microtubules as drug receptors: pharmacological properties of microtubule protein

PART 111. THE PHARMACOLOGY OF ANTIMITOTIC DRUGS

MICROTUBULES AS DRUG RECEPTORS: PHARMACOLOGICAL PROPERTIES OF

MICROTUBULE PROTEIN *

Leslie Wilson

Department of Pharmacology Stanford University School o f Medicine

Stanford, California 94305

In their closing comments on the mechanism of action of colchicine in their book on colchicine,’ Eigsti and Dustin predicted, “It is probably more than mere chance that the unique structure of this tropolone derivative is asso- ciated with so many physiological activities.” Now, almost 20 years later, we know that this situation exists because the structure of colchicine permits it to destroy selectively the function of a unique class of proteins; and we know that a large number of diverse cellular functions depend, either directly or indirectly, upon the proper functioning of these proteins.

Colchicine is one of a number of chemical agents originally known as spindle poisons, which share the property of being able to disrupt the assembly and function of microtubules. The chemical structure of colchicine, along with the structures of the vinca alkaloids vinblastine and vincristine, and podophyllotoxin, is shown in FIGURE 1 . Other compounds exist that disrupt the functions of microtubules, but colchicine, podophyllotoxin, and the vinca alkaloids have played key roles as tools in the investigation of microtubule function. In addition, their biochemical mechanisms of action are the most completely understood. Therefore, I should like to concentrate on the mechanisms of action of these drugs in this monograph.

EFFECTS OF SPINDLE POISONS UNRELATED TO THEIR ACTIONS ON MICROTUBULES

Before discussing how these drugs destroy the function of microtubules, I would like to comment on some of the other actions of these agents. There is no doubt that the antimitotic action of colchicine (as well as many of its other biological effects, such as the alteration of cell shape and inhibition of cellular movement) is due to the disruption of microtubules. It is important to understand, however, that colchicine exerts effects on cells that are not due to disruption of microtubules. The same is true for podophyllotoxin and the vinca alkaloids.

For example, colchicine inhibits nucleoside transport in a number of mammalian cell lines.2 Data obtained by Mizel and Wilson on inhibition of nucleoside transport by colchicine in HeLa cells are shown in FIGURE 2. In each case, the solid circles represent a double reciprocal plot of the transport rates

* This research was supported by United States Public Health Service Grant NS 09335 from the National Institute of Neurological Disease and Stroke, and by Ameri- can Cancer Society Grant (3-95.

213

214 Annals New York Academy of Sciences

Colchicine Padophyllotoxin Vinco Alkaloids

R = CH3 Vinblastine R=CHO Vincristine

FIGURE 1. Chemical structure of colchicine, podophyllotoxin, and the vinca alkaloids vinblastine and vincristine.

FIGURE 2. Inhibition of nucleoside transport by colchicine in HeLa cells. Cells were incubated for 10 min at 37" C with several concentrations of nucleosides in the presence (0) or absence (0) of 2.5x lo-' M colchicine; v=nmoles per 10 min/lV cells. The apparent inhibition constants were: adeno- sine, 6 . 6 ~ M; guanosine, 5 . 0 ~ lo-' M; thymidine, 6.1 x 1 0 4 M; and uridine, 4.5x

M. (From Mizel and Wil- son.' Reprinted by permission of the American Chemical So- ciety.)

Wilson: Microtubules as Drug Receptors 215

of varying concentrations of each nucleoside in the absence of colchicine, and the open circles, in the presence of colchicine. Inhibition is competitive. The inhibition constant for colchicine for all four nucleoside transport systems was approximately 5 x 1W moledliter.

This effect of colchicine does not seem to be due to the disruption of microtubules, since luminocolchicines, derivatives of colchicine that do not bind to tubulin and do not prevent microtubule a~sembly ,~ are as potent as colchicine, and colchicine does not bind to tubulin at O'C, but inhibits nucleoside transport as strongly at 0" C as it does at 37" C.? Thus the notion that colchicine can be used as an unquestioned, highly specific diagnostic tool for the involvement of microtubules does not hold.

Podophyllotoxin can also inhibit nucleoside transport in HeLa cells. This effect of podophyllotoxin, like that of colchicine, seems to be unrelated to an action on microtubules, since picropodophyllotoxin, which has a 1 00-fold lower affinity for tubulin than podophyllotoxin, is as active as podophyllotoxin in the inhibition of nucleoside uptake.41

The actions of the vinca alkaloids on cells are probably more complex than those of colchicine or podophyllotoxin. They seem to affect a variety of unrelated cellular processes.* For example, they inhibit the incorporation of nucleosides into RNA.5- has pointed out that this effect may be due to the inhibition of nucleoside uptake into the cell, as well as to specific inhibition of RNA synthesis. Kingsbury and Voelz8 have shown that vinblastine can cause the aggregation of ribosomes in a procaryotic cell, E. coli, which contains no microtubules.

The question of determining whether specific biological processes involve microtubules is a very important one, but it may be almost impossible to answer it conclusively in the laboratory. If a colchicine-sensitive process does involve the disruption of microtubules, then similar sensitivity should be observed with podophyllotoxin and the vinca alkaloids. By contrast, inactive or less active derivatives should possess little or no activity. Involvement of microtubules in a biological process certainly may be suggested, but is not proven to occur by disruption of function after treatment with colchicine and/or other drugs. Perhaps the best approach is to use a combination of physiological, pharmacological, and morphological methods.

Plagemann

COLCHICINE

Important Features of the Colchicine-Binding Reaction

A great deal of work has been done on the biochemical mechanism of action of colchicine. Much of this work has been described in detail in several recent reviews.3s4s9 Thus, the following will be a summary of several of the more important and interesting characteristics of this reaction.

If one incubates a solution that contains tubulin with tritium-labeled colchicine, the colchicine binds to the tubulin, forming a tight complex. The quantity of complex that forms can be determined accurately by several methods, including gel filtration or adsorption of the complex to discs of DEAE-cellulose impregnated filter paper.'O-16 The binding reaction between tubulin and colchicine has the properties of a biomolecular reaction. Binding is noncovalent, and colchicine is not chemically altered.


The concentration dependence of the binding of colchicine at 37°C to purified tubulin from 13-day-old chick embryo brain is shown in the form of a Scatchard plot" in FIGURE 3. These data have been corrected for the decay of colchicine-binding activity that occurred during incubation of the tubulin with colchicine, but have not been corrected for the decay of binding activity that had taken place during purification of the tubulin. The straight line indi- cates that there is a single affinity class of binding sites. The binding constant

FIGURE 3. Scatchard plot: binding of colchicine to purified chick embryo brain tubulin. Solutions of purified brain tubulin from 15-day-old chick embryos containing 5 . 2 ~ lo-' M vinblastine sulfate (to stabilize the colchicine-binding activity) were incubated with various concentrations of [ucetyl- 'HJcolchicine for 350 min at 37" C (see Wilson and Meza'" for descrip- tion of methods). Binding values were corrected for loss of colchicine- binding activity during incubation with colchicine. No correction has been applied for the loss of colchicine-binding activity during purification of the tubulin. J=the total number of colchicine moles bound per mole tubulin (rnol wt= 115,000).

in this experiment, calculated from the slope of the line, was 2 X lo6 I/mole. The dissociation constant, which is the reciprocal of this value, is 5 x IO-' moles/l. This is the concentration of colchicine that saturates 50% of the colchicine-binding sites. The intercept on the abscissa represents the maximum number of moles of colchicine that can bind per mole of tubulin. In this experiment, the value was 0.62. If one corrects for the decay of colchicine- binding activity estimated to have occurred during purification, this value becomes approximately 1. The same holds true for tubulins from other


l8 Therefore, there appears to be only one high-affinity colchicine- binding site per dimer molecule.

One important characteristic of the colchicine-binding reaction is that the rate of formation of the colchicine-tubulin complex is strongly temperature- dependent. At 0" C, the rate of binding is so slow that no colchicine binding activity can be detected with most tubulins. Interestingly, although the complex does not form at 0" C, when once constituted at 37" C, it is then stable at 0" C.

An interesting feature of the reaction is that the rate of complex formation is very slow. In fact, at low concentrations of colchicine, equilibrium is not reached for several hours at 37" C. Moreover, colchicine forms a remarkably tight complex with tubulin. With chick brain tubulin, the half-time for exchange from the complex of labeled colchicine for unlabeled colchicine is between 25 and 35 hours at 37" C.'?

The colchicine-binding reaction is unaffected by pH (between pH 5.5 and 8.5) and by variations in ionic strength (between 0.1 and 0.5).3 Thus, the interaction between colchicine and tubulin is not electrostatic. Bryan determined the thermodynamic constants of the binding reaction between colchicine and tubulin in sea urchin egg vinblastine crystals. The binding reaction was characterized by a positive enthalpy and positive entropy change, and a large free energy change; lS all of these data are consistent with the notion that colchicine binds in a hydrophobic or nonpolar pocket.

First-Order Decay of Colchicine-Binding Activity

Studies on the colchicine-binding reaction, and the use of colchicine-binding activity to quantitate tubulin, have been complicated by the fact that the colchicine-binding activity of most tubulins is unstable, and decays rapidly in an apparently first-order manner.3* 13, 15, l9! ?O It has been important to understand the decay phenomenon for two reasons: it has enabled us to look directly at the binding reaction after correction for the effects of decay, and it has permitted us to develop an accurate colchicine-binding assay for the quantitation of tubulin.

The results of a typical experiment showing the first-order decay of the colchicine-binding activity of chick embryo brain tubulin, under conditions of optimal stability, are shown in FIGURE 4. In this experiment, the extract was allowed to incubate at 37" C, and every 2 hours an aliquot was removed for a colchicine-binding assay, Each of these points represent the binding activity obtained after a 2-hour incubation with colchicine. The half-time for decay can be calculated from the slope of the line (it was approximately 4 hours in this experiment). The amount of colchicine that would have been bound if no decay had occurred can be determined by a 2-hour extrapolation, which is equal to the time of incubation. We have called this value the initial colchicine binding capacity, and it is independent of the rate of decay.

Some of the features of the decay process are summarized below. First, the rate of decay at 37" C under optimal conditions of stability is about 10 times faster than the rate of dissociation of bound colchicine; if one does not correct for decay, this makes the reversibility of the colchicine-binding reaction appear to be much faster than it really is. Decay itself appears to be completely irreversible. Agents have been found that can slow the rate of decay (among them, GTP and the vinca alkaloids), but none can reverse the loss


PREINCUBATION TIME (minuted FIGURE 4. First-order decay of colchicine-binding activity. A chick embryo brain

supernatant fraction was incubated at 37" C. At the times indicated, aliquots were removed and incubated with 2>( 10-O M [ace~yl-3H]colchicine for 2 hours at 37" C. Bound colchicine was assayed by the gel filtration method. Extrapolation of the line to include the time of the colchicine incubation (back 120 min) yields the initial binding capacity of the tubulin. The half-time is calculated from the slope of the line.

once it has occurred. The rate of decay is highly dependent upon pH, temperature, and the concentration and type of salt that is present in the b ~ f f e r . ~

One very critical parameter that affects the decay rate is the concentration of tubulin itself (FIGURE 5 ) . The decay rate of chick embryo brain tubulin is rapid at low tubulin concentrations. The protein becomes more stable as the tubulin concentration is increased, and reaches a plateau stability at a tubulin concentration of -250 pg/ml.

The biochemical nature of this decay process is not understood. Changes in decay rate may have some functional significance; for example, the activity is stabilized by the addition of guanine n u ~ l e o t i d e s , ~ ~ ~ which are normally bound to the protein. More likely, it represents some form of denaturation. Nevertheless, changes in the decay rate are of paramount practical importance when one is trying to use a colchicine-binding assay to determine the quantity of microtubule protein in tissue extracts. In fact, when corrections for the loss of colchicine-binding activity are not applied, the colchicine-binding values one obtains do not reflect the true quantity of tubulin in that extract.I5 We have found that decay rates differ widely in different tissues, and in the same tissue at different tubulin concentrations. Moreover, in chick embryo brain, Bamburg et a1.Z0 have found that decay rates differ significantly at different stages of development, even at the same tubulin concentration. The use of a time-decay colchicine-binding assay procedure actually yields quite accurate information on the quantity of tubulin in cell and tissue extracts.


Locution of the Colchicine-Binding Site in Assembled Microtubules

An understanding of the colchicine-binding reaction provides only the beginning of an answer to the question of how colchicine disrupts microtubules in vivo. One possible way in which colchicine could disrupt a microtubule would be by attaching to each tubulin dimer directly on the microtubule, so causing the tubulin to dissociate. An alternative possibility is that colchicine binds to soluble tubulin, and prevents microtubule assembly. Previously existing microtubules would then depolymerize at a rate consistent with the normal dissociation rate of the tubulin. We think that colchicine works by some variation of the latter mechanism.

It has recently been possible to demonstrate the presence of and characterize the colchicine-binding site on tubulin from stable outer doublet microtubules from S . purpurutus sperm tails.lG It is important to remember that these microtubules are resistant to the action of colchicine,21 and it was originally thought that this tubulin was different from tubulin derived from cytoplasmic micro-

I- A

; 300 c

; 300 - W

c

J I -l I

9 200

100 I 1 I I 1 I I 0 80 160 240 320 400 400 !KO

I' 100 I I 1 I I 1

0 80 160 240 320 400 400 !KO

NEUROTUBULE PROTEIN CONCENTRATION (pglrnl)

FIGURE 5 . Influence of microtubule (neurotubule) protein concentration on half- time and initial colchicine-binding capacity in embryonic chick brain supernatant fractions. Supernatant fractions obtained from 13-day-old chick embryo brain (containing 42% microtubule protein) were prepared, and time-decay colchicine-binding assays were carried out on samples of different microtubule protein concentration obtained by dilution with phosphate-glutamate buffer (20 mM sodium phosphate, 100 mM sodium glutamate, pH 6.75) to determine initial binding capacity (A) and half-time (0) . Specific activity=dpm bound colchicine per pg total protein in the supernatant fraction. Final concentration of colchicine was 4 . 2 ~ lo-' M. (From Bamburg et a1.I' Reprinted by permission of Neurobiology.)


tubules, in that it did not bind colchicine. We now know that the previous inability to demonstrate the presence of the colchicine-binding site on this tubulin resulted from the use of solubilization procedures that completely destroyed the colchicine-binding activity. This problem was circumvented by modifying the thermal depolymerization procedure of Stephens.?? We found conditions that could be used during depolymerization that preserved appreciable quantities of colchicine-binding activity. By applying corrections for the decay of colchicine-binding activity during depolymerization of the microtubules, and subsequently during incubation with colchicine, we characterized the colchicine-binding activity of this tubulin, and found it to be qualitatively similar to tubulins from labile microtubu1es.l" It then became meaningful to determine whether the colchicine-binding sites were free or blocked in the intact microtubule. In short, no free colchicine-binding sites could be detected on the surface of intact outer doublet microtubules.

The model these data support, if one accepts the assumption that the same situation holds true for labile microtubules, is that the colchicine-binding site is one of the protein interaction sites of tubulin. A tubulin-colchicine complex is incapable of associating with a second tubulin molecule. Thus the ability of colchicine to disrupt assembled microtubules would be dependent upon the stability of the microtubules, or in Dr. InouC's terms, the degree of dynamic eq~ilibrium.?~ The more stable the microtubule, the more resistant it would be to the action of colchicine. The action of colchicine in vivo would have a complex time dependency that involves the rate of binding of colchicine and the rate of depolymerization of existing microtubules.

PODOPHYLLOTOXIN

The colchicine-binding site on tubulin is also the binding site for podo- phyl l~ toxin .~~ 4 ~ 1 s A number of early morphological studies by light microscopy revealed that the antimitotic properties of podophyllotoxin were practically indistinguishable from those of colchicine.? Z 5 The first biochemical evidence that the mechanism of action of podophyllotoxin was very similar to that of colchicine was the finding that podophyllotoxin prevented the binding of colchicine to grasshopper embryo tubulin. Picropodophyllotoxin, an isomer of podophyllotoxin with considerably weaker antimitotic activity, was less potent than podophyllotoxin.'?

In crude chick embryo brain extracts, when podophyllotoxin is added prior to or simultaneously with colchicine, the binding of colchicine to tubulin is prevented in a concentration-dependent manner.'3 That podophyllotoxin com- petes with colchicine for the colchicine-binding site has been shown in experiments with tubulins from several different 16. In all cases inhibition is competitive, as is shown in FIGURE 6, which is a double reciprocal plot of colchicine binding to chick embryo brain tubulin in the absence and in the presence of podophyllotoxin.

Although colchicine and podophyllotoxin share the same binding site on tubulin, the mechanism of the podophyllotoxin-binding reaction is substantially different than that of the colchicine-binding reaction.

For example, we have found that, in contrast to colchicine, podophyllotoxin binds rapidly to tubulin (FIGURE 7A). The reaction appears to reach equilibrium within 5 min at 37" C. In this experiment (carried out by S. Achor in

I

Wilson: Microtubules as Drug Receptors 22 1

our laboratory), podophyllotoxin was incubated with a 100,000 x g supernatant extract of chick embryo brain for the times indicated. [AcetyL3H]- colchicine was added, and the extract was incubated with the labeled colchicine for an additional 15 min at 37" C; then the quantity of bound colchicine was determined rapidly, by a dextran-norite A procedure (see below),

-12 -10 -8 -6 -4 -2 0 2 4 6 8 10 12

I /kCHICfNE (moles/titer x 10-51

FIGURE 6. Competitive inhibition of colchicine binding to tubulin by podophyllotoxin. Aliquots of a 100,000 x g supernatant fraction from a 13-day-old chick embryo brain sonicate (total volume 0.5 ml; each aliquot contained 75 pg tubulin in 20 mM sodium phosphate, 100 mM sodium glutamate, pH 6.75, plus 5 . 2 ~ lo-' M vinblastine sulfate) were incubated with various concentrations of [a~etyl-~H]colchicine (specific activity 171 mCi/mmole) in absence (0) or presence (0) of 2 . 0 ~ M podophyllotoxin for 350 min at 37" C. Binding values were corrected for loss of colchicine-binding activity during the incubation. (From Wilson et a1.3 Reprinted by permission of Federation Proceedings.)

We have also studied the binding of podophyllotoxin to tubulin by back- titration with labeled colchicine after the removal of unbound podophyllotoxin. The method consists of incubating tubulin with podophyllotoxin under various conditions, and then rapidly removing all free podophyllotoxin. The amount of podophyllotoxin-binding at that site is then determined by the use of a colchicine-binding assay. The method for rapid removal of podophyllotoxin that was developed in our laboratory by S. Achor is as follows: A dextran-


4 = 9 0 0 1 80

=OO 15 30 45 60 75 90

c

f 70

h

Reincubation with Fodophyllotonln (15 min.)

0 at 0' A at 37'

9.00

.- f 4 0.04 0 I

0 0

i p 0.02

0

Tima (minuter)

FIGURE 7. Binding of podophyllotoxin to chick embryo brain tubulin. (A) Time dependence of the binding of podophyllotoxin. Aliauots (0.6 ml, 560 pg/ml total protein) of a 100,000 x g supernatant fraction from a 13-day-old chick embryo brain homogenate were incubated at 37" C with 3.6 x lo-' M podophyllotoxin. At the times indicated, 2.0 x 10.' M [~cetyl-~H]colchicine was added, and incubation con- tinued for 15 min. Bound colchicine was determined according to the dextran-norite A assay procedure (see text). (B) Reversibility and temperature dependence of the podophyllotoxin-binding reaction. Aliquots (2.25 ml, 160 pg/ml total protein) of a 100,000 x g supernatant fraction from a 13-day-old chick embryo brain homogenate were incubated at 37" C in the absence of podophylotoxin (O), in the presence of 1 . 4 ~ lo-' M podophyllotoxin at 37" C (A), and at 0" C (0) for 15 min. All free podophyllotoxin was then quickly removed by the dextran-norite A procedure (this took approximately 90 sec). Each sample was then incubated with 1.6 x lo-' M [a~etyl-~H]colchicine at 37" C, and a t the times indicated, 0.4 ml aliquots were removed and bound colchicine determined by the dextran-norite A procedure (see text).


norite A suspension is prepared by mixing a 1.5% (wt/vol) slurry of dextran T 7 0 and norite A ( 1 :5 , wt/wt). A solution that contains tubulin and free and bound podophyllotoxin is then mixed with the dextran-coated activated charcoal suspension for 10 sec. The suspension is then centrifuged rapidly. The dextran-charcoal adsorbs all unbound podophyllotoxin, leaving the tubulin behind in the supernatant solution with its bound podophyllotoxin. In fact, this method can be used to determine directly the binding of any ligand that can be adsorbed by charcoal, and we have used it successfully with colchicine, podophyllotoxin, and vinblastine.

A second difference between the binding mechanisms of podophyllotoxin and colchicine is that podophyllotoxin binding is rapidly reversible, as shown in FIGURE 7B. In this experiment, a chick brain extract was incubated at 37" C with sufficient podophyllotoxin to saturate fully all of the binding sites. Then the free podophyllotoxin was removed, and the colchicine-binding rate determined at 37" C. The line represented by the closed circles is the control (that is, no incubation with podophyllotoxin). The line represented by the closed

TABLE 1

TUBULIN AFFINITIES FOR COLCHICINE AND PODOPHYLLOTOXIN (AT 37' C)

Colchicine Podophyllotoxin KC (P~oPhYllotoxin Source of Purified Tubulin K d Ki Ks (Colchicine)

moles/l moles/l Embryonic chick brain 7.9 x 1 o-7 3.4 x 10-7 0.44 Sea urchin sperm tail outer

doublet microtubules (solubilized) In 1.6 x lo-" 1.3 x 10-a 0.8 1

Adult human brain 1*1 3 .4x lo-' 8.3 x lo-' 2.4 Sea urchin egg vinblastine

crystals'' (at 21" C) 8.5 x 10." 3 x 10" 3.5

triangles was the rate of binding of colchicine after saturation of the binding site with podophyllotoxin and the removal of free podophyllotoxin at 37" C. The exchange of labeled colchicine for podophyllotoxin is rapid, reaching -30% in 30 min. If the tubulin had been presaturated with colchicine instead of podophyllotoxin, no exchange would have been detected during this short period of time (unpublished data). A third difference is that podophyllotoxin binds to tubulin at 0' C. The open circles represent an identical preincubation with podophyllotoxin at 0" C. Appreciable binding of podophyllotoxin occurred; this was shown by the approximately 50% reduction in the rate of colchicine binding as compared with the rate in controls.

Additional evidence that the mechanisms of colchicine and podophyllotoxin differ is derived from examination of the relative affinities of these two drugs for tubulins from different sources (TABLE 1 ) . The Ki/ K,, ratio is a reflection of the relative affinities of these two drugs for tubulin. For chick embryo brain tubulin, this value is 0.44. This means that colchicine is less than half as potent as podophyllotoxin. For sea urchin sperm tail tubulin, this value is 0.81. For adult human brain, the value is 2.4, which means that colchicine is 2.4 times


more potent than podophyllotoxin, and for sea urchin egg vinblastine crystals, the value is 3.5. The ratios differ by a factor of almost 10; this suggests that the molecular mechanisms of the two drugs must be different.

VINCA ALKALOIDS

Treatment of cells or tissues that contain cytoplasmic microtubules with the vinca alkaloids results in disappearance of the microtubules, and the formation of highly regular crystals 27, m (see Wilson et al.3, 4 and Olmsted and Borisy for recent reviews). Vinblastine crystals isolated from unfertilized sea urchin eggs have been found to be composed of tubulin complexed to vinblastine.ls* 2 g 3 30 The finding that vinblastine caused crystal formation in vivo was quickly followed by the demonstration that it could cause the precipitation of tubulin in .vifr0,3~-~~ along with the assumption that the two effects were mechanistically related. However, precipitation in vitro and crystal formation in vivo seem to be produced by different mechanisms.

Vinblastine precipitates tubulin in a concentration-dependent manner; usu- ally high concentrations of vinblastine are required to bring about this effect. This appears, however, to be a non-specific effect of ~ i n b l a s t i n e . ~ ~ For example, we found that vinblastine could precipitate a large number of acidic proteins, including muscle actin, and also nucleic acids (for example, double-stranded DNA). All of the proteins that were vinblastine-precipitable could also be precipitated by calcium ions, and we argued that vinblastine was acting as an "organic cation."

These and other data suggested that there might be a large number of low-affinity binding sites for vinblastine on tubulin; and in fact, Creswell and I 4 3 have recently found that there are between 20 and 30 low-affinity vinblastine-binding sites per mole of chick embryo brain tubulin. These binding experiments were carried out by equilibrium dialysis with vinblastine in the millimolar concentration range; the protein ending up in the form of a vinblastine precipitate.

Several early observations suggested that there must be other binding sites on tubulin with higher affinity than those just described. First, the concentration range in which the vinca alkaloids exert their disruptive effects on microtubules is quite low, lower in fact than the concentration range commonly used for colchicine. The concentration dependence of the inhibition of mitosis in cultured mammalian (EHB) cells by vinblastine, desacetylvinblastine, and vincristine is shown in FIGURE 8. The concentrations required to produce a mitotic index of 50% for all three analogs were approximately the same, 8 x lo-* moles/l. Second, we can draw upon the observation that low concentrations of the vinca alkaloids slow the rate of decay of colchicine-binding activity.l3 Active vinca alkaloids such as vinblastine, vincristine, and desacetylvinblastine possess this property, whereas weaker or inactive derivatives (for example, leurosidine and catharathine) are less able to produce this effect, or do not possess this ability at all.3'

Another important observation, made by Weisenberg and Tima~heff ,~ ; is that low concentrations of vinblastine can cause an aggregation of tubulin, which is seen as an increase in the sedimentation coefficient from 6 S to approximately 14 S. We have also found that low concentrations of vincristine produce the same effect (unpublished data). Weisenberg and Timasheff calcu-

.


lated that the tubulin-vinblastine complex would have an extrapolated sedimentation coefficient of about 9.5 S at zero protein concentration, which is consistent with a dimer of the 6 S tubulin. Thus, the binding of the vinca alkaloids to a postulated class of high-affinity binding sites, which presumably prevents the tubulin from polymerizing, may induce the formation of tubulin dimers. This may be the initial event in the formation of crystals in vivo.

10-0 10-8 0-7 lo- 03 lo-4

VINCA ALKAWID (mdrs/lltrr)

FIGURE 8. Concentration-dependence of inhibition of mitosis in EBH cells by vinca alkaloids. EBH cell cultures (generation time 9 hours) were grown in the presence of different concentrations of vinblastine sulfate ( O ) , vincristine sulfate (O), and desacetylvinblastine sulfate (A) for 8 hours according to the method described by Creswell.3' The mitotic indices were determined on duplicate samples for each drug concentration. A minimum of 1000 cells/sample were scored."

Characterization of the High-Afinity Vinblastine Binding Sites on Chick Embryo Brain Tubitliri

We have characterized the high-affinity vinblastine binding sites of chick embryo brain tubulin by utilizing tritium-labeled vinblastine prepared in our laboratory. The labeled vinblastine, at a specific activity of 107 mCi/mmole, was synthesized by acetylation of desacetylvinblastine with tritium-labeled acetic anhydride, essentially by the method of Greenius et al.38 Purity of the final product was ascertained by several different methods, including column and thin layer chromatography and isotope dilution analysis. Purified chick embryo brain tubulin was used in all experiments.

Gel filtration proved to be unacceptable as a method for the quantitation of vinblastine binding activity, because the vinblastine dissociated from the


tubulin-vinblastine complex as it filtered down the column. Equilibrium dialysis was also useless. Because of vinblastine's large size, the time required to reach equilibrium was excessively long. Another serious problem we encoun- tered was that vinblastine binds tenaciously to glass, plastic, and dialysis membranes.

We finally settled on a DEAE-paper disc assay, identical to that used for the determination of colchicine binding to tubulin.13- 14+ lD This method has also been used successfully with vinblastine by Owellen et al.37 The problem with this method is that its efficiency is low. Approximately 34 of the initially bound vinblastine is lost during the time it takes to carry out the procedure. Nevertheless, the properties and binding constant of the reaction could be determined by this method. Stoichiometry was determined by an independent method (the use of an equilibrium gel filtration column).

The Vinblastine-Binding Reaction

The binding of vinblastine to chick brain tubulin at all temperatures (between 0" and 37" C) is fairly rapid. The results of a typical experiment carried out at 22" C are shown in FIGURE 9. Binding increased to within 80% of

a5

p a4 C

0 C

u) 0

c

Y

= 0.3 z 5 0 a2 C

ii 0. I

O O 30 60 90 120 Time at 22Oc (minutes)

FIGURE 9. Time dependence for the binding of vinblastine to chick embryo brain tubulin. Tubulin from 13-day-old chick embryo brain was purified by a single-step procedure,= modi- fied in that DEAE-cellulose (Whatman DE52) was employed rather than DEAE Sephadex, and 20 mM sodium pyrophosphate buffer was sub- stituted for the 20 mM sodium phosphate used previously, according to the method of Eip- per." The tubulin was eluted from the DEAE-cellulose column by a linear 100-300 mM NaCl gradient; the protein peak appeared at 220-230 mM NaCI. Tritium-labeled vinblastine (specific activity 21.4 mCi/ mmole) was prepared as described by Creswell.= A solution of purified tubulin (560 pg/ml, in 20 mM sodium pyrophosphate and 150 mM NaCI, pH 6.8) was incubated at 22" C. At the times indicated, an aliquot was removed and assayed for bound vinblastine by the DE-8 1 filter-disc method, exactly as employed previously for the determination of bound colchicine?'


FIGURE 10. Scatchard plot of the binding of vinblastine to purified chick embryo brain tubulin. Solutions of freshly purified brain tubulin from 13- to 15-day-old chick embryos in 20 mM sodium pyrophosphate and 150 mM NaCI, pH 6.8 (see the legend to FIG- URE 9) were incubated with various concentrations of labeled vinblastine for 30 min at 37" C. Bound vinblastine was determined by a DE-8 1 paper disc assay procedure, exactly as described previously for the binding of colchicine to tub~lin. '~ The binding constant (K), which was derived from the slope of the straight line (slopex - K O ) was 1 . 2 ~ 1 0 I/mole. The value of q (the extrapolated number of binding sites per mole of tubulin at infinite vinblastine concentration) was 0.41. No corrections have been applied for the loss of bound vinblastine that occurred during the assay procedure.

I 1 I I o 0.1 0.2 0.3 a4 m

Molrr Vinblootinr /Moir Tubulin

maximum within the first 15 min. Binding is not strongly temperature- dependent. At 0" C, maximum binding values were lower by -20%, and at 37" C they were higher by about 20%. Vinblastine binding activity is not appreciably affected by ionic strength, at NaCl concentrations between 5 and 500 mM.

The binding constant for vinblastine at 37" C was determined by analyzing binding values obtained over a wide range of vinblastine concentration by the method of Scatchard;" a plot of this type is shown in FIGURE 10. There appears to be a single class of binding sites. The association constant in this experiment was 1.2 X lo5 I/mole. The concentration of vinblastine that saturates 50% of the binding sites, which is the reciprocal of this value, is 8.5 X lo-'; moles/l. The intercept on the abscissa is the maximum number of moles of vinblastine that can bind per mole of tubulin. In this experiment, the value was 0.41. This does not take into account the amount of vinblastine that dissociated from the complex during the assay. Stoichiometry was determined with the use of an equilibrium column; .lo the number of binding sites per mole of tubulin was found t o be 2 ( to be published). Thus there appear to be two vinblastine-binding sites, each with a similar affinity, per dimer.


28 - FIGURE 11. Inhibition of vin-

24 blastine-binding activity by vincristine. Aliquots of a solution of freshly purified tubulin from - 13- to 15-day-old chick embryo brain in 20 mM sodium pyrophosphate and 150 mM NaCI, pH 6.8, were incubated with different concentrations of labeled vinblastine in the absence (0) and in the presence (0 ) of 1 x M vincristine sulfate for 30 min at 37" C.

5

P Bound vinblastine was deter- -3 - mined by a DE-81 paper disc

assay procedure.'3 The inhibition constant for vincristine was

- - c - I

20 - 0 - P \

t B s - t

-

-

- c

- 1.7 x lo-? moles/l.

-5 0 5 I0 I5 20 25 '/Vlnblartlnr (molor/litrr I WE)

Competitive Inhibition of Vinblastine-Binding Activity by Vincristine and Desacetylvinblastine

Vincristine binds at the vinblastine-binding sites, and can competitively inhibit the binding of vinblastine, as shown in FIGURE 11. The inhibition constant for vincristine in this experiment was 1.7 x 10-5 M. Similar competitive inhibition was obtained with a third vinca alkaloid, desacetylvinblastine; the inhibition constant here was 2.0 X M. Thus these three vinca alkaloids appear to have approximately the same affinity for tubulin. If one compares their relative potencies in the production of mitotic arrest in EHB cells (FIG- URE 8), one finds, similarly, that these three derivatives have approximately the same potencies.

How D o the Vinca Alkaloids Disrupt Microtubules?

I shall return to the question of the effective concentration range of the vinca alkaloids in vivo and the concentrations required for half-maximal saturation in vitro later in this paper. First, I would like to discuss the question of whether the vinblastine-binding sites are free or blocked in assembled microtubules. The study we carried out was essentially identical to that carried out for colchicine; we utilized outer doublet microtubules from sea urchin sperm tails.la Suspensions of purified outer doublet microtubules were incubated with


tritium-labeled vinblastine, at an equimolar ratio of tubulin-vinblastine. The vinblastine concentration was 1.3 X l W M, which would saturate more than 50% of the vinblastine-binding sites in brain tubulin. The vinblastine-binding characteristics of tubulin solubilized from outer doublet microtubules have been worked out only in a preliminary fashion; but we have obtained sufficient data to suggest that the vinblastine-binding characteristics of solubilized outer doublet tubulin are not appreciably different from those of brain tubulin. Outer doublet microtubule suspensions were incubated for various periods of time (between 15 min and 2 hours) and then were centrifuged to remove all microtubules and any bound vinblastine. The amount of radioactivity that remained in the supernatant fraction after centrifugation was determined and compared with the amount of radioactivity present in the original suspension. No vinblastine could be detected pelleting along with the microtubules. The highest quantity of vinblastine that could possibly have been bound in this experiment would have been equal to our pipetting error. If one then calculates a maximum binding constant, utilizing the pipetting error, it would be only 5 x lo? l/mole. Therefore the results are similar to those with colchicine: the vinblastine-binding site is blocked, or the affinity for vinblastine is reduced more than 500-fold in the assembled microtubule. Again, if we permit the assumption that an analogous situation exists for labile microtubules, the data suggest that the vinca alkaloids disrupt microtubules by preventing the assembly reaction, rather than by directly disrupting preformed microtubules.

Let us return now to a discussion of the relationship between the effective in vivo concentration and the binding constant. I t is most interesting that the effective range in vivo is more than 100-fold lower than one would expect on the basis of a dissociation constaut of 1 >( 1 P 5 M. This suggests the possibility that the vinca alkaloids need statistically bind to less than 1 % of the total tubulin in order to disrupt microtubule function half-maximally.

This same phenomenon has been observed by Olmsted and Borisy with colchicine, and Borisy’s group has proposed the idea that colchicine works by “poisoning” microtubule assembly.” It is reasonable to postulate that colchicine, podophyllotoxin, and the vinca alkaloids may all work by this general mechanism. It is easy to visualize the prevention of microtubule assembly by the presence of a colchicine or vinblastine molecule bound at the growing end of a microtubule, or by the attachment of a tubulin-drug complex to the end of a growing microtubule, terminating elongation.

In conclusion, through the efforts of many investigators, we have learned a great deal concerning the mechanism of action of chemical agents that disrupt microtubules. It is certain that further understanding of the molecular mechanisms of action of these agents will make these drugs even more valuable as probes for the study of microtubule function.

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microtubules as drug receptors: pharmacological properties of microtubule protein

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