Antifolate Polyglutamylation and Competitive Drug Displacement at Dihydrofolate ReducãAseas Important Elements in Leucovorin Rescue in L1210 Cells1

[CANCER RESEARCH 46, 588-593, February 1986]

Antifolate Polyglutamylation and Competitive Drug Displacement at Dihydrofolate ReducÃaseas Important Elements in Leucovorin Rescue in L1210 Cells1

Larry H. Matherly,2 Charles K. Barlowe, and I. David Goldman

Departments of Medicine and Pharmacology, Medical College of Virgin/a, Richmond, Virginia 23298

ABSTRACT appear lo represent an importanl factor for the selectivity of leucovorin rescue in vivo. Previous studies from this laboratory have shown that the addition of leucovorin to tumor cells dissociates methotrexate, but not methotrexate polyglutamates, from dihydrofolate reduc INTRODUCTION Ãase(L. H. Matherly, D. W. Fry, and I. D. Goldman, Cancer Res., 43: 2694-2699, 1983). To further assess the importance of The administration of MTX3 in conjunction with a source of these interactions to leucovorin rescue, antifolate growth inhibi reduced folate cofactors enhances the therapeutic efficacy of tion toward L1210 cells in the presence of leucovorin was this agent in tumor-bearing animals (1, 2). The original pharma correlated with the metabolism of (6S)-5-formyl tetrahydrofolate cological premise of this rescue of host tissues from drug toxicity to dihydrofolate as a measure of dihydrofolate reducÃaseactivity. was thai the added tetrahydrofolate would circumvent the MTX Growth inhibition (greater than 95%) by methotrexate (5-10 UM) block at the level of the DHFR, allowing for continued purine and following its intracellular polyglutamylation during a 3-h preex- pyrimidine biosynthesis for cell division. While no obvious basis posure, or by continuous treatment with high levels of the for the demonstraled therapeutic selectivity of this approach is lipophilic antifolate, trimetrexate (1 /UM),was only slightly dimin apparent from these considerations alone, the success of Ihese ished by 10 MMleucovorin (15-25%). High-pressure liquid Chro regimens in experimenlal systems has nonetheless led lo their matographie analyses of the derivatives formed from radiolabeled clinical implementation (3, 4). (6S)-5-formyl tetrahydrofolate under these conditions showed an Primarily because of its chemical stability, rescue protocols have generally used calcium leucovorin [(6fÃ®,S)-5-CHO-FH4]. incomplete conversion to dihydrofolate and metabolism to pre While not a nalural cellular consliluent, the (6S)-isomer of this dominantly 10-formyl tetrahydrofolate. Neither of the antifolates compound is readily transported into cells by the MTX-lelra- interfered appreciably with the metabolism of the folate deriva hydrofolate cofactor carrier (5), and, as depicted in Fig. 1, can tives to polyglutamates. Growth inhibition in the presence of be assimilated into cellular folate pools following its metabolism leucovorin correlated with the accumulation of dihydrofolate initially to (a) 5,10-CH+-FH4 and, subsequently, lo (o) 10-CHO- (1.5-2.2 nmol) from radiolabeled (6S)-5-formyl tetrahydrofolate, FH4, Ihe one carbon donor for purine biosynlhesis, or (c) 5,10- reflecting continued suppression of dihydrofolate reducÃaseac CH2-FH4, Ihe cofactor for thymidylate biosynlhesis. While (6S)- tivity at these drug concentrations. With lower equitoxic levels 5-CHO-FH4 is rapidly melabolized in tumor cells in Ihe presence of the trimetrexate (7.5 nM), the provision of leucovorin allowed (6) and absence (7) of MTX, and can readily support cell division for a restoration of cell growth to a level greater than 90% of (8), il is unclear whelher Ihe rates for these processes for control. Under these conditions, control levels of dihydrofolate cofactor uplake and inlerconversion are sufficienl for cellular (0.2 nmol) were formed from radiolabeled cofactor, consistent replication when DHFR is inactivated by antifolates (9). with sustained dihydrofolate reducÃaseactivity. Several laboratories have reported lhal MTX and reduced These findings support a role for the activation of dihydrofolate folates interacl competitively during rescue, a finding lhat cannot reducÃase as an important component of the reversal of the easily be reconciled soley on the basis of an anabolic role for the effects of diaminoantifolates by leucovorin, presumably by a exogeneously supplied cofactor. While this competitive relation competitive displacement of drug from the enzyme. Since no ship between these folate derivatives has been attributed to displacement occurs in cells which have accumulated methotrex direcl binding interactions al Ihe level of Iheir shared membrane ate polyglutamates, or in the presence of high levels of Irime- Iransport carrier (5, 10), similar interactions have been demon- trexate, il appears that the concenlralion of unbound drug wilhin slraled belween telrahydrofolale cofactors and lipophilic 2,4- cells is a significanl determinant of the extent of this competitive diaminoantifolales which bind to DHFR but penelrate cells by binding interaclion. From these considerations, Ihe high levels of mechanisms distinct from the MTX-telrahydrofolale cofactor car methotrexate polyglutamates thai accumulate in sensitive tu rier (8, 11). This suggesls lhal olher interactions al common mors relative lo bone marrow and gaslrointestinal cells would cellular loci are also involved during Ihe reversal of antifolate action by lelrahydrofolales. Received 6/27/85; revised 10/9/85; accepted 10/11/85. The costs of publication of this article were defrayed in part by the payment of 3The abbreviations used are: MTX, methotrexate; 5-CHO-FH4, 5-formyl tetra page charges. This article must therefore be hereby marked advertisement in hydrofolate; 10-CHO-FH4, 10-formyl tetrahydrofolate; 5,10-CH+-FH4, 5,10-meth- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. enyl tetrahydrofolate; 5,10-CH2-FH4, 5,10-methylene tetrahydrofolate; 5-CH3-FH4, ' Supported by Research Grant CA-16906 and Training Grant CA-09340 from 5-methyl tetrahydrofolate; DHFR, dihydrofolate reducÃase;FH2, dihydrofolate; FH4, the National Cancer Institute, NIH. tetrahydrofolate; GAT, glycine (200 ^M), adenosine (100 Â»IM),and thymidine (10 'Recipient of a Junior Faculty Research Award from the American Cancer fiM); TMQ, trimetrexate (2,4-diamino-5-methyl-6-[(3,4,5-trimethoxyanilino)-methyl] Society. quinazoline); HPLC, high-pressure liquid chromatography.

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Recent interest has focused on a potential role for competitive cells in drug-free medium. TMQ treatment was also for 3 h prior to interactions between MIX and folate cofactors derived from addition of the radiolabeled folate. However, TMQ exposure was contin ued throughout the experiment. [3H]-<6S)-5-CHO-FH4 (5 /IM) was added leucovorin at the level of DHFR as a basis for the reduction of drug activity during rescue (9,12-14). Studies from this labora and radiolabeled intracellular constituents were measured after centrifugation of samples of the cell suspension (8 x 107 cells) at 0Â°C(500 x tory have shown that exogenous tetrahydrofolates in sufficient g for 2 min). The supematants were sampled and analyzed immediately concentrations effect a net displacement of MIX bound to DHFR by HPLC as described below; the cell pellets were washed twice with in cells (13). Significantly, for cells which have accumulated 10 volumes of a 0.85% 0Â°Csaline solution and a portion of the washed appreciable levels of MIX polyglutamates, no net loss of en cells was aspirated into the tip of a Pasteur pipet and extruded onto a zyme-bound antifolate occurs (13). These findings suggest a polyethylene tare. After drying ovenight at 70Â°C, the cell pellets were potential role for MTX polyglutamylation as a selective cellular weighed directly on a Cahn model 4700 electrobalance, placed in scintil determinant of leucovorin rescue in vivo. The present report lation vials, and digested in 0.2 ml of 1 N KOH for 1 h (70Â°C). After explores the molecular relationship between antifolate polyglu neutralization with 0.2 ml of 1 N HCI, radioactivity was measured with a tamylation, binding to DHFR, and the reversal of drug activity by Packard 460C liquid scintillation spectrometer using Readi Solv scintil tetrahydrofolate cofactors. lation cocktail (Beckman, Irving, CA). Corrections for counting efficiencies were made by internal standardization with tritiated toluene. In this fashion net uptake of radiolabeled folate was expressed as nmol of MATERIALS AND METHODS tritium per gram of dried cell pellet. Intracellular water was determined from the difference between the wet and dry weights of the cell pellet, Chemicals. [3',5',7,9-3H]Folic acid was purchased from Amersham less the [14C]inulin space as described elsewhere (5). A value of 0.054 (Arlington Heights, IL). MTX and TMQ (glucuronate salt) were obtained Â±0.005 (SD) ml of intracellular water per 10e cells was determined (n = from the Drug Development Branch, National Cancer Institute, Bethesda, 4). MD. MTX was purified as described previously (15). (6fl,S)-5-CHO-FH4, For the analysis of intracellular folate forms a portion of the washed (6fl,S)-5-CH3-FH4, and (6fl,S)-FH4 were purchased from Sigma Chemical cell pellet was treated with 1 ml of nitrogen-saturated 0.1 M sodium Co. (St. Louis, MO). Standard folates, including 10-CHO-FH4 (16), 5,10- malÃ©ate,pH 7.2, containing 1% 2-mercaptoethanol. The centrifuge tube CH+-FH4 (16), and FH2 (17) were prepared as described previously. was sealed with a serum stopper and the contents were evacuated with [3',5',7,9-3H]-(6S)-5-CHO-FH4 and unlabeled (6S)-5-CHO-FHÂ«were pre a vacuum pump and flushed with nitrogen through a syringe needle. The pared from radiolabeled folie acid and unlabeled FH2, respectively, as cellular suspension was heated in a boiling water bath for 90 s. After described by Moran and Colman (18), except that all procedures were cooling to 0Â°C, the extract was incubated overnight at 32Â°C under conducted under a nitrogen atmosphere as described below. Other nitrogen with a conjugase prepared from chicken pancreas (19). This chemicals were obtained from commercial sources. treatment resulted in an essentially complete (greater than 95%) hydrol Cell Culture. The murine L1210 leukemia was grown in a humidified ysis of the polyglutamate folates to the monoglutamyl forms. A nonen- atmosphere at 37Â°C in RPMI 1640 medium supplemented with 10% zymic conversion of 10-CHO-FH4 to 5-CHO-FH4 (25%) and 5,10-O-T- heat-inactivated dialyzed fetal calf serum, 2 mw i -glutamina, penicillin FH4 (less than 10%) occurred during the extraction and hydrolytic pro (100 units/ml) and streptomycin (100 M9/ml), all purchased from Gibco cedures. While some loss of FH4 was also encountered (approximately Laboratories (Grand Island, NY). Cell folate pools were depleted by 32%) the recoveries of 5-CH3-FH4 and FH2 were essentially quantitative culturing the cells for several weeks in RPMI 1640 without folie acid (greater than 90%). Under these conditions, 5,10-CH2-FH4 appears as (Gibco Laboratories) in the continuous presence of GAT. Inocula were FH4. The degradation products detected both intracellularly and extra- made at 1 x 105 cells/ml and cell transfers were made every 2 days. cellularly following HPLC analysis (see below) were particularly prevalent Cell numbers were determined with a Coulter counter or by direct following extended incubations with the radiolabeled folate (generally 20 microscopic examination. For the growth inhibition experiments, 2.5-ml to 30% of total extracellular and intracellular radiolabel after 3 h). These cultures (1 x 10s cells/ml), pretreated with MTX for 3 h or continously products were attributable primarily to oxidative degradative processes exposed to TMQ, as described in the text, were incubated for 48 h. After during the incubations; the extent of this nonenzymic decomposition was this interval untreated cells had attained a density of approximately 1.5- not reduced by decreasing the oxygen levels to 20%. 2.0 x 106 cells/ml. For analysis of total cellular folate polyglutamates, an additional portion Incubation Techniques and Extraction of Cellular Folates. Cells of washed cells was boiled in 50 ITIM sodium phosphate, pH 7.2, and (approximately 4x10' cell/ml) were incubated in specially designed 1% 2-mercaptoethanol for 3 min followed by the chemical cleavage of flasks under an atmosphere of 95% 02-5% C02 in complete folate-free the folate polyglutamates to the corresponding p-aminobenzoyl polyglu medium containing containing 0.02 mw 2-mercaptoethanol in the pres tamates using the cleavage procedure of Fco, er a/. (20). ence of GAT. MTX treatment was for 3 h followed by resuspension of HPLC Analysis of Folyl Mono- and Polyglutamates. For analysis of the natural folates following the enzymic hydrolysis of the polygluta mates, the extracts were boiled for 90 s followed by centrifugation. Thymidylote Samples were analyzed on an Altex Model 332 gradient liquid Chromat ograph equipped with a Model 210 injector using a 5-^m Spherisorb NAOH NAD* octadecylsilyl column (IBM Instruments, Danbury, CT). The HPLC anal ^â€”->N5-Methyl FH4 ysis consisted of a gradient of from 0 to 2.6% acetonitrile over 2 min followed by an increase to 6.75% acetonitrile from 9 to 14 min. The flow Purines rate was 2 ml/min and 0.5-min fractions were collected and measured for radioactivity as described above. The retention times for the radiola beled cofactors were compared to those for standard folates detected Nlo-Formyl FH4 Â«s Â»N5,N'Â°-Methenyl FH4 by UV monitoring at 280 nm (Table 1). ^-Â»ADP, Pi p-Aminobenzoyl polyglutamates from the reductive cleavage proce dure were analyzed on a 1O^m Spherisorb octadecylsilyl column (Brown- ATP lee Laboratories, Santa Clara, CA) using a linear gradient of from 20 to 5-Formyl FH4 35% acetonitrile in 2.5 rriM tetrabutylammonium phosphate in 5 mw Fig. 1. Pathways of metabolism of (6S)-5-CHO-FH4. sodium phosphate, pH 6.5, over 15 min, followed by an isocratic elution

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Table 1 35% of control) was observed when the leucovorin concentration HPLCretention times for standard folates was increased to 100 MM(not shown). This incomplete abolition time of antifolate activity by leucovorin under these conditions con Derivativep-Aminobenzoic (min)2.67.58.31113.5161717.2trasts with the essentially complete protection afforded drug- acid10-CHO-FH4FH,5-CHO-FH45,10-Or-FH,FH25-CH3-FH45,10-CH2-FH4Retentiontreated cells by continuous exposure to GAT (96.22 Â±8.68% of control at 10 MMMTX, n = 4; also, see rÃ©f.21). Reversal of TMQ Cytotoxicity to L1210 Cells by Leucovorin. While the metabolism of MTX to its polyglutamyl forms inhibits cell division in the presence of folie acid or leucovorin, a greater reversal of the pharmacological effects of equitoxic concentra tions of the lipophilic quinazoline, TMQ, can be achieved by exogenous leucovorin (Fig. 3). TMQ binds avidly to DHFR (8); however, this agent neither competes with tetrahydrofolate co too factors for cell entry (26,27), nor possesses the stuctural require ments for polyglutamylation by the folylpolyglutamate synthetase. The pharmacological effects of TMQ are reversible follow ing a brief drug exposure (data not shown) consistent with the lack of metabolism of this agent to retained derivatives analogous to MTX polyglutamates. However, continuous treatment with this drug potently inhibits the replication of folate-depleted cells incubated with 5 MMfolie acid (Fig. 3). In contrast to the findings with MTX, at concentrations of TMQ (1-10 nM) that inhibit cell growth in the presence of folie acid, drug activity was completely IO"' IO abolished by the addition of 10 MMleucovorin; however, at higher MIX (M) TMQ levels a lesser diminution of the growth inhibition was Fig. 2. Effect of MTX on growth of L1210 cells in the presence of folie acid (5 f!M)or calciumleucovorin(10 >M).L1210 cells were exposed to the indicatedlevels achieved by the reduced cofactor. At 1 MMTMQ, only 14.16 Â± of MTX for 3 h, resuspended into drug-free medium (at 1 x 10scells/ml), and cell 3.76% (n = 4) of control levels of growth was sustained in the growth was monitored after 48 h. The results are expressed as the mean Â±SD presence of 10 MMleucovorin. As observed in the studies with from 4 incubations in 2 separate experiments. MTX, essentially complete protection was afforded by the pro vision of GAT along with the antifolate (91.42 Â±11.42 at 1 MM at 35% acetonitrile for an additional 5 min. The flow rate was 2 ml/min and 0.5-min fractions were collected and measured for radioactivity as TMQ, n = 4; not shown). Metabolism of [3H]-(6S)-5-CHO-FH4 in the Presence of 2,4- described above. Radiolabeled p-aminobenzoyl polyglutamates were identified by comparison of their retention times with authentic standards Diaminoantifolates. To better understand the relationships (Glu,.?) provided by Dr. Barry Shane (University of California at Berkeley). among the extent of reversal of antifolate activities in vitro, The standards were detected by UV monitoring at 254 nm. extracellular drug concentration, and the effects of MTX polyglu tamylation on leucovorin utilization, the radiolabeled (6S)-isomer of 5-CHO-FH4 was prepared and its metabolism was evaluated RESULTS under conditions simulating those in the growth inhibition exper Effect of Leucovorin on the Cytotoxicity of MTX to L1210 iments. Previous reports have described extensive metabolism Cells. Folie acid (5 UM) or leucovorin (50 nw) can meet the of (6S)-5-CHO-FH4 in L1210 cells grown in folate replete medium anabolic requirements for tetrahydrofolates in L1210 cells pre (6, 7). However, in the present studies, as described above, the viously depleted of endogenous folates. Conversely, pretreat tumor cells had previously been depleted of their endogenous ment of these folate-depleted cells with MTX for 3 h followed by folate stores prior to exposure to the radiolabeled cofactor. This their resuspension into drug free medium limits cell division in approach allows a more accurate assessment of tetrahydrofolate the presence of these compounds (Fig. 2). Under these condi tions growth inhibition arises from the accumulation of MTX polyglutamyl derivatives during the initial 3-h exposure to drug (at 10 MMMTX, approximately 5- to 8-fold greater than the level of DHFR). In contrast to unconjugated drug, these derivatives are selectively retained intracellularly in the absence of extracel lular antifolate (15, 21-25). Hence, this metabolism serves to prolong antifolate activity in the absence of continuous drug exposure (21, 22, 24). In Fig. 2, a 50% inhibitory concentration of 0.59 /Â¿Mwas measured in the presence of 5 UM folie acid following MTX pretreatment; this changes only slightly when 10 /Â¿Mofleucovorin is provided as a source of reduced cofactors TMO(M) (50% inhibitory concentration, 2.2 pM). At the most inhibitory Fig. 3. Effect of continuous exposure to TMQ on growth of L1210 cells in the presence of 5 UMfolie acid or calcium leucovorin (10 ^M). Cells (1 x 105/ml)were levels of MTX (greater than 5 tiM), only a partial reversal of drug activity was achieved by the addition of leucovorin (20-25%). continuouslyexposed to the indicatedlevelsof TMQ and cellgrowth was monitored over 48 h. The resultsare expressedas the meanÂ±SDfrom 4 separateincubations Only a slightly greater reversal of drug activity (to approximately in 2 separate experiments.

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Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1986 American Association for Cancer Research. ELEMENTS IN LEUCOVORIN RESCUE metabolism without isotope dilution or metabolic perturbations associated with endogenous folate polyglutamates. Folate-depleted L1210 cells were incubated with 5 MM [3H]- (6S)-5-CHO-FH4, following which cellular folates were extracted under nitrogen, enzymatically hydrolyzed to their monoglutamyl forms, and analyzed by HPLC as described in "Materials and IO-CHO- Methods." Fig. 4 depicts the uptake and metabolism of this FH4 tritiated folate in untreated L1210 cells and cells pretreated with nmol MTX (10 MM)for 3 h so as to form high levels of MIX polyglu tamates, following which extracellular drug was removed. In the untreated cells, predominantly 10-CHO-FH4 accumulated over this interval (65% of total folates), with lower levels of 5-CHO- FH4, and other tetrahydrofolates (5-CH3-FH4 and FH4). Only very low levels of [3H]FH2 were detected intracellularly under these conditions (less than 0.1 UM).The major effect on the intracellular folate distributions following MTX pretreatment was the high Contro) MTX TMQ level of FH2 accumulated (approximately 4 MMby 30 min), con Fig. 5. Total intracellular and extracellular metabolites of [3H]-<6S)-5-CHO-FH4 in L1210 cells. Folate-depleted L1210 cells (1 x 10'), either untreated or treated sistent with the potent inhibition of DHFR by the MTX derivatives. with the antifolates as described in the text, were incubated with 5 MM[3H]-{6S)-5- However, even in the presence of these antifolate derivatives, CHO-FH, for 3 h, followed by quantitation of the intracellular (â€¢)andextracellular folate metabolites (Ãœ)as described in "Materials and Methods." Only the major substantial levels of the various tetrahydrofolates were present derivatives are shown since no significant FH4 or 5-CH3-FH4 was detected extra- as well (75% of cellular folates in the experiment shown). Similar cellularly. findings were obtained in cells continuously exposed to 1 MM TMQ (data not shown). Table 2 After 3 h, a similar distribution of intracellular radiolabeled Effect of antifolate treatment on folate polyglutamylation cofactors was found (Fig. 5). Greater than 70% of these intra Folate-free L1210 cells treated as described in the text were incubated with [3H] -(6S)-5-CHO-FH4 (5 MM)for 3 h, followed by determination of cellular folate polyglu cellular derivatives were present as polyglutamates by 3 h, tamates as described in "Materials and Methods." The data presented are the distributed among cofactor forms containing up to 4 additional mean values from 3 separate experiments. glutamates (Table 2). The total intracellular folate levels were not % of polyglutamate distribution3 significantly affected by drug treatment. Moreover, the polyglu- Total folates Treatment 1 tamyl distribution was only slightly altered, with a small increase (MM) in the tetraglutamyl form at the expense of the other derivatives, None 14.71 14.11 12.63 26.06 29.86 17.33 MTX (10 MM) 14.92 19.66 12.40 17.80 36.67 13.46 in the presence of the antifolates. TMQ (1 MM) 14.74 24.91 11.51 15.82 37.21 10.56 Of particular interest was the finding after 3 h that considerable " The numbers (1-5) designate the total glutamate residues associated with p- aminobenzoate and, hence, the folate derivatives.

CONTROL levels of monoglutamyl metabolites of (6S)-5-CHO-FH4 had ac cumulated extracellularly (Fig. 5). Indeed, 89% of the total folate metabolites derived from [3H]-(6S)-5-CHO-FH4 was present ex- tracellularty as 10-CHO-FH4 in untreated cells following a 3-h incubation. For cells pretreated with 10 MMMTX (3 h) and cells continuously exposed to 1 MM TMQ, a small but significant reduction in the level of 10-CHO-FH4 was observed; extracellular 10-CHO-FH4 under these conditions comprised 71 and 63%, respectively, of the total metabolites. While considerable FH2 was detected both intracellularly and extracellularly in the pres MTX ence of these agents, the total level (extracellular and intracellu lar) of this radiolabeled oxidized derivative was comparatively small, comprising only 24 and 33%, respectively, of the total folate metabolites in the presence of MTX and TMQ. Hence, while the cellular metabolism of (6S)-5-CHO-FH4 is extensive under these conditions, the utilization of this reduced cofactor for thymidylate biosynthesis is apparently limited. Relationship between DHFR Activity and the Reduction of Antifolate Activity by Leucovorin. Fig. 6 presents data which IO 20 30 suggest that the abolition of 2,4-diaminoantifolate activity by MINUTES Fig. 4. Uptake and metabolism of [3HJ-(6S)-5-CHO-FH4. Control cells and cells leucovorin is associated with the activation of cellular DHFR as pretreated with MTX (10 MM)for 3 h were incubated with [3H]-(6S)-5-CHO-FH4. manifested by the maintenance of low levels of FH2. Here the Total cellular tritium and cofactor distributions were determined as described in the extent of cell growth in the presence of 10 MMleucovorin under text. Data are presented for the accumulation of 10-CHO-FH4 (O); 5-CHO-FH, (â€¢); FH2 (A); and other tetrahydrofolates P). The data shown for the accumulation of various conditions is correlated with the total (sum of intracellular 5-CHO-FH4 include the radioactivity cochromatographing with 5,10-CH*-FH4. and extracellular) accumulation of FH2 derived from radiolabeled

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140 PERCENT GROWTH 2.8 critical determinant of the extent of drug displacement is the DIHYDROFOLATE level of free antifolate in the vicintiy of DHFR at the time of 120 2.4 provision of the tetrahydrofolate derivative. Indeed, if this free i 100 2.0 intracellular drug component is elevated, no significant competi tion at the level of DHFR is observed (13). Similarly, in cells which l 80 1.6 have accumulated MTX polyglutamates to a concentration ex |â€” ceeding that of the DHFR, intracellular folates derived from Ã¶ 60 1.2 tr leucovorin do not effectively compete with the drug for enzyme u "- 40 binding (13). 0.8 Consistent with these findings is the observation that the

2O 0.4 metabolism of MTX to polyglutamyl derivatives by L1210 cells limits the extent of rescue achieved by leucovorin in vitro. A 0L similar inverse relationship between the accumulation of intra 10 pM luM MTX TMQ cellular MTX polyglutamates and the reversal of drug activity by Fig. 6. Relationship between reduction of antifolate effects on cell growth by leucovorin has been suggested (31, 32). Moreover, the present leucovorin and DHFR activity. Cells (1x10Â°) were incubated with 5 pM radiolabeled study has demonstrated that an essentially identical effect can (6S(-5-CHO-FH4 for 3 h and the total level of tritiated FH2 (expressed in total nmol be obtained by the continuous exposure of cells to the structur accumulated as the sum of extracellular and mtracellular cofactor) was determined as described in "Materials and Methods." The data reflect the mean Â±SD from ally dissimilar drug, TMQ; however, at lower equitoxic levels of duplicate experiments. The extent of growth inhibition was taken from the results TMQ, a significant diminution of antifolate activity was achieved presented in Figures 2 and 3. with leucovorin. In this report a correlation has been presented between the (6S)-5-CHO-FH4 over a 3-h interval. While only low concentra reduction of antifolate activity in tumor cells by leucovorin and tions of FH2 were formed in untreated cells (0.24 nmol), consid the relative activity of intracellular DHFR. For TMQ, the extent erable levels of this derivative (1.46 nmol) accumulated in the of reduction of cytotoxicity closely paralleled the functional level tumor cells pretreated with MTX. Similarly, at a level of TMQ (1 of DHFR activity, as reflected in the intracellular accumulation of Ã•/M)which rendered cells relatively insensitive to exogenous radiolabeled FH2 from exogenous (6S)-5-CHO-FH4. Hence, leucovorin, high levels of FH2were present (2.16 nmol). However, DHFR reactivation, presumably arising from direct displacement under conditions in which leucovorin appreciably reversed TMQ of enzyme-bound TMQ, analogous to unmetabolized MTX, is a cytotoxicity (7.5 nw and 0.1 Â¿Â¿M),therewas a proportional reduc key event in the reversal of the pharmacological effects of this tion in the level of radiolabeled FH2 detected, suggesting a antifolate by leucovorin. Antifolate displacement from cellular reactivation to DHFR under these conditions. DHFR is not possible for cells which have accumulated apprecia ble levels of MTX polyglutamates (13). Under these conditions DISCUSSION cytotoxicity is sustained even when leucovorin is supplied be cause reactivation of the enzyme is not possible. Similarly, in the This report explores the relationship between tetrahydrofolate continuous presence of high levels of the TMQ, net displacement cofactor metabolism and the pharmacological activities of 2,4- of antifolate from DHFR is not possible, resulting in the expres diaminoantifolates under conditions of leucovorin rescue in vitro. sion of cytotoxicity even when the rescue agent is provided. While the original basis of the apparent selective reversal of MTX On this basis, it seems that the conversion of MTX to its activity by leucovorin in vivo was that an exogenous source of polyglutamyl forms may be an important determinant of the reduced folates, when appropriately administered, would circum selectivity of leucovorin rescue in much the same fashion that vent the drug-induced block at DHFR, considerable experimental this differential metabolism appears to be a key element in the evidence now supports alternative mechanisms for rescue. therapeutic selectivity of MTX. These polyglutamyl derivatives of The finding of competition between MTX and FH2 at the level MTX are synthesized to a much greater extent in susceptible of the intracellular DHFR (28-30) led to the suggestion that tumors than in bone marrow (21) and gastrointestinal cells (33, exogenous leucovorin might increase the intracellular FH2 pool, 34), contributing to a sustained intracellular level of antifolate resulting in a competitive displacement of a small amount of even as the plasma drug concentration falls. In the sensitive host bound antifolate sufficient to reverse the pharmacological effects tissue, conversely, the rapid loss of underivatized MTX parallels of the drug. Further studies from this laboratory demonstrated the plasma drug clearance (33). The elevated levels of MTX that leucovorin, if present in sufficient concentrations, promotes polyglutamates that accumulate in tumor cells would also pre the net dissociation of underivatized MTX bound to DHFR in clude any significant displacement of antifolate from cellular tumor cells (13). While this effect could involve the formation of DHFR by the provision of exogenous tetrahydrofolates. On the cellular FH2 from the added cofactor and consequent displace other hand, the low levels of MTX polyglutamates formed in ment of drug from DHFR by this oxidized derivative, reduced bone marrow and gastrointestinal cells should permit a direct folates have also been found to displace enzyme-bound MTX in displacement of bound antifolate by cellular folates derived from cells even when thymidylate synthase is inhibited by treatment leucovorin, allowing a selective restoration of endogenous tet with 5-fluoro-2'-dexoyuridine preventing the build-up of FH2 (13). rahydrofolate biosynthesis in these tissues. Further, reduced folates displace drug tightly bound to purified The finding in the present study that (6S)-5-CHO-FH4 is exten DHFR (12). sively metabolized to 10-CHO-FH4, even in the presence of the Regardless of the actual folate derivative involved in the re antifolates, with incomplete conversion to FH2, raises the possi placement of the antifolate on the enzyme active site in cells, the bility that tetrahydrofolate utilization is somehow limited under

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Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1986 American Association for Cancer Research. ELEMENTS IN LEUCOVORIN RESCUE these conditions. This contrasts with previous suggestions of a 15. Fry, D. W., Yalowich, J. C., and Goldman, I. D. Rapid formation of polyglutamyl derivatives of methotrexate and their association with dihydrofolate reducÃase rapid interconversion of cofactor forms and maximal depletion of as assessed by high pressure liquid chromatography in the Ehrlich ascites reduced cellular folates following drug exposure (28, 30). While tumor cell in vitro. J. Biol. Chem., 257:1890-1896,1982. no obvious perturbations by the antifolates on either the mon- 16. Rabinowtiz, J. C. Preparation and properties of 5,10-methenyltetrahydrofolic acid and 10-formyltetrahydrofolic acid. Methods Enzymol., 6: 814-815,1963. oglutamate or polyglutamate cofactor distributions are apparent 17. Blakley, R. L. Crystalline dihydropteroylglutamic acid. 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CANCER RESEARCH VOL. 46 FEBRUARY 1986 593

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1986 American Association for Cancer Research. Antifolate Polyglutamylation and Competitive Drug Displacement at Dihydrofolate Reductase as Important Elements in Leucovorin Rescue in L1210 Cells

Larry H. Matherly, Charles K. Barlowe and I. David Goldman

Cancer Res 1986;46:588-593.

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