The Influence of Amino Acids and Antimetabolites on Glycine Retention by Ehrlich Ascites Carcinoma Cells

R. M. JOHNSTONE* AND P. G. SCHOLEFIELDt

(McGill-Montreal General Hospital Research Institute, 3619 University Street, Montreal, P.Q., Canada)

The transport of biologically important sub- Many workers have shown that compounds stances across cell membranes has been studied in such as dinitrophenol, which interfere with the a wide variety of systems. For example, transport availability of energy in the form of adenosine of ions into and out of cells has long formed an triphosphate (ATP), also interfere with the integral part of studies in physiology (8), and it capacity of Ehrlich ascites cells to take up or to has been known since 1913 from the studies of Van transport amino acids (2, 10). However, it was felt Slyke and Meyer (20) that amino acids given to an that the information obtained from this approach animal appear in the tissues at concentrations could well be supplemented by a study of the which are higher than those of the surrounding kinetics of interaction of glycine with other amino medium. The transport of sugars and amino acids acids and with amino acid analogs. Christensen from the mucosal to the serosal side of the small et al. (3) and Tenenhouse and Quastel (17) have intestine in in vitro preparations has also been well already shown that certain amino acids inhibit established (6, 7, 18). the transport of other amino acids into Ehrlich The demonstration of the capacity of isolated ascites cells. Cohen and Monod (4), in their tumor cells to transport actively such substances studies of the displacement of thiogalactosides as ions, sugars, and amino acids (3, 5, 12) has in E. coli, concluded that such displacement is provided the means of more precise studies of this competitive and Tenenhouse and Quastel (17) have general phenomenon. Thus, the results of Chris- reached a similar conclusion with reference to tensen, Riggs, Fischer, and Palatine (3) show that amino acid interactions during the process of glycine may be concentrated in vitro by Ehrlich transport into Ehrlich ascites cells. It has also ascites carcinoma cells to the extent of more than been shown by Riggs, Coyne, and Christensen (14) twelvefold. Later results, obtained by Heinz and that amino acids, previously concentrated, may Mariani (10) using radioactive glycine, showed be displaced from Ehrlich ascites cells on addition that there were possibly three separate reactions of other amino acids. In the studies made by Riggs involved in the establishment of a steady state et al. (14), the amino acids were added at concen- concentration of glycine within the ascites cells. trations of 30-60 mmolar, and the final analyses 1. A specific mechanism for the transport of were made 2 or 3 hours later. A more detailed in- glycine (or other amino acids) across the cell vestigation of this phenomenon was undertaken to membrane. obtain information concerning the factors con- 2. A nonspecific influx and effiux dependent trolling the extent of glycine retention under only on the difference between the concentration various experimental conditions. of amino acid in the medium and that in the cell. Studies by Wiseman and Ghadially (19), in 8. An exchange of glycine in the cell with that which RD3 carcinoma cells were used, and by of the medium without any net flow of glycine, Birt and Hird (1), in which carrot slices were the exchange reaction occurring, according to utilized, showed that methionine is not well con- Heinz and Mariani (10), at a rate which is 5-6 centrated by these preparations but that it is an times as great as that of the rate of actual trans- effective inhibitor of the concentration of other port. amino acids such as glycine. In the present work, * Fellow of the National Cancer Institute of Canada. therefore, methionine has been used extensively t Research Associate of the National Cancer Institute of in studies of glycine retention by Ehrlich ascites Canada. cells. Further, the effects of the antimetabolites Received for publication April 6, 1959. studied on the incorporation of glycine into the 1140

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1959 American Association for Cancer Research. JOHNSTONE AND SCHOLEFIELD--GlycineRetention by Ehrlich Ascites Cells 1141 alcohol-insoluble material (mainly into the pro- not more than 2 per cent of the remaining incuba- tein) has been determined in an attempt to esti- tion mixture. mate the specificity and nature of the effect of the Alcohol-soluble glycine.--The 2-ml. samples were inhibitors. In many cases the influence of added added directly to 3 ml. ice-cold calcium-free glucose was also investigated to assess the relative Krebs-Ringer solution, centrifuged, the super- effects of glycolysis and its consequences, such as natant removed, and the cells resuspended in a glycolytic ATP production, on the processes of further 5 ml. ice-cold calcium-free Krebs-Ring- uptake and incorporation of glycine. er solution. The suspension was again centrifuged, the supernatant removed, and the interior MATERIALS AND METHODS of the tubes dried with paper tissues. To the Ascites cells.--The Ehrlich aseites cells were washed cells was then added 2 ml. 95 per cent harvested from CF1 mice 6 or 7 days after intra- ethanol, and a minimum period of 30 minutes was peritoneal transplantation. After a twenty-fold allowed for completion of the extraction of the dilution with ice-cold isotonic saline, the suspen- alcohol-soluble materials. After centrifugation, an sion was centrifuged for 20 seconds at maximum aliquot (300 gl.) of the supernatant was plated on speed in the six-place head of the International aluminum discs and the radioactivity assayed. A Clinical Model centrifuge. The supernatant, con- Geiger-Mtiller thin end-window tube and Baird taining most of the blood elements, was decanted Atomic "Abacus" Scaler were employed. No cor- and the procedure repeated twice. The packed-cell rection for self-absorption was necessary. volume was then determined by centrifugation of "Alcohol-insoluble" glycine.--The precipitate the loosely packed cells for 2 minutes, and finally remaining after extraction with 95 per cent ethanol the cells were resuspended in sufficient calcium- was washed twice with 5-ml. aliquots of 95 per free Krebs-Ringer solution to yield a 1 : 12 dilution. cent ethanol and once with 5 ml. acetone. The Ten ml. of such a suspension was used per 30 ml. residue was suspended in approximately 0.2 ml. incubation medium. acetone and quantitatively transferred with wash- The S-37 mouse tumor was obtained originally ings to aluminum discs. The precipitate was caused from the Roscoe B. Jackson Memorial Labora- to adhere to the plates by roughening the surface tories and the Ehrlich ascites carcinoma from Dr. and by addition of 0.2 ml. 50:50 (v/v) acetone : J. S. Colter. Both were maintained in the solid water just before complete evaporation of the ace- form by weekly subcutaneous injections and in the tone. The plates were then dried by means of a liquid form by weekly intraperitoneal injections. heat lamp. The dry weight did not exceed 1.5 rag/ At first there was difficulty in growing the S-37 sq cm and was quite consistent among plates in tumor in an ascitic form in CF1 mice, but it took any given experiment. The radioactivity of the readily in DBA mice, and the ascitie tumor thus plates was therefore measured as such, and no at- obtained took readily in CF1 mice. In the experi- tempt was made to apply any correction for self- ments reported here the S-37 ascites were har- absorption. Control experiments indicated that at vested from DBA mice. least 85 per cent of the radioactivity was present Incubation technic.--The incubation medium in the protein and that the specific activity was a calcium-free Krebs-Ringer phosphate solu- (counts/min/mg protein) was not appreciably tion. The cells were allowed to incubate in the altered on isolation of the protein by standard medium for 7 min. at 37 ~ C., and then sufficient methods. 60 mn glycine-l-C ~4 added to yield a final concentration of 2 mM. Approximately 10 gc. was added RESULTS to each 30 ml. of incubation medium. The incuba- The effects of methionine, leucine, and valine.-- tions were performed in Erlenmeyer flasks shaken The results reported by Christensen et al. (3) show aerobically in a Research Specialties Company that glycine entry into Ehrlich ascites carcinoma shaker at 37 ~ C. The rate of shaking varied ac- cells may be decreased by addition to the medium cording to the size of the flasks employed. Thus, a of other amino acids. Further, the results of Riggs shaking rate of 100/rain was needed with 25-ml. et al. (14) concerning the interaction of glycine, capacity flasks, but, to avoid clumping of the cells, tryptophan, and diaminobutyric acid show that this speed was reduced to 60 per minute when previously concentrated amino acids may be dis- 200-ml. capacity flasks were employed. Samples placed by other acids. The present results show (2 ml.) were removed for analysis at intervals as that the addition of many other amino acids leads required. Inhibitors were usually added after 30 to an efitux of glycine from cells which are in min. of incubation in the solid form whenever pos- equilibrium with a glycine-containing medium. sible or as a neutral solution with a total volume of Thus, on addition of sufficient methionine to yield

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1959 American Association for Cancer Research. 114~ Cancer Research Vol. 19, December, 1959 a final concentration of 5 raM, the glycine content tain a measure of the glycine incorporation. It is of cells in equilibrium with a medium containing apparent from the results presented in Table 1 mM glycine fell by approximately 50 per cent as that, in the experiment reported in Chart 1, none shown in Chart 1. This figure also shows that the of the amino acids examined had any effect on addition of leucine and valine under similar ex- glycine incorporation into the alcohol-insoluble perimental conditions had little effect on the fraction. However, a significant stimulation was glycine content of the cells This result is in con- observed on addition of glucose to each of the trast to those of Christensen et al (3), who showed three experimental vessels. Such stimulations of that under similar experimental conditions valine glyeine incorporation frequently occurred, even in inhibited glycine uptake by 35 per cent and the absence of the second amino acid, but could leucine inhibited glycine uptake by 45 per cent. never be shown to be associated with the reversal The inhibitory effect of methionine might be the of an inhibitory effect on glycine incorporation result of a direct effect on the transport of glycine produced by an amino acid or an amino acid ana- or an indirect effect such as, for example, might log. The production of energy via respiration was possibly supplemented by production of ATP I0"0 ' A,B from glycolysis, and this resulted in an increased rate of incorporation of glycine. pmoles 91ycine/rnl. i At this point, it is of interest to note that the packed cells initial rate of glycine entry into the ascites cells in

TABLE 1 5.0 THE EFFECTS OF LEUCINE, VALINE, METHIONINE, AND GLUCOSE ON THE INCORPORATION OF GLYCINE-1-Ct4 c INTO THE ALCOHOL-INSOLUBLEFRACTION OF EHRL1CH ASCITES CELLS DURING INCUBATION AEROBICALLY AT 37 ~ C.

~MOLES GLYCINE INCORPO- i | p RATED//ML PACKED CELLS// 30.-MIN PERIOD 0 50 60 90 INTERVAL FURTHER ADDITIONS Time in mins. (M~.) Flask 3 CHART l.--The effects of leucine, valine, methionine, and I__I glucose on the uptake and retention of alcohol-soluble glycine- 0-30 Nil 0.176 0.190 0.154 1-C 14 by Ehrlich ascites cells during incubation under aerobic 30-60 5 mM L-leucine 0.185 0.164 conditi ons. 5 mM DL-valine A:5 mM L-leucine added at 30 rain. 5 mM DL-methionine 0.190 B:5 mM DL-valine added at 30 min. 60-90 10 mM Glucose 0. ~45 0. 239 0. 259 C: 5 mM DL-methionine added at 30 rain. Glucose was added to all flasks after 60 rain. incubation in The results quoted in this table were from the same experi- amounts sufficient to yield a final concentration of 10 EM. ment as that quoted in Chart 1. The amino acids were added after 30 min. and the glucose 30 min. later. be obtained through a disturbance of the supply of energy. In the experiments quoted in Chart 1 the experiment reported in Chart 1 was 43.6 glucose was therefore added 30 minutes after the gmoles/hour/ml packed cells when measured over addition of methionine, leucine, or valine to supply the first 7 minutes after the addition of glycine. glycolytic energy. The effects of these three amino The rate of glycine incorporation into the alcohol- acids on the glycine level were not influenced on insoluble fraction was constant over the first 60 addition of glucose, and in all subsequent experi- minutes and was approximately equal to 0.35 ments no reversal or potentiation of the effects of gmoles/hour/ml packed cells. The addition of added amino acids or their analogs was ever ob- 10 mM glucose increased the rate to approxi- served on addition of glucose. The inhibitory ef- mately 0.5 gmoles glycine incorporated/hour/ml fects of the amino acids such as methicnine were packed cells. Thus, the rate of glycine incorpora- therefore probably due to a direct effect of these tion is only about 1 per cent of the initial rate of amino acids on some phase of glycine transport. glycine uptake. After free glycine was extracted from the cells The effect of methionine analogs.--In view of the by 95 per cent ethanol, the insoluble material was marked effect of methionine on glycine retention washed, plated, and the radioactivity counted as and the report of Tenenhouse and Quastel (17) described under "Methods and Materials" to ob- that S-ethyl cysteine inhibits the concentration of

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1959 American Association for Cancer Research. JOHNSTONE AND SCHOLEFIELD--GlycineRetention by Ehrlich Ascites Cells 1143 glycine by ascites cells, it was decided to examine added to 9 ml.), methionine causes an effiux of the effect of several methionine analogs on glycine approximately ~5 per cent of the glycine original- retention by the cells. Compounds having the ly present in the cell. If there is competition be- general structure R. CH2. CH(NH2). COOH were tween glycine and methionine for a limited num- examined, where R was CH3. S. CH2-(methionine), ber of sites involved in the transport of methionine, CH3. CH2.S. CH2-(ethionine),CHa. CH2. S- (S-ethyl then the reciprocal of the new equilibrium level eysteine), CHa. CH2. O. CH2-(O-ethyl homoserine), inside the cell should bear a linear relationship to CH3. O. CH2-(O-methyl homoserine), and HOCH~- the concentration of the methionine (compare the (homoserine). When added in amounts sufficient fact that the reciprocal of the velocity of an to yield a final concentration of 5 raM, these com- enzyme-catalyzed reaction obeys a similar law pounds decreased the concentration of glycine in with reference to the concentration of a competi- the aseites cells by more than 50 per cent. Typical tive inhibitor). The results shown in Chart ~ are results obtained in one experiment with ethionine, also plotted by this method, and it is apparent O-ethyl homoserine (the oxygen analog of ethio- that at the lower concentrations such a relation- nine), and S-ethyl cysteine are shown in Table ~. It should be noted that in no case was any sig- 1'25 nificant inhibitory effect on the incorporation of ...... ,..-I B I'0 glycine into the insoluble fraction observed. The 2000 ~X5 results obtained with the other amino acids (meth- C.P.M] 103 semple C.P,M. TABLE

THE EFFECTS OF ETIIIONINE, S-ETHYL CYSTEINE, O- ,ooo / 9 A 0-5 ETnYL HOMOSERINE, AND GLUCOSE ON THE UPTAKE OF GLYCINE-1-C 14 BY EHRLtCH ASCITES CELLS DURING 0-25 INCUBATION AEROBICALLY AT 37 ~ C.

| I |

~MOLES GLYCINE/ML 20 40 PACKED CELLS p moles DL-Methionine added TIM~ (ms.) FURTHER ADDITIONS CHART ~.--The effect of added methionine on the retention Flask l Flask ~ I Flask 3 of glycine by Ehrlieh ascites cells. A: Final level of radioactivity in a $00-/A. aliquot from a 15 4.9 5.3 4.5 total of ~ ml. 95 per cent ethanol used for extraction. 30 6.6 6.2 7.1 B:Reciprocal of the final level of radioactivity plotted 45 5 mM DL-ethionine* 3.7 against the amount of methionine added to 9 ml. incubation 60 3.1 medium. 45 5 mM DL-S-ethyl* cysteine ship occurs. At the highest concentration of 60 2.9 raM) 45 5 mM DL-O-ethyl* 3.6 methionine employed (3.67 the expected homoserine concentration factor (gmoles glycine/ml packed 60 3.6 cells divided by t~moles glycine/ml medium) was 75 10 mM Glucose~ 2.7 2.6 3.5 1.4~ and the observed value was o~.16 compared 90 2.3 i~.l 3.6 with a concentration factor of 4.5 in the absence of methionine. Several attempts were made to * Amino acids added after 30 min. reduce the concentration factor to unity by addi- t Glucose added after 60 rain. tion of excess methionine, but in no case could it be reduced to very much below a value of two. ionine, homoserine, and its O-methyl derivative) The present results, however, do indicate that at were similar to those described above. the lower concentrations of methionine it seems The effect qf methionine at various concentrations. to compete in some way with glycine for the --In all the foregoing experiments the glycine transporting system and that it has a high affinity concentration was ~t mM and that of all the other for this system. amino acids was 5 raM. The effects of several con- At no concentration did methionine seem to centrations of methionine on the extent of glycine have any effect on the rate of glycine incorpora- retention by the ascites cells was also investigated. tion into the alcohol-insoluble fraction. The results obtained in such an experiment are The time oJ addition of methionine.--The present shown in Chart ~. It is apparent that, at a concen- investigations have been directed toward a study tration in the medium of 0.55 mM (4.95 umoles of the attainment of a new lower steady-state level

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1959 American Association for Cancer Research. 1144 Cancer Research Vol. 19, December, 1959 of glycine by ascites cells which had previously Let a = total counts/min added/ml medium; been caused to accumulate glycine. It was of im- Do = gmoles glycine initially present/ml portance to determine whether this level was the medium; same as that reached when the glycine and ~moles nonradioactive glycine added/ methionine were added simultaneously. The re- ml medium; suits shown in Chart 3 indicate that the position M= maximum concentration of glycine of the equilibrium attained is the same whether it within the cells; is approached by an influx of glycine (through m0 ~- initial concentration of glycine within simultaneous addition of glycine and methionine) the cells; or by an efflux of glycine (through pre-packing m --- concentration of glycine inside the with glycine followed by addition of methionine). cells after addition of cold glycine to It would appear, therefore, that, once it has the medium; entered the cells, the glycine is maintained in a K = characteristic constant. state of dynamic equilibrium with that remaining Initial concentration of glycine in the medium in the external medium. ~o M = go umoles/ml = 1--000 I0.0 9 . = A .

moles '~/~,._ "-- " Final concentration - ~0 + ~ M. glycine/ml. / ~.-.-...~ 1000

pocked cells /: ,._..,-- . " 5"0 / .~. "~ " C It has been shown by Cohen and Monod (4) / that, at equilibrium, the concentration of thio-

a b 2000 . A 15 [

i ! | 30 60 90 Time in minutes

CHART &--The retention of alcohol-soluble glycine by C.PM.I Ehrlich ascites cells on addition of methionine at various times. sample [/ A: No methionine added. 5 B: 5 mM DL-methionine added after 30 rain. incubation. 500 ( C: 5 mM DI,-methionine added at zero time.

! ,| | t The effect of glycine.--The studies of Heinz (10, 0 40 80 0 5 I0 11) have shown conclusively that radioactive Tqme in mins. gmolesglycine added glycine may leave ascites cells very rapidly as the CHART 4.--The effect of added nonradioactive glyeine on result of an exchange reaction with nonradio- the level of radioactivity present in Ehrlich ascites cells pre- active glycine in the medium. On addition of extra viously incubated with glycine-l-CTM. a) A:No extra glycine added. nonradioactive glycine to the medium, there must B:~.5 #moles glycine added/ml medium. be a net passage of glycine into the cells, since in- C: 5.0 #moles glycine added/ml medium. crease in the external glycine concentration leads D: 10.0 #moles glycine added/ml medium. to an increase in the internal glycine concentration. b) The reciprocal of the radioactivity present in the cell On the other hand, if there occurs an exchange re- after 70 min. plotted against the amount of nonradioactive glycine added. action or a continued leakage of previously concentrated glycine, then the radioactivity, as galactosides inside Escherichia coli is related to the glycine, in the cells must decrease despite the net concentration in the medium by a mathematical passage of glycine in the opposite direction. The function which is of the same form as the adsorp- results shown in Chart 4a indicate that an effiux tion isotherm. If the same relationship obtains of radioactive glycine does occur and that, in for the equilibrium concentration of amino acids agreement with the results of Heinz and Mariani inside Ehrlich ascites cells, then (10), the rate is much greater than the rate of M 1000K entry of glycine into the cells and is of the same - l+----- order as the rate of exchange diffusion (11). m0 #0 The extent of the decrease in radioactivity of and the glycine within the cells may be calculated as M IO00K - l+------follows: m ~-I-- ~o

On division, these amino acids have been studied by Christen- 1000K sen et al. (3) and by Riggs et al. (15), but it should lq u+u0 be noted that in the present experiments the m IO00K" observation by the former workers that the pres- lq ence of the basic amino acids causes an increase #o in glycine uptake could not be confirmed. In con- Initial specific activity firmation of the results of Rabinovitz et al. (18), a a stimulation of glycine incorporation was always - -- counts/min / umole ; observed on addition of glutamine. In some in- go stances the presence of glutamate led to a similar Final specific activity though much lower stimulation. A further increase in the rate of glycine incorporation was always ob- a counts/min / umole. served when glutamate or glutamine were added g+go in the presence of glucose. Thus The effects of metabolic inhibitors on glycine re- moa Initial radioactivity in cell go mo (u + go) Final radioactivity in cell ma mgo ~+uo q IO00K/ 1000K) _#+gO{l l+----- go k, u + go / Initial radioactivity _ tt + #o + 1000K Final radioactivity go + 100 OK

=1-~ g #0+ 1000K"

Since the initial radioactivity, g0, and K are con- tention and glycine incorporation.--It is now well stant, it follows that the reciprocal of the final established (2, 10, 14) that, in the presence of sub- amount of radioactivity in the cell is proportional stances which bring about a depletion of the cellu- to the number of gmoles extra glycine added, and, lar reserve of ATP, there is a failure of Ehrlich in accordance with this prediction, when the re- ascites cells to concentrate glycine and to incorpo- sults were plotted by this method, the straight- rate it into proteins. The inhibitory effects of line relationship shown in Chart 4 was obtained. The present results emphasize again that the rates TABLE $ of influx and efflux of glycine are in agreement THE EFFECTS OF AMINO ACIDS (5 mM) ON with the concept of a true dynamic equilibrium THE RETENTION OF RADIOACTIVE GLY- between the glycine of the medium and that of the CINE (2 mM) AND ITS INCORPORATION cell. INTO EHRLICH ASCITES CARCINOMA CELLS The effects oJ other amino acids.--The effects of several other amino acids on the retention of Percentage Percentage inhibition Amino acids glycine were also investigated, and the results ob- inhibition of glycine (5 raM) tained are presented in Table 8. The presence of of glycine incorpora- threonine or alanine, like that of methionine, leads retention t ion to a decrease in the amount of glycine within the Threonine 25 0 ascites cells, but these amino acids have no effect Serine 50 40 ~-Phenyl serine 0 25 on the rate of glycine incorporation. The inhibi- O-Ethyl homoserine 50 15 tions of glycine uptake and incorporation observed Alanine 50 0 on addition of serine are probably due, in some /~-Phenyl alanine 0 25 Sodium glutamate 10 0 part, to glycine-serine interconversion. It is, how- Sodium a-methyl ever, of interest to note that the introduction of a glutamate 0 0 j3-phenyl group into the alanine or serine mole- Glutamine 50 --70 Lysine 0 0 cules removes completely the inhibitory effects of Arginine 0 0 these amino acids on glycine retention. Most of

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1959 American Association for Cancer Research. 1146 Cancer Research Vol. 19, December, 1959 dinitrophenol (DNP) and of cyanide, as well as in the cells. Simultaneously there was an immedi- the reversal of these effects on addition of glucose, ate restoration of the incorporation of glycine into are all explicable in terms of ATP production. In the alcohol-insoluble fraction. The rate of incor- view of the great difference in the relative rates poration of glycine into the insoluble fraction after of uptake of glycine and its incorporation into addition of glucose in presence of DNP was con- protein, described above, it was of interest to de- siderably less than the rate of incorporation prior termine the effect of these metabolic inhibitors on to the addition of DNP and less than the rate of the retention of glycine by ascites cells and its in- incorporation in the control (Chart 5b). This effect corporation into the alcohol-insoluble fraction. may be the result of the rapid drop in ptI in The results presented in Chart 5b show clearly that presence of glucose and DNP or to an irreversible on addition of 0.1 mM DNP there was an immedi- effect of DNP on the incorporating system. The ate and complete inhibition of glycine incorpora- results obtained when cyanide was employed were tion into the alcohol-insoluble fraction of the similar to those described above which were ob- ascites cells. The effect of 0.1 mM DNP on the up- tained on addition of DNP. take of glycine was not immediately apparent, Glycine retention by S-37 ascites cells.--The results obtained with the Ehrlich ascites cells have 15,0 0,75 shown wide differences in the capacities of various amino acids to influence the retention of glycine by these cells. It was of interest to determine whether similar relationships would hold when other 10 "0 i 0.50 tumor cells were employed in this form. An ascitic p mole= / gtycine/ml. " \ //'C ~,glyeine~o,es / pocked / TABLE 4 cells / 5-0 THE EFFECTS OF GLUTAMATE, METHIONINE, NONRADIO- ACTIVE GLYCINE, AND GLUCOSE ON THE UPTAKE OF :/ GLYCINE-]-C 14 BY S-37 ASCITES CELLS DURING INCUBA- TION AT 37~ C.

0 60 120 0 60 120 ~tMOLESGLYCINE/ML Time in minutes TIME PACKED CELI~ CHART &--The effect of addition of 0.1 mM DNP at various FURTHERADDITIONS (Mn~.) I times and subsequently of glucose, on (a) the uptake and re- Flask 1 [ Flask Flask 8 tention of glycine-l-C 14 and (b) the incorporation of glycine- 1-C 14 by Erhlich ascites cells. 15 5.5 5.0 5.3 A: No DNP added. 80 6.6 6.9 B:0.1 mM DNP added after 30 min. incubation. C: 0.1 mM DNP added after 6 min. incubation. 45 5 mM glycine* 6.2 In all cases sufficient glucose was added after 80 min. in- 60 6.1 cubation to yield a final concentration in the medium of 10 mM. 45 5 mM DL-methionine* 5.3 60 4.1 45 5 mM L-glutamate* 7.6 but within the next 10 rain. the onset of an exten- 60 7.5 sive efflux of glycine from the cells was observed 75 10 mM glucose~ 5.8 3.6 7.8 (Chart 5a). If the DNP was added at a time when 90 6.0 3.3 8.0 the glyine concentration inside the cells was still increasing at a rapid rate. it had no apparent ef- * Amino acids added after 30 min. fect on this influx for a period of up to 10 rain. (cf. Glucose added after 60 min. the addition of DNP after 7 rain. in Chart 5a). On addition of the DNP at a time when the cells and form of Sarcoma 37 was developed in DBA mice as the glycine-containing medium had come into described under' 'Materials and Methods," and the equilibrium with each other, there was still a lag resultant cell preparation was employed for com- period of 5-10 rain., but after this period there was parative studies. In a typical experiment the ef- again a rapid loss of glycine (cf. the addition of fects of 5 ma~ glutamate, glycine, or methionine DNP after 80 rain. in Chart 5a). Subsequent addi- were investigated and, subsequently, the effects tion of glucose, which yields ATP by glyeolysis, a of 10 mM glucose. It is apparent from the results process which is insensitive to the presence of shown in Table 4 that the effects observed were either DNP or cyanide, led to a reversal of the exactly analogous to those observed with Ehrlich effiux, and glycine once more began to accumulate ascites cells. Thus, addition of glycine led to a de-

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1959 American Association for Cancer Research. JOHNSTONE AND SCHOLEFmLD---Glycine Retention by Ehrlich Ascitss Cells 1147 crease in the amount of radioactivity inside the DISCUSSION cell, methionine caused a rapid and extensive effiux In the present work an attempt has been made of glycine but did not influence glycine incorpora- to determine some of the conditions controlling tion, and glutamate had no effect on the retention the rate of efllux of glycine from Ehrlich ascites of glycine but, in the presence of glucose, stimu- carcinoma cells and to investigate the mechanisms lated glycine incorporation into the alcohol- involved in this effiux. The three factors, suggested insoluble fraction. by Heinz and Mariani (10), to participate in the The site of action of the inhibitory amino acids.-- establishment of an elevated steady-state concen- In the present experiments the passage of radio- tration of glycine within ascites cells should be activity out of ascites cells has been interpreted in considered in this connection. There can be no terms of effiux of glycine (except in the experi- doubt of the existence of an effiux or leakage of ments in which nonradioactive glycine was added). glycine from the cells which is independent of the The efllux resulting from the addition of the amino availability of energy. The present results (Chart acids or metabolic inhibitors may result from an increased rate of effiux due to leakage or to various types of exchange reactions and may also be the o . result of an inhibition of the influx of the radioactive glycine. Since the inhibitory effect of 8 8 methionine was immediately apparent when it p moles was added at the same time as the radioactive glycine/ml. glycine, i.e., when the only possible direction of pocked cells flow of the radioactive glycine could be into the 4 cells, it seemed most likely that methionine had an / inhibitory effect on the uptake of glycine. To determine whether or not methionine also had an 0 40 60 effect on the process of efllux of glycine, cells were Time in rains. allowed to come into equilibrium with a medium containing 3.~ mM glycine-l-C 14 by incubation for CHART 6.--The efflux of glycine from Ehrlich ascites cells. During the first 30 minutes of incubation the cells were allowed 30 min. This higher concentration of glycine was to reach a steady state with respect to their content of glycine- employed to increase the amount of radioactivity 1-C 14. Subsequently they were washed and resuspended in retained by the cells. They were then separated fresh glycine-free media as described in the text. from the medium by centrifugation, washed once O Resuspension made in fresh medium. 9 Resuspension made in fresh medium containing 5 m~s by resuspension in 5 ml. ice-cold calcium-free D~methionine. Ringer solution, and finally made up to a volume /~ Resuspension made in fresh medium containing 0.1 of ~ ml. in the same solution. This ice-cold suspen- mM DNP. sion of cells was then added to 10 ml. fresh incuba- The points near the dotted line represent the total recovery tion medium at 87 ~ C. containing no glycine but of radioactivity from the cells plus supernatant after washing and resuspension. with the additions noted in Chart 6. Samples were taken at intervals, and the radioactive glycine 6) would indicate that this process is potentially present in the cells and in the medium was esti- an extremely rapid one. In agreement with this mated as before. The results obtained (Chart 6) conclusion is the observation (Chart 4) that a new indicate that there was an extensive loss of glycine steady state was rapidly attained when nonradio- from the cells within 1 minute and a further loss active glycine was added. This rapid reaction, which extended over a period of approximately 30 which is of the same order as the exchange reac- min. The presence of 5 mM methionine or of 0.1 mM tions discussed by Heinz (10, 11), coupled with DNP seemed to have little effect on the rate or the process of active transport, would therefore extent of either of these two losses of glycine. The constitute a system capable of maintaining glycine initial rapid loss was not due to leakage of the in the medium and in the cell in a dynamic state glycine during washing, since the total recovery of of equilibrium. The question of the contribution of glycine from the cells and the medium closely ap- exchange reactions, including exchange-diffusion proximated that present in the cells prior to (11), naturally arises. It has been shown conclu- washing. The over-all efftux of glycine was ap- sively by Heinz and Walsh (ll) that the presence parently uninfluenced by the energy level of the of "preloaded" amino acids may greatly stimulate cell, since DNP was without effect on it, as previ- the rate of entry of radioactive glycine, sarcosine, ously indicated by Heinz (9). and alanine. From the definition of "exchange dif-

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1959 American Association for Cancer Research. 1148 Cancer Research Vol. 19, December, 1959 fusion" (11) it would be expected that addition of or to preserve the ascites cells in a condition con- an exchangeable amino acid to cells "preloaded" ducive to amino acid transport. Such a conclusion with another amino acid should also facilitate the would be supported by the fact (16) that 0.1 mM exit of the "preloaded" amino acid. This possibil- DNP causes practically complete Ioss of ATP ity was explored, but the results presented in from ascites cells within 5 minutes. It may be sup- Chart 6 indicate that the presence of methionine posed that the primary function of ATP in this has little or no effect on the rate of exit of "pre- system is to maintain the carrier agent in an effec- loaded" radioactive glycine from the ascites cells. tive form. On the disappearance of ATP from the It can only be concluded that under the present system the carrier agent may be converted to an experimental conditions the process of "exchange inactive form, and the completion of this process diffusion" does not contribute to the efllux of might well take several minutes. During this glycine on addition of methionine. period the agent could continue to cause glycine It is of interest to note that, of the amino acids accumulation, but eventually, when all the agent examined, those which carry a complete net posi- had been converted to an inactive form, active tive or negative charge at neutral pH were without transport would cease, glycine would continue to effect on glycine retention. Thus, both glutamic leak out, and ultimately a new lower level of acid and the basic amino acids did not influence equilibrium would be attained. In this connection, the glycine concentration. Of the neutral amino it is of interest to note that Riggs, Walker, and acids, those which are structurally related to Christensen (15) have shown that potassium ef- methionine, such as the corresponding oxygen flux accompanies glycine influx. If a concentration analog or the corresponding ethyl compounds, of potassium ions is the reaction which directly re- were found to be the most effective inhibitors. On quires ATP, then the uptake of glycine could the other hand, substitution of the carbon chains actually be an exchange with potassium, and the of serine or alanine with a phenyl group complete- lag, observed on addition of DNP, the time re- ly removed the inhibitory effects observed with quired for depletion of potassium ions. these two amino acids. These results point out that specific sites must be involved in the over-all SUMMARY process of glycine transport. The demonstration 1. When Ehrlich ascites carcinoma cells were by Tenenhouse and Quastel (17) that the inhibi- incubated with glycine-l-C TM, the amino acid was tion of glycine accumulation by S-ethyl cysteine concentrated within the cells and an elevated obeys the same law as that obeyed in classical steady-state level was approached in approxi- competitive inhibition points to the same conclu- mately 30 minutes. Addition of alanine, serine, or stion. In the present work, addition of methionine amino acids structurally related to methionine at low concentrations has led to results which are led to a rapid and extensive loss of the previously of the same mathematical form. However, it concentrated glycine from the cells. should be pointed out that such results would also ~. The final equilibrium level of glycine within obtain if specificity were involved only in a process the cell in the presence of methionine was the same of selective adsorption preceding active transport whether it was approached via an influx of glycine of glycine or other amino acids into the cell. (inhibitors added initially) or via an effiux of The question of the nature of this process of glycine (inhibitors added when a steady state had active transport then arises. Of this process little been established in their absence). is known except that it eventually requires ATP 3. The initial rate of glycine-l-C TM uptake into and is markedly decreased when the supply of the ceils was approximately 100 times as great as ATP is reduced. In this connection, it is of interest the rate of glycine incorporation into the alcohol- to note (Chart 5) that, on addition of 0.1 mM insoluble fraction of the cells. DNP, glycine incorporation into the alcohol-in- 4. The decrease in radioactivity of the alcohol- soluble fraction ceased within 1-e minutes but soluble fraction of the ascites cells on addition to glycine continued to be concentrated by the cell the medium of nonradioactive glycine or of for as long as 10 rginutes. The incorporation of methionine may be correlated with the amount of glycine, in which ATP is known to participate di- amino acid added. Methionine appeared to com- rectly, is therefore much more sensitive to change pete with glycine for entry into the cell. in the ATP concentration than is the transport of 5. Studies of the rate of effiux of glycine-l-C 14 glycine. Such a situation would result if ATP did from aseites cells led to the conclusion that, under not participate directly in transport of the amino the present experimental conditions, the only acid but rather served to maintain a carrier system mechanism involved in the net effiuxof glycine

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1959 American Association for Cancer Research. JOHNSTONE AND SCHOLEFIELD--GlycineRetention by Ehrlich Ascites Cells 1149 was passive diffusion. The inhibitory effects ob- Systems. London: Butterworths Scientific Publications, served were due to primary effects on the process 1956. 9. HEINZ,E. The Exchangeability of Glycine Accumulated by of uptake. Carcinoma Cells. J. Biol. Chem., 225:305-15, 1957. 10. HEINZ, E., and MARIANI, H. A. Concentrative Work and ACKNOWLEDGMENTS Energy Dissipation in Active Transport of Glycine into It is a pleasure to thank Prof. J. H. Quastel, F.R.S., for his Carcinoma Cells. J. Biol. Chem., 228: 97-111, 1957. continued interest in this work. It is also a pleasure to thank 11. HEINZ, E., and WALSn, P. M. Exchange Diffusion, Trans- the National Cancer Institute of Canada for their financial port and Intracellular Level of Amino Acids in Ehrlich support of a program of work, of which this represents a part. Carcinoma Cells. J. Biol. Chem., 233:1488-93, 1958. Thanks are due to Dr. L. Berlinguet for a supply of O- 12. MAIZEm, M.; REMINGTON, M.; and TRvscoz, R. Data for methyl- and O-ethyl homoserine. the Calculation of the Rate Coefficients of Sodium Transfer by Mouse Ascites Tumour Cells. J. Physiol., 140:48-60, REFERENCES 1958. 1. BIRT, L. M., and HIRD, F. J. R. Kinetic Aspects of the Up- 13. RABINOVITZ, M.; OI~EN, M. E.; and GREENBERG, D. i. take of Amino Acids by Carrot Tissue. Biochem. J., Role of Glutamine in Protein Synthesis by Ehrlich Ascites 70: 286-92, 1958. Carcinoma. J. Biol. Chem., 222: 879-93, 1956. 2. CHfiISTENSEN, H. N., and RINGS, T. R. Concentrative Up- 14. RIGG8, T. R.; COINS, B. A.; and CHRISTENSEN, H. N. take of Amino Acids by Ehrlich Ascites Carcinoma Cells. Amino Acid Concentration by a Free Cell Neoplasm. J. Biol. Chem., 194:57-68, 1952. Structural Influences. J. Biol. Chem., 209:395-401, 1954. 3. CttRISTENSEN, H. N.; RioGs, T. R.; FmCHER, H.; and 15. Rtoos, T. R.; WALKER, L. M.; and CHmSTENSEN, H. N. PA~TINE, I. M. Amino Acid Concentration by a Free Cell Potassium Migration and Amino Acid Transport. J. Biol. Neoplasm; Relations among Amino Acids. J. Biol. Chem., Chem., 233:1479-95, 1958. 198:1-15, 1952. 16. SCItOLEFIELD, P. G.; CREASER, E. H.; and DE LEON, R. P. 4. COHEN, G. N., and MONOD, J. Bacterial Permeases. Bact. P~ Incorporation by Ehrlich Ascites Carcinoma Cells. Rev., 21:169-94, 1957. Proc. Can. Fed. Biol. Soc., 1:45, 1958. 17. TENENItOUSE, A., and QUASTEL, J. H. Amino Acid Trans- 5. CRANE, R. K.; FIELD, R. A.; CORI, C. F.; and ROBERTS, port into Ehrlich Ascites Carcinoma Cells. Proc. Can. Fed. M. L. Tissue Permeability. I. Penetration of Sugars into Biol. Soc., 1:50, 1958. Ehrlich Ascites Tumour Cells. J. Biol. Chem., 224: 649-62, 18. WmEMAN, G. Absorption of Amino-Acids Using an in 1957. Vitro Technique. J. Physiol., 120:68-7~, 1958. 6. DARLINGTON, W. A., and QUASTEL, J. H. Absorption of 19. WISEMAN, G., and GHADIALLY, F. N. Studies in Amino Sugars from Isolated Surviving Intestine. Arch. Biochem. Acid Uptake by RD3 Carcinoma Cell Suspensions in & Biophys., 43:194-207, 1953. Vitro. Brit. J. Cancer, 9:480-85, 1955. 7. FRIEDIIANDLER, L., and QUASTEL, J. H. Absorption of Ami- 20. VAN SLYKE, D. D., and MEYER, G. M. The Fate of Protein no Acids from Isolated Surviving Intestine. Arch. Bio- Digestion Products in the Body. III. Absorption of Amino chem. & Biophys., 56:424-40, 1955. Acids from the Blood by the Tissues. J. Biol. Chem., 8. HARRIS, S. J. Transport and Accumulation in Biological 16:197-~1~, 1913.

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1959 American Association for Cancer Research. The Influence of Amino Acids and Antimetabolites on Glycine Retention by Ehrlich Ascites Carcinoma Cells

R. M. Johnstone and P. G. Scholefield

Cancer Res 1959;19:1140-1149.

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