Free Amino Acids in Growing and Regressing Ascites Cell Tumors: Host Resistance and Chemical Agents*

EUGENEROBERTS,K.KANOTANAKA,!T. TANAKA,! ANDDAISYG. SIMONSEN

(Departments of Biochemistry and Cytology, Medical Research Institute, City oj Hope Medical Center, Duarte, Calif.)

Previous studies have been made of the pat quent experiments with the Yoshida ascites tumor terns of free or easily extractable ninhydrin-reac- showed that glutamine can be taken up rapidly tive constituents in a variety of animal tumors and utilized by the tumor cells in vivo, but that (7, 13-17). Although each normal tissue in a par these cells possess only limited permeability to ticular species has been found to have a distribu glutamic acid (16). tion of these substances characteristic for that The present report is concerned with studies of tissue, all of the tumors examined have shown a the free or easily extractable amino acids and of similar pattern. Relatively low levels of free or the cytological characteristics of the Yoshida easily extractable glutamine have been observed sarcoma when grown in susceptible (J strain) and in the extract of the tumors. Glutamine was resistant (Wistar) strains of rats and of the effects of sarkomycin, nitromin, and crude podophyllin on the Yoshida ascites tumor and of sarkomycin on the Ehrlich tumor. MATERIALS AND METHODS The procedures for transmission of the tumor, cytological observation, and two-dimensional paper chromatography have all been described previously (14, 17). A reference diagram showing the relative positions of the detectable constitu ents on the paper chromatograms is shown in Chart 1. RESULTS AND DISCUSSION CHART 1.â€”Diagram showing constituents on chromato- graiHs: 1, phenylalanine; 2, tyrosine; 3, leucine and isoleucine; Free amino acids of the Yoshida sarcoma grown 4, valine; 5, ethanolamine; 6, methionine (methionine sulfone) ; in J strain or Wistar rats.â€”The Yoshida sarcoma 7, taurine; 8, proline; 9, a-amiiio-n-butyric acid; 10, threonine; is usually carried in J strain rats, a strain in which 11, serine; 12, cystine (cysteic acid); 13, alenine; 14, histidine; the tumor has a high degree of transplantability 15, /3-aIanine and/or glycerylphosphorylethanolamine; 16, glu (approximately 95 per cent takes). A failure of the tamine; 17, glycine; 18, glutamic acid; 19, arginine; 20, lysine; Â¿1,ethanolamine phosphoric ester; 22, aspartic acid; 28, gluta- latter strain of rats to breed properly for a period thione (oxidized) ; 24, glutathione. made it necessary to transmit the tumor in Wistar rats. The tumor grows in the latter animals for not detected at any time after transplantation in several days after inoculation and then begins to the solid or ascites forms of the C1498 leukemia undergo regressive changes, finally disappearing grown in a susceptible line of mice (C57BL/ completely. The tumor persists before regressing 10-H-2b) but appeared in these tumors when they for longer periods when grown in young than in began to regress in a resistant subline (C57BL/ adult Wistar rats. However, it is possible to main 10-H-2d) which differed by one gene (13). Subse- tain the tumor indefinitely in the Wistar rats by * This investigation was supported by research grant C- serial transplantation at intervals of approximate ly 5 days.1 In this manner the Yoshida sarcoma has 2568 from the National Cancer Institute, United States Public Health Service. been maintained in animals of the Pacific Farms t Present address, Department of Zoology, Hokkaido Wistar strain (WP) for more than 60 generations. University, Japan. 1This also was observed previously for the Yoshida sarcoma Received for publication June 18, 1956. by N. Mitsuoka in unpublished observations. 970

Preliminary Chromatographie examination of the WP rat. Subsequently, similar observations extracts of the cells and fluid of the Yoshida sar were made in three additional WP rats. coma grown in the WP rats revealed the presence A cytological analysis of the tumor cells in the of glutamina in some samples, while this amino two rats is shown in Table 1, and smears showing acid was not noted in any of the samples obtained the appearance of the cells at 4 and 7 days, re from J strain rats bearing the same tumor. It was, spectively, are shown in Figures 1-4. In the WP therefore, of considerable interest to follow the rat a high rate of cell division was maintained free amino acids in the cells and fluid on successive through the 6th day after transplantation (see days after transplantation of the Yoshida sarcoma Fig. 1). On the 7th day no mitotic cells were in both the WP and J strain rats and to attempt to found, and the percentage of damaged cells and

TABLE1 CYTOLOGICALANALYSESOFGROWINGANDREGRESSINGTUMORSINRATS

TlllEDayÂ« PERCENTAGEDISTRIBUTIONSor CELL CATEGORY aftertransplanta NO.CELLSEXAMINED1*53124812211718850119312521185122011571240126312151197117011511178126011541123127213901227125315451456Restingtumorrelis81.780.173.738.20.081.479.280.581.586.585.081.682.584.681.080.9MitotictumorcellÂ»2.23.93.803.13.9403.72.41.01.91.40.20.30.1Damagedtumorcells00.22.320.0000.20.10.30.60.40.52.1.i.l5.0Nontumorcells*16.115.820.241.810015.616.915.314.710.812.516.115 TBEAT- MENT STRAIN tion456784.567891011121814Hours Xone WPf

None

613.113.614.0

afterinjection(before)ÃŽ4612(before)12172436TOTAL

XitrominÃ® 86.8 2.8 0 10.4 85.8 3.2 0.1 10.9 86.5 1.0 Â¿.1 10.4 89.9 0.2 a.l 7.8 80.0 0.0 7.3 12.7 84.0 2.3 0.0 13.7 82.6 0.1 5.5 11.8 79.2 0.1 6.5 14.2 70 5 0.8 3.4 25.8 69.9 2.2 15 26.4 * These are various types of leukocytes (especially monocytes and basophils in regression) and lymphocytes. t Pacific Animal Farms, Wistar strain. Ã•Methylbis(/}-chloroethyl)amine-N-oxide hydrochloride; intraperitoneal dose, 30 mg/kg.

correlate the findings and cytological observations, content of leukocytes increased sharply (Fig. 2). since in the previous study with C1498 leukemia At this time a number of the tumor cells were in mice it was found that the beginning of regres found to be undergoing disintegration. At 8 days sion of the tumors grown in the resistant subline after transplantation no further tumor cells were was accompanied by the appearance of free detectable in the peritoneal fluid of the WP rat, glutamine (13). the cellular content consisting almost entirely of Yoshida sarcoma cells were transplanted from leukocytes. In the J strain rat tumor cells began a Wistar rat into a rat of the Wistar strain (240 growth immediately after inoculation, and the gm.) and one of the J strain (275 gm.). Samples of mitotic frequency was at the highest level between tumor were removed by peritoneal puncture be 4 (Fig. 3) and 7 (Fig. 4) days after transplantation. ginning with 4 days and continuing until death at The cytological features of the tumor cells during 14 days in the animal of the J strain or until regres this period resembled those observed for the 4- and sion occurred at 8 days after transplantation in 5-day cells in the WP rat. The number of mitotic

figures decreased after the 8th day. An accumula ing up to the 7th day of transplantation, and the tion of a large amount of ascitic fluid took place glutamic acid content of the fluid remained ap during this period. Toward the end of the life span proximately the same throughout the period of the J strain rat there was an increase in the per studied (Figs. 6, 8, 10, and 12). Although the centage of damaged, degenerating cells which tumor grown in WP rats was used for some of the were characterized by pyknotic masses of nuclear subsequent experiments, the results were em substance, and there was a concomitant decrease ployed only in those instances in which it was in the mitotic index. The content of leukocytes, shown that the glutamine content of the control which was minimal at 8 days, remained relatively cells and fluid was low on the day of the experi constant throughout the period of observation, ment and when the condition of the tumor was ranging between 10.8 and 16.9 per cent of the total judged to be optimal by cytological criteria. cell count. Effect of sarkomycin on the Yoshida osciles tu The patterns of free amino acids found at 4 and mor.â€”Sarkomycin, a weak antibiotic, has been 5 days in the cells grown in both strains of animals, found to have a marked destructive effect on cells which were not distinguishable cytologically at of the Ehrlich ascites tumor and to be somewhat this time, were similar with the exception of the less effective on the Yoshida tumor and the MTK- finding of a larger amount of glutamine in the cells sarcoma II (18, 22). Evidence has been presented grown in the WP rat (Figs. 5 and 7) than in the J which indicates that the active material is 2- strain rat (Figs. 13 and 15). Glutamine is ordinari methylene-3-oxocyclopentanecarboxylic acid (4). ly not found in this tumor in the J strain when the Preliminary results in this laboratory indicated transplantation is made from the same strain. On that sarkomycin (preparation RC-903,5F76, Bris the 6th day (Fig. 9) the amount of glutamine in tol) was rapidly effective against cells of the the tumor grown in the WP animal increased Yoshida tumor when an intraperitoneal dose of above that found on the previous 2 days, while approximately 30 mg/100 gm rat was employed. in the J strain (Fig. 17) there was a decrease in the A WP rat weighing 175 gm. was employed for amount of glutamine to a barely detectable level. the experiment on the 5th day after inoculation Relative increases in the amounts of valine, leu- of the Yoshida tumor. After removal of the control cine, and isoleucine and decreases in taurine and sample, 1 ml. of physiological saline containing threonine content were found in the tumor cells of 50 mg. of sarkomycin was injected intraperitoneal- the WP rat on the 7th day after transplantation ly, and samples of tumor were removed at 15-, (Fig. 11), some of which changes may be attribut 30-, 45-, 60-, 120-, and 300-minute intervals. The able to the infiltration with leukocytes (Table 1). control sample showed a high mitotic rate (4.4 Glutamine was not detected in any samples of per cent) and only few damaged cells. The chro- tumor cells in the J strain rat after the 7th day, matograms of the cells and fluid showed typical and the glutamic acid level decreased after the 8th patterns of free amino acids, small amounts of day after transplantation (Fig. 19), the patterns glutamine being noted in the cells but not in the of amino acids found between the 8th and 14th fluid. In the sample examined 30 minutes after the days being the same as those usually found in this injection of the sarkomycin, most of the tumor tumor carried serially in the J strain rats (17). Al cells showed cytoplasmic blebbing. Chromosomal though glutathione was present in the cells of both abnormalities such as clumping and irregular the WP and J strain rats in easily detectable scattering at metaphase were noted in cells under amounts from the 4th through the 7th days, the going mitosis. The extracts of the cells showed a content of this peptide decreased after the 4th day greatly elevated content of glutamine, and this and fell to an undetectable level after the 8th day amino acid appeared in the fluid at 15 and 30 in the J rat. minutes. No other significant changes from the Glutamine was detectable in the ascitic fluid of control chromatograms were noted at this time. the J rat in small amounts through the 5th day The cellular damage increased with time, cells of and then decreased to undetectable amounts (Figs. the 60-minute samples showing severe cytoplasmic 14, 16, 18, 20). On the 8th and successive days distortion as a result of blebbing, with irregular there appeared to be an increase in the content of processes or atypical amoeboid protrusions. Mitot alanine by comparison with the levels of this ic arrest occurred at metaphase in increasing amino acid on the first 7 days after transplanta numbers of cells, and nuclear abnormalities were tion. The content of glutamic acid fell to a barely found in resting cells and at early prophase. The detectable level by the 13th day after transplanta nuclei began to exhibit a coarse ultrastructure and tion. Glutamine was found in the ascitic fluid in despiralized or broken down chromonemata. The in the WP rat in all the samples studied, increas number of cells undergoing normal mitosis de-

creased in number. The chromatograms of cells incubated with 0.5 ing. of DL-glutamine-2-Cu and fluid showed no notable changes from the 15- (approximately 1.5 //c) for 10 minutes at 37Â°C., minute sample up to 120 minutes after the ad following which the cells were separated quickly ministration of the sarkomycin. At 300 minutes by centrifugation and the cells and fluid extracted the cells and fluid both showed a further increase in the usual manner. The rat was then given an in glutamine content over the control sample. At injection of 30 mg/100 gm of sarkomycin, and 120 and 300 minutes the degenerative changes in another sample of ascites was removed at 55 the tumor cells were even more marked than at minutes and incubated with glutamine-2-C14 as previous times. More than 50 per cent of the described for the control sample. Chromatograms tumor cells observed showed pyknotic aggrega and radioautographs were prepared in the usual tions of chromatin following inhibition at meta- manner. phase. No normal mitotic figures were observed, The results are shown in Figs. 21-28. Compari the abnormalities observed including bridge forma son of the chromatograms of the cells and of the tion of sticky chromosomes and lagging chromo fluid before and after sarkomycin injection re somes at anaphase and telophase. Observations vealed little change from the control with the with phase microscopy showed that damage also exception of decreases in aspartic acid and gluta occurred to the mitochrondria after treatment thione contents, and of the finding of a new un with sarkomycin, the fine granules or thread-like identified ninhydrin-reactive substance to the left shapes in the untreated tumor cells changing of glutamic acid and underneath glycine, which progressively after treatment into rounded forms, has been found only occasionally in Yoshida tu which subsequently clumped together and eventu mor cells after treatment with sarkomycin and ally aggregated into deformed masses of these de invariably in Ehrlich cells. Preliminary evidence generated structures. After 300 minutes the intra- indicates that this may be an addition product of peritoneal cellular reaction became marked, and glutathione and sarkomycin. Previous suggestions no further cytological or chemical studies were have been made of interactions of sarkomycin and made. sulfhydryl compounds (23, 24). Changes in glu Chromatograms of acid hydrolysates of the tamine level which might have occurred as a re picric acid filtrates of the extracts of the cells sult of treatment were obscured by the glutamine showed that there was no marked accumulation of which was added to the incubation mixture. peptidic material after treatment with sarko The radioautograph of the extract from the mycin, and they were remarkably similar after control cells in Fig. 23 shows that there was a treatment with sarkomycin to those found in the rapid cellular uptake of glutamine and a con control sample. Glutamine, ethanolaminephos- version of the glutamine to glutamic acid. It is not phoric ester, and glycerylphosphorylethanolamine likely that the glutamic acid in the cells was first disappeared after hydrolysis, and ethanolamine formed from glutamine in the ascitic fluid and appeared prominently on all of the chromato then taken up by the cells, since it has been shown grams. In addition, relatively large amounts of in a previous study (16) that the Yoshida tumor cystine (cysteic acid) were noted in all of the cells are only very slightly permeable to glutamic hydrolysates but not in the original extracts. The acid, while glutamine enters the cells readily. release of cystine was probably not attributable Radioactive areas corresponding to succinate, entirely to the breakdown of glutathione, since aspartate, and glutathione were also detected; only traces of the latter peptide were found in the this showed the rapid utilization of the carbon of unhydrolyzed filtrates. Increases in glutamic and the glutamine. In addition, pyrrolidone carboxylic aspartic acid contents also were noted on hy acid (tentatively identified) was observed as well drolysis. The contents of the other ninhydrin- as some as yet unidentified materials above glu reactive constituents did not appear to be changed tamine. In the fluid (Fig. 24) there appeared to be appreciably upon hydrolysis. more radioactivity in the glutamine than in glu Experiment on the influence of sarkomycin on tamic acid, while in the cell the reverse was found. gluiamine metabolism of Yoshida ascites tumor.â€” It is not certain whether glutamine was con An experiment was then performed in which the verted to glutamic acid in the fluid as well as in effect of treatment with sarkomycin in vivo was the cells or whether the radioactive glutamic acid tested on the metabolism of radioactive glutamine found in the fluid was solely the result of the action by the Yoshida tumor in a short in vitro incuba of the cells upon glutamine with a subsequent tion. A sample of the tumor was removed from a liberation of the glutamic acid into the fluid. Un Wistar rat on the 5th day after transplantation by published experiments in this laboratory have peritoneal puncture, and 0.5 ml. of the ascites was shown the presence of phosphate-activated glutam-

inase activity in both the cells and ascitic fluid. cells, and a marked decrease in the content of The results with the tumor after treatment with glutathione. Valine and the leucines increased in sarkomycin indicated that the cells still possessed amounts, and the new ninhydrin-reactive material the capacity to take up glutamine and to convert which was mentioned in the previous section ap it to glutamic acid (Figs. 25-28). Somewhat more peared to the left of glutamic acid on the chroma- radioactivity in the area of pyrrolidone carboxylic togram and persisted through the period of study. acid and the unknown substances above glutamine The only change from the control sample found in was noted in the autographs of both the cells and the fluid was the appearance of a trace of glu fluid of the treated tumor. The results are in keep tamine. ing with the interpretation that the effect of The Chromatographie results obtained at the sarkomycin resulting in the increased content of 10- and 20-minute intervals (Figs. 36, 37) were easily extractable glutamine in the tumor cells is very similar to those found at the 5-minute period not necessarily related to an altered ability of the with the exception that taurine appeared in the tumor cells to take up exogenous glutamine and to fluid in considerably larger amounts. However, convert it to glutamic acid. the cytological damage progressed rapidly. The Effect of sarkomycin on tiie Ehrlich osciles tumor. degree of cytoplasmic blebbing became remark â€”¿Theextensively studied Ehrlich ascites tumor able, and many cytoplasmic buds were formed on (8,10,19,20) has been found to be highly sensitive the cell surfaces. The number of cells with the to the action of sarkomycin (22), tumor growth chromonema-like structures attained a maximum being remarkably inhibited by administration of at 10 minutes after treatment (Fig. 35), and at 20 2.5â€”4.0mg. of the crude preparation per mouse minutes coalescence of these structures and de with little toxicity to the host. It was of interest generation were occurring in many of the ab to compare the time sequence of changes occurring normal nuclei. in the Ehrlich tumor with that observed in the The cells in the 40-minute sample (Fig. 38) Yoshida ascites tumor. showed extensive cytoplasmic damage as well as A relatively large amount (0.4 ml.) of tumor prominent karyorrhexis in both resting and divid ascites showing active multiplication was adminis ing states. Disintegration of nucleus and cyto tered intraperitoneally to 10 A/He mice averaging plasm appeared to be occurring simultaneously in 35 gm. in body weight. Smear preparations from many instances. The clear ascitic fluid showed peritoneal punctures showed a pronounced growth elevations in the levels of glutamine, aspartic acid, of the tumor within 48 hours after inoculation with taurine, and valine and decreases in alanine and numerous mitotic figures present, the appearance glycine contents by comparison with the control being similar in all of the mice. The Ehrlich cells sample (Fig. 40). At this time the glutamine, are larger than those of the Yoshida tumor and valine, leucine and isoleucine, tyrosine, and lysine possess highly chromatophiHc nuclei. At this time contents of the cells (Fig. 39) were markedly ele a pooled sample of tumor was obtained for analysis vated over the control levels, and the other from two of the mice (see Figs. 29, 30, and 31, re ninhydrin-reactive constituents were unchanged. spectively, for control smear and chromatograms Virtually identical cytological and Chromato of cells and fluid) and the remaining eight mice graphie results were obtained when the Ehrlich were given intraperitoneal injections of 3.5 mg. of tumor carried in C57BL mice was studied before sarkomycin. Pooled samples then were obtained and 30 minutes after injection of 3.5 mg. of from each of two mice at 5,10, 20, and 40 minutes, sarkomycin on the 3d day after transplantation of respectively, after the injection. the tumor. Alterations in the tumor cells were noted All the damaged cells had disappeared from the within 5 minutes after the administration of the ascitic fluid, and a relatively small number of sarkomycin (Fig. 32), a large number of cells normal-appearing tumor cells was found between showing small blisters on cytoplasmic surfaces. 60 and 120 minutes after the injection of the Some of the mitotic cells, especially those at sarkomycin. metaphase, showed marked nuclear abnormalities Chromatograms of acid hydrolysates of the ex characterized by clumped or sticky chromosomes. tracts of the cells and fluid showed the absence of Resting nuclei also appeared to be affected; they appreciable amounts of peptidic material both in showed chromonema-like structures resembling the control samples and at various times after in those found at prop hase. The chromatograms made jection of sarkomycin. Increases in content of of the tumor cells (Fig. 33) and ascitic fluid (Fig. ninhydrin-reactive constituents over the unhy- 34) at this time showed the appearance in cells of drolyzed samples in the cell extracts were largely glutamine, which was not found in the control attributable to hydrolysis of glutamine, ethanol-

amine phosphoric ester, glycerylphosphoryletha- sarcoma, exerting an antitumor effect comparable nolamine, and glutathione and to glutamine and to to the original nitrogen mustard but showing con glycerylphosphorylethanolamine in the extracts of siderably less toxicity (5, 25). the fluid. The large increases in cystine content All of the experiments to be described were after hydrolysis were comparable to those ob carried out with tumor-bearing rats of the WP served with the Yoshida sarcoma and could not be strain at 4 and 5 days after transplantation of the attributed solely to glutathione. The newly ob tumor. Preliminary experiments established that served ninhydrin-reactive material to the left of a dosage level of 30 mg/kg was suitable. Larger glutamic acid on the chromatograms (spot Xi) amounts produced a hemorrhagic condition which disappeared on acid hydrolysis. made subsequent biochemical and cytological The maintenance of high levels of easily ex- study impossible. In two experiments in which the tractable amino acids in the presence of such samples of tumor were obtained before adminis marked destruction of cellular architecture as tration of the nitromin and 4 hours after the injec observed above indicates that these amino acids tion, it was found that no significant changes in may not be in simple solution in the cytoplasm the free amino acid patterns of either the cells or and nucleoplasm, but rather that they may be fluid had occurred, with the exception of a slight bound by loose linkages to some cellular con increase in the content of glutamine in the cells. stituents or structures from which they can be A study was then made of the cytological liberated by the gentle procedure of extraction changes and the distribution of free amino acids which is employed. The rapid increase in gluta in cells and fluid in a rat at 2, 4, 6, and 12 hours mine content of the tumor cells after the injection after treatment and in another animal at 12, 17, of sarkomycin and the rather slow and relatively 24, and 36 hours after the administration of nitro small change in the content of this amino acid in min. The data in Table 1 show that by 4 hours the ascitic fluid suggest that the glutamine, which there was a sharp decrease in the number of was not observed in the control cells, may have mitotic cells observed, and at 12 hours no mitotic come from some intracellular source. figures could be found in the sample examined. Comparison of molecular models of sarkomycin During this period, although the number of and glutamine.â€”The chief antitumor constituent mitotic figures was decreasing, those figures which of sarkomycin, a weak antibiotic, is believed to were observed appeared to be normal. Con- be 2-methylene-3-oxocyclopentanecarboxylic acid comitantly, a number of the resting cells showed (4). The hydrogÃ©nation product, 2-methyl-3- abnormal-appearing nuclei, even at 4 hours. The oxocyclopentanecarboxylic acid, retains the anti- damaged cells showed pyknotic nuclear disinte tumor activity but loses the antibacterial effect. gration. At 12 hours considerable cellular debris Examination of the molecular models of glutamine was evident from the degenerating cells. The and the hydrogenated compound revealed a close leukocytic content of the ascites during this period resemblance between the rigid structure of did not appear to be abnormally high. The amino sarkomycin (Fig. 41) and one aspect of one of the acid patterns of the cells and fluid exhibited a re possible configurations of glutamine (Fig. 42) and markable constancy, showing little change from suggested the possibility that one action of sarko the control with the exception of a significant in mycin might be an interference with some phase crease in the free glutamine level. The relative in of glutamine metabolism by the tumor cells. The crease in the level of glutamine appeared to be opposite sides of the sarkomycin (Fig. 43) and the greater in the cells than in the fluid and was glutamine (Fig. 44) molecules differ, but substitu particularly evident in the 12-hour sample. tion of the methyl group on carbon-2 and the hy The results with the second rat studied showed drogen on carbon-1 with amino groups would give that the loss of mitotic figures persisted for 24 a compound, l,2-diamino-3-oxocyclopentanecar- hours, but that by 36 hours the number of boxylic acid, the structure of which (Fig. 45) bears mitoses approached the control percentage. How a remarkably close resemblance in all aspects to ever, the mitotic figures exhibited a variety of the glutamine structure examined. It would be of abnormalities which were characterized by the interest to study the biological properties of this breakdown of the chromonemata at prophase, compound, which to our knowledge has not yet disruption of the fragmented chromosomes, ir been synthesized. regular scattering of chromosomes, chromosome The effect of nitromin on the Yoshida tumor.â€” bridges and lagging at ana-telophases, multipolar Nitromin (methylbis(/3-chloroethyl)amine-N-ox- division, and irregular cytoplasmic division, etc. ide hydrochloride) has been found to be an effec (Figs. 46-48). After 24 hours there was an increase tive agent in the chemotherapy of the Yoshida in the number of leukocytes, and after 36 hours

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1956 American Association for Cancer Research. 976 Cancer Research the large number of leukocytes present precluded observations were made for the control sample and further study. At 17, 24, and 36 hours after the for those obtained at 15, 30, 45, and 60 minutes injection of nitromin the increase in glutamine after the injection. The chromatograms of both content in both the cells and fluid was greatly the extracts from the cells and the fluid were accentuated over that observed at the earlier time virtually identical at all the time intervals studied, intervals. The content of free lysine also seemed to the only change being the appearance of a small be somewhat increased. Otherwise, little change amount of glutamine in the 15-minute sample of was observed from the control cells with regard to the cell extract; it persisted for the remaining time the other constituents detected on the chromato- intervals without increase in amount. In the case grams. of the ascitic fluid, the 15-minute sample showed The effect of this chemical agent appeared to be the presence of taurine, glutamic acid, aspartic primarily upon the nucleus of the tumor cells and, acid, ethanolaminephosphoric ester, and glyceryl- in general, corresponded to that found by Yoshida phosphorylethanolamine in small but detectable (25). The effects observed on the free amino acid amounts, while these constituents were not noted patterns were largely restricted to the finding of on the chromatogram of the control sample of an increased content of free glutamine. fluid. There was a considerable increase over the The influence of crude podophyllin on the Yoshida control in the content of taurine at 60 minutes sarcoma.â€”Cytological effects of crude podophyllin and subsequent time periods. In addition, glycine and four of the constituents isolated therefrom (6) appeared to be increased at 60 minutes. The only have been made with the Yoshida sarcoma and sample of ascitic fluid which contained even a the MTK sarcoma (11,12, 21). The results showed trace of glutamine was that at 60 minutes. In that the inhibitory effect on tumor growth pro general, the changes in the patterns of amino duced by these substances was associated with acids in the cells were minimal. The changes ob chromosomal or nuclear damage as well as with served in the fluid suggested a slight leakage of cytoplasmic damage. some of the intracellular constituents. Experiments were performed with the Yoshida Three WP strain rats ranging in weight from sarcoma carried in one J strain and three WP 135-200 gm. were given injections of podophyllin strain rats. An aqueous solution was made in all on the 5th day after transplantation of the tumor, instances of the crude podophyllin (Resin Podo- two of the rats receiving 15 mg/kg and one rat 10 phyllum Merck) after quick immersion in absolute mg/kg. The tumor cells in the WP animals ap alcohol. The amounts of podophyllin used in each peared to be more sensitive to the action of instance were administered in 1 ml. of physio podophyllin than those grown in the J strain rat; logical saline. The doses employed ranged from cytoplasmic protrusions or blebbing were in evi 10 to 15 mg/kg. All of the injections produced dence even 15 minutes after the injection of the hemorrhage after a period, and the animals died drug. The cytoplasmic damage was progressive 7-24 hours after the administration of the drug. and appeared to be more marked at every stage Cytological and chemical observations were made than in the case of the J rat. There appeared to be on the tumor cells before and after treatment but a close similarity between the cytological effects before the hemorrhage ensued. of podophyllin and those produced by sarkomycin. In the J strain rat a control sample of tumor However, the type of damage was in striking con was removed on the 8th day after transplantation trast to that produced by nitromin, which was and a 15 mg/kg dose was injected intraperitoneal- largely characterized by nuclear damage. Podo ly. After the injection a number of tumor cells phyllin produced very little change in the amino showed metaphase arrest. The chromosomes were acid patterns of the cells and the fluid at the early scattered or irregularly condensed. The number of time intervals (15, 30, and 60 minutes) after the cells showing these findings increased progressively administration. The only difference appeared to with time, and the chromosomes appeared shorter be the slight increase in the glutamine content of and thickened. At 30-45 minutes many irregular the cells and fluid over that found in the controls. processes or atypical ameboid protrusions ap However, this increase was not progressive. In one peared in the cytoplasm in a number of the cells, of the rats at 3J hours there appeared to be an in while at 60 and 120 minutes all cells in metaphase crease in the quantity of glutamine and glutamic showed chromosome clumping or other degenera acid as well as of cystine and taurine in the ascitic tive changes such as the formation of deformed, fluid. The contents of valine, leucine, and iso- rounded, or bizarre bodies. Only a few normal leucine were also elevated. In another animal slight mitoses were noted at this time, and an increasing decreases in alanine and aspartic acid and in number of damaged cells was found. Chemical creases in glutamic acid contents were observed in

the cells at 90 minutes. The only change noted in metabolism of the tumor cell probably lies in a the content of the ninhydrin-reactive constituents careful evaluation of the rates of the various in the ascitic fluid was a small increase in gluta- processes by which carbon and nitrogen enter mine over that found in the controls. glutamine and by which they are used for various The results with podophyllin show that marked synthetic processes in tumors and normal cells. cytological damage can be produced in the tumor This would involve the study of both the easily cells with relatively little change occurring in the extractable and bound forms of intracellular and patterns of the easily extractable amino acids, and extracellular glutamine in living systems under suggest that the properties of these cells which are various conditions such as have been outlined in responsible for maintaining high concentrations of this communication. these constituents in relatively constant propor Increases in the concentrations of valine, leu- tions may be quite resistant to some cytotoxic cine, and isoleucine have been noted frequently in agents. affected tumor cells following the rise in glutamine GENERAL COMMENT level. Increases in these latter amino acids also are found in the early stages of the sterile autolysis Whether tumor growth is affected by host re of tissues.3 It is possible that transpeptidation re sistance, by chemicals having differing cytological actions involving glutamine or the activation of effects, such as sarkomycin or nitromin, or by physical means such as x-ray,2 the first change ob intracellular peptidases or proteinases may ac count for the increase in these amino acids. served in the pattern of easily extractable or free amino acids is an increase in the amount of free SUMMARY or easily extractable glutamine, and amino acid which is either undetectable or present in very 1. A description was given of the cytological small amounts in most transplantable tumors characteristics of Yoshida ascites tumor cells and studied. Glutamine plays important roles in the of the content of ninhydrin-reactive constituents de novo syntheses of nucleic acids (2, 3) and pro detected on two-dimensional paper chromato- teins (1), and it is possible that any one of a num grams prepared from extracts of the cells and ber of ways of inhibiting the production of nucleic ascitic fluid at various times after transplantation acids and proteins in cells may cause the accumu into a susceptible rat (J strain), in which the tumor lation of glutamine. grew progressively and killed the animal in 14 The experiments reported in the present com days, and into a resistant rat (Wistar), in which munication raise the question of the source of the the tumor grew initially but regressed com glutamine which accumulates under the above pletely within 8 days. At all times after trans conditions. The experiments with sarkomycin plantation the content of free or easily extractable would seem to suggest that the source of the glutamine was higher in the cells grown in the re glutamine is intracellular. Intracellular glutamine sistant rat. The chemical findings generally were could arise by the synthesis of glutamine, libera similar to those observed previously in compa tion from substances in which the glutamine is in rable experiments with mice bearing the C1498 peptide linkage through the o-amino or a-carboxyl leukemia. group, or removal from a site to which glutamine 2. Detailed results were given of the sequential is bound by adsorptive or ionic forces. At present changes in the cytological features of the cells and it seems unlikely that glutamine is being synthe of amino acid patterns of extracts of the cells and sized in the tumors at a rate which is sufficiently fluid of the Yoshida sarcoma in rats after the ad rapid to account for the appreciable accumulation ministration of sarkomycin, nitromin, or crude which occurs very shortly after treatment with podophyllin and of the Ehrlich ascites tumor in sarkomycin in both the Ehrlich and Yoshida mice after the injection of sarkomycin. Patterns of ascites tumors (9), although it is possible that the cytoplasmic and nuclear damage produced by agent may accelerate glutamine synthesis and sarkomycin and podophyllin were similar to each decrease utilization simultaneously. The intra other but differed from that observed with nitro cellular source of glutamine may be some bound min, an agent whose initial action is primarily form which either has not been detected in the upon the nucleus. All of the above substances pro extracts or which is not extracted by the methods duced increases in the content of free glutamine in employed in the present study. the tumor cells and ascitic fluid, with sarkomycin The resolution of the problems of the sources of showing the most marked increase most rapidly. the easily extractable glutamine and its roles in 8E. Roberts, R. T. Jordan, and D. G. Simonsen, unpub 1W. D. Kaplan and T. Tanaka, in preparation. lished.

Other changes in ninhydrin-reactive constituents mal and Neoplastic Tissues. J. Nat. Cancer Inst., 16: also were described. 347-52, 1954. 10. LOVE,R.; KOPROWSKI,H.; and Cox, H. R. Cytological 3. A consideration of both the cytological and Studies on the Ehrlich Ascites Tumor before and after chemical changes produced by sarkomycin sug Injection with Bunyamwera Virus. Cancer Research, 13: gested that the glutamine which appears rapidly 350-57, 1953. after treatment may be intracellular in origin. An 11. MAKING,S., and NAKANISHI,Y.The Cytological Effect of experiment with DL-glutamine-2-C14 showed that Chemicals on Ascites Sarcomas. IV. A Phase Microscopy Study of the Damage to the Tumor Cells by Podophyllin. treatment with sarkomycin in vivodid not destroy Cytologia, 20:89-95, 1955. the ability of Yoshida tumor cells to take up 12. MAKINO,S., and TANAKA,T. The Cytological Effect of labeled glutamine in vitro rapidly and to convert Chemicals on Ascites Sarcomas. I. Partial Damage in Tumor Cells by Podophyllin, Followed by Temporary Re it to glutamic acid. gression and Prolongation of Life of Tumor-Bearing Rats. 4. A similarity of certain aspects of the molec J. Nat. Cancer Inst., 13:1185-1200, 1953. ular models of sarkomycin and glutamine sug 13. ROBERTS,E., and BORGES,P. R. F. Patterns of Free gested the possibility that sarkomycin might be Amino Acids in Growing and Regressing Tumors. Cancer interfering with some phase of utilization of Research, 16:697-99, 1955. 14. ROBERTS,E., and FRANKEL,S. Free Amino Acids in Nor glutamine in the susceptible cells. mal and Neoplastic Tissues of Mice as Studied by Paper Chromatography. Cancer Research, 9:645-48, 1949. ACKNOWLEDGMENTS 15. â€¢¿â€”â€¢â€”â€¢â€”.Further Studies on Free Amino Acids in Normal We wish to express our gratitude to Drs. A. R. Menotti and and Neoplastic Tissues. Ibid., 10:237, 1950. 0. Chester Stock for generous samples of sarkomycin, to Dr. 16. ROBERTS,E., and TANAKA,T. Free Amino Acids of the Morton Rothstein for the synthesis of the DL-glutamine-2-C14, Yoshida Ascites Tumor. Cancer Research, 16:204-10, and to Mr- Richard Ray for all of the photographic work in this 1956. paper. 17. ROBERTS,E., and TISHKOFF,G. H. Distribution of Free REFERENCES Amino Acids in Mouse Epidermis in Various Phases of 1. BARRY,J. M. Use of Glutamine by the Mammary Gland Growth as Determined by Paper Partition Chromatog for the Synthesis of Casein. Nature, 174:815-16, 1954. raphy. Science, 109:14-16, 1949. 2. GOLDTHWAIT,D.A.; PEABODY,R. A.; and GREENBERG, 18. SASAKI,M.The Cytological Effect of Chemicals on Ascites G. R. Glycine Ribotide Intermediates in the de Nono Sarcomas. V. Effects of Cortisone and Sarkomycin on the Synthesis of Inosinic Acid. J. Am. Chem. Soc., 76:5258- MTK-sarcoma II. J. Fac. Sci. Hokkaido Univ. Ser. VI, 59, 1954. Zool., 12:433-41, 1956. 3. HAHTMAN,S.C.; LEVENBERG,B.;and BUCHANAN,J.M. 19. SEEGER, P. G. Untersuchungen am Tumoraszites der Involvement of ATP, 5-Phosphoribosylpyrophosphate and Maus. I. Mitteilung; Vitalfarbarkeit der Asziteszellen. L-Azaserine in the Enzymatic Formation of Glycinamide Arch. f. Exper. Zellforsch, 20:280-335, 1937. Ribotide Intermediates in Inosinic Acid Biosynthesis. J. 20. SUGIURA,K.Effect of Various Compounds on the Ehrlich Am. Chem. Soc., 77:501-3, 1955. Ascites Carcinoma. Cancer Research, 13:431-41, 1953. 4. HOOPEH,I. R.; Cheney, L. C.; CRON, M. J.; FAKDIG, il. TANAKA,T.; KAXO, K.; TONOMURA,A.; OKADA,T. A.: O. B.; JOHNSON,D.A.; JOHNSON,D.L.; PALERMITI,F.M.; and UMETANI,M. The Cytological Effect of Chemicals on SCBMITZ,H.;and WHEATLEY,W.B. Studies on Sarkomy Ascites Sarcomas. III. Damage Induced by Podophyllo- cin. Antibiotics & Chemother., 6:585-95, 1955. toxin, Alpha-peltatin, Beta-peltatin and Quercetin. Gann, 5. ISHIDATE,M.; KOBAYASHI,K.; BARERAI,Y.; SATO,H.; 46:15-26, 1955. and YOSHIDA,T. Experimental Studies on Chemotherapy 22. UMEZAWA,H.;YAMAMOTO,T.;TAKEUCHI,T.; OSATO,T.; of Malignant Growth Employing Yoshida Sarcoma ORAMI,Y.; YAMAOKA,S.;OKUDA,T.; NITTA, K.; YAGI- Animals. II. The Effect of N-Oxide Derivatives of Nitro SHITA,K.; UTAHAHA,R.; and UMEZAWA,S.Sarkomyein, gen Mustard. Proc. Jap. Acad., 27:493-500, 1951. an Anti-cancer Substance Produced by Streptomyces. 6. KELLY,M. G., and HARTWELL,J. L. The Biological Ef Antibotics & Chemother, 4:514-20, 1954. fects and the Chemical Composition of Podophyllin. A 23. YAMAMOTO,T., and KOMEUI, T. Some Considerations Review. J. Nat. Cancer Inst., 14:967-1010, 1954. upon the Mode of Action of Sarkomycin. Gann, 46:520-2-2, 7. KIT, S., and AWAPAHA,J.Free Amino Acid Content and 1955. Transaminase Activity of Lymphatic Tissues and Lym- 24. YAMAMOTO,T.;YAMAOKA,S.;YMEZAWA,H.;TAKEUCHI, phosarcomas. Cancer Research, 13:694-98, 1953. T.; and NITTA, K. Anti-tumour Effects of Sarkomycin. 8. KLEIN, G. The Use of the Ehrlich Ascites Tumor of Mice Gann, 46:503-5, 1954. for Quantitative Studies on the Growth and Biochemistry 25. YOSHIDA,T. Studies on an Ascites (Reticulo-endothelial of Neoplastic Cells. Cancer, 3:1052-61, 1950. cell?) Sarcoma of the Rat. J. Nat. Cancer Inst., 12:947-62, 9. LEVIXTOW,L. The Glutamyltransferase Activity of Nor 1952.

FIGS. 1-4.â€”Photomicrographs of smears of Yoshida sar coma cells at 4 and 7 days after transplantation in a WP rat (Figs. 1 and 2, respectively) and in a J strain rat (Figs. 3 and 4, respectively). Smear preparations stained with acetic dahlia and aceto-orcein X500.

f ? r Ã¯% â€¢¿a,

â€¢¿

f -

Â¿& ww

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1956 American Association for Cancer Research. FIGS. 5-12.â€”Chromatograins of extracts of cells and fluid at various times after transplantation of the Yoshida sarcoma into the Wistar (WP) rats. Figs. 5, 7, 9, and 11 are chromatograms of extracts of cells, and Figs. 6, 8, 10, and l'i are corre sponding chromatograms of extracts of ascites fluid obtained 4, 5, 6, and 7 days, respectively, after transplantation. Specific at tention is directed to glutamine, 16; glutamic acid, IS: glutathione, 24; valine, 4; the leucines, 3; taurine, 7; and threonine, 10, by arrows on the figures.

4* - â€ž¿

8 t

9 16 18, io â€¢¿ â€¢¿*

.11 12

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1956 American Association for Cancer Research. FIGS. 18-20.-â€”Chroniatograms of extracts of cells and fluid at various times after transplantation of the Yoshida sarcoma into 3 strain rats. Figs. 13,15,17, and 19 are chromatograms of extracts of cells and Figs. 14, 16, 18, and 20 are corresponding chromatograms of extracts of ascites fluid obtained at 4, 5, 6, and 7 days, respectively, after transplantation. Glutamine, 16; glutamic acid, 18; glutathione, 24; valine, 4; and the leucines, 8, are identified by the arrows.

I**-3 15 16 *~4" %

Â»Â«. ., 18

Â»

19 20

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1956 American Association for Cancer Research. FIGS. 21-28.â€”Chromatograins of extracts of Yoshiila sar coma cells (Fig. 21) and fluid (Fig. 22) and corresponding radioautographs (Figs. 28 and 24, respectively) after 10-niinute in cubation of control tumor with DL-glutaniine-2-C14as described in the text. C'hromatograms of extract of cells (Fig. 25) and fluid (Fig. 26) and corresponding radioautographs (Figs. 27 and 28, respectively) in a similar experiment with tumor from the same animal 55 minutes after the intra peritoneal injection 22:of sarkomycin. glutamine, Arrows 16; glutathione, point to glutamic 24; succinic acid, acid, 18; aspartic SI"(.'('; acid, pyrrolidone carboxylic acid, PC; possible condensation product of glutathione and sarkomycin, Xi; other unknowns, XÂ«andXÂ¡.

SUCC, /

16 23 24

Â«

25 26

27 28

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1956 American Association for Cancer Research. Fias. 29-40.â€”Photomicrographs of smears (X500) (Figs. 29, 32, 35, and 38) and of chromatograms of extracts of cells (Figs. 30, 33, 36, and 39) and fluid (Figs. 31, 34, 37, and 40) of the Ehrlich ascites tumor grown in A/He mice before and at 5, 10, and 40 minutes, respectively, after the intraperitoneal administration of sarkomycin.

I I <0

esi Â»lÃl co * â€¢¿r. t

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1956 American Association for Cancer Research. FIGS. 29-40.â€”Photomicrographs of smears (X500) (Figs. 29, 32, 35, and 38) and of chromatograms of extracts of cells (Figs. 30, 33, 36, and 39) and fluid (Figs. 31, 34, 37, and 40) of the Ehrlich ascites tumor grown in A/He mice before and at 5, 10, and 40 minutes, respectively, after the intraperitoneal administration of sarkomycin.

> \

co co

Â» * ^ co I Ã 4 â€¢¿:â€¢ 4 : -.- **< Ã‰l Â»

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1956 American Association for Cancer Research. FIGS.41-45.-â€”Molecularmodels of hydrogenated sarkomycin (a-methyl-S-oxocyclopentanecarboxylic acid) (Figs. 41 and 43), of glutamine (Figs. 42 and 44), and of l,2-diamino-3-oxocyclopentanecarboxylic acid (Fig. 45). FIGS. 46-48.â€”Photomicrographs (X2000) of Yoshida tu mor cells in a \VP rat taken at 36 hours after the injection of nitromin showing typical abnormalities in mitoses.

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1956 American Association for Cancer Research. Free Amino Acids in Growing and Regressing Ascites Cell Tumors: Host Resistance and Chemical Agents

Eugene Roberts, K. Kano Tanaka, T. Tanaka, et al.

Cancer Res 1956;16:970-978.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/16/10_Part_1/970

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/16/10_Part_1/970. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.