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

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

Martin Kanje, Johanna Deinum, Margareta Wallin, Per EkstrÃ¶m,Anders EdstrÃ¶m,and Beryl Hartley-Asp2

Department of Zoophysio/ogy, University of Lund, S-223 62 Lund Â¡M.K., P. Â£.,A. E.J; Department of Medical Physics, University of GÃ¶teborg, Box 330 31, S-400 33 GÃ¶teborg[J. D.Â¡;Department of Zoophysiology, University of GÃ¶teborg,Box 250 59, S-400 31 GÃ¶teborg[M. W.]; and AB Leo, Research Laboratories, Box 941, S-251 09 Helsingborg [B. H-A.J, Sweden

ABSTRACT MATERIALS AND METHODS

Estramustine phosphate (0.01 to 0.5 HIM),an estradici mustard Chemicals. Estramustine (estradiol 3-[A/,A/-bis(2-chloroethyl)carbamate] 17-hydroxy), estramustine phosphate (estradiol 3-[A/,N- derivative used in the therapy of prostatic carcinoma, inhibited bis(2-chloroethyl)carbamate] 17/3-phosphate), and estradiol 17ff-phos- the assembly of brain microtubule proteins in vitro and disassem phate were synthesized by AB Leo, Helsingborg, Sweden. Estramustine bled preformed microtubules. In the presence of estramustine phosphate was dissolved in distilled water, and its concentration was phosphate, the minimum microtubule-protein concentration suf estimated from the absorbance at 274 nm using the extinction coefficient ficient for the assembly of microtubules was increased. Low of estramustine, Et% = 17.7.3 In all the experiments with the drug, either concentrations of taxol (20 U.M)completely reversed the inhibition Ca2+- and Mg2+-free solutions or solutions containing EDTA were used of assembly by estramustine phosphate. The effects were spe to avoid precipitation of an insoluble Ca2+: or Mg2+:estramustine phos cific to estramustine phosphate since neither estradiol 170- phate complex. EDTA was used as the chelator because the buffer contained 0.5 mw MgSO4 but no added Ca2*. Estramustine was dis phosphate, the hormonal moiety of the drug, nor nornitrogen solved in Cremophor ELethanol (1:9) before dilution in standard frog mustard, the alkylating moiety, had any effect on assembly. Ringer's solution. Cremophor EL was kindly provided by Dr. K-E. Falk, Estramustine phosphate (0.1 to 0.5 HIM) was also found to AB Hassle, MÃ¶lndal,Sweden. reversibly inhibit fast axonal transport in the frog sciatic nerve. Taxol was a gift from Dr. M. Sufness at the NIH, Bethesda, MD. L- The nerve content of adenosine triphosphate, adenosine diphos- [4,5-3H]Leucine (130 Ci/mmol, 1 mCi/mL) was obtained from The Radi- phate, and adenosine monophosphate was not significantly af ochemical Centre, Amersham, England. All other chemicals were reagent fected by estramustine phosphate. grade. Our results suggest that the cytotoxic action of estramustine Microtubule Proteins. Microtubule proteins were prepared from bo phosphate could be dependent partially on an interaction with vine brain in the absence of glycerol by 2 or 3 cycles of assembly- microtubules, probably via the microtubule-associated proteins. disassembly in the presence of 0.5 mw MgSCu (2,19). In the first cycle, ethyleneglycolbis(2-aminoethyl ether)-W,N'-tetraacetic acid was present to complex free Ca2+. The final pellet, which contains approximately 80% tubulin (5), was stored in liquid nitrogen. Prior to use, the pellet was INTRODUCTION resuspended in buffer [100 mw piperazine-/V,A/'-bis(2-ethanesulfonic acid), 0.5 row MgSOÂ«,with 1 DIM GTP at pH 6.8]. After incubation at Estramustine phosphate (estradici 3-[W,A/-bis(2-chloro- 4Â°Cfor 30 min, the sample was clarified by centrifugation at 35,000 x g ethyl)carbamate] 17/3-phosphate| is active against advanced for 30 min at 4Â°C.Protein solutions were stored in liquid nitrogen after prostatic carcinoma (14). The mechanism of action of the drug drop freezing (5). Protein Concentration. Microtubule protein concentration was deter is not fully understood and cannot solely be ascribed to its mined by Bio-Rad protein assay using Coomassie Brilliant Blue based antigonadotropic properties (10, 20). It is cytotoxic in human on the method of Bradford (3) with tubulin as a standard. The concentra prostatic carcinoma cells in culture (13) and induces mitotic arrest tion of the standard was determined from E27B= 1.2 mg"1 -cm*2 and a at metaphase (12). These metaphases have contracted chro molecular weight of 110,000 (23). mosomes, which no longer are aligned in the metaphase plane. Assembly. Assembly of microtubule proteins in 100 rriM piperazine- Other drugs which exhibit this property, such as colchicine, N,A/'-bis(2-ethanesulfonic acid), 0.5 mw MgSCu, 1 mw EDTA, and 1 nriM vinblastine, and nocodazole (23), are inhibitors of the assembly GTP was started by addition of 50 n\ microtubule proteins at 4Â°Cto 300 ti\ buffer at 37Â°C, and the increase in turbidity was monitored continu of microtubules, the main component of the mitotic spindle. Hitherto, a direct effect of estramustine phosphate on microtu ously by the change in absorbance at 350 nm (5). Estramustine phos bules in vitro or on other microtubule-dependent processes, such phate or an equivalent amount of water was added from a stock solution either to the buffer or to the protein. It was found that the addition of 1 as fast axonal transport, has not been studied. mM EDTA did not inhibit microtubule assembly. Taxol was added from a In the present study, the inhibitory effect of estramustine 3 mM stock solution in dimethyl sulfoxide. phosphate on the assembly of isolated brain microtubules and Axonal Transport. The sciatic nerve together with the eighth and on fast axonal transport is reported. The results indicate that the ninth dorsal ganglia were dissected from frogs (Ã±aÃ±atemporaria). A cytotoxicity of estramustine might be caused by an interaction ligature was placed on the nerve 30 mm from the ganglia. Treated and with microtubule proteins. control nerve preparations were from the same frog. The nerves were incubated in an apparatus where the ganglia could be separated from ' Part of this work has been supported by grants from the National Research the nerve by silicon grease barriers (8). Frog Ringer's solution devoid of Council, Harald Jeanssons Stiftelse, Magnus Bergvalls Stifteise, Torsten and Ca2+ and Mg2+ with or without estramustine phosphate was added to Ragnar SÃ¶derbergs Stiftelse, Kungliga Hvitfeltska Ã¶verskotts Fonden, Lars Hiertas the nerve compartment. The absence of these cations in the Ringer's Minne, and AB Leos Research Foundation. 1 To whom requests for reprints should be addressed. Received 4/23/84; revised 9/19/84. 1/11/85; accepted 1/24/85. 3AB Leo, personal communication.

CANCER RESEARCH VOL. 45 MAY 1985 2234

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1985 American Association for Cancer Research. EFFECT OF ESTRAMUSTINE PHOSPHATE ON MICROTUBULES solutions does not affect axonal transport (6) in sheated frog sciatic nerves. The ganglia were exposed to [3H]leucine containing standard frog Ringer's solution. After incubation for 17 h at 18Â°C,the nerve was cut into 3-mm segments and analyzed for protein-incorporated radioac tivity as described previously (8). The transport rate was determined using 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+-free Ringer's solution with or without the drug. Adenosine Nucleotide Determination. Nerves were incubated for 17 h at 18Â°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 by trioctylamine in 1,1,2-trichlorotrifluoroethane (21) prior to injection into a Polyanion SI column (Pharmacia) attached to a VarÃan5000 high-pres sure liquid Chromatograph. Nucleotides were eluted by a linear phosphate gradient, 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 and quantified using a nucleotide standard. Electron Microscopy. Negatively stained microtubule specimens for 20 25 electron microscopy were prepared from S-iÂ¿\protein samples. Fixation TIME (min) was performed with 1 drop of Karnovsky's solution (17), after which the Chart 1. Time course of assembly of brain microtubules. Estramustine phos specimen was washed with distilled water and stained with 1% uranyl phate (EMP) (0.42 mM) was added initially (Trace A) or at steady state level of acetate. Embedded microtubule specimens were prepared after centrif- assembly (Trace B at arrow). After complete disassembly, 20 /JMtaxol was added ugation at 35,000 x g for 30 min at 35Â°C. The pellets were fixed with (Trace B at arrow). Microtubule assembly (1.8 mg/ml protein) was started by raising the temperature from 10 to 37Â°Cand was monitored by the increase in absorbance Karnovsky's solution followed by 1% osmium tetroxide in 0.1 M cacc- at 350 nm against time. The assembly buffer contained 0.1 M piperazine-W,N'- dylate buffer (17). The pellets were dehydrated in a graded series of bis(2-ethanesulfonic acid) at pH 6.8, 0.5 mM MgSO4, 1 mw GTP, and 1 mw EDTA. acetone and embedded in Epon. Thin sections were made on a LKB ultramicrotome and were double stained with uranyl acetate and lead citrate. The specimens were viewed in a Zeiss 109 electron microscope. Nerves exposed to estramustine phosphate were fixed in 2.5% glu- --a taraldehyde (16) followed by 1% osmium tetroxide. Subsequently, the nerves were dehydrated in a graded series of ethanol, block stained in 1% phosphotungstic acid and 0.5% uranyl acetate, and embedded in A EMPat "steady state" Epon:Araldite. â€¢E2 50- RESULTS

Effects of Estramustine Phosphate. Estramustine phosphate inhibited the assembly of microtubule proteins in a concentration- dependent manner (Charts 1 and 2). The rate and extent of assembly was less in the presence of estramustine phosphate. 50 100 50 200 500 1000 Furthermore, estramustine phosphate induced rapid disassem bly of microtubules at steady state (Chart 1). No difference in drug (p M ) the level of assembly was found whether estramustine phos Chart 2. Microtubule assembly at different concentrations of estramustine phos phate (EMP). The steady state level of assembly was measured after preincubation phate was added initially or at steady state (see Chart 2) or if for 10 min at 10Â°Cwith estramustine phosphate, nornitrogen mustard (nor-HW2), the microtubule proteins were preincubated with the drug for 30 or estradici 17fj-phosphate (Â£2),and furthermore after addition of estramustine min at 4Â°C.The electron micrographs (not shown) of microtu phosphate to preformed microtubules (A). The level was expressed in percentage of the AAaso of the control, since different protein preparations were used. The bules disassembled to approximately 50%, taken directly after microtubule protein concentration was 2.1 mg/ml, and the conditions were as the addition of estramustine phosphate, showed normal but described in the legend to Chart 1. fewer microtubules with extending projections. The extent of microtubule assembly in the presence of increas tion sufficient for assembly of microtubules, was increased. ing amounts of estramustine phosphate showed a nonlinear Low concentrations (20 MM) of the microtubule-promoting dose-response relationship (Chart 2). The same curve was ob drug, taxol, were able to reinduce assembly of completely inhib tained if the disassembly level was plotted. ited microtubule proteins to the same or higher levels than that Addition of estramustine phosphate at a constant concentra of the control (Chart 1). The electron micrographs of the pellet tion to preformed microtubules induced the same change in of the taxol-induced microtubules showed the presence of many turbidity independent of the protein concentration. If the turbidity aberrant forms of microtubules as compared to the microtubules was plotted against different microtubule protein concentrations, formed in the absence of the drug (Fig. 1). it could be seen that with a constant concentration of estramus The effect on microtubule assembly was specific for estra tine phosphate a linear relationship remained with the same mustine phosphate since neither estradici 1/Â¿(-phosphate nor slope as for the control but shifted to the right (Chart 3). Thus, nornitrogen mustard had any effect on microtubule assembly at in the presence of estramustine phosphate the critical protein comparable concentrations (see Chart 2). Furthermore, 1 mw concentration, i.e., the minimum microtubule protein concentra estradiol 17/3-phosphate did not compete with estramustine

CANCER RESEARCH VOL. 45 MAY 1985 2235

mMestramustine phosphate, followed by washing with standard Ringer's solution and subsequent assay. In electron micrographs of the sciatic nerves treated with the drug, microtubules could 0.4- still be observed (not shown). From the above experiments, in which ligatures were used, differentiation of the effect of the drug on the rate of transport or on the amount of transported materials is not possible. How ever, the pulse-labeling experiment (Chart 4) showed that there 0.3- was little difference in the total amount of transported material but a reduced transport rate in the presence of 0.5 mM estra mustine phosphate. 0.2- Energy Metabolism. In orderto excludea metaboliceffectof estramustine phosphate as a cause of the inhibited axonal transport, the levels of ATP, ADP, and AMP were measured in nerves which had been incubated with the drug. As can be seen 0.1- in Table 2, the adenosine nucleotide composition was not signif icantly influenced by a 17-h incubation with either estramustine phosphate or estramustine.

DISCUSSION 0123 The mechanismof action of the anticancer agent estramustine MICROTUBULE PROTEIN (mg/ml) phosphate is not known. In humans, the drug is rapidly dephos-

Chart 3. Microtubule assembly at different protein concentrations in the pres ence of estramustine phosphate. The steady state level of microtubule assembly was measured after addition of estramustine phosphate (â€¢.OP.M;â€¢,84 >IM; O, 151 Â¡Â¡M)topreformed microtubules at 37Â°C.The conditions were as described in the legend to Chart 1. 6-

Table 1 Effects of estramustine phosphate and estramustine on the accumulation of 3H- labeled protein in front of a ligature Accumulation in drug-treated nerve8 (%)

0.1 RIM 0.5 rriM 81 Estramustine phosphate 59Â±5C25Â±6C 34Â±6C Estramustine Estradici 170-phosphate 82 Â±23 (NS)d Nornitrogen mustard57Â±56'c 87 Â±23 (NS) Values are expressed as the accumulation in the drug-treated nerve in per centage of that in the control nerve. 6 Mean Â±standard average. CP = 0.001. " NS, not significant. phosphate for the same binding site, because in its presence the inhibitory action of the drug on microtubule assembly was un 12 24 36 48 changed. Furthermore, electron micrographs of microtubules Distance along axon (mm) assembled in the presence of 0.5 ITIMconcentrations of either Chart 4. Frog sciatic nerve ganglia were pulse-labeled with[3H]leucine for 2 h estradici 17/3-phosphateor nornitrogen mustard showed perfect and subsequently transferred to Ringer's solution Â±0.5 mM estramustine phos microtubules (not shown). phate (EMP). After 10 h incubation, the protein-incorporated radioactivity (y axis) Axonal Transport. Estramustinephosphateat 0.5 and 0.1 was measured in successive 3-mm segments (x axis). rriM reduced the accumulation of labeled material in front of a ligature to 25 and 57%, respectively, of the control values. Table 2 Effects of estramustine phosphate on the adenosine nucleotide content of frog Estramustine was equally effective as an inhibitor of axonal sciatic nerve transport (Table 1). Furthermore, the effect was specific because The nerves were incubated for 17 h at 18Â°Cin Ringer's solution, free of Ca2* neither 0.5 mw estradici 170-phosphate nor 0.5 mw nornitrogen and Mgz+ Â±0.1 HIM drug. The nucleotide content was analyzed as described in "Materials and Methods." The mean concentrations of ATP, ADP, and AMP (^mol/ mustard had any effect on axonal transport (Table 1). mg, wet weight) were 0.95 Â±0.04, 0.25 Â±0.06, and 0.16 Â±0.06, respectively. The accumulation of radioactive material in the presence of Drug-treated:control ratio 0.1 ITIMestramustine phosphate was more suppressed [31 Â± 5% (SD), P < 0.05, N = 8] in nerves preincubated at 1Â°Cfor 3 mMestramustine mMestramustine0.92 phosphate1.02 h than in nerves which had not been pretreated by cooling. Â±0.16a AMP Â±0.15 The decrease in axonal transport by 0.5 ITIMestramustine ADP 1.02 Â±0.26 0.97 Â±0.13 phosphate was reversible. Axonal transport did not differ from ATP+0.1 1.00 Â±0.17+0.1 0.82 Â±0.28 the control after 5 h preincubation at 4Â°Cinthe presence of 0.5 5Mean Â±SD,W= 6 (Student's t test).

CANCER RESEARCH VOL. 45 MAY 1985 2236

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1985 American Association for Cancer Research. EFFECT OF ESTRAMUSTINE PHOSPHATE ON MICROTUBULES phorylated releasing estramustine, which is readily oxidized to Together with the observed inhibition of mitosis (12) and the estromustine (11). Thus, on hydrolysis, both estradici and es recent observation that estramustine inhibits microtubule-de trone are released to exert their hormonal effect on the prostatic pendent pigment granule movement in squirrel fish erythro- tumor (10). However, estramustine itself does not compete with phores,5 the results suggest that estramustine phosphate exerts estradici for the estrogen receptor site (9, 20), indicating a lack its cytotoxic effect by interaction with microtubules in the intact of classical hormone effect. Estramustine does not induce DNA cell also. strand breaks either, indicating a lack of alkylating activity, in spite of the nornitrogen mustard moiety of estramustine (22). ACKNOWLEDGMENTS However, the observation that estramustine inhibits mitosis in prostate tumor cells in culture (12) implicates an involvement of Thanks are due to Inger Antonsson for expert technical assistance. microtubules in its mode of action. In the present study, we studied the effect of estramustine REFERENCES phosphate on the assembly of cold-labile microtubules from brain. Brain tissue contains relatively large amounts of microtu 1. Avila, J., Garcini, E. M., Wandosell, F., Villasante, A., Sogo, J. M., and Villanueva, N. Microtubule associated protein MAP2 preferentially binds to dA/ bules. Cold-labile microtubules from different tissues have been dT sequence present in mouse satellite DNA. EMBO J., 2: 1229-1234, 1983. shown to have the same general properties with respect to 2. Borisy, G. G., Olmsted, J. B., Marcum, J. M., and Allen, C. Microtubule assembly in vitro. Fed. Proc., 33: 167-174, 1974. different assembly inhibitors. We found that estramustine phos 3. Bradford, C. A rapid and sensitive method for the quantitation of microgram phate inhibited brain microtubule assembly in vitro at physiolog quantities of protein utilizing the principle of protein-dye binding. Anal. ically relevant concentrations and induced rapid disassembly of Biochem., 72: 248-254,1976. 4. Deinum, J., Sjirskog, L. Wallin, M., and DahlbÃ ck, E. Molecular weight preformed microtubules. The effect on microtubules in vitro was dependency heparin inhibition of microtubule assembly in vitro. Biochim. specific for estramustine phosphate because neither nornitrogen Biophys. Acta, 802: 41-48, 1984. mustard nor estradiol 170-phosphate, the 2 parent moieties, 5. Deinum, J., Wallin. M., and Lagercrantz, C. Spatial separation of the two essential thiol groups and the binding-site of the exchangeable GTP in brain affected microtubule assembly or disassembly. Neither could the tubulin. A spin label study. Biochim. Biophys. Acta, 677: 1-8, 1981. 2 parent moieties potentiate the effect of estramustine phos 6. EdstrÃ¶m,A. J. Effects of Ca(ll) and Mg(ll) on rapid axonal transport of proteins phate on microtubule assembly. in vitro in frog sciatic nerve. J. Cell Biol., 67: 812-818,1974. 7. EdstrÃ¶m, A., Hanson, M., Wallin, M., and Cederholm, B. Inhibition of fast Although estramustine has a colchicine-like effect on mitosis, axonal transport and microtubule polymerisation in vitro by colchicine and preliminary results indicate that the drug does not interfere with colchiceine. Acta Physiol. Scand., 707: 233-237, 1979. the binding of colchicine to tubulin. Our experiments suggest 8. EdstrÃ¶m, A., and Mattson, H. J. Fast axonal transport in vitro in the sciatic that estramustine phosphate interacts with MAPs.4 Hence, es system of the frog. Neurochemistry, 79: 205-221,1972. 9. Forsgren, B., and Bj0rk, P. Specific binding of estramustine to prostatic tramustine phosphate increased the critical protein concentration proteins. J Urol., 23 (Suppl.V 34-38. 1984. 10. Fritjofsson, A., Morten, B. J., Hogberg. B., Rajalakshmi, M., Cekan, S. Z.. and required for the assembly of microtubules, in contrast to colchi Dczfalusy, E. Hormonal effects of different doses of estramustine phosphate cine, vinblastine, and nocodazole (23), which specifically bind to [Estracyt(R)] in patients with prostatic carcinoma. Scand. J. Urol. Nephrol., 75: the tubulin dimer. This effect is also exhibited by drugs which 37-44, 1981. 11. Gunnarsson, P. O., Plym Forshell, G., Fritjofsson, A., and Morten, B. J. Plasma have a direct effect on the MAPs-dependent nucleation phase of concentration of estramustine phosphate and its major metabolites in patients assembly and which bind to MAPs, such as DNA (1) and heparin with prostatic carcinoma treated with different doses of estramustine phos (4). The reversal of the estramustine phosphate-induced inhibi phate (Estracyt). Scand. J. Urol. Nephrol., 75: 201-206, 1981. 12. Hartley-Asp, B. Estramustine induced mitotic arrest in two human prostatic tion of assembly by taxol is not possible in the presence of carcinoma cell-lines Du145 and PC-3. Prostate. 5: 93-100, 1984. tubulin-binding drugs such as colchicine (18), which is further 13. Hartley-Asp, B.. and Gunnarson, P.-O. Growth and cell survival following treatment with estramustine. nor-nitrogen mustard, estradiol and testosterone support for an interaction of estramustine phosphate with MAPs. Furthermore, we have found (24) that 3H-labeled estramustine of a human prostatic cancer cell line. J. Urol., 727: 818-822,1982. 14. Jonsson.G.. Hogberg, B., and Nilsson. T. Treatment of advanced prostate phosphate bound predominantly to MAPs and moreover that carcinoma with estramustine phosphate [Estracyt(R)]. Scand. J. Urol. Nephrol., 77:231-238, 1977. addition of MAPs reversed the inhibition of assembly induced by 15. Kadohama, N., Kirdani, R. Y., Madajewicz, S., Murphy, G. P., and Sandberg, estramustine phosphate. However, the relatively high molar ratio A. A. Estramustine. Metabolic pattern and possible mechanisms for its action (compared with the concentration of MAPs) of estramustine in prostatic cancer. NY State J Med.. 79: 1005-1009, 1979. 16. Kanje, M., EdstrÃ¶m, A., and Hansson, M. Inhibition of rapid axonal transport phosphate needed for complete inhibition of assembly and the in vitro by the ionophores X537A and X23187. Brain Res., 204: 43-50, 1981. nonlinear dose-response curve (Chart 2) suggest a weak inter 17. Karnovsky, M. J. Formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol., 27: 137A, 1965. action, which is also indicated by the reversibility of the effect on 18. Kumar, N. J. Taxol-induced polymerization of purified tubulin. J. Biol. Chem., axonal transport. 256: 10435-10441, 1981. Axonal transport, a microtubule-dependent process, was used 19. Larsson, H., Wallin, M., and EdstrÃ¶m, A. Induction of a sheet of polymer of tubulin by Zn(ll). Exp. Cell Res., 700: 104-110. 1976. to assess the effect of estramustine phosphate and estramustine 20. LeClercq, G., Heuson, J.-C., and Deboei, M. C. Estrogen receptor interaction in an intact cellular system. In agreement with the inhibition of with Estracyt(R) and degradation products, a biochemical study on a potential the drug on microtubule assembly is the finding that estramustine agent in the treatment of breast cancer. Eur. J. Drug Metab. Pharmacokinet., 2: 77-84, 1976. phosphate and estramustine inhibited axonal transport in the 21. Pogolotti, A. L, and Santi, D. V. High-pressure liquid chromatography-ultravi- sciatic nerve. This also demonstrates that the phosphate group 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- contributes only by increasing the solubility of the drug. The Asp, B. Cytotoxicity of a steroid-nitrogen mustard derivative through non-DNA inhibition occurred without significant alterations of the nerve targets. Mol. Pharmacol., 24: 324-328, 1983. content of ATP, ADP, and AMP. The inhibition was reversible, 23. Wallin, M., and Deinum, J. Tubulin. In: A. Laitha (ed.), Handbook of Neuro chemistry, Vol. 5, pp. 101-126. New York: Plenum Publishing Corp., 1983. and low temperature intensified the effect of the drug, which is 24. Wallin, M.. Deinum, J., and Friden, B. Interaction of estramustine phosphate comparable to the effects of colchicine on the sciatic nerve (7). with microtubule-associated proteins. FEBS Lett., 779: 289-293, 1985.

4 The abbreviation used is: MAPs, microtubule-associated proteins. sM. E. Steams and K. D Tew. personal communication.

CANCER RESEARCH VOL. 45 MAY 1985 2237

'o o

m -

Â».

'- O

â€¢ '-,'â€¢ * t &JHâ€¢ *. '* "vv 'â€¢

Fig. 1. Electron micrograph of embedded microtubules. A, microtubules; B, microtubules in the presence of 20 UM taxol; C, microtubules in the presence of 0.42 mw estramustine phosphate induced by 20 MMtaxol. The conditions were as described in the legend to Chart 1. x 90,000. 2238 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1985 American Association for Cancer Research. .-V-, .v.,'.-,â€¢.- "t

EFFECT OF ESTRAMUSTINE PHOSPHATE ON MICROTUBULES

â€¢

v-. Â«..i * v * Â«

4 \

CANCER RESEARCH VOL. 45 MAY 1985 2239

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1985 American Association for Cancer Research. Effect of Estramustine Phosphate on the Assembly of Isolated Bovine Brain Microtubules and Fast Axonal Transport in the Frog Sciatic Nerve

Martin Kanje, Johanna Deinum, Margareta Wallin, et al.

Cancer Res 1985;45:2234-2239.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/45/5/2234

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/45/5/2234. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.