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[CANCER RESEARCH 54, 75-84, January 1, 1994]

Inhibition of Microtubules and Cell Cycle Arrest by a New 1-Deaza-7,8-

dihydropteridine Antitumor Drug, CI 980, and by Its Chiral Isomer, NSC 6138631

C o n c e p c i o n de Ines , D a n i e l L e y n a d i e r , I sabe l B a r a s o a i n , 2 V i n c e n t P e y r o t , P a t r i c k G a r c i a , C l a u d e t t e B r i a n d ,

G r e g o r y A . R e n e r , a n d C a r r o l l T e m p l e , Jr.

Centro de Investigaciones Biologicas, Consejo Superior de Investigaciones Cientificas, Velazquez 144, 28006 Madrid, Spain [C. I., L B.]; Groupe de Recherche sur les Interactions des Proteines en Pharmacologie, Facult~ de Pharmacie, 27Bd. Jean Moulin, 13385 Marseille Cedex 5, France [D. L., V. P., P. G., C. B.]; and Organic Chemistry Research Department, Southern Research Institute, Birmingham, Alabama [G. A. R., C. T.]

ABSTRACT

CI 980 (NSC 613862; [S-(-)]) and NSC 613863 [R-(+)] are the two chiral isomers of ethyl 5-amino 1,2-dihydro-2-methyl-3.phenylpyrido[3,4- b]pyrazin-7-ylcarbamate (NSC 370147), which is a mitotic inhibitor with in vivo and in vitro activity against murine multidrug-resistant sublines. We have characterized the inhibition of in vitro microtubule assembly by the S (CI 980) and R (NSC 613863) enantiomers, their actions on the cytoplasmic microtubule network of epithelial-like PtK2 cells, and on the cell cycle of different human and murine leukemias and PtK2 cells. As- sembly of purified tubulin, or tubulin plus microtubule-associated pro- teins, into microtubules was substoichiometrically inhibited by both compounds, which also induced a slow depolymerization of preassembled microtubules. Half inhibitory concentrations were 0.4-0.7/~M and 1.6-2.1 /zM for the S and R isomers, respectively. Excess of both drugs induced polymerization of liganded tubulin into abnormal polymers similar to colchicine. The cytoplasmic microtubules of PtK2 cells were disrupted by both compounds in a concentration- and time-dependent manner, which was observed by indirect immunofluorescence microscopy and quantified by an enzyme-linked immunoassay of cytoskeletal tubulin. Half inhibitory concentrations were 6 nM S isomer, 100 nM R isomer, and 1 pM colchicine. Twenty ma S isomer or 500-700 nra R isomer gave nearly maximal effect. At these concentrations, half maximal microtubule depolymerization took place after 2 h of treatment. After drug removal, slow microtubule assembly and nearly complete reorganization of the cytoplasmic microtubules of PtK2 cells were observed (24 h). One nM S enantiomer or 25 nM R enantiomer induced mitotic arrest in 8 h in U937, HL60, and EIA leukemias. PtK2 cells also stopped in mitosis after a 24-h incubation with 50 nM R isomer or 5 nM S isomer. The inhibition of cell division was irreversible in the leukemic cells, while PtK2 cells partially resumed growth. Although the interactions of CI 980 with microtubules in vitro are not very different from other drugs, it is a most potent cellular microtubule and mitotic inhibitor.

I N T R O D U C T I O N

Several 1,2-dihydropyrido[3,4-b]pyrazines were prepared as inter- mediates for the synthesis of certain antifolates (1, 2); they did not function as analogues of folic acid but inhibited mitosis and caused cultured cells to accumulate in metaphase (3). Therefore, new compounds of this type have been synthesized and tested as inhibitors of cell proliferation and anticancer agents. Ethyl 5-amino-l,2-dihydro- 2-methyl-3-phenylpyrido[3,4-b]pyrazin-7-ylcarbamate, 2-hydroxy- ethanesulfonate, hydrate (NSC 370147) was very active in inhibiting proliferation of cultured L1210 cells and in increasing the mitotic index. It also inhibited pig brain microtubule polymerization and the

Received 7/16/93; accepted 10/29/93. The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grant PB 87-0230 from the Direccion General de Investigacion Cientifica y Tecnica, French-Spanish grants, and grants from Association pour la Recherche sur le Cancer Federation de Centres de lutte Contre Cancer.

2 To whom requests for reprints should be addressed.

binding of [3H]colchicine to purified tubulin and slightly enhanced the binding of [3H]vincristine to tubulin. NSC 370147 was synergistic with vincristine in killing cultured L1210 cells and increasing life spans of mice bearing P388 leukemia (4). Moreover, this compound had good antitumor activity against several tumors in culture and also in mice as well as against murine leukemic sublines that were resistant to most drugs used in cancer therapy (5, 6).

NSC 370147 is a racemic compound. Its two chiral isomers, NSC 613862 [S-(- ) ] (CI 980) and NSC 613863 [R-(+) ] (Fig. 1), have been prepared and present significant differences in inhibiting the proliferation of L1210 (7-10) and U937 cells (11). CI 980 is presently undergoing Phase I clinical trials (12). Frequently, one isomer of a drug may produce a desired effect while another may be less active, inactive, or even produce some undesired effect. Microtubule inhibitors are not an exception. Inversion of optical activity of the acetamide group of colchicine abolishes its activity (13), and isomerization of podophyllotoxin (c is- lactone) results in a 10-fold loss of activity (14). It has been shown that both isomers CI 980 [S-(-)] and NSC 613863 [R-(+) ] are able to bind to purified tubulin with equilibrium disso- ciation constants of approximately 0.2 ~M and 0.3 p,M, respectively, apparently overlapping the trimethoxybenzene locus of the colchicine binding site. Their different fluorescence properties upon binding to tubulin have suggested a different orientation for the isomers in the same locus (15).

In order to gain insight into the mechanism of action of CI 980 and NSC 613863, we have studied their effect on in vitro microtubule polymerization and their action on the cytoplasmic microtubule network and cell division by using PtK2 cells as well as human and murine leukemias.

MATERIALS AND M E T H O D S

Chemicals, Culture Media, and Antibodies. The synthesis of NSC 613862 (CI 980) and NSC 613863 has been reported (8). Stock solutions were made in DMSO 3 and stored at -20~ Their concentrations were measured spectrophotometrically using extinction coefficients E374 nm 15100 and 15400 M -1 cm -1 for the S and R compounds, respectively (8). MTC was a gift from Dr. T. J. Fitzgerald (16). Its concentration was measured spectrophotometrically using the extinction coefficient •343 n m : " 17600 M-I"cm -1 (17). GTP, disodium salt, and colchicine were from Fluka. Podophyllotoxin was from Aldrich Chemical Co. Dulbecco's modified Eagle's medium, RPMI-1640, FCS, L-glutamine, nonessential aminoacids, and antibiotics were from Flow Laboratories (Irvine, United Kingdom). Propidium iodide, DM1A anti-tubulin monoclonal antibody, and goat-antimouse polyvalent immunoglobulins (fluo- resceine-conjugated) were from Sigma Chemical Co. Rabbit- antimouse immunoglobulins (peroxidase-conjugated) were from Dakopatts (Copenhagen, Denmark).

3 The abbreviations used are: DMSO, dimethyl sulfoxide; MTC, 2-methoxy-5-(2',3',4- trimethoxyphenyl)-2,4,6-cycloheptatrien-l-one; MTP, microtubule protein; FCS, fetal calf serum; MES, 4-morpholineethanesulfonic acid; PEM, 100 mM piperazine-N,N'-bis(2- ethanosulfonic acid), 1 mM EGTA, and 1 mM MgC12, pH 6.8; PBS, phosphate-buffered saline; BSA, bovine serum albumin.

75

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H H I I R2

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NSC61::5865(R ( + ) ) "R 1 = H ; R2 =CH3 Fig. 1. Chemical structures of CI 980 and NSC 613863.

Microtubule Protein and Purified Tubulin. MTP was purified from pig brain according to the method of Shelanski et al. (18) through three cycles of in vitro assembly (37~ and disassembly (4~ as described previously (19). The MTP solution was stored at -80~ Calf brain tubulin was purified by ammonium sulfate fractionation and ion-exchange chromatography, stored in liquid nitrogen, and prepared for use as described previously (17, 20, 21). Tubulin concentrations were determined spectrometrically at 275 nm in 6 M guanidine hydrochloride (E275n m = 1.09 liter-g-l'cm-1; Ref. 22) or in 0.5% sodium dodecyl sulfate in neutral aqueous buffer (E275n m = 1.07 liter'g -1" cm-1; Ref. 17). Microtubule protein concentrations were determined using the Bio-Rad Coomassie assay (23) with BSA and purified tubulin standards.

Mierotubule Assembly. The in vitro polymerization of microtubules was monitored by turbidity at 350 nm in a Beckman DU 70 spectrophotometer with a thermostated 1-cm light path cell at 37~ For MTP, the polymerization

0 . 6

buffer was 100 mM 2-(N-morpholino)ethanesulfonic acid, 0.5 mM MgCI2, 1 mM ethylene-bis(oxyethylenenitrilo)tetraacetic acid, and 1 mM GTP, pH 6.7. The buffer solution used to polymerize purified tubulin consisted of 10 mM sodium phosphate, i mu ethylene-bis(oxyethylenenitrilo)tetraacetic acid, 8 mu MgCI2, 3.4 M glycerol, and 0.1 mM GTP, pH 6.8. The reaction was started by a temperature shift from 4 to 37~ R and S enantiomers containing samples and their controls had 1% residual DMSO. Samples were fixed with 0.5% glutar- aldehyde, adsorbed to Formvar coated grids, negatively stained with 1.5% uranyl acetate, and examined under a Jeol 1200 electron microscope (Labo- ratoire de Microscopie Electronique, Facult6 de M6decine, Marseille, France). The assembly reaction of tubulin into abnormal polymers induced by colchicine and both R and S enantiomers was followed turbidimetrically at 475 nm in 10 mM sodium phosphate, 16 mM MgCI2, and 0.1 mM GTP, pH 7.0, at 37~ This wavelength was chosen in order to minimize light absorption by the

compounds (22). Cell Lines. U937 monocytic and HL60 promyelocytic human leukemias

and EL-4 C57B1 benzanthracene-induced murine lymphoma were grown in RPMI-1640 supplemented with 2 mM glutamine, 10% FCS, and antibiotics at 37~ in a humidified atmosphere containing 5% CO2. PtK2 potoroo epithelial- like kidney cells (American Type Culture Collection, Rockville, MD) were grown in Dulbecco's modified Eagle's medium supplemented with 10% FCS, 2 mu glutamine, 0.1 mu nonessential aminoacids, and antibiotics.

Immunofluoreseence Experiments. PtK2 cells were plated onto 9 x 9 mm glass coverslips (Bellco) at a density of 100,000-200,000 cells/ml, cultured for 1 day, and then treated with the drugs in culture medium for the desired concentration and time. Residual DMSO in cultures was 0.1% (v/v). The coverslips were processed as described previously (24) with modifications. In brief, the coverslips were washed with PEM containing 4% polyethylene glycol 8000 (Sigma). The cells were lysed with 0.5% (v/v) Triton X-100 and 1 mM GTP in PEM for 90 seconds at room temperature; then the coverslips were washed with PEM-polyethylene glycol-1 mu GTP. The coverslip-at- tached cytoskeletons were fixed with 3.7% (w/v) formaldehyde-l% DMSO in PEM for 30 min and kept in PBS at 4~ Alternatively, the complete cells were fixed with 3.7% formaldehyde (10 min at room temperature), methanol (10

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Fig. 2. Turbidity time course of in vitro microtubules assembly. Twenty /./,M tubulin (a) with added 0.3 ~M R isomer (b) or with 0.3 ~M S isomer (c). Inset, in a different experiment, the plateau absor- bance values of tubulin polymerization were measured without ligand 01), in presence of 0.5 ktu R isomer (~), or in the presence of 0.5 p,u S isomer (A).

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EFFECTS OF CI 980 AND NSC 613863 ON MICROTUBULES AND CELL DIVISION

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min at -20~ and finally acetone (30 s at -20~ The fixed cytoskeletons or cells were incubated for 1 h with DM1A monoclonal antibody reacting with ot-tubulin (diluted 1:400) in PBS containing 10 mg/ml BSA at 37~ After washing twice with PBS and gentle agitation (10 min), the coverslips were overlaid with fluoresceinated goat-antimouse immunoglobulins (diluted 1:100 in PBS-BSA-0.02% NAN3), incubated for 45 rain at 37~ washed with PBS in the dark, and mounted with antifading solution. The cytoskeletons or cells were photographed on Kodak Tri-X film using a Zeiss Axioplan microscope equipped with epiilumination, 63x Plan-Apochromat and Plan-Neofluar 40x objectives, and the appropriate filters for fluorescein.

Determination of Assembled Tubulin in Cytoskeleton. Quantification of tubulin in cytoskeletons was made by an enzyme-linked immunoassay modified from the procedure of Nieto (25). Cells were plated at 100,000/ml (0.1 ml) in 96-well plates, cultured for I day, and then treated in triplicate with the drugs for the desired time. They were then lysed and fixed as above. The plates were blocked with 0.4% gelatin for 1 h at 37~ After the plates were incubated with DM1A antibody (diluted 1:3200 in 0.05% gelatin PBS) for 1.5 h at room temperature, they were washed three times with 0.075% Tween 20 in PBS. Then peroxidase-conjugated rabbit-antimouse immunoglobulins (1:1000 in

0.05% gelatin PBS) were incubated for 45 min at room temperature. The plates were washed five times in 0.075% Tween-PBS and developped with 1,2- phenylendiamine and HaO2. The absence of undesirable background or cross- reaction was verified by omitting the first antibody, by the use of an irrelevant monoclonal antibody (P1C3 against a neutrophil membrane protein; Ref. 26), and by using wells without cells.

Determination of DNA Content. Flow cytometric analysis was performed as described (27) with modifications. Ceils were plated at 100,000- 300,000/mL and incubated with drugs. One ml of resuspended cells was centrifuged, and the cell pellet was resuspended in 350/xl of RPMI-1640. Then 125 /xl of RPMI-1640 containing 0.2% Nonidet P40, followed by 25 /.d of 1 mg/ml propidium iodide, were added. After 30 min at room temperature, the cells were analyzed in a Coulter Epics CS cytofluorometer.

RESULTS

Effects of CI 980 and NSC 613863 on the Assembly of Purified Tubulin and Microtubule Protein. Fig. 2 shows the effects of these R and S enantiomers on the turbidimetric time course of assembly of microtubules from purified tubulin (2 mg/ml). Half inhibitory concentrations were 1.6/XM for the R isomer and 0.4/XM for the S isomer. The half inhibitory concentrations for microtubule protein (1 mg/ml) were 2.10 /~r~ and 0.75 ~u for R and S isomers, respectively (not shown). Therefore, in both cases a clear inhibition was noticed in the presence of substoichiometric R or S isomer concentrations. However, the S enantiomer was 3-4-fold more active than the R isomer. The process was also examined at varying protein concentrations with a fixed R or S concentration (0.5/xr~). The results, shown in Fig. 2, inset, indicated an increase of the protein concentration required for the assembly (abscissa intercept of the plot, 1.2 mg/ml in the presence of R isomer and 1.5 mg/ml in the presence of S isomer). Electron microscopy examination (not shown) indicated that the polymers formed under these conditions were microtubules.

Fig. 3A displays the results of adding 20 /xM R or S isomer to assembled tubulin preparations after the turbidity plateau had been reached. A difference in the behavior of both compounds was noticed. While the R isomer induced a small decrease in turbidity, the same concentration of S isomer induced a marked turbidity increase. Elec- tron microscopic examination showed microtubular structures and some aggregates for the R compound, while for the S enantiomer some microtubules remained visible and large amorphous structures appeared. Polymers formed in the presence of S isomer could be depo- lymerized by cooling to 13~ for 15 min, and the residual turbidity observed was due to protein aggregates. Rewarming to 37~ induced microtubule assembly in the control. With the R isomer, a weak turbidity increase took place (no microtubules were observed), whereas the S isomer induced a much larger turbidity, which was again cold reversible.

Formation of the tubulin-colchicine complex is known to induce protein conformational changes which result in GTPase activity (28, 29) and polymerization into abnormal polymers (22, 30). Like colchicine, the two compounds CI 980 and NSC 613863 induce GTP hydrolysis when they bind to tubulin (15). Therefore, and in view of the above results, it was of interest to examine their ability to induce the formation of polymers that are different from microtubules in glycerol-free buffer. As shown in Fig. 3B, Trace b for R and Traces c and d for S, both isomers, after a lag time, produced an increase of turbidity which was reversible upon cooling to 13~ and was protein concentration dependent. The inset displays a critical protein concentration plot showing that the S enantiomer needs less tubulin (0.52 mg/ml) than the R isomer (1.00 mg/ml) to induce abnormal polymer formation. Negatively stained, fixed preparations under the electron microscope showed the absence of microtubules and the formation of

77




Fig. 4. Depolymerization of PtK2 cell microtubules by R and S isomers. Cells were incubated for 4 h at 37~ without drugs (ll), with colchicine (IS]), with R isomer (O), or with S isomer (O). Cytoskele- tal tubulin was quantified by enzyme-linked immu- nosorbent assay (see "Materials and Methods"). Data, mean ___ SD of four independent experiments made in triplicate.

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large, filamentous structures, similar to the results described for the polymerization of the tubulin-colchicine complex (22, 31).

Depolymerization of the Cytoplasmic Microtubules of PtK2 Cells by CI 980 and NSC 613863. We first determined the dose curve of both isomers on the cytoplasmic network of PtK2 cells by immunoassay tubulin determination in cytoskeletons and also by immunofluorescence microscopy. The first method permitted quantification of microtubule inhibition (Fig. 4), while the second permitted visualization of the microtubule network morphology (Fig. 5). The depolymerization by the S isomer is significant at 3 nM, while the effect of the R isomer is significant at 50 nM. The immunoassay data correspond with the observation by indirect immunofluorescence. The half inhibitory concentrations for the S and R isomers were 6 nM and 100 nM, respectively, while that of colchicine was 1 /~M. The effect increased gradually until reaching maximal depolymerization of microtubules. Both drugs were more clearly active than colchicinc. We next studied the time course of the microtubule depolymerization by 20 nMS and 700 nM R isomers. Figs. 5 and 6 show the results for both drugs. The effect of the R isomer was significant 30 min after treatment, and the effect of the S isomer was noticeable 15 rain after incubation. Half maximal inhibition was reached after two h treatment with both R and S isomers, and inhibition was nearly maximal at 4 h. Then both drugs slowly increased their effect with time until 24 h. In reversibility experiments (Figs. 7 and 8), microtubules of cells treated with R isomer began to repolymerize after 15 rain, while PtK2 cells treated with S isomer required more than 30 min. In the reverting PtK2 cells, bright microtubule organizing centers in their cytoskeletons were observed. Half-recovery times were about 1 and 2 h, respectively. Then the reversion continued, and at 24 h of incubation, the R isomer-treated cells had totally reverted, while those treated with the S isomer had not completely recovered. This experiment shows again that the S isomer is more active than the R isomer, since its reversion is slower and is still not completed 24 h later.

Mitotic Arrest of Leukemic and Epithelial Cell Lines by C1980 and NSC 613863. The effect of R and S isomers on different human (U937 and HL6O) and murine (EL-4) leukemias and on PtK2 cells was studied. Cells were incubated for 17 h with different concentrations of drugs. It was found that 1 nMS enantiomer or 25 nM R enantiomer fully stopped the cell cycle in mitosis in U937 (Fig. 9A), HL60, and EIA cell cultures (results not shown). This inhibition of cell proliferation occurred in a very narrow concentration range, un- like the depolymerization of cytoplasmic microtubules which took place in a wider range of concentrations (Fig. 4). There was an accu- mulation of cells that had probably finished their S phase but did not proceed through mitosis. The PtK2 cells were more resistant to these drugs; nevertheless, they were also stopped in mitosis by concentrations of 5 nM and 50 nM S and R isomers, respectively (Fig. 9B). The PtK2 cell line presented some polyploidies both in control and treated cells, and with longer drug incubation periods, both octoploid and tetraploid cells appeared. The time course of this cell arrest was studied next. In these experiments, it was found that U937, HL60, and EL4 cells were maximally arrested after 8 h of treatment (Fig. 10). PtK2 cells were arrested after 24 h of treatment (results not shown). To examine if the effect was reversible, cells were incubated for 24 h in the absence or presence of 5 nMS isomer or 50 nm R isomer. Then some were washed and their recovery was studied, while other cells were not washed. As a reversible control drug, we used 250 nu MTC. In PtK2 cell cultures treated with MTC, R and S isomers were partially recovered on day 5 after washing (Table 1). Much debris was found in unwashed cells as well as tetraploid and octoploid cells (results not shown). On the other hand, with U937, HL60, and EL-4, no reversibility was found with either 25 nM R or 1 nMS isomers nor with 50--100 nM MTC (results not shown). In some experiments, U937 and HL60 cultures treated with R isomer and washed had around 10% of the control of viable cells 5 days later (results not shown). In summary, the effect of R and S enantiomers

78




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on cell division was found to be essentially irreversible in the leukemic cells, while they were partially reversible in PtK2 cells.

enantiomers on preformed microtubules diverged. The S isomer was able to induce the formation of thermosensible anomalous structures under microtubule assembly conditions much more markedly than the R isomer. This difference in the behavior of both enantiomers could be related to the critical protein concentration required to form abnormal structures in the glycerol-containing microtubule polymerization buffer. In the absence of glycerol, the critical protein concentration necessary for abnormal polymerization was two times higher for the R isomer than for the S isomer.

Effects of CI 980 and NSC 613863 on Cytoplasmic Microtu- bules. The S and R enantiomers induce a maximal disruption of the cytoplasmic microtubule network of PtK2 cells with the S enantiomer being approximately 16 times more active than the R enantiomer. These drugs are much more powerful than the usual inhibitors of cellular microtubules such as colchicine, MTC, and nocodazole (Fig.4; Refs. 38 and 39). The assembly of the microtubules of PtK2 cells after the S and R enantiomer removal is considerable slower than with MTC (38), nocodazole (39, 40), MDL 27048 (35), or Colcemide (41) and similar to that of colchicine (41). Complete microtubule repolymerization requires 24 h of incubation for the R enantiomer and longer periods for the S enantiomer.

Effects of CI 980 and NSC 613863 on Cell Division. Our results with human (U937 and HL60) and murine (EL4) leukemias indicate that as little as 1 nMS or 25 nM R enantiomers arrested these cells in mitosis with CI 980 being the most active proliferation inhibitor. The growth arrest was obtained after 8 h of incubation in the presence of the drugs. The high degree of mitotic arrest found after a single cell cycle time of exposure to these drugs is remarkable. It is similar to that found in HeLa cells in response to Vinca alkaloids (42, 43), colchicine (44), and nocodazole (45). We have also found that the inhibition of cell proliferation and mitotic arrest occur at concentrations of drugs

DISCUSSION

Interactions of CI 980 and NSC 613863 with Tubulin and Mi- crotubules in Vitro. Both S and R isomers inhibited substoichiometrically the in vitro assembly of microtubules in a similar way to known microtubule inhibitors such as colchicine, podophyllotoxin, vinblastine, or MDL 27048 (32-35). This inhibition was related to an increase in the critical concentration required for tubulin and MTP polymerization, and the S enantiomer was approximately 2--4-fold more powerful than the R enantiomer.

In a previous report (15), both R and S enantiomers were shown to bind at the colchicine site and compete with podophyllotoxin, sug- gesting an overlap of the ring A (trimethoxyphenyl) binding subsite, which is common to podophyllotoxin and colchicine. Both enantiomers and colchicine induced the tubulin GTPase activity, whereas podophyllotoxin did not. Preliminary results obtained using tropolone methyl ether as a blocking agent of the ring C (tropolone) subsite appeared to indicate that this part of the colchicine site is also involved in the binding of both compounds.

In the present study, it has been shown that both ligands induced the formation of anomalous tubulin polymers, which is characteristic of colchicine ligands such as MTC, allocolchicine, and biphenyl colchicine analogues (17, 31, 36, 37). It has been proposed that in order to induce the abnormal tubulin polymers, colchicine analogues have to bind with their C rings in proper orientation into the corresponding subsite of the colchicine binding site (37). The present results indicate that the two enantiomers act in the same manner as colchicine at stoichiometric concentrations, perturbing the normal protein-protein interactions involved in microtubule formation, which supports the hypothesis that they also bind to the ring C locus. The action of both

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of PtK2 ceils. Cells were incubated without drugs (l), 700 nM R isomer (O), or 20 nMS isomer ((3) at 37~ for 4 h. Then cells were washed three times with fresh culture medium and incubated in drug-free medium during different periods of time.

80




t i~ . N. FiIIl~_' C(~tlr~,u of r11i~-'Ft}WhLIIC r~_'pOl> Tl/cri/tttilH1 I~>IIL~'~', ila~ .% J',~m~.'r rcnl~)~.ttl. [~11'~- ~ ~.'clJ~, \~ L'rc tr~_'tttcd \,,ill1 7(,~() i~xl R <~i 21) T/\l >,' i s~mcrs Ic~r 4 h at 37~ T h e n the d r u g w a s

rcn'~c~vc.'d ;_[Tll.I t h e \ w e r e ct l l tLlrccl iT1 l'rosh r l lcc i iun l clcir ing d i t t i . ' rc i l t t ime' p<_'rit~d,,: ~<,il~plct~' <.'ell,, w~'rc l ixcct and pic~ccs<,cd For im l l l t l l l ~ l l i t l o rcscc r l c r o [ m i c r o l u b u l c s . Ba#; 10 #am. A, cc l l s :~t i i l~lc l i cil[t:r .$' ismnlcr re' i l l ()\ ~tl. T h i , is ~tlso rcl~rc'scntati~ c_' oH ~ h~I i,, ~t~',cr~ cd x~ i th ih~_' f7 i~,on?c.'r :tt 15 n ] in ~illd ~.~, i th i.hu .S' i,>c~mcr ~ll 15 ~inct : i lso 3(1 n l in . /7+ ce l l s i~t 1 h a f te r S i s o m e r rcn l t ) \ ~ll. T h i s is zil,,c) rc'l~lc<,cnl~Hi\ c tH \\h~l~. i~, sct_'il ~ i l h t lw 17 i , ,~mml .111 it~iiT t i l te r cl iu 7 rul~l~)\tt l . ( . cel ls {ll lime.' 3 h t i f lo r .~ i - ,~nlcr ic.'rll~)~,~il. T h i s is a lso o b s e r v e d w i t h the <7 i s o m e r :tt 2 - 2 4 h ~ti]ct w i t h the R i:-,t)i]~c,i ;1{ [ 4. h. 1). c{_'ll <, 24 h zdl~..r f7 i,,c~mc.'r lC..lllt~\ <11.



EFFECTS OF C1 980 AND NSC 613863 ON MICROTUBULES AND CELL DIVISION

0

to

o

0

..Q

E D Z

i f}

(D

L A

L

Fluorescence intensity B

o

0

o

E D z

Fluorescence intensity

Fig. 9. Dose effect of R and S isomers on cell division. U937 (A) and PtK2 cells (B) were incubated for 17 h in the absence (a, b, g, and h) or in the presence of the following drug concentrations: R isomer, 1 (c), 25 (e and i), or 50 (k) nM; and S isomer, 0.5 (d), 1 (jr), 2.5 ( j ) or 5 (1) riM. The relative DNA content per cell was measured by fluorocy- tometry (see "Materials and Methods") of 5000 cells.

much lower than those needed for a complete depolymerization of cytoplasmic microtubules. This could indicate that the inhibition of proliferation by the R and S enantiomers, as well as by MTC, colchicine, and nocodazole, could be due to perturbation of the spindle microtubule dynamics and not due to depolymerization of the microtubules, as demonstrated by Jordan et al. (42, 46) for HeLa cells and Vinca alkaloids. During 17 h of exposure of the cultures to R and S enantiomers, cells did not multiply, and following the washing of the drugs, very little or no reversibility in the inhibitory effect was found. Similar results were obtained with MTC that seem to be irreversible in these leukemias, although this inhibitor has been found to be reversible with K562 myeloblastic leukemia (unpublished results). Previous results with the racemic NSC 370147 (47) indicated that the inhibition of L1210 proliferation after 12 h exposure was irreversible 24 h after drug removal. On the other hand, exposure of PtK2 cells to 5 nM S or 50 nM R isomers (during the 24-h incubation period) resulted in a partially reversible growth arrest. Cells arrested in mitosis for extended periods of time can die or return to interphase without the separation of sister chromatids, resulting in polyploidy (45). After a long exposure of the cell culture to these drugs, a high proportion of polyploid cells were found. This could affect the normal reversion of the cell growth after removal of the drug, which was much slower than

82

that obtained with MTC (38) and not complete. The differences found in reversibility with the different human and murine leukemias and Ptk2 cells might be due to differences in sensitivity to induction of apoptosis by microtubule-disrupting agents (48) in some of these cell lines. Studies are in progress to examine this possibility.

Mechanisms of Action of C1980 and NSC 613863. There are two puzzling observations in this study: (a) while both enantiomers bind to tubulin and inhibit microtubule assembly in vitro not very differ- ently from known microtubule inhibitors, the inhibitory concentrations in the cells examined are orders of magnitude smaller; and (b) the binding to tubulin is weakly selective for the S enantiomer, and this compound inhibits microtubule assembly in vitro 3-4 times more powerfully that the R enantiomer. However, the cellular microtubule inhibition and the mitotic arrest are strongly chiral selective by a factor of - 2 0 in favor of the S compound. This selectivity is in qualitative agreement with previous results (7, 8,), although in our experimental conditions, the differences in activity between the S and R enantiomers are less pronounced.

The difference between the inhibitory activity of the S and R compounds in vitro and in cells is reminiscent of results with HeLa cells and Vinca alkaloids (42, 46, 49, 50). HeLa cells incubated with vincristine and vinblastine accumulate significantly higher concentrations intracellularly, in some cases 200-fold higher than the concentration in the external medium (42). Recent results with the S enantiomer and L1210 cells indicate that it rapidly reached intracellular concentrations in considerable excess of that in culture medium, and a model involving passive diffusion followed by significant reversible binding to intracellular or cell membrane components has been suggested (10). The in vitro affinity of tubulin for this compound does not seem to account for such a mechanism. Another possible explanation could be the metabolism of both compounds. However, a recent study has indicated that both the activity and the difference in potency between the R and S enantiomers are retained in major hy- droxylated metabolites (9). Other possible explanations could be sought in the following hypotheses that: (a) high sensitivity to these compounds by a particular subset of cellular tubulin molecules is not well represented in the brain preparations used; (b) there exists cellular factors which modulate the intrinsic sensitivity of microtubules to these compounds; (c) there is an additional, sensitive cellular target, different from tubulin, which would be chiral selective for CI 980 more than for the R isomer, causing the inhibition of microtubules; or (d) CI 980 could have an additional target that does not inhibit microtubules but inhibits cell division, i.e., C1980 could have multiple and independent mechanisms of action.

Table 1 PtK2 cell cycle distribution following R- and S-isomer exposure and removal

Percentage of cells in specific cell cycle phases following treatment with 50 nM R- or 5 nM S-isomer for 24 h and then removal of the drug (time 0). Results of a representative experiment out of 3 are shown here.

Time Treatment (h) G1 S (G2 + M)

Control 0 58 12 30 R-isomer 0 11 7 82 S-isomer 0 11 8 81 MTC 0 8 13 79






A B C 3h

L 6 h

~ k . Fig. 10. Time course of the effect of R and S isomers on cell division. Control (Column A), 25 nM R isomer (Column B), and 1 n~ S isomer (Column 0 C) treated U937 cells were incubated for 3, 6, 8, and ~ 8 h ~D 16 h. r

Z

16h

k,

3h 3h

6h

A

6h

A 8h

A 16h

8h

.A 16h

Fluorescence intensity

In conclusion, the compound CI 980 was found to be a most potent mitotic and microtubule inhibitor by causes not exclusively attribut- able to its intrinsic binding to tubulin. CI 980 is presently being tested in clinical trials (12) and could be of great interest in cancer therapy due to its superior or equivalent in vitro activity to vincristine and its full activity against vincristine-resistant tumors.

ACKNOWLEDGMENTS

We thank Drs. Jos6 Manuel Andreu and Juan Miguel Nieto for discussion,

Dr. T. J. Fitzgerald for MTC, Dr. Santiago Abarca and Tom,is Huelamo

for technical assistance, and Pedro Lastres for expert assistance with flow cytometry.

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1994;54:75-84. Cancer Res Concepcion de Ines, Daniel Leynadier, Isabel Barasoain, et al. Its Chiral Isomer, NSC 6138631-Deaza-7,8-dihydropteridine Antitumor Drug, CI 980, and by Inhibition of Microtubules and Cell Cycle Arrest by a New

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