Methotrexate inhibition of CCRF-CEM cultures of human lymphoblasts

Europ. J. CancerVol. 11, pp. 771 782. Pergamon Press 1975. Printed in Great Britain

Methotrexate Inhibition of CCRF-CEM Cultures of Human Lymphoblasts * DEWAYNE ROBERTS and ELLEN V. WARMATH Laboratories of Pharmacology, St. Jude Children's Research Hospital, 332 North Lauderdale, P.O. Box 318, Memphis, T N 38101, USA A b s t r a c t - - T h e biochemical basis for methotrexate ( M T X ) inhibition of human

lymphoblasts was examined with the CCRF-CEM cell line. Methotrexate inhibition of growth by cultures of this human lymphoblast cell line was prevented by a combination of hypoxanthine, thymidine and deoxycytidine and was reversed competitively by folinic acid. Within 5 rain M T X inhibited the incorporation of isotopically-labeled deoxyuridine, hypoxanthine and glycine into D NA, although thymidine incorporation continued normally for more than 90 min before becoming inhibited. In media supplemented with thymidine plus deoxycytidine, 2- to 5-fold more M T X was requiredfor 50~o inhibition of growth than in regular media. In media supplemented with hypoxanthine, or thymidine, deoxycytidine and 5-fluorodeoxyuridine, folinic acid competitively reversed M T X inhibition. At 0"02 and 0"01 # M M T X , inhibition of dihydrofolate reductase (DHFR) required 6-16 hr to develop, and deoxyuridine incorporation into D NA was not inhibited. In media containing dialyzed serum 0"08 llM M T X inhibited DHFR within 60 min and deoxyuridine incorporation within 85 rain. After preincubation with 0"08 ItM M T X for 3 hr, cells recoveredtraces of DHFR in 20 min. Simultaneously, deoxyuridine incorporation into DNA started to recoverand attained control rate in 60 min with less than 10% recovery of DHFR activity. These data indicate: (a) CCRF-CEM cells have 10- to 20-fold more DHFR than requiredfor cell division. (b) Growth inhibition of cultures resultedfrom thymidine deficiency. (c) M T X did not selectively inhibit either thymidine or purine biosynthesis. Consideration of these factors suggests that M T X inhibition of cell division resultedfrom the inhibition of DHFR.

INTRODUCTION

also requires deoxycytidine to maintain growth in the presence ofthymidine [7]. Although the oncolytic activity of M T X is generally attributed to an inhibition of DHFR, at higher drug concentrations, thymidylate synthetase and a number of other enzymes are inhibited also [1, 8-11]. D H F R catalyzes the reduction of folate or dihydrofolate to tetrahydrofolate, and inhibition of D H F R by M T X blocks the synthesis of tetrahydrofolate, which is required for synthesis of precursors of DNA, RNA and protein. Interest in secondary sites of inhibition by M T X has been stimulated by the general lack of correlation between the drug's oncolytic activity and the inhibition of D H F R [12]. Condit and Mead observed that folinic acid accumulated in the liver after folate was administered to mice. This accumulation was prevented by M T X [13], however, the drug did

M T X is an analog of folic acid and an inhibitor of cell division. Folate and tetrahydrofolate derivatives reverse the inhibition of microbial cultures and cell cultures by M T X , block the drug's intoxication of rodents and humans, and interfere with the drug's oncolytic activity [1-4]. Growth inhibition of cell cultures by M T X is reversed by products of tetrahydrofolate-dependent reactions. Thymidine and a purine for nucleic acid synthesis and, with some cell cultures, glycine for protein synthesis, are required to reverse M T X inhibition [5, 6]. A human lymphoblast cell line, CCRF-CEM, Accepted 26 May 1975. *Supported by Research Grants CA-I 1148, CA-12732, and CA-08480 from the National Cancer Institute, NIH, and by ALSAC. 771

772

DeWayne Roberts and Ellen V. Warmath

not block conversion of dihydrofolate to folinic acid. These studies were recently confirmed and extended with new methodology [14]. Prolongation by M T X of the lives of tumorinoculated mice is correlated with greater M T X uptake by ascites tumor cells but not with the level of D H F R activity in these cells [15, 16]. With D H F R activity inhibited in mouse ascites tumors by M T X administration, only partial inhibition of deoxyuridine incorporation into DNA is observed in vitro with tumor cells [17]. The further addition of M T X to these murine ascites tumor cells increased the inhibition of deoxyuridine incorporation by a sensitive cell line, L1210, and an insensitive tumor line, P329, but not of a MTX-resistant variant of L1210. M T X inhibition of D H F R from leukemia cells is observed for both MTX-sensitive and insensitive human leukemias [12]. The early studies of human leukemia by Winzler et al. indicated that low concentrations of M T X more effectively inhibit formate incorporation into thymine of DNA than into purlnes of RNA. Therefore, a differential sensitivity of various one-carbon pathways to M T X has been considered [18]. A series of studies relates M T X action to an inhibition of thymidylate synthetase [1], to an elevation of thymidylate synthetase activity and recovery from drug action [19, 20], or to an elevation of thymidylate synthetase activity with resistance [21, 22]. However, a pharmacologically important relation between D H F R and M T X is indicated by: (a) the stoichiometric inhibition of D H F R at pH 6.1 and the very significant, although slightly less effective, inhibition of the enzyme at higher pH [23-25] ; (b) in vivo titration of enzyme activity to which drug retention is attributed [25]; and (c) the correlation of M T X resistance with elevation of D H F R activity in association with more rapid recovery of enzyme activity after M T X inhibition

[2, 26, 27]. Discrepancies appear to exist between the inhibition of D H F R by M T X and the drug's oncolytic effectiveness [19]. Although acute lymphocytic leukemia is more responsive to M T X than acute myelogenous leukemia, D H F R activity of lymphoblasts and myeloblasts was inhibited equally by M T X [28]. After treatment with M T X , the recovery of D H F R activity in leukocytes from patients with leukemia was not correlated with the sensitivity of their disease category to the drug [12, 28, 29]. However, a more pronounced inhibition of deoxyuridine incorporation was observed after M T X administration to patients with acute lymphocytic leukemia [12].

The studies that follow with a human lymphoblast cell line examine the biochemical basis for discrepancies between inhibition of DHFR, DNA synthesis and cell division by MTX.

MATERIAL AND METHODS Materials

Thymidine- (3H-methyl), deoxyuridine-6all, hypoxanthine-8-3H, and glycine-l-~4C were obtained from Schwarz-Mann, Orange, N.J. Folic-G-3H acid was obtained from Amersham/Searle Corp., Arlington Heights, Ill., and was purified by chromatography on Whatman No. 1 paper with 0.01 M sodium phosphate buffer, pH 7.0. Eagle's minimal essential medium with Earle's salts and Lglutamine for spinner cultures was obtained from Grand Island Biological Company, Grand Island, N.Y. Fetal calf serum was purchased from Grand Island Biological Company, or from Flow Laboratories, Inc., Rockville, Md. Folinic acid, calcium leucovorin for injection, was a product of Lederle Laboratories. M T X was a gift of the Lederle Laboratories, and a single batch was used for these studies. Nonlabeled thymidine, deoxyuridine, hypoxanthine and glycine were obtained from Calbiochem, La Jolla, California. Cell line and culture conditions

The C C R F - C E M cell line in these studies was derived from the blood of a patient with acute leukemia that developed from lymphosarcoma [30]. This lymphoblast line was maintained in Eagle's minimal essential medium with Earle's salt solution lacking CaC12 and supplemented with 10 times the phosphates, with L-glutamine and with 10% fetal calf serum. The medium contained methionine and choline as potential sources of one-carbon units as well as folic acid. Presumably, glycine as well as low concentrations ofpurines, thymidine and reduced folates were present in the serum complement. The culture cannot be maintained indefinitely with media containing dialyzed fetal calf serum. In the presence of M T X , the cultures would grow normally if the medium was supplemented with 100 pM hypoxanthine, 10/~M thymidine, and 100/~M deoxycytidine. Deoxycytidine prevented the initial inhibition of growth by thymidine which would otherwise have delayed growth of cultures by approximately 18 hr. The cells grew unattached to the flask, or in suspension as spin cultures.

M TX Inhibition of CCRF-CEM Cells Isotopic studies Incorporation of thymidine-(3H-methyl) and deoxyuridine-6-3H into D N A was assayed by collecting the cells on a glass fiber filter and repeatedly washing them with cold 0"9% NaC1 solution followed by cold 5% trichloroacetic acid. The damp filter was dropped into a counting vial containing 10ml of liquid scintillation medium. After standing for approximately 20 min, the contents were vigorously mixed and counted by liquid scintillation technique. Hypoxanthine-8-3H was primarily incorporated into RNA. A 2-hr hydrolysis at 37 ° in 1 N N a O H was adequate to remove the R N A when the remaining TCA-insoluble material was collected on a glass filter. After repeated washing with 0.9% NaC1 solution and 5~, TCA, the precipitate with D N A was assayed by liquid scintillation techniqne to measure incorporation into DNA. Assay of glycine-l-lgC incorporation into adenine of the nucleic acids required separation of the R N A and D N A followed by separation of the purine and pyrimidine bases by twodimensional paper chromatography. The cells from a 100-ml culture were collected by centrifugation at 500 g for 5 min, and the cell pellet was washed three times with 4 0 m l 0.9% NaC1 solution and five times with 5 ml of 5% TCA. The precipitate was resuspended in 1 ml of 1 N K O H and incubated for 2 hr at 37 ° . After the suspension was chilled in an ice bath, 1 ml of 1 N HCl-15% T C A was added, and the precipitate, which contained DNA, was collected by centrifugation for 5 min at 500 g. The ribotides from R N A in the supernatant fluid were hydrolyzed to release the purines. One-half ml of 10~/o HCIO4 was added to the supernatant fluid, and the precipitate of K C 1 0 4 was packed by centrifugation. The supernatant fluid was removed, and 260/~1 of 12 N HC1 was added to the fluid. The purine nucleotides were hydrolyzed by heating at 100 ° for 1 hr to release the bases. Adenine was isolated by the same technique used for DNA. The precipitate with D N A was washed three times with 5 ml of 5% TCA, then resuspended in 0"35 ml of 1 N HC1 and heated for 1 hr in a boiling water bath. The remaining insoluble material was collected by centrifugation and discarded. The supernatant fluid was evaporated to dryness at 60-65 ° . The residue was dissolved in water and placed along a 1-cm front on Whatman No. 1 paper and chromatographed, descending, with saturated aqueous butanol plus 0.01 volume of15 N N H 4 O H [31].

773

The adenine was located by co-chromatography with a standard in another region of the chromatogram. After the strip with adenine was cut from the original chromatogram, the adenine was rechromatographed in a second dimension with isopropanol-HCl-water [31] The adenine was eluted with water from the second chromatogram, and the eluate was evaporated to dryness. The adenine was redissolved in water and identified by the ratio of absorbancy at 260 and 280 m#, after correction for background absorbancy with a corresponding region from a blank chromatogram. Radioactivity was measured by liquid scintillation technique.

Assay of DIJFR The isotopic assay with folate-3H was used to assay D H F R [32]. The cells from 40-ml cultures were collected by centrifugation for 5 rain at 500g. The cell pellets were resuspended with 20 ml of 0.9% NaC1 and recollected by centrifugation, after which the cells were washed with 10 or 20 ml and then with 3 ml of 0.9% NaC1 solution. The final cell pellet was resuspended in 0.2 or 0.5 ml of 0.01 M Tris, p H 7"5, and then disrupted by sonic oscillation. An aliquot of homogenate was removed for assay of protein [33], and the remainder was centrifuged for 2 5 m i n at 20,000g. The supernatant fluid was frozen at - 2 0 ° until thawed for assay of D H F R activity.

RESULTS

Recovery of CCRF-CEM cultures from M T X inhibition C C R F - C E M cultures were tested for loss of viability after 4, 8, 12 and 24 hr of inhibition by 10 pM M T X . Viability was assayed by the increase in cell number after the medium was supplemented with 100 pM hypoxanthine, 10 ~M thymidine, and 100 gM deoxycytidine (Fig. 1). When these metabolites were added to the media, cultures rapidly resumed cell division after exposure to M T X for 4, 8 or 12 hr. After 24 hr of exposure to M T X , a delay followed the addition of metabolites before growth resumed. These results indicate that after prolonged exposure to M T X a large proportion of the cells in C C R F - C E M cultures were capable of cell division when the medium was supplemented with thymidine, hypoxanthine and deoxycytidine.

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Fig. I. Reversal of inhibition of C E M cultures by M T X . Spin cultures were initiated with 0.60 million C E M cells/ml in medium supplemented with 10 l t M M T X . At O, 4, 8, 12 or 24 hr, 100 # M hrpoxanthine, 100 l~M deoxycytidine, and 10 I t M thymiaine were added to one culture. r

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Fig. 2. Inhibition by M T X of CE.~I cultures supplemented with hypoxanthine or with thymidine and deoxycytidine. Three groups of cultures were inoculated with 0.60 million C E M cells/ml. Duplicate cultures in each group received from 0.001 l t M to 1 l t M M T X . One group of cultures was supplemented with 100 l l M deoxycytidine and 10/tM thymidine ( TdR + CdR). The second group was supplemented with I0 l t M hypoxanthine (HX). Another group was diluted with water. Growth was measured at 19 hr and at 43 hr. The 43-hr growth is presented to show that growth was not prevented by an initial lag.

Methotrexate sensitivity of CCRF-CEM cultures supplemented with hypoxanthine or thymidine By adding (a) M T X and hypoxanthine or (b) MTX, thymidine and deoxycytidine to media, the drug's specific effect relative to the missing metabolite, thymidine or hypoxanthine respectively, was monitored by changes in growth rate of cultures as the drug concentration varied. Hypoxanthine supplementation permitted the study of M T X effect on thymidine metabolism. Conversely, when the medium

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Fig. 3. Inhibition by M T X of 6-H3-deoxyuridine incorporation into D NA. Deoxyuridine, 1.13 ltCi (21.8 Ci/mmole), was added at time 0 to 2-ml aliquots of a C E M culture. The cells with deoxyuridine were incubated at 37 ° for various periods of time. These aliquots were supplemented with 0.1 to 10 l t M M T X or with water. At termination of incubation, 2 ml of cold 0.9o/o NaCl was added, and the cells immediately flushed onto a glass .fiber filter where they were washed with cold 0.9% NaCl followed by cold 5% trichloroacetic acid before counting by liquid scintillation.

thymidine plus deoxycytidine supplementation, approximately 8-fold more M T X was required for 50% inhibition of growth than in unsupplemented medium. This study was repeated three times, and although the quantitative relation varied in the studies, a similar qualitative relation was observed between hypoxanthine, thymidine and MTX. In contrast to other studies, hypoxanthine supplementation did not enhance M T X toxicity of CCRF-CEM cultures. These results indicate that growth of CCRF-CEM cultures was less sensitive to purine deficiency than to thymidine deficiency.

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Sensitivity o f purine and thymidine biosynthesis de novo to M T X

Purine biosynthesis was measured by glycine incorporation into adenine of RNA or DNA. This incorporation requires N 5, N 1°-methenyltetrahydrofolate and N ~°-formyltetrahydrofolate for biosynthesis of purine nucleotides [9]. Deoxyuridine incorporation into DNA requires N 5, N~°-methylenetetrahydrofolate for the conversion of deoxyuridine to thymidine 5"-monophosphate prior to incorporation into DNA [9]. In less than 30 min, 0.5 to 10 #M MTX completely inhibited deoxyuridine incorporation Table 1. Methotrexate inhibition of 1-14C-glycine incorporation into adenine of nucleic acid Methotrcxate

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after its simultaneous administration with 10/~M thymidine, 100 #M deoxycytidine, and 0.05 to 0.5/zM MTX to cultures. Equivalent inhibition of glycine incorporation into both RNA and DNA was observed as expected if the drug were blocking de novo synthesis of purines (Table l). By comparison with glycine incorporation in the controls, minus MTX, the pronounced inhibition of glycine incorporation, approximately 85% during the 1-hr incubation with 0"5/~M MTX, is compatible with a depletion of the tetrahydrofolate pool within 5 to 10 min after drug addition. Comparison of the effect of MTX on deoxyuridine and on glycine incorporation suggests that within the limits of experimental error, thymidine and purine synthesis were equally sensitive to the drug. If the synthesis of DNA continues beyond 5-15 rain, then thymidine or purine pools must be adequate to compensate for the block of de novo synthesis. z

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into DNA (Fig. 3). Extrapolation from the drug inhibited incorporation to the control rate indicates that the tetrahydrofolate cofactor pool was probably depleted in 5-15 min. Three hr after the addition of 0.1 #M MTX, deoxyuridine incorporation was incompletely inhibited. Glycine incorporation into adenine of DNA appeared as sensitive to MTX inhibition as deoxyuridine incorporation, but because of the low specific activity of 1-~4C-glycine, incorporation was not measured earlier than 1 hr

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Fig. 4. Requirements for 8-3H-hypoxanthine incorporation into DNA by CEM cultures in the presence of 10 ltM ,~I TX. A series of 2-ml aliquots from a CEM culture with 0.645 million cells/ml were incubated for various intervals with 0.72 ltCi 8-3H-hypoxanthine, 23"5 Ci/mmole, at 37°C. One half of the aliquots were supplemented with 10/tM ,~I TX. One group of MTX-supplemented cultures and one control group were also supplemented with 10/,tM thymidine ( TdR) plus 100 ltAar deoxyeytidine (CdR). Incubation was terminated by the addition of cold 0.9% NaCl and centrifugation at 500 g oCor 5 min at 4°. The cells were washed once with 0.9°o NaCl before adding 1 ml of 1 N NaOH and incubating for 2 hr at 37°. After chilling the tubes in an ice bath, 1 ml of 1 N HCl-15% trichloroacetic acid was added, and the acid-insoluble residue was collected on glass fiber filters and washed with 5% trichloroacetic acid. Hypoxanthine incorporation into DNA was assayed by liquid scintillation counting.

Maintenance o f physiologically active pools o f deoxynucleoside triphosphates

Thymidine and hypoxanthine can be incorporated into DNA only as long as adequate pools of all four deoxynucleoside triphosphates are present in the cells. When the medium is supplemented with hypoxanthine, depletion of thymidine triphosphate will terminate incor-

776

D e W a y n e Roberts and Ellen V. Warmath

poration into DNA. Supplementation of the medium with thymidine should prevent termination of hypoxanthine incorporation. Within 5 min after the addition of 10 #M MTX, hypoxanthine-8-3H incorporation was severely inhibited, although traces of hypoxanthine continued to be incorporated throughout the 90-min study (Fig. 4). As expected, if hypoxanthine incorporation were limited by a lack of thymidine triphosphate, supplementation of the medium with thymidine and deoxycytidine would restore hypoxanthine incorporation to the rate for unsupplemented cultures. However, addition of 10 #M thymidine and 100 #M deoxycytidine to control cultures

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cultures, hypoxanthine stimulated thymidine incorporation, and the subsequent rate of incorporation exceeded the control rate. In other studies, inhibition of thymidine incorporation developed simultaneously from 90 min after the addition of 0"5 and, 10/~M MTX to cultures. This study indicates that treatment with MTX does lead to a delayed purine deficiency and to the inhibition of thymidine incorporation into DNA. However, a pronounced purine deficiency did not develop unless thymidine and deoxycytidine were added with MTX to the cultures.* Folinic acid reversal o f M T X

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reduced hypoxanthine-83H incorporation into DNA. The pools of tetrahydrofolate cofactors and of thymidine triphosphate were inadequate to maintain DNA synthesis at control rates for more than 5 min. Time to the onset of a purine deficiency was examined after the addition of tritiated thymidine, deoxycytidine, and 10 #M MTX (Fig. 5). Two hr after these additions, thymidine incorporation into DNA was reduced approximately 30 ~o. Thymidine incorporation was further reduced by M T X as the experiment continued, although thymidine utilization by the controls was linear for more than 8 hr and continued for 12-14 hr. Hypoxanthine supplementation after 8 hr did not alter thymidine utilization by the controls. However, in the MTX-treated

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with dialyzed fetal calf serum, cultures were preincubated for 18 hr with 0.1 #M MTX to reduce the size ofintracellular and extracellular tetrahydrofolate pools. These cultures were used to study the relation between folinic acid and MTX with respect to the incorporation of deoxyuridine-6-3H into DNA. The medium was supplemented with 100 #M hypoxanthine to circumvent the inhibition of purine biosynthesis by MTX. After 18hr preincubation with 0.1 pM *Unpublished observation.

M T X Inhibition qf CCRF-CEM Cells M T X , deoxyuridine incorporation was approximately 13 % of the maximum rate observed with folinic acid supplementation (Fig. 6A). Although deoxyuridine incorporation was stimulated by folinic acid, the higher concentrations of folinic acid were also inhibitory. This inhibition was prevented by increasing the concentration of M T X . The maximum rate of deoxyuridine incorporation was observed with 50 or 100/~M folinic acid plus 1 ~M M T X . As the concentration of M T X was increased, inhibition of deoxyuridine incorporation became more pronounced, and folinic acid less effectively reversed the inhibition. The reciprocal plot of tblinic acid concentration vs the reciprocal of deoxyuridine incorpora-

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purines was the remaining growth-limiting reaction inhibited by M T X (Fig. 7). As the concentration of folinic acid was increased, inhibition of cell division by M T X was reduced and, conversely, increasing the concentration of

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tion at various concentrations of M T X indicated a competitive relation between the concentration of folinic acid and M T X (Fig. 6B). With the biosynthesis of thymidine 5'monophosphate as the growth-limiting reaction in the presence of M T X , a competitive relation was observed between folinic acid and M T X , rather than product replacement.

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The relation between folinic acid and M T X on growth of C C R F - C E M cultures was also examined with conditions where the biosynthesis of purines was rate-limiting. In order to block thymidylate synthetase and to reduce the possibility that an abortive oxidation of N s, N~°-methylenetetrahydrofolate was occurring, 0.1 /~M 5-fluorodeoxyuridine was added to inhibit the enzyme. Inhibition of cell division by 5-fluorodeoxyuridine was reversed by supplementing the medium with 10/~M thymidine plus 100 FtM deoxycytidine. With these concentrations of pyrimidines, the biosynthesis of

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Figf. 8A and 8B. The e~'ect of ~llTX, 0.01 to 0.08 i~/lI, on DHFR activi~ and on deoxyuridine incorporation into D N A of CCRF-CEM cultures. At 2-hr intervals aliquots were assayed for D H F R activity, Fig. 8A. Deoxyuridine, 1 lzM and 1 t~Ci/ml, was added at initiation of the study, Fig. 8B. The study was initiated with 0-57 million cells/ml.

M T X reduced the effectiveness of folinic acid. Approximately the same relation was observed at 18 hr as at 42 hr. A plot of the reciprocal for folinic acid concentration versus the reciprocal of the increase in cell number indicated a competitive relation between folinic acid and M T X .

778

De Wayne Roberts and Ellen K Warmath

Correlation between M T X inhibition o f D H F R and o f deox}'uridine incorporation

These data indicate that methylation of deoxyuridine 5'-monophosphate and de novo synthesis of purines are of approximately equivalent sensitivity to M T X . This is the expected response if M T X were inhibiting a single site such as DHFR. The results in Fig. 8A indicate that D H F R activity was inhibited in less than 2 hr by 0-08/~M M T X . Deoxyuridine incorporation into DNA was partially inhibited at 2 hr, and inhibition increased with time (Fig. 8B). With 0.04 pM M T X , there was a greater delay in the inhibition of D H F R and deoxyuridine incorporation. At 0.02 and 0.01 pM M T X , 6 and 16 hr exposure respectively, were required to reduce D H F R activity below the sensitivity

for depletion of"tetrahydrofolate" or thymidine nucleotide pools. However, two additional factors could contribute to the discrepancy: (1) D H F R is assayed at pH 6-1, and additional drug trapped intracellularly, but not enzymebound, during isolation of the cells may bind to the enzyme after sonic disruption and before assay, or (2) The kinetics of enzyme-drug

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MINUTES

Fig. 9. Development of the inhibition of dihydrofolate reductase aclivity and deoxyuridine incorporation by 0.08 #M M T X . The culture contained 0.54 million ceUs/ml and 1 fl:~l deoxyuridine, 1 #Ci/ml.

t ~~J°°8;v of the assay, and at these drug levels M T X did not inhibit deoxyuridine incorporation. The absence of an inhibition of deoxyuridine incorporation at a time when D H F R activity cannot be measured after addition of 0-01 and 0"02/~M M T X , indicates a possible dissociation of M T X effect on these two parameters (Fig. 8B). However, this dissociation could indicate that undetected levels of D H F R were adequate to support deoxyuridine incorporation into DNA by whole cells. The effect of 0.08 t~M M T X on the inhibition of D H F R and deoxyuridine incorporation was studied in greater detail (Fig. 9). The inhibition of D H F R preceded by approximately 25 min an inhibition ofdeoxyuridine incorporation. With 10/~M M T X , less than 5 rain were required to establish an inhibition of deoxyuridine or hypoxanthine incorporation (Figs. 3 and 4). Therefore, only a fraction of the delay between inhibition of D H F R and deoxyuridine incorporation can be attributed to time required

O

20

40

MTX

60

80

I00

120

MINUTES

B

Figs. lOAand 10B. Recovery of dihydrofolate reductase activity and of deoxyuridine incorporation after inhibition by ~ITX. CCRF-CEM cultures were preincubated for 3 hr with 0.08 # M 3ITJt2 The cells were collected by centrifugation at 500 g and resuspended, 0.55 million cells/ml, in the original volume of fresh media containing 10% dialyzed fetal calf serum. The recovery of DHFR activ#y is presented in A and deoxynridine incorporation in B. Deoxyuridine concentration was 1 p M with 1 pCi/ml.

binding may change in cells when the level of free D H F R is reduced [25]. If unbound drug were present intracellularly, time would be required for efflux of the drug and if most of the drug bound to D H F R is retained, then recovery of D H F R activity will result from synthesis of enzyme [7].

M TX Inhibition of CCRF-CEM Cells Recovery from M T X inhibition of DHFR activity and incorporation of deoxyuridine MTX, 0.08 I~M, was added to CCRF-CEM cultures, and after 3 hr the cells were collected by centrifugation and resuspended in fresh medium containing dialyzed fetal calf serum. D H F R activity was detected between 20 and 40 min after resuspension in MTX-free medium (Fig. 10A). Two hr after resuspension of the cells, D H F R activity was approximately onesixth the level in control cultures. The recovery of deoxyuridine incorporation into DNA occurred betweert 20 and 40 min after resuspension of the cells, and by 60 min the rate of deoxyuridine incorporation paralleled the control cultures (Fig. 10B). At 60 rain D H F R activity was less than 10% of the level in control cultures. A similar pattern for recovery of D H F R activity and for restoration of deoxyuridine incorporation was observed also with cells after 18hr of preincubation with 0.08/~M MTX.

DISCUSSION M T X inhibition of growth by CCRF-CEM cultures was reversed by supplementing the media with products of reactions that require tetrahydrofolate cofactors. Thymidine plus deoxycytidine was more effective than hypoxanthine in preventing the inhibition of growth by low concentrations of MTX, and CCRF-CEM cultures could resume cell division with little or no delay after inhibition by the drug for up to 24hr. Supplementation with deoxycytidine was required to prevent an initial inhibition by thymidine. Thymidine plus deoxycytidine was more effective than hypoxanthine in preventing inhibition by M T X of growth. However, methylation of deoxyuridine with subsequent incorporation into DNA and incorporation of glycine into adenine nucleotides of RNA or DNA were inhibited equally by MTX. The greater efficacy of thymidine relative to hypoxanthine in preventing the inhibition of growth is attributed to larger pools of purines than of thymidine. Within 5 min after the addition of 10 l~.~i MTX, a thymidine deficiency limited the incorporation of hypoxanthine into DNA, and this inhibition correlated with the rapid inhibition of deoxyuridine incorporation into DNA by this concentration of drug. The incorporation of thymidine into DNA, which requires purine deoxynucleoside triphosphates, conti~med normally for 90 min and, further-

779

more, in other confirmatory studies, M T X was unable to cause a pronounced purine deficiency unless the medium was supplemented with thynfidine and deoxycytidine. Folinic acid reversed MTX's inhibition of deoxyuridine incorporation into DNA. Although the larger concentrations of folinic acid inhibited deoxyuridine incorporation, a competitive relationship was observed between M T X and folinic acid at selected concentration ratios. Folinic acid also reversed the MTXinduced inhibition of purine biosynthesis. These studies indicated that M T X was inhibiting folate metabolism in CCRF-CEM cells. The competitive relation between M T X and folinic acid is attributed to competition for uptake by the cells [34-36]. Competition between M T X and folinic acid at some locus specifically related to either thymidine or purine biosynthesis appears unlikely since the competition was demonstrated in media that was supplemented with either thymidine or hypoxanthine. In addition, the intracellular concentration of free drug for the studies presented in Fig. 9, 10A and 10B was probably too low for significant inhibition of thymidylate synthetase or any other enzyme inhibited by the drug [9, 20]. The most likely locus for M T X inhibition of folate metabolism was DHFR. Inhibition of D H F R would lead to a depletion of tetrahydrofolate cofactors in ceils synthesizing thymidine 5'-monophosphate, and a depletion of these cofactors would simultaneously inhibit de no~o synthesis of thymidine and purine nucleotides [9]. If M T X inhibited the growth ofcuhures as a result of an inhibition of DHFR, then a dose- and time-dependent relation should exist between the inhibition of D H F R activity and dcoxyuridine incorporation into DNA. Interpretation of the data from Fig. 8, 9 and 10 on the relation between inhibition of D H F R and deoxyuridine incorporation requires consideration of several factors. The intracellular concet:.tration of unbound M T X is generally assumed to be vanishingly small if uninhibited D H F R activity exists in the cell. Furthermore, the rate of inhibition of D H F R activity is assumed to approximate the rate of M T X uptake until the enzyme is nearly saturated [25]. However, the formation of the drugenzyme complex is reversible [25], and the affinity between M T X and D H F R can be modified by thymidylate synthetase [37]. In vitro, affinity between D H F R and M T X is lower at physiological pH than at pH 6"1 [38], and as the level of free enzyme is reduced, the

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DeWayne Roberts and Ellen V. Warmath

kinetics of inhibition shift from "stoichiometric" toward a more classical, equilibrium type reaction [25]. This is generally observed as persistence of low levels of activity on titration of D H F R by inhibiting with M T X [25, 38]. In addition, dihydrofolate, the in vivo substrate, competes more effectively with M T X than folate [38]. In designing the present studies, the stoichiometric relationship between the drug and the enzyme was emphasized by selecting an assay with folate as substrate at pH 6-1. The apparent discrepancy between the inhibition of D H F R activity by 0.01 tiM and 0"02/~M M T X and the absence of an inhibition of deoxyuridine incorporation into DNA by these concentrations is attributed to a persistence of D H F R activity in the intact cell. Observation of a similar discrepancy between the inhibition of D H F R activity and deoxyuridine incorporation into DNA with L1210 cells led to the present studies with a human lymphoblast culture [17]. Goldman and Sirotnak and Donsbach have extended the original observation and observed respectively that the intracellular concentration of M T X for L-cells and L1210 cells must be slightly in excess of the stoichiometric equivalent ofintracellular D H F R before a pronounced inhibition of deoxyuridine incorporation occurs [39, 40]. The kinetics for inhibition of D H F R and deoxyuridine incorporation into DNA as well as recovery from drug inhibition were therefore examined in greater detail (Fig. 9 and 10). An approximately 25-min lag was observed between inhibition of D H F R activity and of deoxyuridine incorporation into DNA by 0.08 t*M

M T X . By extrapolation from the data in Fig. 9, continued uptake of M T X after the disappearance of D H F R activity and prior to inhibition of deoxyuridine incorporation would only permit the intracellular accumulation of M T X equivalent to 30% of the initial D H F R activity. This lag is attributed to the shift in kinetics of drug inhibition as D H F R activity becomes vanishingly small, and utilization of pre-existing pools of tetrahydrofolate cofactors and thymidine nucleotides. However, the slight excess of drug expected to accumulate intracellularly in the 25 min between the disappearance of D H F R and the inhibition of deoxyuridine incorporation is considered to be less than the concentration required to effectively inhibit thymidylate synthetase activity in homogenates of C C R F - C E M cells [20]. Time required for efflux of M T X , slight dissociation of the drug-enzyme complex, possibly synthesis of new enzyme and restoration of tetrahydrofolate cofactor and thymidine triphosphate pools contribute to the 20-min lag before the initial recovery of deoxyuridine incorporation into DNA and D H F R activity (Fig. 10). The complete recovery of the normal rate for deoxyuridine incorporation into DNA prior to full recovery of D H F R activity suggests that CCRF-CEM cells normally contain at least 10-20 times more enzyme activity than required to maintain growth of the culture. The present information is compatible with a single locus for M T X inhibition of CCRFCEM cultures. We suggest that inhibition of D H F R by M T X is responsible for the inhibition of growth by CCRF-CEM cultures.

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