cytotoxic effects of maleuric acid and the structurally related...

Cytotoxic Effects of Maleuric Acid and the StructurallyRelated Compounds on Ehrlich Ascites Tumor Cells*

TADASHI A. OKADA AND EUGENE ROBERTS

(Department of BiOchemistry, City of Hope Medical Center, Duarte@ California)

SUMMARY

The cytotoxic effects on Ehrlich ascites tumor cells of a large variety of compoundsstructurally related to maleuric acid (N-carbamyhnaleamic acid) have been studied.The most effective compounds of this series were ethylenebis(maleurate), and themethyl, @9-hydroxyethyl, allyl, 2-chloroethyl, isopropyl, and propargyl esters of maleuric acid (MA), all producing pyknotic chromatin masses as well as severe cytoplasmic blebbing and mitotic abnormalities at all stages of mitosis. Structural requirementsfor activity in this series of compounds are discussed. The effective esters destroyed theintegrity of the spindle fibers and were effective in preventing the entry of preprophasecells into mitosis. Glutathione administered simultaneously counteracted the cytotoxiceffects of the above effective compounds.

In previous papers it was shown that maleuricacid (N-carbamylmaleamic acid) produced mitoticinhibition in plant cells and extensive morphological abnormalities in Ehrlich ascites tumor cells(7, 8). Intraperitoneally administered maleuricacid (MA) was found to be far more effective inproducing abnormalities in tumor cells than wereisomolar amounts of maleic acid and N-ethylmaleimide. It was also ascertained by autoradiographic technics that MA prevented the incorporation of tritiated thymidine into deoxyribonucleicacid in tumor cells (9). In the present report a further study has been carried out to explore theeffects on Ehrlich ascites tumor cells of a largevariety of compounds structurally related to MA.

MATERIALS AND METHODS

A mouse tumor, Ehrlich ascites tumor (hypotetraploid strain), was used to study the effect ofvarious compounds.1 The ascites tumor was transmitted serially in mice of the Swiss/HaICR strain,weighing 18â€”20gm., fed on a stock diet of Purina

SThisworkwassupportedinpartby researchgrant#C-%568 from the National Cancer Institute, National Institutes of Health, Public Health Service.

1 All the compounds tested in this report were obtained from

Naugatuek CoemicalDivision, U.S. Rubber Company, Naugatuck, Conn.

Received for publication July 30, 1962.

chow and water. Some of the compounds were dissolved in distilled water. In the case of chemicalsinsoluble in water, an aqueous suspension was prepared by using a glass homogenizer. Intraperitoneal injections of freshly prepared aqueous solutions or suspensions of compounds were usuallybegun on the 5th day after transplantation of thetumor, at which time the tumor was well established, and active proliferation of the tumor cellswas taking place in the peritoneal cavity of theanimals. All compounds were tested in single injections of 3 mg. and in quantities isomolar withS mg. of maleuric acid.

At various intervals, indicated in each experiment, samples of tumor ascites were removed byperitoneal puncture, squashed with acetic dahlia,and examined cytologically. The numerical dataand the following descriptions were calculated onthe basis of a count of approximately 500 interphase and dividing cells for every sample.

For the observations of the mitotic apparatus,wet-smeared slides were fixed in Carnoy's solution(absolute methanol: acetic acid, 3 : 1) for 3 minutesand run through graded alcohol to water. After thecustomary Feulgen staining, slides were put indilute fast-green acetic acid solution for 30 seeonds. Then, the materials were covered with coverglasses and sealed with paraffin balsam (6).

217

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EFFECTIVECHEMICALSLESSEffECTIVE I'REMICIL$INEffECTIVE CHEMICALS

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Less effective:N-(2-Chloroethylcarbamyl)maleamic acid2-EthoxyethylmaleurateN-OctadecylmaleurateN-AmylmaleurateN-(Phenylcarbamyl)maleamic acidBenzylmaleurateMaleuricacid

Ineffective chemicals:Methylsodium N-carbamylsulfosuccinamateN-CarbamylfumaramicacidN-Carbamylsuccinamic acidN-ButylmaleurateIsopropyl N-tert.butyl maleurateN.Dodecyl maleurateGeranylmaleurate0

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Cancer Research Vol. 23, February 1963218

The autoradiographic procedures for the studyof DNA synthesis were described previously (9),each animal being given 10 [email protected] tritiated thymidine (T-ll3) (1.9 c/mmole) simultaneously withthe test chemical, or in the different time intervalsbefore or after injection of the chemical.

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CHART 1.â€”Effectiveness of maleuric acid and derivativeson morphology of Ehrlich ascites cells.

TABLE 1

APPEARANCE OF ABNORMAL MITOTIC CELLs AT VAiuous TIMEs AFTER SINGLE INJECTIONSOF MALEURICAcm AND RELATED CoxrpouNDs*

S The values represent the averages obtained in from five to ten animals.

t Normalmitoticfigures.@ Abnormal mitotic figures.

RESULTS

Twenty compounds used in this study are classified into three groups according to the degree oftheir effectiveness in producing cellular abnormalities as follows : (a) effective, (b) less effective, and(c) ineffective compounds (Chart 1). Also, thevalues for mitotic inhibition with twenty cornpounds are shown in Table 1.

Effective compounds.â€”Compounds of this groupshowed severe damaging activity on tumor cells,such as blebbing of cell surface, pykuotic chromatin masses, and abnormalities of chromosomes.However, only methyl N-carbarnylmalearnate(MCM) showed essentially the same activity at3 mg. as at a reduced dose of 1 mg/animal. Thefollowing description of sequential cytologicalchanges is based upon observations made on cellsfrom mice receiving a single dose of 3 mg. of MCM.

Both cells in interphase and in various mitoticstages showed visible signs of damage at 30 mmutes after the injection. There was severe distor

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OKADA AND R0BERTsâ€”Cytotoxic Effects on Ehrlich Ascites CelLi 219

tion of the cytoplasm due to blebbing of cell surfaces. Marked disappearance of normal-appearingtumor cells, interphasic and mitotic, was observedat 1 hour. In almost all the mitotic cells there wereobserved stickiness and clumping of chromosomesand pyknotic chromatin masses or other degenerative changes which appeared to increase progressively with time (Figs. 1, 3). At the 6- to 10-hourinterval chromosomes in most of the abnormalmitotic cells became pyknotic chrornatin masses.In addition to these abnormalities, some mitoticcells with numerous c-rnitotic chromosomes werefound. At 20â€”24hours there were still no normalmitotic cells, and interphasic cells still showed distortion of the cytoplasm. Cells at anaphase andtelophase were not observed at all during the entire experimental period. However, at a dose of 2mg. a few cells at anaphase and telophase werefound in the ascites at 12â€”24hours after theinjection.

Le88 effective compounds.â€”Compounds in this

group did not induce cells with a pyknotic chromatin mass at doses isornolar with 3 mg. of maleuric acid.

Both the cells in interphase and dividing stageshowed visible signs of damage within SO minutesafter the injection of the chemicals. There wassome distortion of the cytoplasm due to blebbing.Some decrease occurred in the number of normalappearing dividing cells, and agglutination ofchromosomes at metaphase was observed. As inGroup 1, marked disappearance of normal-appearing tumor cells in interphase and dividing stageswas observed at 1 hour, and almost all the mitoticcells showed stickiness and clumping of chromosomes, which appeared to increase progressively

with time (Figs. 2, 4). At the 2- to 3-hour intervalclumping was observed between the sticky chromosornes at anaphase. The clumped chromosomesat metaphase showed a ringlike arrangement. Inaddition to chromosome aberrations, numerousmicronuclei were observed in cells at telophase. Itappeared possible that some of the mitotic cellswere able to complete cytoplasmic cleavage despite the abnormalities in the chromosomes. At4â€”6hours the total number of mitotic cells observed was very small, and the typical cytoplasmicabnormalities still were observed. Subsequently,there was a gradual increase in the dividing cells,both normal and abnormal. After 12 hours thecells in interphase again showed a normal appearance.

From all the findings with both the effective andless effective compounds (Table 1), it is apparentthat, when the number of carbons in the substitu

ent (R.1) esterified to the carboxyl group of maleuric acid exceeded three, the cytotoxic activity ofthe compounds decreased. Moreover, when a substituent (H2) replaced one of the hydrogens of theamide group of maleuric acid, cytotoxic activity ofthe compounds also decreased. Furthermore, N-carbamylfumaramic acid, N-carbamylsuccinarnic acid, and methyl sodium N-carbarnylsulfosuccinamate (see Chart 2 for structures) are ineffective compounds. N-Carbamylfumaramic acid isthe trans-form of maleuric acid. N-Carbarnylsuccinarnic acid and methyl sodium N-carbamylsulfosuccinamate do not have a double bond. Seemingly, there is considerable specificity required inthe structure of this class for the production ofmaximal cytotoxic effects.

The most remarkable difference between thetwo groups of compounds designated as effectiveand less effective is the occurrence of the cells withpyknotic chromatin masses after treatment with

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the former group of substances. However, when,5 jig. of colchicine was injected simultaneouslywith 3 mg. of MA into animals, many cells withpyknotic chromatin masses were induced in theascites (Fig. 6) . MA (Figs. 2, 4) or colchicine (Fig.

5) alone at these dose levels did not produce thiskind of abnormality. Furthermore, it is knownthat coichicine is a well known inhibitor of spindlefiber formation, whereas MA induces sticky chromosomes. Therefore, it is conjectured that the for

rnation of pyknotic chromatin masses may havebeen caused by the combined destruction ofspindle fibers and the production of sticky chromosomes.

Observations on the spindle fibers.â€”In an attempt to analyze the effects of MA and other cornpounds on tumor cells in further detail, observations were made of the spindle fibers of the cells.

At prophase and early metaphase, spindle fibersin untreated tumor cells were not observable. In

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Cancer Research Vol. 23, February 1963220

untreated tumor cells at metaphase spindle fiberswere observed clearly, running from chromosomesat metaphase plate to poles (Figs. 7, 13). At thesame time, continuous fibers also could be seen

from one pole to the other in the cytoplasm by

changing the focus of the microscope. The contourregion of the spindle fibers is denser than the innerpart. The number of spindle fibers is difficult tocount because of their proximity to one another.The connections between spindle fibers and chromosomes are not observable because of the high

contrast of the Feulgen-stained chromosomes.When the daughter chromosomes begin to moveapart from one another toward the poles, thekinetochores of the daughter chromosomes areseen clearly attached to spindle fibers. In the case

of MA and other less effective compounds, thespindle fibers in the treated cells appeared normalwithin the first 3 hours after injection, although thechromosomes in the same cells showed abnormalities (Figs. 7â€”12). In some of the cells, several

groups of chromosomes took their positions at thepole of the cells, but the spindle fibers still wereseen clearly to run from poles to the chromosomes.With time the chromosomes clumped more tightlyand moved closer to one pole. Meanwhile, thespindle fibers in these cells changed their rnorphological features. For example, the length of thespindle fibers from one pole to the chromosomesbecame shorter. Also, the diameter from one edgeof the spindle fiber to the other became very narrow. Sometimes chromosomes in the same cellswere far from the main region of spindle fibers.About 10 hours after injection some of the abnormal mitotic cells at metaphase showed a tendencyto become anaphase cells. In these cells interzonalfibers floating in the cytoplasm were observed.

Since clumped and sticky chromosomes in mostof the abnormal mitotic cells became pyknoticchromatin masses rapidly during the first hourafter injection of MCM and the other effectivecompounds, observations were made mainly onsamples obtained at 5-, 10-, 15-, 20-, 30-, and 60-minute intervals. At 5 minutes after injections ofthe chemical the spindle fibers and the chromosomes were still normal in shape and took positionsin the same place as in the control cells. After 10â€”1@5minutes the spindle fibers and the chromosomesof most of the mitotic cells began to show abnormality. The chromosomes tended to show stickiness and to clump together. The spindle fibers be

came slender. Sometimes one or two chromosomesor chromatids were found to be located in thepolar region of cells (Fig. 14). At 20â€”SOminutesthe spindle fibers became more irregular in theirmorphological configuration (Figs. 15, 16). They

showed disturbance in orientation such as asymmetrical location of both central poles and fibers.In some cells they were either partially or totallydetached from the chromosomes. In other cellsthey became separated from one another, appearing as though in multipolar division. However,from the observation of the same material usingacetic dahlia staining, cells in multipolar divisionwere seen only rarely in the ascites. Subsequently,chromosomes became more clumped, appearinglike pyknotic chromatin masses. Chromosomes inthese cells still maintained their position midwaybetween poles.

About 60 minutes after the injection of MCMthe spindle fibers began to break down into fragments and to dissolve. They still showed the tracerunning from the polar center to the chromosomes. At the same time some of the cells showedspindle-shaped opaque regions around the chromosomes. Fragmented spindle fibers were scatteredat random in the cytoplasm, and chromosomes inthe same cells became pyknotic chromatin masses.Thereafter, the number of abnormal mitotic cellswith a pyknotic chromatin mass and withoutspindle fibers kept increasing up to 1@hours afterthe injection.

The effect of methyl N-carbamylmoleamate on theincorporation of tritiafed thyniidine.â€”Intraperitoneal injection of 3 mg. and 5 rng. of MA into micebearing Ehrlich ascites tumor simultaneously, orat various times prior to the injection of tritiated thymidine (T-H3), prevented the appearance ofthe label in DNA of interphasic or mitotic cells atlevels detectable by the autoradiographic procedures employed (9).

In the present experiment the effects of the injection of 2.5 mg. of methyl N-carbamylmaleamate (MCM) simultaneously or at different timeintervals before or after the injection of T-H3 wereobserved (Table 2).

In control experiments rapid incorporation ofT-H3 took place into interphase cells, approximately 50 per cent of the cells being labeled 1 hourafter injection of this isotope, the earliest time atwhich a sample was taken (Table 2). Thenceforth,there was a slight variation of doubtful statisticalsignificance in the percentage of labeled cells up to24 hours. The minimal period between the timeexogenously supplied thymidine was incorporatedinto DNA and the identification of labeled mitoticcells was S hours. Maximal labeling of mitotic cellswas attained at 12 hours (Table 2). When the drugwas given simultaneously with the labeled nucleoside, or 24 hours earlier, a decreased incorporationof isotope into interphase cells was found only atthe 1-hour period, while there was a normal rate



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OKADA AND ROBERTSâ€”CytOtOXiC Effects on Ehrlich Ascites Cells 221

of appearance of label in the cells obtained fromanimals that had received MCM 30 minutes afterthe injection of T-H3. When MCM was injected30 minutes before T-H3 the number of labeledinterphase cells at the 1-hour interval was approximately 16 per cent of normal, and the percentageincreased to approximately 60 per cent at the latertimes. When the administration of T-H3 was preceded by 3 hours by the injection of MCM thetotal number of labeled interphase cells was approximately 60 per cent of the control at all timeintervals studied.

Observations of the percentage of labeled mitotic cells (normal and abnormal) clearly showedthat MCM blocks entry of preprophase cells intomitosis (Table 2). In all sequences of injection ofMCM and T-H3 the effect was evident. Very few

Similar experiments were carried out, in thepresent study, with MCM and $-hydroxyl N-carbamylmaleamate. When 25 mg. of GSII was injected intraperitoneally into tumor-bearing mice simultaneously with 3 mg. of these chemicals thecytoplasmic blebbing and chromosomal abnormalities usually produced by this dose of either of these

chemicals were prevented completely. However,when 25 mg. of GSH was injected intraperitoneally into tumor-bearing animals at 3 hours afterinjection of MCM there was no protective action.An experiment was then performed in which 2.5mg. of MCM was injected simultaneously withGSH (neutralized solution) at 25-, 20-, 15-, 10-, or5-mg. levels. With the injection of GSH at 25- and20-mg. levels virtually complete protection couldbe observed. In the animals given GSH in 15-, 10-,

TABLE S

S PERCENTAGES OF INTERPHASIC (1) AND MITOTIC (M) CELLs WITH LABEL AT

VARious TIMES AFTERINJECTION OFTHYMIDINE-H3

S Each individual figure is the mean value of determinations made in S animals.

t Tritium-labeled thymidine.@ Methyl N-carbamylmaleamate.

labeled mitotic cells were noted at any time whenthe injection of MCM preceded the administrationof T-H3, even though relatively large proportionsof the interphase cells had become labeled. Evenwhen the agent was given 30 minutes after thenucleoside and normal proportions of interphasecells were labeled there was a remarkable inhibition of the appearance of isotope in mitotic cells.

From the observations described above it wouldappear that MCM might prevent tumor cells fromentering into mitosis by destroying spindle fibersor spindle fiber substances after the cells completetheir DNA synthesis.

Protective action of glutathione (GSH) .â€”It wasshown in a number of experiments in a previousstudy that the intraperitoneal injection of 25 mg.of GSH into tumor-bearing mice simultaneouslywith 3 mg. of MA completely prevented the occurrence of morphological abnormalities in the tumorcells usually produced by this dose of MA (8).

or 5-mg. doses there was a considerable decreasein the frequency of cells with pyknotic chromatinmasses, although the chromosomes still showed

abnormalities. A further experiment was carriedout in which 2.5 mg. of MCM was administeredto tumor-bearing animals simultaneously withT-H3 and GSH. Samples were obtained from treated animals at 3, 6, 12, and 924hours after the injection of the chemicals. The number of interphasiccells with label was the same as the control. Inaddition, mitotic cells with labeled chromosomeswere observed at 3 hours after injection of thechemicals, and the number of labeled mitotic cellsremained the same as in the controls during theentire experimental period.

DISCUSSION

Some of the compounds structurally related toMA were found to be much more effective thanMA in producing abnormalities in Ehrlich ascites



222 Cancer Research Vol. 23, February 1963

tumor cells. These are the esters of maleuric acid:methyl-N-carbamylmaleamate, @-hydroxyethylN-carbamylmaleamate, allyl maleurate, 2-chloroethylmaleurate, isopropyl maleurate, ethylenebis(maleurate),and propargyl maleurate.It is possible the effective chemicals may be more activethan MA itself because of more rapid entry intothe cells and more effective reaction with some essential sulfhydryl compounds within the cells.

Unlike MA the effective esters exhibited theireffects on the spindle fibers in the tumor cells.Spindle fibers in the cells treated with the effectivechemicals disappeared, and pyknotic chromatinmasses were observed in the cytoplasm. On theother hand, the spindle fibers in the cells treatedwith MA and the other less effective chemicalsappeared normal, although chromosomes in thesame cells showed abnormalities.

Gaulden and Carlson (2) studied the effects ongrasshopper neuroblast cells of colchicine at various dose levels and stated that the formation of

pyknotic chromatin mass could be induced by colchicine at certain dose levels. In the present experiment it was pointed out that a small dose of colchicine injected simultaneously with MA producedrelatively large numbers of ascites tumor cells withpyknotic chromatin masses. MA or colchicinealone, in the amounts used, did not produce thiskind of abnormality. Coichicine is an inhibitor ofspindle fiber formation (1), inducing c-mitoticcells, and MA produces sticky chromosomes (7, 9).Thus, from these data it seems possible that thosesubstances which produce cells with pyknoticchromatin masses might have effects on bothspindle fibers and chromosomes.

It was shown in the present study that, when25 mg. of glutathione (GSH) was injected intraperitoneally into tumor-bearing mice simultane

ously with 3 mg. of MCM, the cytoplasmic blebbing and chromosomal abnormalities usually produced by this dose of MCM were prevented cornpletely. However, when the dose of GSH wasreduced, c-mitotic cells or cells with sticky chromosornes were observed in the ascites. Therefore, itseems that the formation of pyknotic chromatin

masses in cells treated with MCM is caused by thedestruction of spindle fibers in addition to the pro

duction of sticky chromosomes.

In the past few years there have been some convincing demonstrations of the role of protein SHgroups in the mitotic apparatus (5). The experiments of Kawamura and Dan (4) and Kawamura(3) suggested that the formation of the mitoticapparatus in sea urchin eggs involves the assemblyof proteins having a higher SH concentration thanthe other proteins of the cytoplasm. It appearspossible that the destruction of spindle fibers byMCM results from interaction of MCM with SHgroups in the cells. In order to clarify this assumption further experiments with tritiated MCM arein progress, as well as chemical studies of interaction of MCM with protein and nonprotein sulfhydryl groups.

The survival of the animals employed in thisstudy was not decreased or increased significantlyover that of the untreated controls. This suggeststhat the drugs at the dose used in this report werenot particularly toxic and that probably only arelatively small proportion of the tumor cells distributed throughout the tissues of the animalswere permanently damaged by single doses of thechemicals studied.

REFERENCES1. EIGSTI, 0. J., and DusrIN, P. Colchicine. 1st ed. Ames,

Iowa: The Iowa State College Press, 1957.S. GAULDEN, M., and Caaj.aoN, J. Cytological Effects of

Colchicine on the Grasshopper Neuroblast in vitro withSpecial Reference to the Origin of the Spindle. Exp. CelllIes.,2:416â€”38,1951.

3. [email protected]@,N. Cytochemical and Quantitative Study ofProtein-bound Sulfhydryl and Disulfide Groups in Eggs ofArbacia during the First Cleavage. Exp. Cell lIes., 20:127â€”38,1960.

4. KAWAMURA,N., and DAN,K. A Cytochemical Study of theSulfhydryl Groups of Sea Urchin Eggs during the FirstCleavage. J. Biophysic. Biochem. Cytol., 4:615â€”20, 1958.

5. MAzIA,D. Mitosis and the Physiology of Cell Division. TheCell, 3:77â€”894. New York: Academic Press, 1961.

6. OKADA,T. A. Cytological Observations of the InterzonalConnection in Ehrlich Ascites Tumor Cells during Mitosis.Exp. Cell Res., 24:600â€”603, 1961.

7. OKADA, T. A., and ROBERTS, E. Antimitotic Action ofMaleuric Acid. Proc. Soc. Exp. Biol. Med., 99:329â€”32, 1958.

8. . Cytological Analysis of Effects of Maleuric Acid onEhrlich Ascites Tumor Cells. Cancer lies., 20: 1154â€”59,1960.

9. SISKEN,J. ; OKADA,T. A. ; and ROBERTS,E. Autoradiographic Studies of Antimitotic Action of Maleuric Acid.Proc. Soc. Exp. Biol. Med., 101:460â€”63, 1959.

FIGS. 1-6.â€”Appearance of Ehrlich ascites tumor cells aftertreatment with MCM (1 and 3), MA (S and 4), colchicine (5),and coichicine plus MA (6) (see text). 1, 2, 5, and 6, X1000; 3and 4, X400.



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FIG. 13.â€”An untreated cell at metaphase.FIG. 14.â€”1O minutes after the injection of MCM, mets

phase, showing a chromosome at the pole.FIG. 15.â€”At 20â€”30minutes, spindle fibers show separation

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1963;23:217-222. Cancer Res Tadashi A. Okada and Eugene Roberts Compounds on Ehrlich Ascites Tumor CellsCytotoxic Effects of Maleuric Acid and the Structurally Related

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