[CANCER RESEARCH 45, 2234-2239, May 1985]

Effect of Estramustine Phosphate on the Assembly of Isolated Bovine BrainMicrotubules and Fast Axonal Transport in the Frog Sciatic Nerve1

Martin Kanje, Johanna Deinum, Margareta Wallin, Per Ekström,Anders Edström,and Beryl Hartley-Asp2

Department of Zoophysio/ogy, University of Lund, S-223 62 Lund ¡M.K., P. £.,A. E.J; Department of Medical Physics, University of Göteborg, Box 330 31, S-400 33Göteborg[J. D.¡;Department of Zoophysiology, University of Göteborg,Box 250 59, S-400 31 Göteborg[M. W.]; and AB Leo, Research Laboratories, Box 941, S-25109 Helsingborg [B. H-A.J, Sweden

ABSTRACT MATERIALS AND METHODS

Estramustine phosphate (0.01 to 0.5 HIM),an estradici mustardderivative used in the therapy of prostatic carcinoma, inhibitedthe assembly of brain microtubule proteins in vitro and disassembled preformed microtubules. In the presence of estramustinephosphate, the minimum microtubule-protein concentration suf

ficient for the assembly of microtubules was increased. Lowconcentrations of taxol (20 U.M)completely reversed the inhibitionof assembly by estramustine phosphate. The effects were specific to estramustine phosphate since neither estradiol 170-

phosphate, the hormonal moiety of the drug, nor nornitrogenmustard, the alkylating moiety, had any effect on assembly.

Estramustine phosphate (0.1 to 0.5 HIM) was also found toreversibly inhibit fast axonal transport in the frog sciatic nerve.The nerve content of adenosine triphosphate, adenosine diphos-

phate, and adenosine monophosphate was not significantly affected by estramustine phosphate.

Our results suggest that the cytotoxic action of estramustinephosphate could be dependent partially on an interaction withmicrotubules, probably via the microtubule-associated proteins.

INTRODUCTION

Estramustine phosphate (estradici 3-[W,A/-bis(2-chloro-ethyl)carbamate] 17/3-phosphate| is active against advanced

prostatic carcinoma (14). The mechanism of action of the drugis not fully understood and cannot solely be ascribed to itsantigonadotropic properties (10, 20). It is cytotoxic in humanprostatic carcinoma cells in culture (13) and induces mitotic arrestat metaphase (12). These metaphases have contracted chromosomes, which no longer are aligned in the metaphase plane.Other drugs which exhibit this property, such as colchicine,vinblastine, and nocodazole (23), are inhibitors of the assemblyof microtubules, the main component of the mitotic spindle.Hitherto, a direct effect of estramustine phosphate on microtubules in vitro or on other microtubule-dependent processes, such

as fast axonal transport, has not been studied.In the present study, the inhibitory effect of estramustine

phosphate on the assembly of isolated brain microtubules andon fast axonal transport is reported. The results indicate that thecytotoxicity of estramustine might be caused by an interactionwith microtubule proteins.

' Part of this work has been supported by grants from the National Research

Council, Harald Jeanssons Stiftelse, Magnus Bergvalls Stifteise, Torsten andRagnar Söderbergs Stiftelse, Kungliga Hvitfeltska överskotts Fonden, Lars HiertasMinne, and AB Leos Research Foundation.

1To whom requests for reprints should be addressed.

Received 4/23/84; revised 9/19/84. 1/11/85; accepted 1/24/85.

Chemicals. Estramustine (estradiol 3-[A/,A/-bis(2-chloro-ethyl)carbamate] 17-hydroxy), estramustine phosphate (estradiol 3-[A/,N-bis(2-chloroethyl)carbamate] 17/3-phosphate), and estradiol 17ff-phos-

phate were synthesized by AB Leo, Helsingborg, Sweden. Estramustinephosphate was dissolved in distilled water, and its concentration wasestimated from the absorbance at 274 nm using the extinction coefficientof estramustine, Et% = 17.7.3 In all the experiments with the drug, eitherCa2+- and Mg2+-free solutions or solutions containing EDTA were usedto avoid precipitation of an insoluble Ca2+: or Mg2+:estramustine phos

phate complex. EDTA was used as the chelator because the buffercontained 0.5 mw MgSO4 but no added Ca2*. Estramustine was dis

solved in Cremophor ELethanol (1:9) before dilution in standard frogRinger's solution. Cremophor EL was kindly provided by Dr. K-E. Falk,

AB Hassle, Mölndal,Sweden.Taxol was a gift from Dr. M. Sufness at the NIH, Bethesda, MD. L-

[4,5-3H]Leucine (130 Ci/mmol, 1 mCi/mL) was obtained from The Radi-

ochemical Centre, Amersham, England. All other chemicals were reagentgrade.

Microtubule Proteins. Microtubule proteins were prepared from bovine brain in the absence of glycerol by 2 or 3 cycles of assembly-

disassembly in the presence of 0.5 mw MgSCu (2,19). In the first cycle,ethyleneglycolbis(2-aminoethyl ether)-W,N'-tetraacetic acid was presentto complex free Ca2+. The final pellet, which contains approximately 80%

tubulin (5), was stored in liquid nitrogen. Prior to use, the pellet wasresuspended in buffer [100 mw piperazine-/V,A/'-bis(2-ethanesulfonic

acid), 0.5 row MgSO«,with 1 DIM GTP at pH 6.8]. After incubation at4°Cfor 30 min, the sample was clarified by centrifugation at 35,000 x gfor 30 min at 4°C.Protein solutions were stored in liquid nitrogen after

drop freezing (5).Protein Concentration. Microtubule protein concentration was deter

mined by Bio-Rad protein assay using Coomassie Brilliant Blue based

on the method of Bradford (3) with tubulin as a standard. The concentration of the standard was determined from E27B= 1.2 mg"1 -cm*2 and a

molecular weight of 110,000 (23).Assembly. Assembly of microtubule proteins in 100 rriM piperazine-

N,A/'-bis(2-ethanesulfonic acid), 0.5 mw MgSCu, 1 mw EDTA, and 1 nriM

GTP was started by addition of 50 n\ microtubule proteins at 4°Cto 300ti\ buffer at 37°C, and the increase in turbidity was monitored continu

ously by the change in absorbance at 350 nm (5). Estramustine phosphate or an equivalent amount of water was added from a stock solutioneither to the buffer or to the protein. It was found that the addition of 1mM EDTA did not inhibit microtubule assembly. Taxol was added from a3 mM stock solution in dimethyl sulfoxide.

Axonal Transport. The sciatic nerve together with the eighth andninth dorsal ganglia were dissected from frogs (ñañatemporaria). Aligature was placed on the nerve 30 mm from the ganglia. Treated andcontrol nerve preparations were from the same frog. The nerves wereincubated in an apparatus where the ganglia could be separated fromthe nerve by silicon grease barriers (8). Frog Ringer's solution devoid ofCa2+ and Mg2+ with or without estramustine phosphate was added tothe nerve compartment. The absence of these cations in the Ringer's

3AB Leo, personal communication.

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EFFECT OF ESTRAMUSTINE PHOSPHATE ON MICROTUBULES

solutions does not affect axonal transport (6) in sheated frog sciaticnerves. The ganglia were exposed to [3H]leucine containing standardfrog Ringer's solution. After incubation for 17 h at 18°C,the nerve was

cut into 3-mm segments and analyzed for protein-incorporated radioac

tivity as described previously (8). The transport rate was determinedusing pulse-labeling of the ganglia for 2 h with Ringer's solution contain

ing [3H]leucine followed by incubation for 10 h in Ca2+-, Mg2+-freeRinger's solution with or without the drug.

Adenosine Nucleotide Determination. Nerves were incubated for 17h at 18°C,blotted on filter paper, weighed, dropped into liquid nitrogen,

and crushed. Nucleotides were extracted with 10% trichloroacetic acid.The extract was neutralized by extracting the trichloroacetic acid bytrioctylamine in 1,1,2-trichlorotrifluoroethane (21) prior to injection into aPolyanion SI column (Pharmacia) attached to a Varían5000 high-pres

sure liquid Chromatograph. Nucleotides were eluted by a linear phosphategradient, 10 to 850 mw, pH 7.0, at a flow rate of 1 ml/min during 15 min.ATP, ADP, and AMP were detected by their absorbance at 254 nm andquantified using a nucleotide standard.

Electron Microscopy. Negatively stained microtubule specimens forelectron microscopy were prepared from S-i¿\protein samples. Fixationwas performed with 1 drop of Karnovsky's solution (17), after which the

specimen was washed with distilled water and stained with 1% uranylacetate. Embedded microtubule specimens were prepared after centrif-ugation at 35,000 x g for 30 min at 35°C. The pellets were fixed withKarnovsky's solution followed by 1% osmium tetroxide in 0.1 M cacc-

dylate buffer (17). The pellets were dehydrated in a graded series ofacetone and embedded in Epon. Thin sections were made on a LKBultramicrotome and were double stained with uranyl acetate and leadcitrate. The specimens were viewed in a Zeiss 109 electron microscope.

Nerves exposed to estramustine phosphate were fixed in 2.5% glu-

taraldehyde (16) followed by 1% osmium tetroxide. Subsequently, thenerves were dehydrated in a graded series of ethanol, block stained in1% phosphotungstic acid and 0.5% uranyl acetate, and embedded inEpon:Araldite.

RESULTS

Effects of Estramustine Phosphate. Estramustine phosphateinhibited the assembly of microtubule proteins in a concentration-

dependent manner (Charts 1 and 2). The rate and extent ofassembly was less in the presence of estramustine phosphate.Furthermore, estramustine phosphate induced rapid disassembly of microtubules at steady state (Chart 1). No difference inthe level of assembly was found whether estramustine phosphate was added initially or at steady state (see Chart 2) or ifthe microtubule proteins were preincubated with the drug for 30min at 4°C.The electron micrographs (not shown) of microtu

bules disassembled to approximately 50%, taken directly afterthe addition of estramustine phosphate, showed normal butfewer microtubules with extending projections.

The extent of microtubule assembly in the presence of increasing amounts of estramustine phosphate showed a nonlineardose-response relationship (Chart 2). The same curve was ob

tained if the disassembly level was plotted.Addition of estramustine phosphate at a constant concentra

tion to preformed microtubules induced the same change inturbidity independent of the protein concentration. If the turbiditywas plotted against different microtubule protein concentrations,it could be seen that with a constant concentration of estramustine phosphate a linear relationship remained with the sameslope as for the control but shifted to the right (Chart 3). Thus,in the presence of estramustine phosphate the critical proteinconcentration, i.e., the minimum microtubule protein concentra

20 25

TIME (min)

Chart 1. Time course of assembly of brain microtubules. Estramustine phosphate (EMP) (0.42 mM) was added initially (Trace A) or at steady state level ofassembly (Trace B at arrow). After complete disassembly, 20 /JMtaxol was added(Trace B at arrow). Microtubule assembly (1.8 mg/ml protein) was started by raisingthe temperature from 10 to 37°Cand was monitored by the increase in absorbanceat 350 nm against time. The assembly buffer contained 0.1 M piperazine-W,N'-

bis(2-ethanesulfonic acid) at pH 6.8, 0.5 mM MgSO4, 1 mw GTP, and 1 mw EDTA.

50-

--a

A EMPat "steady state"

•E2

50 100 50 200

drug (p M )500 1000

Chart 2. Microtubule assembly at different concentrations of estramustine phosphate (EMP). The steady state level of assembly was measured after preincubationfor 10 min at 10°Cwith estramustine phosphate, nornitrogen mustard (nor-HW2),or estradici 17fj-phosphate (£2),and furthermore after addition of estramustinephosphate to preformed microtubules (A). The level was expressed in percentageof the AAaso of the control, since different protein preparations were used. Themicrotubule protein concentration was 2.1 mg/ml, and the conditions were asdescribed in the legend to Chart 1.

tion sufficient for assembly of microtubules, was increased.Low concentrations (20 MM) of the microtubule-promoting

drug, taxol, were able to reinduce assembly of completely inhibited microtubule proteins to the same or higher levels than thatof the control (Chart 1). The electron micrographs of the pelletof the taxol-induced microtubules showed the presence of many

aberrant forms of microtubules as compared to the microtubulesformed in the absence of the drug (Fig. 1).

The effect on microtubule assembly was specific for estramustine phosphate since neither estradici 1/¿(-phosphate nor

nornitrogen mustard had any effect on microtubule assembly atcomparable concentrations (see Chart 2). Furthermore, 1 mwestradiol 17/3-phosphate did not compete with estramustine

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EFFECT OF ESTRAMUSTINE PHOSPHATE ON MICROTUBULES

0.4-

0.3-

0.2-

0.1-

0123

MICROTUBULE PROTEIN (mg/ml)

Chart 3. Microtubule assembly at different protein concentrations in the presence of estramustine phosphate. The steady state level of microtubule assemblywas measured after addition of estramustine phosphate (•.O P.M;•,84 >IM;O,151 ¡¡M)to preformed microtubules at 37°C.The conditions were as described in

the legend to Chart 1.

Table 1Effects of estramustine phosphate and estramustine on the accumulation of 3H-

labeled protein in front of a ligatureAccumulation in drug-treated nerve8 (%)

0.1 RIM 0.5 rriM

Estramustine phosphateEstramustineEstradici 170-phosphateNornitrogen mustard57±56'c

59±5C25±6C 34±6C82 ±23 (NS)d

87 ±23 (NS)

Values are expressed as the accumulation in the drug-treated nerve in percentage of that in the control nerve.

6 Mean ±standard average.CP = 0.001." NS, not significant.

phosphate for the same binding site, because in its presence theinhibitory action of the drug on microtubule assembly was unchanged. Furthermore, electron micrographs of microtubulesassembled in the presence of 0.5 ITIMconcentrations of eitherestradici 17/3-phosphateor nornitrogen mustard showed perfectmicrotubules (not shown).

Axonal Transport. Estramustinephosphateat 0.5 and 0.1rriM reduced the accumulation of labeled material in front of aligature to 25 and 57%, respectively, of the control values.Estramustine was equally effective as an inhibitor of axonaltransport (Table 1). Furthermore, the effect was specific becauseneither 0.5 mw estradici 170-phosphate nor 0.5 mw nornitrogenmustard had any effect on axonal transport (Table 1).

The accumulation of radioactive material in the presence of0.1 ITIMestramustine phosphate was more suppressed [31 ±5% (SD), P < 0.05, N = 8] in nerves preincubated at 1°Cfor 3

h than in nerves which had not been pretreated by cooling.The decrease in axonal transport by 0.5 ITIMestramustine

phosphate was reversible. Axonal transport did not differ fromthe control after 5 h preincubation at 4°Cin the presence of 0.5

mMestramustine phosphate, followed by washing with standardRinger's solution and subsequent assay. Inelectron micrographs

of the sciatic nerves treated with the drug, microtubules couldstill be observed (not shown).

From the above experiments, in which ligatures were used,differentiation of the effect of the drug on the rate of transportor on the amount of transported materials is not possible. However, the pulse-labeling experiment (Chart 4) showed that therewas little difference in the total amount of transported materialbut a reduced transport rate in the presence of 0.5 mM estramustine phosphate.

Energy Metabolism. In orderto excludea metaboliceffectofestramustine phosphate as a cause of the inhibited axonaltransport, the levels of ATP, ADP, and AMP were measured innerves which had been incubated with the drug. As can be seenin Table 2, the adenosine nucleotide composition was not significantly influenced by a 17-h incubation with either estramustinephosphate or estramustine.

DISCUSSION

The mechanismof action of the anticancer agent estramustinephosphate is not known. In humans, the drug is rapidly dephos-

6-

81

12 24 36 48Distance along axon (mm)

Chart 4. Frog sciatic nerve ganglia were pulse-labeled with[3H]leucine for 2 hand subsequently transferred to Ringer's solution ±0.5 mM estramustine phos

phate (EMP). After 10 h incubation, the protein-incorporated radioactivity (y axis)was measured in successive 3-mm segments (x axis).

Table 2

Effects of estramustine phosphate on the adenosine nucleotide content of frogsciatic nerve

The nerves were incubated for 17 h at 18°Cin Ringer's solution, free of Ca2*and Mgz+ ±0.1 HIM drug. The nucleotide content was analyzed as described in"Materials and Methods." The mean concentrations of ATP, ADP, and AMP (^mol/

mg, wet weight) were 0.95 ±0.04, 0.25 ±0.06, and 0.16 ±0.06, respectively.

Drug-treated:control ratio

AMPADPATP+0.1

mMestramustine0.92±0.16a

1.02 ±0.261.00 ±0.17+0.1

mMestramustinephosphate1.02

±0.150.97 ±0.130.82 ±0.28

5Mean ±SD, W= 6 (Student's t test).

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phorylated releasing estramustine, which is readily oxidized toestromustine (11). Thus, on hydrolysis, both estradici and estrone are released to exert their hormonal effect on the prostatictumor (10). However, estramustine itself does not compete withestradici for the estrogen receptor site (9, 20), indicating a lackof classical hormone effect. Estramustine does not induce DNAstrand breaks either, indicating a lack of alkylating activity, inspite of the nornitrogen mustard moiety of estramustine (22).However, the observation that estramustine inhibits mitosis inprostate tumor cells in culture (12) implicates an involvement ofmicrotubules in its mode of action.

In the present study, we studied the effect of estramustinephosphate on the assembly of cold-labile microtubules from

brain. Brain tissue contains relatively large amounts of microtubules. Cold-labile microtubules from different tissues have beenshown to have the same general properties with respect todifferent assembly inhibitors. We found that estramustine phosphate inhibited brain microtubule assembly in vitro at physiologically relevant concentrations and induced rapid disassembly ofpreformed microtubules. The effect on microtubules in vitro wasspecific for estramustine phosphate because neither nornitrogenmustard nor estradiol 170-phosphate, the 2 parent moieties,affected microtubule assembly or disassembly. Neither could the2 parent moieties potentiate the effect of estramustine phosphate on microtubule assembly.

Although estramustine has a colchicine-like effect on mitosis,preliminary results indicate that the drug does not interfere withthe binding of colchicine to tubulin. Our experiments suggestthat estramustine phosphate interacts with MAPs.4 Hence, es

tramustine phosphate increased the critical protein concentrationrequired for the assembly of microtubules, in contrast to colchicine, vinblastine, and nocodazole (23), which specifically bind tothe tubulin dimer. This effect is also exhibited by drugs whichhave a direct effect on the MAPs-dependent nucleation phase ofassembly and which bind to MAPs, such as DNA (1) and heparin(4). The reversal of the estramustine phosphate-induced inhibition of assembly by taxol is not possible in the presence oftubulin-binding drugs such as colchicine (18), which is furthersupport for an interaction of estramustine phosphate with MAPs.Furthermore, we have found (24) that 3H-labeled estramustine

phosphate bound predominantly to MAPs and moreover thataddition of MAPs reversed the inhibition of assembly induced byestramustine phosphate. However, the relatively high molar ratio(compared with the concentration of MAPs) of estramustinephosphate needed for complete inhibition of assembly and thenonlinear dose-response curve (Chart 2) suggest a weak interaction, which is also indicated by the reversibility of the effect onaxonal transport.

Axonal transport, a microtubule-dependent process, was usedto assess the effect of estramustine phosphate and estramustinein an intact cellular system. In agreement with the inhibition ofthe drug on microtubule assembly is the finding that estramustinephosphate and estramustine inhibited axonal transport in thesciatic nerve. This also demonstrates that the phosphate groupcontributes only by increasing the solubility of the drug. Theinhibition occurred without significant alterations of the nervecontent of ATP, ADP, and AMP. The inhibition was reversible,and low temperature intensified the effect of the drug, which iscomparable to the effects of colchicine on the sciatic nerve (7).

4The abbreviation used is: MAPs, microtubule-associated proteins.

Together with the observed inhibition of mitosis (12) and therecent observation that estramustine inhibits microtubule-dependent pigment granule movement in squirrel fish erythro-phores,5 the results suggest that estramustine phosphate exerts

its cytotoxic effect by interaction with microtubules in the intactcell also.

ACKNOWLEDGMENTS

Thanks are due to Inger Antonsson for expert technical assistance.

REFERENCES

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2. Borisy, G. G., Olmsted, J. B., Marcum, J. M., and Allen, C. Microtubuleassembly in vitro. Fed. Proc., 33: 167-174, 1974.

3. Bradford, C. A rapid and sensitive method for the quantitation of microgramquantities of protein utilizing the principle of protein-dye binding. Anal.Biochem., 72: 248-254,1976.

4. Deinum, J., Sjirskog, L. Wallin, M., and Dahlbàck, E. Molecular weightdependency heparin inhibition of microtubule assembly in vitro. Biochim.Biophys. Acta, 802: 41-48, 1984.

5. Deinum, J., Wallin. M., and Lagercrantz, C. Spatial separation of the twoessential thiol groups and the binding-site of the exchangeable GTP in braintubulin. A spin label study. Biochim. Biophys. Acta, 677: 1-8, 1981.

6. Edström,A. J. Effects of Ca(ll) and Mg(ll) on rapid axonal transport of proteinsin vitro in frog sciatic nerve. J. Cell Biol., 67: 812-818,1974.

7. Edström, A., Hanson, M., Wallin, M., and Cederholm, B. Inhibition of fastaxonal transport and microtubule polymerisation in vitro by colchicine andcolchiceine. Acta Physiol. Scand., 707: 233-237, 1979.

8. Edström, A., and Mattson, H. J. Fast axonal transport in vitro in the sciaticsystem of the frog. Neurochemistry, 79: 205-221,1972.

9. Forsgren, B., and Bj0rk, P. Specific binding of estramustine to prostaticproteins. J Urol., 23 (Suppl.V 34-38. 1984.

10. Fritjofsson, A., Morten, B. J., Hogberg. B., Rajalakshmi, M., Cekan, S. Z.. andDczfalusy, E. Hormonal effects of different doses of estramustine phosphate[Estracyt(R)] in patients with prostatic carcinoma. Scand. J. Urol. Nephrol., 75:37-44, 1981.

11. Gunnarsson, P. O., Plym Forshell, G., Fritjofsson, A., and Morten, B. J. Plasmaconcentration of estramustine phosphate and its major metabolites in patientswith prostatic carcinoma treated with different doses of estramustine phosphate (Estracyt). Scand. J. Urol. Nephrol., 75: 201-206, 1981.

12. Hartley-Asp, B. Estramustine induced mitotic arrest in two human prostaticcarcinoma cell-lines Du145 and PC-3. Prostate. 5: 93-100, 1984.

13. Hartley-Asp, B.. and Gunnarson, P.-O. Growth and cell survival followingtreatment with estramustine. nor-nitrogen mustard, estradiol and testosteroneof a human prostatic cancer cell line. J. Urol., 727: 818-822,1982.

14. Jonsson.G.. Hogberg, B., and Nilsson. T. Treatment of advanced prostatecarcinoma with estramustine phosphate [Estracyt(R)]. Scand. J. Urol. Nephrol.,77:231-238, 1977.

15. Kadohama, N., Kirdani, R. Y., Madajewicz, S., Murphy, G. P., and Sandberg,A. A. Estramustine. Metabolic pattern and possible mechanisms for its actionin prostatic cancer. NY State J Med.. 79: 1005-1009, 1979.

16. Kanje, M., Edström, A., and Hansson, M. Inhibition of rapid axonal transportin vitro by the ionophores X537A and X23187. Brain Res., 204: 43-50, 1981.

17. Karnovsky, M. J. Formaldehyde-glutaraldehyde fixative of high osmolality foruse in electron microscopy. J. Cell Biol., 27: 137A, 1965.

18. Kumar, N. J. Taxol-induced polymerization of purified tubulin. J. Biol. Chem.,256: 10435-10441, 1981.

19. Larsson, H., Wallin, M., and Edström, A. Induction of a sheet of polymer oftubulin by Zn(ll). Exp. Cell Res., 700: 104-110. 1976.

20. LeClercq, G., Heuson, J.-C., and Deboei, M. C. Estrogen receptor interactionwith Estracyt(R) and degradation products, a biochemical study on a potentialagent in the treatment of breast cancer. Eur. J. Drug Metab. Pharmacokinet.,2: 77-84, 1976.

21. Pogolotti, A. L, and Santi, D. V. High-pressure liquid chromatography-ultravi-olet analysis of intracellular nucleotides. Anal. Biochem., 726: 335-345,1982.

22. Tew, K. D., Ericksson, L. C., White, G., Wang, A. L., Schein, P. S., and Hartley-Asp, B. Cytotoxicity of a steroid-nitrogen mustard derivative through non-DNAtargets. Mol. Pharmacol., 24: 324-328, 1983.

23. Wallin, M., and Deinum, J. Tubulin. In: A. Laitha (ed.), Handbook of Neurochemistry, Vol. 5, pp. 101-126. New York: Plenum Publishing Corp., 1983.

24. Wallin, M.. Deinum, J., and Friden, B. Interaction of estramustine phosphatewith microtubule-associated proteins. FEBS Lett., 779: 289-293, 1985.

sM. E. Steams and K. D Tew. personal communication.

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1985;45:2234-2239. Cancer Res   Martin Kanje, Johanna Deinum, Margareta Wallin, et al.   Frog Sciatic NerveBovine Brain Microtubules and Fast Axonal Transport in the Effect of Estramustine Phosphate on the Assembly of Isolated

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