[CANCER RESEARCH 34, 371-377, February 1974]

Stereochemical Characteristics of the Folate-Antifolate Transport Mechanism in L1210 Leukemia Cells1

Francis M. Sirotnak and Ruth C. Donsbach Memorial Sloan-Kellering Cancer Center, New York. New York 10021

SUMMARY versus normal cells. This apparently occurs because of the larger potential for concentrative uptake of drug by the The rate of influx, extent of concentrative uptake, and the tumor cells after the serum concentration has fallen to a rate of efflux (loss) by active transport in L1210 leukemia low level. Although the significance of these findings to anti cells has been compared for the pteridine antifolates, amino- folate treatment of human leukemia remains to be deter pterin and methotrexate, eight related quinazoline analogs, mined, the potential for therapeutic exploitation of this and two pyrimidine derivatives. The data reveal a difference physiological site seems obvious. in the Stereochemical specificity for influx and efflux. Influx The manner by which antifolates penetrate tumor cells is preferential in the order pteridine, quinazoline, and py has been of interest to a number of workers (3, 5, 6-11, 15- rimidine. Influx of aminopterin is more rapid than that of 17, 20, 21, 23, 27, 28). There is extensive evidence (6-9, 16, methotrexate. L-Glutamylquinazolines were taken up faster 17, 20, 21, 23, 27, 28) in vitro indicating that uptake in than L-aspartylquinazolines, but influx of a D-glutamyl- L1210 cells occurs by active transport. In most other tumor quinazoline was slower than the corresponding D-aspartyl cells, uptake has been shown (3, 5, 17) to at least resemble derivative. Influx of the quinazolines was faster when there an active transport process. was a methyl- or chloro- substitution at position 5. Influx of The characteristics of uptake in L1210 cells demonstrated the pyrimidines was also faster when a methyl group was at in vitro closely approximate that seen in the animal (24, 25). position 6. Michaelis constants (Km) for influx of the various analogs varied from 1.42 x 10~6Mto over 10"4 M. Individ In another aspect of these studies, the Stereochemical re quirements of the transport mechanism in L1210 cells were ual Vmax values were essentially the same (1.87 to 2.22 examined. Measurements were made of the rate of influx nmoles/min/g dry weight). The relationship between the and extent of concentrative uptake, as well as the rate of values for initial velocity of influx (v), the Km and Vmaxob efflux (loss) of a variety of folate analogs. The results of pre tained with each analog are in agreement with that predicted liminary studies comparing aminopterin, methotrexate, and by the Michaelis-Menten equation and is consistent with the methasquin have been reported from our laboratory (23). notion that differences in rates of influx are attributable to Comparisons between methotrexate and methasquin (20) differences in the affinity of the carrier for the system. Ef and methotrexate and a pteroate analog (16) have also been flux is preferential in the order pteridine, pyrimidine, and made elsewhere. A more extensive kinetic analysis involving quinazoline. Efflux of aminopterin and methotrexate occurs a number of individual Stereochemical differences among at the same rate. Both aspartyl- and glutamylquinazolines analogs is presented here. efflux at about the same rate, but the D-aspartyl and D- glutamyl forms efflux more rapidly than the corresponding L forms. A methyl, and particularly a chloro, substitution MATERIALS AND METHODS at position 5 of the quinazoline reduces the rate of efflux. The extent of concentrative uptake observed for each analog The maintenance and transplantation of the ascitic L1210 directly reflects the relative magnitude at which the influx line (V) in vivo has been described (14). Methotrexate and and efflux processes operate and may be the physiological aminopterin were supplied by Lederle Laboratories, Pearl parameter most relevant to therapeutic efficacy. River, N. Y. The quinazoline and pyrimidine analogs were provided by Parke Davis and Co., Detroit, Mich. Amino pterin and methotrexate were purified by chromatography INTRODUCTION (22). The final purity of all drug samples was evaluated bioautographically (4). The dihydrofolate reducíasecon Recent findings (24, 25) from this laboratory attribute the tent of the LI 210 cells was determined by titration inhibition selective activity of methotrexate during therapy of the with methotrexate or methasquin (30). L1210 leukemia to a greater persistence of drug in tumor Enzyme Assay for Antifolate. The content of drug in cell- free extracts was determined by titration (30) with a par ' This work supported in part by Grant CA-08748 from the National tially purified (29) dihydrofolate reducíasefrom a high- Cancer Institute and Grant BC-108 from the American Cancer Society. level recombinant strain of Diplococcus pneumoniae (26). Received June 20, 1973; accepted November 7, 1973. The details of the routine tube assay have already been

FEBRUARY 1974 371

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1974 American Association for Cancer Research. Francis M. Sirotnak and Ruth C. Donsbach described (23). All but 2 of the antifolates (see legend of the total uptake at 37°)provides a measure of drug rapidly Table 1) titrate the microbial enzyme to about 80% inhibi adsorbed on the cell surface. This is essentially a tempera tion. Both D-deazaaminopterin and D-quinaspar titrate the ture-independent process (9, 23). In agreement with the enzyme only to about 60% inhibition. However, in our ex known lipophobic character of the folate analogs, the rate perience a good level of reliability in assays for both com of passive diffusion occurring within the concentration range pounds can still be obtained by using additional sample (2.2 n\i or less) used during these studies would be ex replicates. The range for the amount of drug detectable in pected to be negligible (2, 18). This was confirmed (6) for this assay varies between 0.05 and 0.5 ng. methotrexate by an estimation made at 37°at an external Antifolate Uptake by I 1210 Cells. The harvesting of cells concentration (100 ßM)wellin excess of that necessary to has been described (23, 27). Usually 2 x IO7cells in 1 ml saturate the carrier mechanism. Similar measurements of (A6oo = 3.0) of suspending medium (pH 7.5) were incu the diffusion at 37°of methotrexate were also made in the bated with drug. The uptake of drug at 37°corrected for current study at an external concentration of 500 ¿IM.The drug associating with cells at 0°was used as a measure of rate of diffusion at 0.45 JÕM(theconcentration used in the uptake by active transport. Uptake at 0°(about 1 to 2% of rate determinations shown in Table 1) calculated from

Table 1 The uptake of folate antagonists by LI210 leukemia cells

Substituents

of influx' ring acid (nmole/min/ Compound"AminopterinMethotrexateD-Deaza-aminopterinDeazaaminopterin5-Methyldeaza-aminopterin5-Chlorodeazaami-nopterinD-QuinasparQuinasparMethasquinChlorasquinNSCstructure"2,4-Diami-nopteridine2,4-Diaminop-teridine2.4-Diamino-quinazoline2,4-Diamino-quinazoline2,4-Diamino-quanazoline2,4-Diamino-quinazoline2.4-Diamirio-quinazoline2,4-Diamino-quinazoline2,4-Diamino-quinazoline2,4-Diamino-quinazoline2,4-Diamino-pyrimidine2,4-Diamino-pyrimidineAmino5 6 10moietyL-GlutamylCH, g drywt)0.7100.2300.0410.2080.2370.3500.1750.0470.0680.0760.00330.0047Km"(X10"M)1.424.9557.206.655.952.777.6538.3027.2023.50>100>100

L-GlutamylD-GlutamylL-GlutamylCH3

L-GlutamylCl

i.-GlutamylD-AspartylL-AspartylCH3

L-AspartylCl

L-Aspartyl*

110180NSC L-Aspartyl•

110191Basic CHj L-AspartylRate

" Aminopterin, /V-|p-|((2,4-diamino-6-pteridinyl)methyl]amino|benzoyl|-L-glutamate; methotrexate. A'-(p-|((2,4-diamino- 6-pteridinyl)methyl]methylamino|benzoyl|-L-glutamate; D-deazaaminopterin, Ar-|p-|[(2,4-diamino-6-quinazolinyl)methyl]- amino|benzoyl|-i)-glutamate, hemihydrate; deazaaminopterin, N-\p |[(2,4-diamino-6-quinazolinyl)methyl]amino|benzoyl](- L- glutamate: 5-methyldeazaaminopterin, W-|/>-|[(2,4-diamino-5-methyl-6-quinazolinyl)methyl]amino|benzoyl|-L-glutamate, diso- dium, tetrahydrate; 5-chlorodeazaaminopterin, Ar-(/j-|[(2,4-diamino-5-chloro-6-quinazolinyl)methyl]amino|benzoyl|-L-glutamate, hemihydrate; D-quinaspar, /V-(p-{[(2,4-diamino-6-quinazolinyl)methyl]amino|benzoyl|-D-aspartate, disodium, heptahydrate; quinaspar, /V-|/7-|[(2,4-diamino-6-quinazolinyl)methyl]amino|benzoyl|-L-aspartate; methasquin, /V-(/>-|((2,4-diamino-5-methyl- 6-quinazolinyl)methyl]amino|genzoyl|-L-aspartate. disodium, pentahydrate; chlorasquin, /V-|p-|[(2,4-diamino-5-chloro-6-quina- zolinyl)methyl]amino|benzoyl|-L-aspartate, dihydrate; NSC 110180, Ar-(p-|(/)-(2,4-diamino-5-pyrimidinyl)benzoyl]amino|ben- zoyl|-L-aspartate; NSC 110191,/V-jp-|[p-(2,4-diamino-6-methyl-5-pyrimidinyl)benzoyl]amino|benzoyl|-L-aspartate. "p-Aminobenzoyl moiety attached at position 9 of the pteridinyl ring and benzylaminobenzoyl attached at position 5 of the pyrimidines. ' The initial rate of uptake at 37°corrected for uptake at 0°.Rate = nmoles/min/g dry weight (drug],,«,!,.]= 0.45 M.Values are an average of 4 to 6 replicate experiments, with a standard deviation of less than 30%. Each compound was always compared to methotrexate run as an internal control in the same experiment. " Michaelis constant (molar). Values are an average of 5 to 8 replicate experiments, with a standard deviation of less than 30%. Each compound was always compared to methotrexate run as an internal control in the same experiment. Precautions used in deter mining rates for true initial velocity were the same as described previously (6, 15). ' Not applicable, since this is a 5-arylpyrimidine (see above).

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1974 American Association for Cancer Research. Antifolate Transport in L/210 Leukemia Cells these data was 0.00075 to 0.001 nmole/min/g dry weight. The initial rate of temperature-dependent influx of the The suspending medium consisted of 107 HIMNaCl, 5.3 various analogs, at a concentration of 0.45 ¿tM,isshown in mM KC1, 26.2 mM NaHCO3, 1.9 mM CaCl2, 1 mM Table 1. The relative rate of influx among the group varied MgCl2-6H2O, 10 mM glucose, and 10 mM Tris-HCl. Ex more than 200-fold. Influx of aminopterin was the most posure to drug was terminated by the addition of 10 ml rapid, while influx of the pyrimidine analogs, NSC 110180 of cold 0.02 M potassium phosphate-0.14 M NaCl, pH 7.5. and NSC 110191 was the least rapid. The rate of influx of Cells were washed free of drug by resuspension twice in the methotrexate, and the related L-glutamylquinazoline, de- same solution to a final volume of 1 ml. The cell number azaaminopterin, was only one-third to one-fourth as rapid was determined by an absorbance measurement using a as that of aminopterin. The L-aspartylquinazoline analogs standard curve relating microscopic cell count to absorb were transported at one-fifth the rate of the corresponding ance (27). Drug was removed from the cells by heat extrac L-glutamylquinazolines. 5-Chlorodeazaaminopterin and the tion, and cellular debris was discarded after centrifugation. 5-chloro-L-aspartylquinazoline (chlorasquin) were trans Kinetic Analysis of Antifolate Influx. The Michaelis con ported at a rate almost twice that of the corresponding un- stant (Km) for the transport system was determined by the substituted derivatives. A somewhat smaller increase in the method of Lineweaver and Burk (19). In this system, it rate of influx occurred with 5-methyl-deazaaminopterin and represents the external drug concentration needed for half- methasquin. The 6-methyl 2,4-diaminopyrimidine analog saturation and is used as a relative measure of the affinity of (NSC 110191) had a higher rate of influx when compared the carrier component for drug. The initial rate of influx at to the unsubstituted 2,4-diaminopyrimidine (NSC 110180). 37°(minus uptake at 0°)was measured at varying concen The influx of the D-glutamylquinazoline, D-deazaamino- trations of each antifolate. The time of incubation was ad pterin, occurred at only one-fifth the rate obtained with justed to allow for measurement of uptake below the di- L-deazaaminopterin. In contrast, the rate of influx of the hydrofolate reducíasecontent. Since no free drug exists D-aspartylquinazoline (o-quinaspar) was 4-fold greater internally in this range, uptake is essentially unidirectional than that of the corresponding i.-aspartyl form (quinaspar). and a valid measure of influx. The necessity for this pre Kinetic Analysis of Antifolate Influx. Saturation (Mi- caution has been stressed in earlier reports (9, 23). chaelis-Menten) kinetics for temperature-dependent uptake Measurements of Efflux by L1210 Cells. The efflux of the was demonstrated for all of the analogs except the 2 pyrimi various antifolates from LI210 cells was measured (9, 23) dine analogs (NSC 110180 and NSC 110191). The apparent by preloading the cells with drug (usually to an intracellular Michaelis constant (Km) derived for each analog is shown in level 3 to 4 times the dihydrofolate reducíasecontent) and Table 1. As anticipated from the data on the rate of influx, washing the cells free of drug with cold 0.02 M potassium the transport mechanism appears to exhibit the greatest phosphate-0.14 M NaCl, pH 7.5. The cells were then re- affinity for aminopterin (Km = 1.42 x 10"6 M)and some suspended in fresh medium and incubated at 37°for vary what less for methotrexate (Km = 4.95 x 10"6 M) and the L-glutamylquinazolines (Km = 2.77 to 6.65 x 10~8M). The ing periods of time. mechanism seems to have even less affinity for the D- aspartylquinazoline (Km = 7.6 x 10~6 M) and the L- aspartylquinazolines (Km = 23.5 to 38.3 x 10"6 M) and RESULTS least for the D-glutamylquinazoline (Km = 57.2 x 10~6M). No Km value could be obtained with the 2 pyrimidine ana The Influx of Antifolates by 1.1210 Cells. The folate ana logs because of the extremely high concentrations appar logs used during these studies include the pteridines, amino- ently necessary to saturate the system. The maximum rate pterin and methotrexate, 8 quinazolines, and 2 pyrimidine of influx obtainable (Vmax)with the pteridine and quanazo- derivatives. In accordance with their gross stereochemical line analogs was calculated graphically or from the basic similarity, the 3 groups appear to compete for the same Michaelis-Menten equation, v = (Vmax-S)/(K.m + S), where transport mechanism(s). This conclusion is based on a prior v is initial velocity of influx and S is [drug]externai-Values demonstration of inhibition of the rate of influx at 37°of for each analog are approximately the same, varying from methotrexate-3H by methasquin (20, 23) and of inhibition 1.87 to 2.22 nmoles/min/g dry weight. of the rate of influx of some of these drugs (sometimes by The Intracellular Concentration of Antifolate at Steady kinetic analysis) by the natural folates folie acid, 5-methyl- State. The kinetics and extent of concentrative uptake for tetrahydrofolic acid, and 5-formyltetrahydrofolic acid (7-9, each folate analog were measured by incubating L1210 cells 17, 21, 23, 27). A similar measurement of the inhibition of at 37°with drug (0.45 or 2.2 fiM)until the free intracellular influx of the pyrimidine analogs at 37°by normal folates drug concentration was at equilibrium (steady state). This was also made during the current studies. Like prior results, usually requires a period of 40 to 60 min. The data shown inhibition of the rate of influx of these analogs was shown to in Table 2 and Charts 1 and 2 were corrected for uptake at be competitive or resemble a competitive process and de 0°.This value is usually very small and has been shown (9, monstrable only at saturating or near-saturating external 23) to represent mainly adsorption on the cell surface. The concentrations. The extent to which the influx of each class kinetics of concentrative uptake of the various analogs of antifolate was inhibited by normal folates was in good differed considerably. The accumulation of the pteridines quantitative agreement with the relative rate and kinetics (Chart 1) is similar. Uptake occurs at essentially a constant of influx observed for each. rate (only influx occurs) until the dihydrofolate reducíase

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Table 2 The concentrative uptake of folate antagonists by Li 210 leukemia cells

drugCompound"AminopterinMethotrexateD-DeazaaminopterinDeazaaminopterin5-Methyldeazaaminop-terin5-Chlorodeazaaminop-terinD-QuinasparQuinasparMethasquinChlorasquinNSCIntracellular

wt)21.915.912.931.244.841.651.380.135.522.935.340.32.21.6Freec(UM)11.127.465.6216.9025.2022.7029.1946.8019.4511.7519.3322.39Ratio,(drug)liu.rn.,/(drug)»tern»5.063.392.5537.5911.4010.6366.5021.258.855.358.8010.18

110180NSC 110191(drugJe.uMT.ai6(MM)2.22.22.20.452.22.20.452.22.22.22.22.211.011.0Total(nmolesdry °SeeTable 1 for structural details. ' External drug concentration. ' Based on a volume of 0.005 ml for the water content of the intracellular free space in 2 x IO7 cells (20). The amount of drug bound to dihydrofolate reducíase(3.75 nmoles/g dry weight) was substracted from the total amount accumulated intracellularly. Values are based on 4 to 6 replicate experiments with a standard deviation of less than 30%.

60r

40 [drug]ext = 2.2 u M

dea/a .iminopterin

aminopterín

methotrexate

60

Chart I. The rate of concentrative uptake at 37°of pteridine and py- rimidine antifolates in LI210 leukemia cells. The uptake of each analog was always related in 4 to 6 replicate experiments to the uptake of metho 60 trexate. Total uptake corrected for uptake at 0°.ext,external.

Chart 2. The rate of concentrative uptake at 37°of quinazoline anti level is reached. The rate then gradually diminishes as the folates in L1210 leukemia cells. The uptake of each analog was always re steady-state level is approached. With the exception of lated in 4 to 6 replicate experiments to the uptake of methotrexate. Total D-deazaaminopterin, quinaspar, and possibly D-quinaspar, uptake corrected for uptake at 0°.ext,external.

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the kinetics of uptake for the quinazoline analogs appears 25 r

(Chart 2) to be quite different. Uptake of drug continues at •¿D-dea/a-aminopterin a constant rate to internal concentrations far greater than o deaza-aminopterin the drug equivalence of the dihydrofolate reducíasecon A 5-methyl, deaza- aminopterin A 5-chloro. deaza aminopterin tent. In these cases linear kinetics of uptake continue against •¿D-quinaspar a sizable concentration gradient (see below). This is dra O quinaspar matically apparent in the case of 5-chlorodeazaaminopterin X methasquin where drug is accumulated to nearly 14-fold the dihydro -+•chlorasquin folate reducíaselevel. At an exlernal concenlralion of 2.2 ¿tM,the total accumulation of the pyrimidine analogs (Chart 1) did not reach the dihydrofolate reducíaselevel. The extenl of concenlralive uplake observed for each fo- lale analog varied over a wide range. The inlernal concen- Iralions of drug, al Ine sleady-slale levels shown in Charts 1and 2, are given in Table 2. At an exlernal concentration of 2.2 /iM, uptake of ihe pleridine (Charl 1), aminoplerin, and melholrexale was at the least concentrative (3- to 5- fold higher internal concentralion). Uplake of all of the quinazoline analogs (Chart 2), excepl D-deazaaminoplerin and quinaspar, was considerably more concenlralive (al leasl 8.8-fold higher inlernal concenlralion). 5-Chloro- deazaaminoplerin was concenlrated to Ihe grealesl exlent. Al an exlernal concenlralion of 2.2 ¡J.M,iheinlernal level al equilibrium was over 21 limes grealer. Al an external concenlralion of 0.45 ¿tM,ihe difference was 70 times 60 grealer. Al ihe same exlernal concenlration (0.45 jtM), deazaaminoplerin was concenlraled almosl 40-fold. Con- t (min) cenlrative uptake of the pyrimidine analogs, NSC 110180 Chart 4. The efflux of quinazoline antifolates by L12IO leukemia cells. and NSC 110191, could not be demonstraled al ihese con The data shown are based on the average of 3 to 5 replicate experiments in centrations. which the efflux of methotrexate was measured as an internal control. The Efflux of Folate Analogs by L1210 Cells. Previous studies (8, 9, 17, 23) have shown thai the loss of folate ana logs from L1210 cells is temperalure dependent. This is in 20 agreemenl wilh ihe idea lhal the same carrier is ulilized for bolh influx and efflux. A similar lemperature dependence for efflux of all of the analogs has been observed during the current study. The efflux of the various folate analogs from L1210 cells in drug-free medium at 37°isshown in Charts •¿aminopterin o methotrexate 3 and 4. Efflux of both the pteridine and pyrimidine analogs A NSC 110. 180 A NSC 110. 191 (Chan 3) occurred very rapidly. The inlracellular drug level was al ihe dihydrofolale reducíaselevel within 10to 15 min after incubation was initiated. As reported earlier (8, 9, 23), further loss of drug with lime occurred al an almosl imper- ceplible rale. Efflux of all of Ihe quinazoline analogs oc curred (Charl 4) al a much slower rate. The internal level of only D-deazaaminopterin, deazaaminopterin, and D-quin aspar reached enzyme level during ihe 60-min efflux period. Since Ihe decrease in concenlration of free drug in L1210 cells approximates a 1si-order process, il was possible more accuralely lo quanlilale Ihe relalive rale of efflux for each analog. A /i/2 value (lime required for a 50% decrease in concenlralion) was first calculaled from a linear semilog- arilhmic plol of ihe internal concentration of free drug at

t (min) various time inlervals. Values derived for each analog are given in Table 3. The rale conslanl for efflux for each is ob- Chart 3. The efflux of pteridine and pyrimidine antifolates by L1210 lained from ihe expression k = In 2//i/2. These values are leukemia cells. The data shown are based on the average of 3 to 5 replicate experiments in which the efflux of methotrexate was measured as an inter also given in Table 3. A relalive difference in efflux rale of nal control. aboul 20-fold was demonslraled for Ihe various analogs.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1974 American Association for Cancer Research. Francis M. Sirotnak and Ruth C. Donsbach Table 3 Experiments showing a rapid loss of aminopterin and The rale of efflux affatale antagonists by LI 210 leukemia cells methotrexate suggest an interaction with the efflux mecha constant' nism which again is most efficient in the case of the pteridine Compound"AminopterinMethotrexateD-DeazaaminopterinDeazaaminopterin5-(min-1)0.2390.2020.0470.0340.0270.0110.0530.0190.0170.0180.1470.158analogs. However, in contrast to that seen during influx, the (min)2.93.414.520.526.263.813.136.240.739.84.54.4Ratemethyl group at A"°has little effect on efflux. Moreover, both of the pyrimidine analogs efflux almost as rapidly as the pteridines, suggesting a nearly equivalent affinity for the same system. The quinazoline analogs as a group appear to Methyldeazaaminopterin5-ChlorodeazaaminopterinD-QuinasparQuinasparMethasquinChlorasquinNSChave the lowest affinity for the efflux mechanism. These are lost from the L1210 cells slowly, with little difference ob served between the L-aspartyl and L-glutamyl derivatives. Both D-aspartyl and D-glutamyl analogs efflux more effi ciently than the corresponding L forms. Whereas a methyl 110180NSC or chloro substitution at position 5 of the quinazoline ring 110191l¿ was found to potentiate influx, the opposite is true for °Structural details are given in the legend of Table 1. efflux. "Time required for internal concentration of free drug to decrease by It is vividly apparent that both influx and efflux play a one-half. Values are averages of 3 to 5 replicate experiments with a stand significant role in determining the internal level of free drug ard deviation of less than 30%. achievable at a specific external concentration. Moreover, ' k = In 2/t*. the kinetics and extent of concentrative uptake actually ob served for each analog quantitatively reflects the magnitude Aminopterin had the highest rate of efflux (k = 0.239), at which each can operate. For example, when both influx followed by methotrexate and the 2 pyrimidine analogs, and efflux are relatively rapid (aminopterin) or slow (meth- NSC 110180 and NSC 110191. Efflux of D-deazaamino- asquin), fairly high intracellular levels are achievable at pterin and D-quinaspar was slower, but at nearly the same relatively low extracellular concentrations. When influx is rate. The rate of efflux of the L-aspartylquinazoline analogs slow and efflux is rapid, as in the case of the pyrimidine was less than one-tenth the rate observed for aminopterin. analogs (NSC 110180 and NSC 110191), only low intra Two of the L-glutamylquinazolines, deazaaminopterin and cellular levels are possible. On the other hand, when influx 5-methyldeazaaminopterin effluxed somewhat faster than the corresponding L-aspartyl forms. The rate of efflux for is rapid but efflux is slow, phenomenally high intracellular levels are possible, as with 5-chlorodeazaaminopterin. the 5-chlorodeazaaminopterin was the lowest of the entire Knowledge as to general therapeutic relevance of achiev group (k = 0.011). ing high steady-state levels of free drug in tumor cells will require further study of these drugs at a pharmacological DISCUSSION level. As suggested (8, 23), the rate of influx alone is prob The function of the antifolate transport mechanism in ably an inadequate parameter for evaluating possible ther L1210 leukemia cells exhibits a considerable degree of di apeutic efficacy. This, in fact, has proved to be the case with versity with respect to stereochemical specificity. Moreover, methasquin, which was relatively inert during experiments the data reveal a difference in the stereochemical basis of measuring influx but is a fairly effective antileukemic agent the influx and efflux processes. Based on the respective (12). apparent Michaelis constants for influx, the mechanism Although we have not directly demonstrated competition seems to exhibit the greatest affinity for the 2,4-diamino among various folate analogs for the efflux mechanism, it is analog (aminopterin) of folie acid. The affinity was some assumed that each analog utilizes the same carrier for this what less in the case of the 2,4-amino, /V'-methyl analog purpose. This is probably true, if both influx and efflux oc (methotrexate). The poor affinity of the mechanism for folie cur by a single carrier, since some evidence has been pro acid itself has already been demonstrated (8, 9, 17, 23, 29). vided (Refs. 7 to 9, 17, 20, 21, 23, and 27, and in the present On the other hand, 5-formyltetrahydrofolate (citrovorum study) which indicates that all of the analogs compete for factor) and 5-methyltetrahydrofolate are probably trans the same influx mechanism. Additional evidence for the in ported by this mechanism to about the same extent as volvement of a single carrier for influx and efflux comes methotrexate (8, 9, 17, 21, 23, 27). The mechanism has re from a demonstration of both countertransport (9, 21) and a duced affinity for quinazoline derivatives and a severely transtimulation effect (7) in the L1210 system. The latter reduced affinity for the corresponding pyrimidine analogs. was based on data showing that methotrexate influx is more Interaction with the L-aspartyl analogs is poor in compari rapid in cells preloaded with 5-formyltetrahydrofolate. We son to the L-glutamyl analogs. The opposite seems to be have confirmed this finding and have also obtained the same true with regard to analogs bearing D-glutamyl or D- result in connection with the influx of the quinazolines and aspartyl moieties. The affinity of the mechanism is greater pyrimidines (F. M. Sirotnak and R. C. Donsbach, unpub for quinazoline analogs bearing a methyl or especially a lished results). chloro group at position 5. The system has greater affinity The large spectrum of interaction between folate analogs for a pyrimidine analog, if a methyl group is substituted at for the transport carrier is quite different from that seen position 6. with the target enzyme, dihydrofolate reducíase.All of the

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1974 American Association for Cancer Research. Antifolate Transport in L1210 Leukemia Cells analogs examined here are excellent inhibitors of the en 12. Hutchison, D. J. Quinazoline Antifolates: Biologic Activity. Cancer zyme in L1210 cells (Ref. 13; F. M. Sirotnak and R. C. Chemotherapy Rept., 52: 697-705, 1968. Donsbach, unpublished results) with dissociation constants 13. Hutchison, D. J., Sirotnak, F. M., and Albrecht, A. M. Dihydrofolate (K,) in the vicinity of 10~" M. The greater stereochemical Reducíase Inhibition by the 2,4-Diaminoquinazoline Antifolates. flexibility of the interaction with enzyme undoubtedly re Proc. Am. Assoc. Cancer Res., 10: 41, 1969. 14. Hutchison, D. J., Robinson, D. L., Marlin, D., lllensohn, O. L., and lates to the multisite nature (1) of the contact demonstrated Dillenberg, J. Effecls of Selected Cancer Chemolherapeulic Drugs on with this class of inhibitor. 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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1974 American Association for Cancer Research. Stereochemical Characteristics of the Folate-Antifolate Transport Mechanism in L1210 Leukemia Cells

Francis M. Sirotnak and Ruth C. Donsbach

Cancer Res 1974;34:371-377.

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