Response of Jensen Sarcoma Cell Cultures to Some Analogs, Homologs, and Peptides of Arginine, Ornithine, and Citrulline*

Home , Aspartic acid, Canaline, Glutamic acid

PATB. WHITE,CARROLLW.SMITH,ANDPAULF. KRUSE,JR.

(Biomedicai Division, The Samuel Roberti Noble Foundation, Inc., Ardmore, Oklahoma)

SUMMARY Previous studies with canavanine led to the present testing of the effects of canali ne and other compounds related to arginine, ornithine, and citrulline on Jensen sarcoma cell cultures. Canaline inhibited proliferation of the tumor cells in vitro by 50 per cent at 0.1 mM concentration. The inhibition was not competitive with ornithine, but it was prevented by pyridoxal phosphate and glutamic acid semialdehyde. The toxicity of canaline probably rested, therefore, in "carbonyl-trapping" properties of its substi tuted hydroxylamine structure. In related studies, the o-hydroxy and a-keto analogs of arginine could neither inhibit proliferation nor support it in the absence of arginine. Two lower and one higher (homoarginine) homologs of arginine were similarly ineffective, as were a num ber of other related compounds. Two lower homologs each of citrulline and ornithine and one higher (homocitrulline) of citrulline could not inhibit cellular proliferation appreciably. In contrast to the above results with arginine analogs and homologs, several arginine-containing peptides and protamines not only could supplant arginine in the culture medium but also markedly stimulated proliferation. L-Prolyl-L-phenylalanyl-L-arginine and two protamines, clupeine and salmine, most stimulated proliferation, producing ca. 50 per cent more new cells than in control culture with free arginine. L-Valyl-L- arginine and L-prolyl-L-arginine supported proliferation but did not stimulate it appre ciably. L-/3-Methylaspartyl-L-arginine supported proliferation but did not inhibit it. D-Alanyl-L-arginine could only partially spare the arginine requirement, while L-phenylalanyl-L-citrulline was incapable of replacing arginine.

The competitive inhibition of growth and incor- arginine, ornithine, and citrulline was tested. Also, poration into the protein of tumor cell cultures it was of interest to compare the growth-promot- were reported previously (20, 23) for the arginine ing properties of a number of arginine peptides analog canavanine. Since canavanine can also and protamines with that of arginine alone, yield the corresponding oxygen-containing analog of ornithine, canaline (18), one purpose of the MATERIALS AND METHODS present study was to test the effect of this com- Chemicals.â€”The following compounds were pound on similar cell culture systems; it proved to synthesized by the procedures referred to: i/-cana- be a potent growth inhibitor in vitro. line (14, 35) ; L-homoarginine (39) ; 2-keto-5-guani- In addition, the response to a variety of other dinovaleric acid (28) ; L-2-amino-4-ureidobutyric analogs, homologs, and "metabolic relatives" of acid (38); DL-glutamic acid semialdehyde (29, 44). * Portions of this study were presented at meetings of the A solution of the semialdehyde (in equilibrium SouthwestSection,Am.Assoc.for CancerResearch,Oklahoma With A'pyrrolme-o-carboxylic acid) was prepared City, November, 1961,and the Tissue Culture Assoc.,Wash- fresh each day of use. Physical constants of these ington, D.C., May, 1962. compounds agreed with literature values, and all Received for publication March 6,1963. analyzed correctly for C, H, and N (the semialde- 1051

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1963 American Association for Cancer Research. 1052 Cancer Research Vol. 23, August 1963 hyde as the 2, 4-dinitrophenylhydrazone hemi- laides, Parke, Davis and Co. The protecting sulfate).'DL- 2-Amino- 6-guanidinaminocaproic acid groups, carbobenzoxy and nitro, were removed was synthesized by condensation of 5-bromo-l- following an acidic hydrogÃ©nationprocedure de pentene with sodiodiethylacetamido malonate; rived from Berse, Piche, and Uchiyama (2); in the condensation product was hydrolyzed in most cases the peptide was isolated and recrys- NaOH solution, acidified, and the resultant tallized as the acetate. For subsequent analytical 2-acetamido-6-heptenoic acid was ozonized. The purposes the peptides were characterized by nin- ozonization reaction mixture was treated with an hydrin color yield (compared with leucine) (30), equivalent of aminoguanidine, hydrogenated at as well as by optical rotation and ionophoretic room temperature and pressure, with palladium mobilities (see below). All other compounds used in this study were TABLE1 obtained commercially; in some cases the degree INHIBITIONOF PROLIFERATIONOFJENSEN of purity was established by appropriate analyses. SARCOMACELLCULTURES Cell cultures.â€”Suspensions of freshly excised 7- BYL-CANALINE* to 8-day-old Jensen sarcoma cells were prepared and cultured in T-15 flasks according to procedures cent of pro outlined by McCoy and Neuman (27), with Me m\iL-Canaline:0.10.20.40.6-i.OL-Ornithine:00.10.10.10.1Pvridoxal00.20.2DL-GIutamicaldehyde:Ã®00.2.0.23.00.10.51.23.0phosphate:0.40.20.4acidConcentration liferation in con trol Medium7af5020609157565559707982864753dium 7a, which differs from Medium 5a (25) only in increased levels of the basic amino acids and decreased hydroxyproline content (21). Initial in oculum was ca. 200,000 cells/flask, and, prior to stoppering, each culture was gassed for 10 seconds with 5 per cent CC>2,75 per cent Nj, and 20 per cent Ãœ2.Thecultures were established by incuba tion at 37Â°for ca. 48 hours, and at this time the "cell take" was determined in representative cul tures, followed by replenishment of the spent media in the remaining cultures with fresh media containing the test compounds. In this way, the initiation and termination of the experiments were conducted in an exponential phase of the Jensen sarcoma proliferation cycle in vitro (22). From two setni-0.40.20.4Per to eight T-15 replicate cultures were used for each compound tested. Compounds tested as growth inhibitors were introduced at 0.4, 1.2, and 2.4 HIMconcentrations, unless otherwise stated, in replicate flasks followed "Test compounds introduced in log phase of proliferation; by 48 hours' incubation. Control cultures in com for culture conditions see text. t Calculated by dividing the number of new cells produced plete Medium 7a were present in each experiment. in test compound cultures by those produced in nonsupple- Compounds tested for their capacity to supplant mented Medium 7a control cultures (X100); from 1.6-2.4 arginine in the growth medium were introduced population doublings occurred in control cultures in 48 hours. Ã•Calculated on basis of L-isomer. at the time of medium change, usually in 0.4, 2.0, and 10.0 miviconcentrations in Medium 7a minus on charcoal used as catalyst, and the resultant arginine. Controls in these experiments included 2-acetamido-6-guanidinaminocaproic acid was cultures in complete Medium 7a as well as ones in isolated, purified, and hydrolyzed in HC1 to yield medium lacking arginine or (with some peptides) the desired product. The latter analyzed well for another dietary essential amino acid. The experi C, H, and N. A sample of L-octopine was the gift ments were conducted over 96 hours with an of Dr. M. Winitz, National Institutes of Health. intervening change of medium at 48 hours. Samples of N"-nitro-L-arginine and protected Analytical procedures.â€”Inall experiments the cultures were chilled at 4Â°C.to free the cells from arginine (and one citrulline) peptides were kindly supplied by Dr. J. II. Dice and Dr. E. D. Nico- the glass, and whole cell counts were made with 1Microanalyses were performed by Schwarzkopf Micro- a Coulter Electronic Counter; the cell count data analytical Laboratory, Woodside, N.Y.; Alfred Bernhardt were calculated as arithmetic means of from six Laboratory, MUlheitn (Ruhr), West Germany; and Galbraith to eight cell counts. The average deviation from Laboratories, Inc., Knoxville, Tenn. the mean was Â±5.4per cent. In some instances an

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1963 American Association for Cancer Research. WHITE et al.â€”Jensen Sarcoma Cell Cultures 1053 indication of the degree of population viability was Of the other substances tested (at 0.4, 1.2, and obtained with trypan blue staining (11). 2.4 niM cone., in replicate flasks), with the excep In studies with peptides and protamines, iono- tion of arcaine none proved to be effective growth- phoretic mobilities of the compounds and their inhibitors; these included L-arginine amide, L-argi- constituent amino acids were first measured at nine methyl ester-HC1, benzoyl-L-arginine, nitro- 5 pH's in a Kensington Scientific Corp. Model 50 L-arginine, agmatine, DL-2-amino-6-guanidinami- Electrophoresis Apparatus and calculated as ama nocaproic acid, and L-octopine. The degree of ranth units (47). Next, the peptides and polymers effectiveness of arcaine was related to the presence were dissolved in Earle's solution with and without of arginine in the medium; in complete Medium 5 per cent dialy/ed, pooled human serum and 7a, containing 0.4 HIMcone. L-arginine, the cells incubated 2.5 and 24 hours at 37Â°C.Aliquots were could achieve as much as 50 per cent of control ionophoresed, and the degree of hydrolysis, if any, growth in the presence of 1.2 HIMcone, of arcaine of the peptide or protamine was estimated by (1, 4-diguanidinobutane). If, however, cells in means of color reactions with ninhydrin (30) and Medium 7a minus arginine were treated with as isatin (37). little as 0.4 mM cone, of arcaine, a severe cyto- TABLE2 INCAPACITYOFSOMEANALOGSANDHOMOLOGSOFARGININETOINHIBITORSUPPORT PROLIFERATIONOFJENSENSARCOMACELLCULTURES*

PER CENT OF CONTROLPROLIFERATIONIN MEDIUM 7af

MEDIUM:AXALOO, L-arginine0.4227 minus

(mu):INCUBATIONHOMOLOGcose,

(DATS):Analog:L-2-Hydroxy-5-guanidinovalericTIME

acid acidHomolog:L-2-Amino-3-guanidinopropionic2-Keto-5-guanidinovaleric 105Ila104931.22112101108108912.421089610496967a361301848 95932.02313111011461157210.0248381364258013

acidL-2-Amino-4-guanidinobutyric acidL-Homoarginine7a0.42100

* See footnote, Table 1. t See footnote, Table 1; Medium 7a minus L-arginine cultures showed 15 and 7 per cent of 7a control culture proliferation in 2 and 4 days, respectively; from 3.0-4.4 population doublings occurred in control cultures in 96 hours.

RESULTS toxicity was elicited, and rapid cell disintegration Analogs, homologs.â€”Table 1 summarizes the ensued. Several of the above compoundsâ€”i.e., experiments with L-canaline. This substance benzoyl-L-arginine, nitro-L-arginine, agmatine, and proved to be more effective as a growth-inhibitor L-octopineâ€”were also tested for their capacity to than L-canavanine. In contrast to the competitive replace arginine in the culture medium; none of type relationship of canavanine and arginine (20), these were effective. In addition, combinations of the inhibition by canaline was not competitive ornithine plus uracil or the keto analog of arginine with ornithine. The inhibition was, however, pre could not spare the arginine requirement. vented by an equimolar quantity of pyridoxal Next, homologs of ornithine and citrulline were phosphate and partially so by similar additions tested as growth-inhibitors, with the results shown of glutamic acid semialdehyde. Both of these in Table 3. In comparison with an inhibitor such compounds were slightly toxic at 0.4 m.Mwhereas as canaline (Table 1), all five compounds were in ornithine was not appreciably toxic at 3.0 HIM. effective, although the higher concentrations of The results of testing two analogs and three 2, 3-diaminopropionic acid elicited some inhibitory homologs of arginine for their capacity to inhibit properties. or support proliferation are shown in Table 2. Peptides, protamines.â€”-Table4 gives characteri None of the five compounds could serve in either zation data for six peptides and two protamines, capacity. including estimates of their stability in cell-free,

TABLE 3 serum-containing Earle solution. All compounds INABILITYOF SOME HOMOLOGSOFORNITHINEAND analyzed well for C, H, and N. The ninhydrin ClTRULLINETOINHIBITPROLIFERATIONOFJENSEN color yield varied widely, with those peptides con SARCOMACELLCULTURESAPPRECIABLY* taining N-terminal proline having very low color (yellow) values, whereas the N-terminal D-alanyl PEB CENT OF CONTROL peptide gave 61 per cent more intense purple color PROLIFERATION IN than did leucine. These results are similar to MEDIUMTAt those reported for color yields of other N-terminal MEDIUM:HOMOLOG proline (19) and alanine (32) peptides. From the data in Table 4, the usefulness of electrophoresis (mil):INCUBATIONCONC. at 5 pH's to characterize peptides is emphasized. Whereas the mobilities of several peptides at (DATS):HomologsTIME several pH's may be experimentally indistinguish ofornithine:DL-2,8-Diaminopropionic acid}L-2,4-Diaminobutyric able, the 5-pH profile was distinctly different for acidHomologs each. The two protamines were, of course, ex ofcitrulline:L-2-Amino-S-ureidopropionic pected to show similar profiles of mobilities. Both acidL-2-Amino-4-ureidobutyric of these broke down somewhat upon incubation acidL-Homocitrulline7a0.429884827583l.i268878072812.423682796568with serum proteins, but there was no evidence of free arginine. The tripeptide, L-prolyl-L-phenyl- * See footnote, Table 1. alanyl-L-arginine, was the only peptide showing appreciable hydrolysis; the value of 30 per cent t See footnote, Table 1. after 24 hours' incubation was estimated from the ÃŽSeefootnote, Table 1. TABLE4 CHARACTERIZATIONOFSOMEPEPTIDESANDPROTAMINESANDTHEIRSTABILITYINCELLCULTUREMEDIUM*

tOpticalrotation TT%

inE.S.+hydrolysis MobilityifpH serum||Hours2.5tr00010Smallei24100trtr30â€¢pep PROTAMINEL-/3-Methylaspartyl-L-arginine-H2OD-Alanyl-L-argininePEPTIDE OR buffer3.333595465488076194.7146475142727605.9244454841697807.2-of ÃŽD+

0.1- 4454346377374- acetateL-Valyl-L-arginine 5.7+16.8-26.0**-15 acetate-HaOL-Prolyl-L-arginine acetateL-Prolyl-L-phenylalanyl-L-arginine ace tateClupeine 3-65.0-59 86463-41STABILI sulfateSalmine sulfateL-Phenylalanyl-L-citrulline- 5+27.4yieldsÂ§N0.561.611.120.170.081.24Electrophoretic tides nofreeargii0ine0

HjOCHARACTERIZATION 99.3-3511246-

* Earle solution containing 5 per cent dialyzed, pooled human serum and 190 jug/ml of penicillin and streptomycin but without free amino acids and vitamins. t See footnote 1 in text; all peptides analyzed well for C, H, and N. ÃŽMeasuredas the following per cent concentrations in water: L-^-methylaspartyl-L-arginine, 7.2; D-alanyl-L-arginine, 3.1; L-valyl-L-arginine, 3.1; L-prolyl-L-arginine, 2.6; L-prolyl-L-phenylalanyl-L-arginine, 2.8; clupeine and salmine, 1.2; L-phenylalanyl-L-citrulline, 3.4. Literature (13) value, L-prolyl-L-arginine, â€”28.6at 27Â°C.(2.6 per cent in water). Â§Ninhydrin (30) color yield compared with that of L-leucine = 1.00. # Determined in a Kensington Scientific Corp. Model 50 Electrophoresis Apparatus; for composition of buffers see (47); fig ures are amaranth units (47). From 20 to 60 ng. of peptide or protamine were spotted and data are arithmetic mean of four to ten determinations each with average deviation from the mean usually less than Â±2Am units. || See footnote*; 5.0 mg. of peptide or protamine was dissolved aseptically in 0.25 ml. of medium and incubated aj and 24 hours at 37Â°C.;in control tests no hydrolysis was found in the absence of serum; figures are estimations to the nearest 10 per cent, cal culated from size and color intensity of the arginine spot produced, if any; tr = trace. ** Temperature = 27Â°C.

arginine spot. Whether the resultant prolyl- two aldehydes, pyridoxal phosphate and glutamic phenylalanine was also split was not investigated. acid semialdehyde, were effective in preventing Since the peptides and protamines were rela the inhibition. A method for identification of tively stable under the above conditions, they carbonyl compounds of biological interest through were next tested for capacity to supplant arginine their reaction with canalino was reported by in the culture medium. The results are shown in Walker (46). Table 5, from which it is readily apparent not Since no other arginine analogs tested proved only that arginine-containing peptides and prota to be potent inhibitors of proliferation (in com mines could support proliferation, but also that plete culture medium), they will not be discussed some of them greatly stimulated it. The stimula- further here, except to note that, whereas agma- TABLE5 CAPACITYOFSOMEPEPTIDESANDPROTAMINESTOSUPPORTORSTIMULATE PROLIFERATIONOFJENSENSARCOMACELLCULTURES*

PEE CENT OF CONTROL PROLIFERATION IN MEDIUM ?Af

MEDIUM:PEPTIDE, L-arginine0.11130 minus

(mu):LNCUHATIOXFRÃ“TAMETECONC.

(DAYS):Peptide,TIME

protamine:L-/3-Methylaspartyl-L-arginine H2OD-Alanyl-L-arginine â€¢ acetateL-Valyl-L-arginine acetate -H2O L-Prolyl-L-arginineacetateL-Prolyl-L-phenylalanyl-L-arginine 10411715414641291091541431360.Â«ÃŒ945712595115146152214113401321251501521345O.s21239411314215841151181371471540.4t9182981111161561452541076811713814314014250.6288111105138138490128182142150 ace-tate{Clupeine

sulfateÂ§Salmine sulfateL-Phenylalanyl-L-citrulline â€¢H20#7a

* See footnote, Table 1. t See footnote, Tables 1 and 2; peptides were tested first at 0.2 and 0.4 mM; stimulatory peptides were tested further at 0.1 0.3, 0.6 mM. ÃŽThe dipeptide L-prolyl-L-phenylalanine did not stimulate proliferation in complete Medium 7a; the tripeptide supported but did not stimulate proliferation in 7a minus arginine and phenylalanine. Â§mM Cone, based on 7.1 per cent S and 70 per cent arginine; at a.O mM clupeine and salmine gave 76 and 51 per cent, re spectively, of control proliferation. # L-Phenylalanine-L-citrulline supported proliferation in Medium 7a minus phenylalanine. tion was greatest with the tripeptide or either of tine proved inhibitory to green monkey cells (43), the two protamines. The citrulline peptide could it did not affect the Jensen tumor cell proliferation not replace the nutritive requirement for arginine. appreciably. We have been unable to find any previously DISCUSSION reported biological study with the two lower Analogs, homologs.â€”Relatively low concentra homologs of arginine; in the present case neither tions, 0.2-0.4 mM, of canaline effectively stopped of these, nor homoarginiiie, exhibited any capacity growth of the sarcoma cells in vitro. Canaline has to impede or support proliferation. Also, it was of been reported to be an effective inhibitor of interest to note that the sarcoma cells did not growth of L. arabinosus (42) but not of L. fermenti possess the enzymatic machinery f necessary to (41, 45) or two strains of E. coli (45). In the present utilize either the hydroxy or keto analog of argi study the inhibition was not affected by added nine. This result was similar to that found by ornithine, and this indicated that the toxicity was Jacquez et.al. (15), in which they found a number possibly due to potent "carbonyl-trapping" prop of mouse and rat tumors unable to transaminate erties of the substituted hydroxylamine structure. phenylpyruvic acid. Eagle (5) also reported that Support for this conjecture was obtained when cell cultures derived from human and mouse

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1963 American Association for Cancer Research. 1056 Cancer Research Vol. 23, August 1963 tissues were unable to utilize the keto analog of In previous nutritional studies (26) it was arginine. found that citrulline could not spare the arginine The lower homolog of ornithine, 2, 4-diamino- requirement in Jensen sarcoma cells. Walker butyric acid, has recently received considerable carcinosarcoma 256 cells behaved similarly (20), attention since its discovery (34) as an agent although most cell strains and lines are able to responsible for lathyrism. However, it exhibited no utilize citrulline (5, 25, 31, 43). In the present appreciable physiological activity in the present study L-phenylalanyl-L-citrulline could not sup cell system, nor did the lower homolog, 2, 3-diami- port proliferation in the absence of arginine but nopropionic acid. Similar results were obtained could do so when only phenylalanine was absent. with three homologs of citrulline. Homocitrulline It is known that peptides and amino acids may be was recently (10) found present in urine of infants transported into a cell by different mechanisms and young children, but its physiological signifi (24), and it is of interest to note that citrulline is cance is as yet obscure. impermeable to some cell types (33). Thus, the Peptides, protamines.â€”It has previously been present results furnished circumstantial evidence reported by Eagle (6) that dipeptides containing that the lack of proliferation of Jensen cell cultures leucine, isoleucine, phenylalanine, or tyrosine sup in the presence of citrulline was probably based ported proliferation of mammalian cells in vitro upon their inability to convert it to arginine in the absence of the constituent amino acids. Similarly, in the present study all the arginine intracellularly. peptides, except D-alanyl-L-arginine, completely To us the most interesting result in these studies spared the requirement of arginine for prolifera was the pronounced stimulation of proliferation by tion. The two protamines could do likewise, and three of the peptides and two protamines. Similar the preliminary stability studies in Earle solution phenomena have been observed frequently (8) in containing serum indicated that these replacement bacteriological systems, but to our knowledge they capacities were primarily the net result of cellular have not been observed previously in tumor activity and probably not simple hydrolysis in systems. A number of proposals have been set fresh culture fluid per se. Whether the cleavage forth to explain the efficacy of peptides to support took place intracellularly or by peptidases ex bacterial proliferation better than their constit pressed into the surrounding medium by the cells uent-free amino acids. Included among these, for was not determined. Actually, both mechanisms example, are avoidance of antagonisms among are possible, since tumor cells are known to release free amino acids (17), bypassing extensive degra a considerable number of proteolytic enzymes (1) dation of a free amino acid (9, 40), different uptake and also to exhibit high peptidase activities intra mechanisms for peptides (17, 24), transpeptidation cellularly (12). In preliminary tests with DL-j8-methylaspartic reactions involving energy transfer from one pep tide for formation of another (7), and participation acid, no inhibition of proliferation was observed. intact in protein synthesis (7). The present studies Therefore, it was not unexpected that the peptide L-/3-methylaspartyl-L-arginine failed to suppress do not provide any substantial clue as to which of these, or others, was responsible for the observed proliferation of the tumor cells, although the stimulation of the tumor cell cultures. With analog has been reported (49) to be a potent in respect to the cancer problem, however, the pres hibitor of an E. coli mutant. It was of interest to find that o-alanyl-L-arginine was stable except in ent results do suggest several possibilities for further studies. Chief among these, perhaps, is the the presence of the tumor cells. This peptide did possibility that cancer cells may possess an en not support good proliferation, however; but hanced capacity to concentrate and utilize pep whether its extent of cleavage was so slight as to tides;2 in turn, this capacity might be employed furnish only an insufficient supply of arginine or for the transport and intracellular localization of whether the peptide itself was inhibitory could constituent metabolic anatagonists. not be determined readily by the response type experiments of this study. In any event, the ACKNOWLEDGMENTS physiological activity in tumor cells of peptides The authors wish to thank Miss Jorena Wicker for her containing amino acid antagonists or optical anti excellent technical assistance. podes is a relatively unexplored area, although 2Wiseman and Ghadially (48) have postulated that the such studies have been made in other systems metabolic superiority of tumor cells in vivo rests largely in (e.g., 3, 4, 16, 36). their enhanced amino acids concentrating power.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1963 American Association for Cancer Research. Response of Jensen Sarcoma Cell Cultures to Some Analogs, Homologs, and Peptides of Arginine, Ornithine, and Citrulline

Pat B. White, Carroll W. Smith and Paul F. Kruse, Jr.

Cancer Res 1963;23:1051-1058.

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