the “Ministkre des Af- faires Etrangeres.” The suggestions of Dr. J.C. Meunier are acknowledged.

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rhazinilam mimics the cellular effects of taxol by different mechanisms of action

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