Synthesis of Methotrexate Polyglutamates in L1210 Murine...
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[CANCER RESEARCH 37, 408-412, February 1977] SUMMARY The ability of L1210 mouse leukemia cells and of a mutant methotmexate-nesistant cell line (L1210/MTX) to synthesize methotrexate polyglutamates was studied. Host DBA/2 mice were treated with methotmexate, after which Ieukemic cells were harvested from ascitic fluid and levels of metho trexate and metabolites in them were determined by Sepha dex G-15 gel chromatography. The level of methotrexate in Li2iO/MTX cells was 12.5 times greaten than that in L1210 cells, reflecting the increased level of dihydrofolate meduc tase that characterizes this mutant cell line. Synthesis of methotrexate polyglutamates in each cell line required a dose of methotrexate (2.4 mg/kg) 10 times greater than the dose that yielded extensive methotrexate polyglutamate synthesis in rat liver and kidney in previous studies. Total methotnexate polyglutamates synthesized in 4 hr with this dose were the same in each cell line, demonstrating that this metabolism was not affected by differences in the level of dihydrofolate meductase. Methotrexate polyglutamates comprised 47 ±20% of the total methotrexate in L1210 cells. Methotrexate diglutamate was the predominant form. Levels of methotmexate monoglutamate and diglutamate were similar in L1210/MTX cells, whereas methotrexate monoglutamate was the predominant metabolite in host liver, kidney, and small intestine. These differences may reflect differences in substrate preference of pteroylpoly glutamate synthetase in these different tissues. Twenty-four h r after methotmexate ad mi n istration , total methotrexate in Li210 cells was one-third of that at 4 hn; but the proportion of metabolites was the same, presumably reflecting cell death and division rather than loss of a freely exchangeable portion of intracellular methotmexate present at the earlier time. The affinity of methotrexate polyglutamates for dihy drofolate reductase was found to be similar to that of meth otnexate, providing evidence that these metabolites may be as potent antagonists of folate metabolism as is methotrex ate itself. Recent studies indicate that inhibition of folate metabolism in cells requires their exposure to high levels of methotrexate in order to achieve intracellular levels of methotnexate greater than needed to bind to dihydnofalate reductase. Such conditions conform to those required for synthesis of methotrexate polyglutamates. Thus, these me 1 This work was supported by grants from the Medical Research Council of Canada and the Cancer Research Society. 2 Requests for reprints should be addressed to Division of Haematobogy, Montreal General Hospital, 1650 Cedar Avenue, Montreal, Quebec, Canada H3G1A4. Received July 8, 1976; accepted November 1, 1976. tabolites may play a specific molein inhibiting folate metabo lism, distinct from the antifolate potential that they appear to share with methotnexate. INTRODUCTION MTX,3 an analog of the vitamin folic acid (pteroylglutamic acid), is an important anticancer agent. It has proved to be particularly effective in the treatment of choniocarcinoma (18) and of acute lymphoblastic leukemia in children (ii). It has also proved to be of benefit in the treatment of a wide variety of other malignant diseases. Recently, adjuvant them apy of osteogenic sarcoma in children using exceedingly large doses of MTX followed by citnovorum factor rescue has been rewarded with long-term remissions (14). MTX is a potent inhibitor of the enzyme dihydrofolate reductase (EC 1.5.1 .3) and thereby of folate metabolism. Classically, its cytotoxic action has been attributed to this inhibition (3). However, recent studies have identified a mole for a component of intracellular MTX in excess of that portion which is tightly bound (presumably to dihydmofolate meductase)in contributing to the inhibition of folate metabo lism (9, 12, 21). It has been suggested that binding of MTX to targets other than dihydnofolate neductase (9, 16) or the attainment of mane complete inhibition of dihydrofalate neductase by this excess intracellular MTX (12) may be critical to the action of this agent and explain the added benefit seen clinically with high-dose MTX treatment (14). Synthesis of poly-'y-glutamyl metabolites of MTX has been reported from a number of laboratories (2, 5, 10, 13, 19, 24, 25). Detailed study of this metabolism of MTX has been carried out in mattissues (2, 24, 25). In this animal, dose dependent synthesis of both MTX(+G1) and MTX(+G@1) has been observed in liven and kidney (24, 25), whereas almost no such synthesis occurred in small intestine and thymus. In this report, these studies have been extended to the metabolism of MTX in L1210 murine leukemia cells, since these cells are sensitive to MTX (21) and have served often for the study of the activity of MTX. In addition, MTX metab alism was studied in a MTX-resistant mutant strain, L12i0/ MTX, characterized by a high level of dihydrofolate reduc tase (7), to see whether differences in its metabolism were present. A preliminary report of these studies has been presented (23). 3 The abbreviations used are: MTX, methotrexate; MTX(+G1), methotrex ate monoglutamate; MTx(+G,), methotrexate diglutamate; MTx(+G,), meth otrexate hexaglutamate. 408 CANCER RESEARCH VOL. 37 Synthesis of Methotrexate Polyglutamates in L1210 Murine Leukemia Cells1'2 V. Michael Whitehead Research Institute, Montreal General Hospital, Montreal, Quebec, Canada on March 26, 2019. © 1977 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from
Transcript of Synthesis of Methotrexate Polyglutamates in L1210 Murine...
[CANCER RESEARCH 37, 408-412, February 1977]
SUMMARY
The ability of L1210 mouse leukemia cells and of a mutantmethotmexate-nesistant cell line (L1210/MTX) to synthesizemethotrexate polyglutamates was studied. Host DBA/2mice were treated with methotmexate, after which Ieukemiccells were harvested from ascitic fluid and levels of methotrexate and metabolites in them were determined by Sephadex G-15 gel chromatography. The level of methotrexate inLi2iO/MTX cells was 12.5 times greaten than that in L1210cells, reflecting the increased level of dihydrofolate meductase that characterizes this mutant cell line. Synthesis ofmethotrexate polyglutamates in each cell line required adose of methotrexate (2.4 mg/kg) 10 times greater than thedose that yielded extensive methotrexate polyglutamatesynthesis in rat liver and kidney in previous studies. Totalmethotnexate polyglutamates synthesized in 4 hr with thisdose were the same in each cell line, demonstrating thatthis metabolism was not affected by differences in the levelof dihydrofolate meductase. Methotrexate polyglutamatescomprised 47 ±20% of the total methotrexate in L1210cells. Methotrexate diglutamate was the predominant form.Levels of methotmexate monoglutamate and diglutamatewere similar in L1210/MTX cells, whereas methotrexatemonoglutamate was the predominant metabolite in hostliver, kidney, and small intestine. These differences mayreflect differences in substrate preference of pteroylpolyglutamate synthetase in these different tissues. Twenty-fourhr after methotmexate ad mi n istration , total methotrexate inLi210 cells was one-third of that at 4 hn; but the proportionof metabolites was the same, presumably reflecting celldeath and division rather than loss of a freely exchangeableportion of intracellular methotmexate present at the earliertime. The affinity of methotrexate polyglutamates for dihydrofolate reductase was found to be similar to that of methotnexate, providing evidence that these metabolites may beas potent antagonists of folate metabolism as is methotrexate itself. Recent studies indicate that inhibition of folatemetabolism in cells requires their exposure to high levels ofmethotrexate in order to achieve intracellular levels ofmethotnexate greater than needed to bind to dihydnofalatereductase. Such conditions conform to those required forsynthesis of methotrexate polyglutamates. Thus, these me
1 This work was supported by grants from the Medical Research Council
of Canada and the Cancer Research Society.2 Requests for reprints should be addressed to Division of Haematobogy,
Montreal General Hospital, 1650 Cedar Avenue, Montreal, Quebec, CanadaH3G1A4.
Received July 8, 1976; accepted November 1, 1976.
tabolites may play a specific molein inhibiting folate metabolism, distinct from the antifolate potential that they appearto share with methotnexate.
INTRODUCTION
MTX,3 an analog of the vitamin folic acid (pteroylglutamicacid), is an important anticancer agent. It has proved to beparticularly effective in the treatment of choniocarcinoma(18) and of acute lymphoblastic leukemia in children (ii). Ithas also proved to be of benefit in the treatment of a widevariety of other malignant diseases. Recently, adjuvant themapy of osteogenic sarcoma in children using exceedinglylarge doses of MTX followed by citnovorum factor rescuehas been rewarded with long-term remissions (14).
MTX is a potent inhibitor of the enzyme dihydrofolatereductase (EC 1.5.1 .3) and thereby of folate metabolism.Classically, its cytotoxic action has been attributed to thisinhibition (3). However, recent studies have identified a molefor a component of intracellular MTX in excess of thatportion which is tightly bound (presumably to dihydmofolatemeductase)in contributing to the inhibition of folate metabolism (9, 12, 21). It has been suggested that binding of MTXto targets other than dihydnofolate neductase (9, 16) or theattainment of mane complete inhibition of dihydrofalateneductase by this excess intracellular MTX (12) may becritical to the action of this agent and explain the addedbenefit seen clinically with high-dose MTX treatment (14).
Synthesis of poly-'y-glutamyl metabolites of MTX has beenreported from a number of laboratories (2, 5, 10, 13, 19, 24,25). Detailed study of this metabolism of MTX has beencarried out in mattissues (2, 24, 25). In this animal, dosedependent synthesis of both MTX(+G1) and MTX(+G@1)hasbeen observed in liven and kidney (24, 25), whereas almostno such synthesis occurred in small intestine and thymus.In this report, these studies have been extended to themetabolism of MTX in L1210 murine leukemia cells, sincethese cells are sensitive to MTX (21) and have served oftenfor the study of the activity of MTX. In addition, MTX metabalism was studied in a MTX-resistant mutant strain, L12i0/MTX, characterized by a high level of dihydrofolate reductase (7), to see whether differences in its metabolism werepresent. A preliminary report of these studies has beenpresented (23).
3 The abbreviations used are: MTX, methotrexate; MTX(+G1), methotrex
ate monoglutamate; MTx(+G,), methotrexate diglutamate; MTx(+G,), methotrexate hexaglutamate.
408 CANCER RESEARCH VOL. 37
Synthesis of Methotrexate Polyglutamates in L1210 MurineLeukemia Cells1'2
V. Michael Whitehead
Research Institute, Montreal General Hospital, Montreal, Quebec, Canada
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MTX Polyglutamates in L1210 Cells
MATERIALS AND METHODS
Male adult DBA/2 mice bearing L1210 and L1210/MTXleukemias in ascitic form were obtained from Dr. I. Wodinsky, Arthur D. Little, Inc., Cambridge, Mass. They were meplaced after 6 months. Leukemias were maintained byweekly transfer to DBA/2 mice (8). For study, mice wereusually killed with ether on Day 5, and cells were harvestedfrom the penitoneal cavity by rinsing it with 2 to 3 ml ofsterile 0.9% NaCI solution. Cells from 2 to 4 mice werepooled. Grossly hemorrhagic specimens were discarded.Cells were washed 3 times with sterile 0.9% NaCI solutionand finally suspended in 4 volumes of 100 mM sodiumphosphate buffer, pH 7.0. Duplicate cell counts were madein a Coulter Counter, Model S (Coulter Electronics,Hialeah, Fla.) and were usually in the range of 1 to 4 x 108cells/mb. Cells frequently clumped together during cellwashing. This was not corrected by addition of hepanin tocell suspensions. Therefore, cell counts may sometimeshave been falsely low. Combined cytacnits and cell countswere carried out on suspensions of Li210 and L1210/MTXcells on 9 and 16 occasions, respectively. The mean cellvolume of Li2iO cells (553 ±162 fI) did not differ significantly from that of Li2iO/MTX cells (737 ±272 fI). Themean cell volume of Li210 and L1210/MTX together was671 ±254 fI. Final cell suspensions were heated in boilingwater for 10 mm and centrifuged, and the supemnatant wasstored at —20°.Preparation of extracts of host mouse tissues was carried out as previously described (24).
3',5',-9-[3H]MTX (Amensham/Searle Co., Don Mills, Ontania, Canada) was mixed with MTX (Ledenle ProductsDept., Cyanamid of Canada Ltd., Montreal, Quebec, Canada) to obtain a specific activity of 0.1 to 0.3 @Ci/@g,andthe mixture was purified by Sephadex G-i5 gel chromatography (Pharmacia, Uppsala, Sweden) as previously descnibed (24). [3H]MTX was usually administered to mice as asingle i.p. injection 4or24 hr pniorto harvesting of leukemiccells. MTX and metabalites in heated extracts of leukemiccells and of host mouse tissues were separated by chromatography on columns of Sephadex G-i5 (0.9 x 55 cm), andwere identified by cochnomatography with authentic standards. MTX(+GJ, MTX(+G@), and MTX(+G13)were kindlysupplied by Dr. C. M. Baugh and Dr. M. G. Nair of theDepartment of Biochemistry, University of South Alabama,Mobile, Ala. (17). Results were expressed as ng MTX or MTXequivalent per g tissue or i0@cells. Unlike rat tissues (24,25), chramatograms of extracts of mouse liver, kidney, andsmall intestine contained a number of 3H-Iabeled materialsin addition to but largely distinct from MTX and MTX palyglutamates. These materials were not present in the injected E3HIMTX.They were not identified. These contaminants accounted for as much as 20% of the 3H in somechromatograms, and they affected quantitation of MTX andMTX polyglutamates from time to time. Fortunately, theywere neverdetected in chromatograms of L1210 and Li 210/MTX cells (Chart 1).
The binding affinity of MTX palyglutamates fan dihydrofolate reductase was compared to that of MTX using thesingle-step variation of a new ligand-binding radioassay farMTX (15). This assay used dihydrofolate reductase as
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Chart 1. Sephadex G-15 gel chromatograms of extracts of L1210 cells (A)and L1210/MTX cells (B), prepared 4 hr after a dose of [3H]MTX (2.4 mg/kg)administeredi.p. to host DBA/2 mice. Extractswere cochromatographedwith MTX (—. ) MTX(+G,) (- - - -), and MTX(+G,) ( ).
binder, [3H]MTX as label, and Dextran T-iO-coated charcoal(Pharmacia) to separate bound from unbound reactants.The ability of MTX polyglutamates to compete with [3H]MTXfor binding to dihydrofolate neductase was measured bysubstituting them for MTX in standard curves performed bydirect competitive binding (15). Studies were carried out ondihydrofolate reductase partially purified from guinea pigliver (4, 15) and on dihydrofolate reductase present in bysates of Li2iO and L12i0/MTX cells (1).
3H in samples was counted in an lsocap/300 Liquid Scmtillation System (Nuclear-Chicago Corp., Des Plaines, Ill.)The chromatographic behavior of unlabeled standards wasdetermined spectrophotometnically on a Beckman ModelDK2 recording spectrophotometem. The inhibitory biobogical activity of MTX and metabalites was measured with thefolate assay organism Lactobacillus Casei (American TypeCell Culture 7469) (26).
RESULTS
Synthesis of MTX Polyglutamates in L1210 and L1210/MTX Cells and in Host DBA/2 Mouse Tissues. Sephadex G15 gel chromatograms of heated extracts of Li210 andLi2iO/MTX cells revealed 2 3H-Iabeled fractions in additionto MTX (Chart 1). These fractions were inhibitory to growthof L. casei. They corresponded in volume of elution tometabolites of MTX previously characterized as MTX(+G1)and MTX(+G@)in rat liven and kidney (24, 25). The identity ofthese fractions as MTX(+G@)and MTX(+G1) was confirmedby cochnomatography with authentic standards (17) (Chart1).
Characteristic of MTX metabolism in L12i0 cells was thelarge proportion of MTX polyglutamates and the predominance of MTX(+G@)(Chart 1A). In 6 studies of pooled Li 210cells, MTX polyglutamates comprised 47 ±20% of the totalintracellular MTX, and the level of MTX(+G@1)was 4 timesgreaterthan that of MTX(+G1) (p < 0.05) (Table 1). The levelof MTX in Li2iO/MTX cells was 12.5 times greater than thatin L1210 cells (p < 0.001) (Chart 1B; Table 1). The level of
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Levels of MTX and metabolites in L1210 and L1210/MTX cells and in host mousetissuesMice
receiveda single i.p. injection of [3H]MTX(2.4 mg/kg) and were sacrificed 4hrlater.Cells from 2 to 4 mice were pooled and extracts were prepared. Kidney extractswerealsopooled. Results are expressed as ng MTX or equivalent per 10@cells or gtissue(±
S.D.).Levels
of MTXand metabolites in cells andtissuesTissue
No. of studies MTX MTX(+G,) MTX(+G,)TotalL1210
6 281± 162 47±22 196±145 524±237L1210/MTX7 3511 ±1483 166 ±97 112±104 3789 ±1554Liver6 1130 ± 270 370 ±96 64 ± 48 1564 ±312Kidney4 464 ± 108 67 ±16 Oa 537 ±125Small
intestine 6 1131 ± 710 35 ±13 18 ± 8 1184 ± 706
The dose of [3H]MTX was divided and administered as 6 or9injectionsat 15-mmintervals. Four hr after the 1st injection,cellsfrom
2 to 4 mice were harvestedand pooled and extractswereprepared(see“Materialsand Methods―).Resultsare expressedasng
MTX or MTX equivalent per [email protected]
of MTXand metabolitesincells(ng/i0 cells)
Doseof[3H]MTX MTXMTXCells(mg/kg) MTX (+G,) (+G,)TotalLi210
0.16 93 5 31010.24277 ‘12 82972.40282 68 290640L1210/MTX
0.16 723 4 17280.244632 8 746472.401754 215 366 2335
V. M. Whitehead
Table I
a MTX(+G2) was absent from 3 pooled kidney extracts and present (22 ng/g) in 1.
total MTX polyglutamates in L1210/MTX cells (278 ±197ng/109 cells), however, did not differ significantly from thetotal in L12i0 cells (243 ±161 ng/109 cells) (Table 1). Thusboth MTX-sensitive and -resistant L1210 cells appeared tohave a similar capacity to synthesize MTX polyglutamates,although in the latter case both MTX(+G1) and MTX(+G@)were present in roughly equal amounts. In host liver thelevel of MTX(+G@)was about 6 times that of MTX(+G@)(p <0.001). Total MTX and metabolites in mouse kidney wereabout one-third of those in liver (p < 0.001). In mouse smallintestine, despite a mean level of intracellular MTX comparable to that in liver (Table 1), total MTX polyglutamatesconstituted only 6 ±3% of the total. Again, MTX(+G1) wasthe larger component (p < 0.01). Thus metabolism of MTXappeared to be distinctive in each of the 5 tissues studied(Table i).
Synthesis of MTX polyglutamates in L1210 and L1210/MTX cells was dose dependent. The dose of MTX requiredappeared to be 10 times greater than that which resulted inextensive synthesis of MTX polyglutamates in rat liver andkidney (24, 25). Thus with divided s.c. doses of [3H]MTXtotaling 0.16 and 0.24 mg/kg, almost no metabolism of MTXwas noted , whereas a divided s.c. dose of 2.4 mg/kg yieldedlarge quantities of both MTX(+G1) and MTX(+G@)(Table 2).These experiments served to define an appropriate dose of[3H]MTX for subsequent studies.
Levels of MTX and MTX palyglutamates were compared inpooled Li 210 and Li 210/MTX cells 4 and 24 hr after a singlei.p. dose of [3H]MTX (2.4 mg/kg). This study was undertaken in part to determine whether a discernible labile intracellular MTX component was present 4 hr after the dose, asconcluded by Simotnak and Donsbach (20), and in part toexamine the possibility that cells surviving for 24 hr mighthave metabolized MTX differently from those not surviving.In 3 paired studies, the mean total 3H in pooled Li210 cellsat 24 hr was 34% of that at 4 hr (p < 0.01) (Table 3). Meanlevels of MTX, MTX(+G1), and MTX(+G@)were reduced to39, 30, and 28%, respectively, of levels at 4 hr. Thus themewas no significant shift in proportion of MTX and metabobitesat the later time. Studies with Li2iO/MTX cells showeda reduction in level of total MTX and metabolites at 24 hr to62% of that at 4 hr. Again, no major shift in proportion ofMTX and metabolites was discerned.
For comparison, levels of total 3H in host liven, kidney,and small intestine were measured 4 and 24 hr after MTXadministration in 12 leukemia-bearing mice. Total 3H in liven
Table 2
Effectsof doseof MTXon synthesisof MTXpolyglutamatesin L1210and L1210/MTXcells
fell 78%, in kidney it fell 48%, and in small intestine it fell92%. Differences at these times were all highly significant (p< 0.001). Total levels at 24 hr corresponded to levels found
previously in mattissues (24, 25), suggesting that total intracellular MTX was artificially and temporarily elevated at 4 hr,particularly in liver and small intestine.
Comparison of the Affinity of MTX and MTX Polyglutamates for Dihydrofobate Reductase. The powerful antifolate activity of MTX has been equated with its exceptionallyhigh affinity for the enzyme dihydrofolate reductase (3). Toobtain information regarding the relative potency of MTXpolyglutamates as falate antagonists, their binding affinityfar dihydnofolate neductase was compared to that of MTX.Results obtained with dihydrofolate reductase contained inlysates of L12i0 and Li2iO/MTX cells are shown in Chart 2.They have been expressed as 1/fractian of [3H]MTX bound,related to the quantity of unlabeled MTX or MTX polyglutamates present. With lysatesfrom Li210 cells, 50% reductionin binding of [3H]MTX was obtained with 1.i pmoles MTXand 1.2 pmabes MTX(+G@)[ratio of 2 (Chart 2.4)]. With lysatefrom L12i0/MTX cells, 50% reduction in binding of [3H]MTXwas obtained with 1.0 pmale MTX, 1.4 pmoles MTX(+GJ,1.6 pmales MTX(+G@), and 1.0 pmole MTX(+G11).Theseresults showed that the affinities of MTX polyglutamates fordihydrofalate reductase were similar if not identical to that ofMTX. Similar results were obtained using dihydnofalate meductase partially purified from guinea pig liver (not shown).
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Table3Levelsof MTXand metabolitesin L1210and L1210/MTXcells 4 and 24hr after a singlei.p.injection
of (3HJMTX (2.4 mg/kg) into host DBA/2miceResults
are expressed as ng MTX or equivalent per [email protected]
of MTXand metabolites in cells (ng/i09 cells)No. of Time
Cells studies (hr) MTX MTX(+G,) MTX(+G2)TotalLi2iO
• 3 4 387 ±143 66 ± 6 275 ±169 727 ±8i324 149 ± 28 20 ± 8 77 ± 31 246 ±66Li2iO/MTX
2 4 3602±765 277 ±29 248 ± 50 4127 ±844224 2199± 4 154±36 211± 52 2563± 84
MTX Polyglutamates in L1210 Cells
sensitive to MTX toxicity, might backthis metabolic pathwayas well. Indeed, almost no metabolism of MTX occurred ineither Li 2i 0 or Li 210/MTX cells with doses previously usedin the rat (Table 2). However, this may have been due in partto failure of MTX to accumulate in sufficient amount inascitic fluid after s.c. injection (20). When a dose of MTX(2.4 mg/kg) was used that was comparable to the doserecommended for the reduction of the L12i0 leukemia cellmass (3.0 mg/kg host weight) (8), extensive metabolism ofMTX was indeed seen, both with divided s.c. administrationand with single i.p. doses. Synthesis of MTX pobyglutamatesalso occurred in mouse small intestine with these doses.Thus, sensitivity to MTX toxicity did not equate with failureto synthesize MTX palyglutamates. Nor could resistance toMTX toxicity, which characterizes the L1210/MTX mutantcell line, be attributed to differences in MTX pobyglutamatesynthesis, since the total quantity of MTX polyglutamateswas the same in both cell lines.
MTX(+G@1)was the predominant metabolite in Li2iO cells.However, MTX polyglutamates larger than MTX(+G1), e.g.,MTX(+G3), did not separate well from it under the conditions of chromatography used in the present study. Theserequire a large column to be visualized (unpublished abservation). Therefore, it is possible that L12i0 cells synthesizedsome MTX polyglutamates containing more than 2 additionab -y-glutamyb residues (Chart iA). In contrast, the majormetabolite of MTX in other tissues studied to date has beenMTX(+G1). Each of the tissues studied here showed a distinctive pattern of distribution of MTX and metabolites. Itmay be speculated that the predominance of MTX(+G@)inLi210 cells reflects a difference in the enzyme pteroybpolyglutamate synthetase in this cell with regard to substratepreference. Further, it is possible that differences in metabobism of MTX in different tissues may reflect differences inpteroylpolyglutamate synthesis in these tissues as well. Levebs of total MTX pobyglutamates in Li2iO and Li2iO/MTXcells were the same despite the high level of MTX in thelatter, reflecting no doubt the high level of dihydrofolatereductase that characterizes this mutant (7). It was concbuded that synthesis of MTX polyglutamates was not noticeably affected by this bargedifference in level of dihydrofobate reductase.
The concept of “free―intracellular MTX, or of bevels ofMTX in cells in excess of that required to bind to dihydrofolate reductase (9, 12, 21), has taken on importance as aresult of new therapeutic initiatives, with the use of highdose MTX (14), and following a number of studies whichhave credited this intracellular portion of MTX with a criticalrole in the inhibition of folate metabolism (9, 12, 16, 21).
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Chart2.Comparisonof theaffinityof MTXandMTXpobyglutamatesfordihydrofobate reductase present in lysates of (A) L1210 cells and (B) L1210/MTX cells, using a radiochemical bigand-binding assay for MTX (1, 15).Incubation mixtures consisted of 0.9 ml of 0.01 M potassium phosphate, 0.15M KCI buffer (pH 6.0) containing 1 mg albumin and 0.1 @moIeNADPH per ml,0.1 ml MTX or MTX polyglutamate solution containing 2.2 to 90.0 pmoles/ml,and 0.1 ml of [3H]MTX containing 15 pmoles/mb. Incubation was started withthe addition of 0.1 ml binder (lysate) preparation (binding capacity, 1 to 1.2pmoles MTx). After 10 mm of incubation at 4°,the reaction was stopped bytheadditionof 1.0mlofasuspensionof2.5%charcoaland0.5%DextranT10. Standard curves prepared with MTX (—, •),with MTX(+G,) (- - - -),with MTX(+G,) (—. _.), and with MTx(+G6) ( , 0) are shown.
These studies were carried out at pH 6.0. Essentially identicab results were obtained when the affinities of MTX andMTX(+G@)far dihydrofolate reductase from Li 210/MTX andguinea pig liver were compared at pH 7.0 and 8.0.
DISCUSSION
MTX polyglutamates were previously identified in rat tissues (2, 24, 25). In these studies, MTX(+GJ and MTX(+G1)were recognized as 3H-labeled materials that produced folate-reversible inhibition of growth of the fobate assay organisms L. casei and Streptococcus fecalis. They were identified as pobyglutamates by the demonstration that they chromatognaphed as MTX after incubation with a source ofpteroylpalygbutamate hydmobase(‘y-glutamybcarboxypeptidase, falate conjugase) (2, 24, 25). Their identities wereestablished by cochromatography using authentic standards of MTX(+G@)and of MTX(+G11)(17). The demonstration of synthesis of MTX(+G1) and of MTX(+G@) in bothL12i0 and Li2iO/MTX cebls(Chart i)extendsthe number oftissues in which such metabolism of MTX has been found tooccur (2, 5, 10, 13, 19, 24, 25). Not surprisingly, synthesis ofMTX polyglutamates was also found in host mouse tissues(Table 1), confirming previous observations (22).
The present results were not entirely anticipated. Because rat small intestine did not synthesize MTX pabyglutamates (24, 25) (and small intestine is exquisitely sensitive toMTX toxicity), it appeared likely that L12i0 cells, themselves
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Sirotnak and Donsbach (20) reported levels of MTX in Li 210cells 4 hr after an i.p. dose of MTX (3.0 mg/kg) to be greaterthan the bevel of dihydrofolate reductase. This bevel hadfallen by 24 hr, and the fall was attributed to the lass offreely exchangeable MTX present within these cells at theearlier time. If this were so, one would have expected adisproportionate fall in level of MTX compared to MTXpolyglutamates in Li210 cells at 24 hr in this study. In fact,the proportion of MTX and metabolites was the same atboth times. These results do not define a so-cabled “labile―portion of MTX at 4 hr, nor do they explain the subsequentfall. It is possible but unlikely that MTX and metaboliteswere released from living Li2iO cells at the same rate.However, this was not seen with rat liver (24, 25). It is morelikely that the fall in bevelof MTX and metabolites reflectedthe combined effect of cell death and of division and growthof the surviving population.
Jacobseta!. (13) studied the ability of MTX(+GJ to inhibitthe activity of dihydrofobate reductase purified from L12i0
cells. They found it to be as potent as MTX. In fact, itappeared to be a more potent inhibitor than MTX whenstudied at pH 8.5. Further, they showed that growth of
Li2iO cells in culture was inhibited equably by MTX andMTX(+G1) and that MTX(+G3) had antitumor activity simibar to that of MTX in viva in prolonging the life-span ofBALB/c x DBA/2 F1mice bearing the Li2iO leukemia. Thedemonstration in this study that MTX pobyglutamates boundas tightly to dihydmofolate reductase as did MTX (Chart 2)provides further evidence that these metabobites are as patent antifolates as is MTX and that decreased membranetransport of MTX polyglutamates appears to be the majorreason for their lesser toxicity in whole-cell systems such asS. feca!is (17). These results also demonstrate the insensitivity of the MTX binding site on dihydrofolate neductaseregarding the number of glutamyb residues present. A simibar observation has been made regarding this enzyme and
poly-y-glutamyb derivatives of dihydrofolate (6). Lastly, thisstudy showed that measurement by ligand-binding madioassay (15) of total tissue MTX and metabobites can be carriedout with a single standard curve and without the need toisolate constituents.
It has been speculated that binding of MTX to intracellularsites of bower binding affinity than that for dihydrofobatereductaseisrequiredforinhibitionoffobatemetabolism(9).Suggested targets have been thymidybate synthetase (EC2.1 .1.b), and the fobate-dependent enzymes involved in puminesynthesis. Present results would suggest that MTX polyglutamates comprise part of this component of intracelbulamMTX.
ACKNOWLEDGMENTS
The author thanks Marlene Dupont, Maureen McCormack, and VivianeNicolet for technical assistance; Charles Baugh and M. G. Nair for the kindprovisionof MTXpolyglutamates;and Bart Kamenfor his advicewith theradioassay.
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