transport and metabolism of methotrexate in normal and...

[CANCERRESEARCH39,735-743,March197910008-5472/79/0036-0000 $02.00

Transport and Metabolism of Methotrexate in Normal and ResistantCultured Rat Hepatoma Cells1

John Galivan

Division of Laboratories and Research, New York State Department of Health, Albany, New York 12201

ABSTRACT

The transport and metabolism of methotrexate (MTX)have been examined in monolayer cultures of a cell linederived from the Reuben H35 hepatoma. The H35 cells havea doubling time of approximately 19 hr and cannot growwhen MTX is present at 3 x 10@ M. The cells exhibit linearuptake of MTX which lasts for 4 to 6 hr and reaches a steadystate after approximately 16 hr. The uptake was saturablewith a maximal rate of 1.1 nmol/min/g cell protein at anMTX concentration of 18 p.M or higher. 5-Methyltetrahydro

folate markedly inhibited uptake at equimolar concentrationbut folic acid exerted no effect at 5-fold excess. Once insidethe cell, MTX was rapidly converted to its polyglutamatederivatives with a conversion in excess of 90% when theintracellular concentration was greater than 2 pM. Resistantsublines were developed by culturing H35 cells in increasing concentrations of MTX. These cells are similar to theparentcellsinlevelsofdihydrofolatereductase,thymidylatesynthetase, and the extent that MTX can be converted to itspolyglutamate derivatives. They appear to differ from normal cells in not having the capacity for the extended linearuptake that is observed with normal cells. As a result, thereis a greatly reduced concentration of MTX and its polyglutamates at steady state. These results suggest that the cellsbecome resistant as a result of a stable change in thetransport system for MTX, but the mechanism of this process is not yet understood.

INTRODUCTION

The relative ability of the antifolate, MTX,2 to inhibit cellgrowth is a product of several properties of the cell uponwhich it is acting. These include the capacity of MTX to betransported into the cell and be maintained at a concentration which is sufficient to inhibit the target enzyme dihydrofobate reductase (EC 1.5.1 .3). Although MTX has beenshown to be a stoichiometnic inhibitor of purified dihydrofolate reductase (29), studies with intact cells have mdicated that the intracellular levels of MTX must be in excessof that which is tightly bound to the enzyme to effectivelyinhibit cell growth (21, 22, 30, 55). A further complicationarises from the fact that MTX can be converted to itspolyglutamate derivatives in normal and tumor cells (2, 9,27, 45, 50, 52, 61 ). The MTX polyglutamates appear to be atleast as effective as is MTX itself as inhibitors of dihydrofo

1 Supported in part by Grant NIA AG00207 from the National Institute on

Aging.2 The abbreviations used are: MTx, methotrexate; PteGIu, folic acid; 5-

CIt,-H@PteGIu,5-methyltetrahydrofolate; C)) methotrexate diglutamate;1. methotrexate triglutamate; PBS, 0.9% NaCl solution with 10 [email protected]

potassium phosphate, pH 7.4.Received August 11, 1978; accepted November 14, 1978.

late reductase (32, 60). However, they may enhance thetoxicity of MTX by having a longer half-life within the cell orby being more inhibitory to other folate-utilizing enzymes.Thymidylate synthetase (EC 2.1 .1.45) has been invoked as apossible site of MTX action to accommodate the expenimentabdiscrepancies mentioned above (8, 21, 44).

The acquisition of resistance to MTX toxicity has beenobserved in a number of mammalian cell lines. Acquiredresistance has been related to elevated bevelsof dihydrofolate reductase (4, 13, 15, 26, 33, 39), structurally altereddihydrofolate reductase (14), and an impairment in transbocation of MTX across the cell membrane (3). The firstmechanism is the most thoroughly investigated and appears due to the presence of multiple copies of the genecoding for this enzyme (1, 10, 34). Resistance to MTX as aresult of an altered transport system is less well defined.Studies to date have dealt primarily with the kinetics ofinflux of the drug, and changes in the Vm@,(46) and affinity(K1) (31, 57) of the transport system for MTX have beenimplicated as factors which contribute to resistance.

The present study was undertaken to examine the transport of MTX in a continuous cell line derived from theReuben H35 hepatoma (47, 48). These cells are highlysensitive to MTX but acquire resistance when cultured inprogressively higher bevelsof MTX. The level of the targetenzyme dihydrofolate reductase was only slightly elevatedin the resistant cells, but their capacity to transport MTXrelative to normal cells was greatly reduced. In the courseof these studies, it was found that H35 cells rapidly converted MTX to its polyglutamate derivatives and that theresistant cells appear to share this property.

MATERIALS AND METHODS

Materials. Swims Medium, S-77, folic acid-free SwimsMedium S-77, horse serum, and fetal calf serum wereobtained from Grand Island Biological Co. , Grand Island,N. Y. MTX (Lederle Laboratories, Pearl Riven, N. Y.) and

PteGlu (Sigma Chemical Co. , St. Louis, Mo.) were purifiedby DEAE-celbubose column chromatography prior to use(17). 7,8-Dihydrofolate was synthesized by the procedure ofBlakely (5), and 5,6,7,8 Tetrahydnofolate was prepared bythe catalytic hydrogenation of PteGIu (41). 5-CH,-H4 PteGIuwas synthesized by the method of Gupta and Huennekens(25). The concentrations of solutions of PteGlu derivativesand MTX were determined from their respective extinctioncoefficients (6, 17). [5-3H]dUMP (Schwarz/Mann, Orangeburg, N. V.) was purified by Dowex 1-formate chromatography and byophilization (41). [U-'4C]-3-O-Methyl-D-glucosewas obtained from New England Nuclear, Boston, Mass.[3',5',9-3H]MTX (Amersham/Searle Corp., Arlington Heights,III.) was purified on DEAE-cellubose (17). The resulting

MARCH 1979 735

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J. Ga!ivan

material exhibited a single radioactive spot when subjectedto cellulose thin-layer chromatography (Polygram Cal 300UV@M; Bninkmann Instruments, Inc., Westbury, N. V.) and

developed with either 0.1 M potassium phosphate, pH 6.0,or 5% acetic acid saturated with isoamyl alcohol adjustedto pH 9 with ammonia (52).

Cell Cuftures. H-11-E-C3cells derivedfrom the ReubenH35 hepatoma (47, 48) (referred to herein as H35 cells) werekindly provided by Joyce Becker and Dr. V. A. Potter of theMcArdle Laboratory at the University of Wisconsin. Thecells were grown as monolayers on 60-mm Falcon dishes ina 5% CO2 atmosphere. They were subcubtuned weekly andplated at a density of 2 x 10@cells/dish. The mediumconsisted of Swims Medium 5-77 supplemented with 20%horse serum, 5% fetal calf serum, and 4 mr,i glutamine.Media changes were done routinely at 72 and 120 hr afterplating. Cells were released from the dishes with 0.05%trypsin, and cell counting was carried out with a ZBICoultercounter. Cell stocks of normal and resistant lines werestored in liquid nitrogen. Assays for Mycop!asma contamination (38) were conducted routinely on cultures and frozensamples and were negative.

Development of MIX .resistant Cell Lines. Resistantlines were developed by initially adding MTX at 10@ Mthrough a culture period of one week. The concentrationwas then raised by 3-fold, and cells were grown for 2 to 3weeks in the elevated level of MTX until their growth curveequaled that of the normal cells. This was repeated until theMTX concentration reached 3 x 10@ M, and the cells thatgrew normally at this level are designated H35R1. A moreresistant cell line developed by a similar procedure ismaintained in 2 x 10-s M MTX and is referred to as H35RIII.The resistant line used in these studies, unless otherwisenoted, is H35A1. The growth curves for all cell lines wereaccomplished by trypsinization of duplicate plates at theindicated time points and counting each sample with theCoulter counter at settings of 1/amplification = 1, 1/apertune current = 2 with the lower threshold at 9 and the upperthreshold at 100.

Enzyme Assays. Cell-free extracts were prepared byscraping the plates with PBS containing 20 mM 2-mercaptoethanob. Extracts were prepared with a Dounce homogenizer, and the homogenate was centrifuged at 105,000 x gfor 1 hr at 4Â°in a Beckman L2-65B centrifuge. The resultingsupernatant was used for enzyme assays. Thymidybate synthetase was assayed by 3H release from [5-3HJdUMP (40)and dihydnofolate reductase was assayed spectrophotometnically (7). The protein concentration of cell extracts wasmeasured by the method of Lowry et a!. (42).

Determination of Intracellular MTX Levels with Dihydrofolate Reductase. Followingthe incubationperiod, theplates were chilled to 0Â°and washed 4 times with ice-coldPBS. The cells were then removed from the plates in thesame solution with a rubber policeman. The contents of 4to 10 plates were pooled, and the cells were broken with aDounce homogenizer. The extract was centrifuged at105,000 x g for 60 mm. An aliquot of the supennatant wasassayed enzymically for MTX (54) using dihydrofolate reductase from MTX-nesistant Lactobaci!lus casei (24), whichwas kindly provided by Dr. G. Maley of this laboratory.Another aliquot was boiled for 10 mm and centrifuged at

10,000 x g for 20 mm, and the pellet was washed with 1 mlof 0.9% NaCI solution, combined with the supernatant andassayed for MTX. The difference between the amount ofMTX in the heated extracts and that found in unheatedextracts was designated bound MTX. The MTX concentration was based upon measurements of intracellular volumeas determined by the procedure of Kletzian et a!. (36), andit was found that 1 mg of cellular protein was equal to0.00315 ml of intracellular water. Cell protein was determined by removing the cells from identical duplicate plateswith 2 successive washes of 1 ml of 1 N NaOH and measuring for protein by the procedure of Lowry et a!. (42).

Uptake of MTX by Normal and Resistant H35 Cells.Transport studies were routinely conducted on Day 5 of theculture cycle, a time which corresponds to early stationaryphase.The medium was changed to 2 ml of Swims S-77containing 4 mM glutamine without serum or PteGbu 1 hrprior to addition of [3',5',9-3H]MTX. The specific activityvaried between 1 x 10@and 6 x 10@dpm/nmol dependingupon the experiment. [3',5',9-3HIMTX was then added, andattheindicatedtimes,theplateswere incubatedat37Â°ina5% CO2 incubator. In all cases, duplicate plates were usedfor each time point. To terminate uptake, the plates werecooled to 0Â°and washed 4 times with ice-cold PBS. Thecells were removed with two 1-mbsuccessive washes of 1 NNaOH at room temperature. An abiquot of this sample wasused for measuring cellular protein (42), another aliquotwas neutralized with an equal volume of 1 N HCI, and thecontent of radioactivity was measured in 15 ml of Aquasol(LS-250 liquid scintillation system ; Beckman Instruments,Inc., Fullerton, Calif.). The results presented are the average of duplicate plates. Deviation from the average was lessthan 5% in 95% of the samples, and the average deviationof a typical experiment with 24 duplicates was 2.4%. Thevalues obtained thus are referred to as Cell MTX and areexpressed as cell protein, nmol/g, or intracellular H2O,nmol/ml (MM). When rates and kinetic constants weredetermined, the curves were derived by regression analysisas described previously (18).

Isolationand Identificationof MTX PolyglutamatesfromH35 Cells. The H35 cells were preincubated with [3',5',9-3H]MTX at the concentrations and times indicated. Theplates were then washed with 4 successive washes of 4 mlof ice-cold PBS, and the contents were combined in two 1-ml aliquots of PBS. The samples were boiled for 10 mm andthen centrifuged at 10,000 x g for 10 mm in a Sorvall AC-2Bcentrifuge. The precipitate was washed with 1 ml of thesame solution, and the supernatants were pooled andbyophilized. The residue was dissolved in 0.5 ml of 0.02 MNH4HCO3,0.5 @molof carrier MTX was added, and chromatography was conducted on an 0.9- x 50-cm SephadexG-15 column (60) with 0.02 M NH4HCO)as the eluting buffer.The column was standardized with MTX(G2) and MTX(G:C)which were kindly provided by Dr. C. M. Baugh of theDepartment of Biochemistry of the University of SouthAlabama, Mobile, Ala. The standards were located in thecolumn effluent by measuring the absorbance at 302 nm.The cell extracts were measured for radioactivity and expressed as dpm/fraction. In all experiments, the recovery ofradioactivity in the effluent was greater than 85% of thatpresent in the dissolved residue.

736 CANCERRESEARCHVOL. 39



MTX was added at the indicated concentrations to culturesofH35cells at 0 time in the culture cycle. Cell growth wasmeasuredthrough

the culture cycle with duplicate points every 24 hrasdescribedin â€˜â€˜Materials and Methods.' â€T̃he data presented areameasure

of the cells attached at 96 hr as a percentage ofthenumberof cells at 0 time. In this experiment, the average of the2observations

is reported, and the mean deviation from this is3%.MTX

Cellgrowth(nM)(%)0.05300.35051.06403.0

66010.024030.01550.0

0

48 96 44

Transport and Metabolism of MTX

Polyglutamate derivatives of MTX were hydrolyzed usingpancreatic conjugase. The enzyme was prepared from dehydrated chicken pancreas (Difco Laboratories, Detroit,Mich.)accordingtothemethod of Eigenand Schockman(12). Standard MTX polyglutamates or chromatographic

fractions from Sephadex G-15 gel filtration were incubatedovernight at 23Â°with an equal volume of purified conjugase.Complete hydrolysis of MTX(G) ) and ptenoylheptaglutamateoccurred under these conditions when analyzed by thinlayer chromatography (53).

RESULTS

The Effect of MTX on the Growth of H35 Hepatoma Cells.H35 cells grown in monolayer culture had a doubling timeof approximately 19 hr during log phase. The effect ofadding MTX at midbog is depicted in Chart 1 where it can beseen that the addition of 10 flM MTX at 72 hr causes nochange in the growth curve, while 30 nM MTX causes celldetachment after a lag of about 24 hr. MTX had no effect ongrowth at concentrations up to 3.0 nM when added at thetime of plating, but cell growth was inhibited 50% by 10 nMMTX, and the cells were not viable in 30 flM MTX (Table 1).H35AI cells had the same growth curve in 0.3 pM MTX asdid the normal cells in MTX-fnee media (Chart 1). Thestudies that will be reported here deal primarily with thisresistant subline, although cultures derived from these cellshave become resistant to levels of MTX as high as 2 @M.

Levels of Dihydrofolate Reductase and ThymidylateSynthetasein Normal and ResistantH35 Cells. Acquiredresistance to MTX in many mammalian systems has beenshown to be due to commensurate increments in the targetenzyme dihydnofobatereductase(4,13,15,26,33,39),andseveral reports have demonstrated that thymidylate synthetase levels are elevated by MTX (7, 11, 37, 49, 51). Thus, the

Table1Effect of MTXon the growth of H35cells

levels of both enzymes were measured in normal andresistant cells throughout the culture cycle. The activities inextracts of the normal cells appear to be similar to thoseobserved in other mammalian systems (37, 46, 49). Dihydrofolate reductase did not fluctuate greatly during the growthcycle, but the activity of thymidybate synthetase was muchhigher during log phase. The activity of dihydrofolate reductase was 15 to 100-fold higher than was thymidylatesynthetase throughout the culture cycle which has beenobserved in several cell lines (46, 59). Small differenceswere noted when the enzyme activities were measured inextracts of the resistant cells. Dihydrofolate reductase activity was 1 .7-fold higher in H35R1 cell extracts on Day 5 and

only 2.3-fold higher in H35R111cell extracts. By contrast,synthetase activity in H35RI cell extracts was reduced by 60to 70% during bog phase. This effect is probably not due tothe direct inhibition of thymidylate synthetase by MTX in theextracts since H35RI cells grown in the absence of MTX forseveral generations also showed a reduced level of enzymeactivity. The level of dihydrofolate reductase in the sameextracts was equal to that in extracts of normal cells.Although the altered levels of both enzymes may aid inreducing the toxicity of MTX to the resistant cells, it doesnot appear to be of sufficient magnitude to be the primarysite of MTX resistance.

Transport of MTX Into H35 and H35R Cells. Measurements of the uptake of MTX into normal and resistant cells,however, were associated with changes which correlatedwell with MTX resistance. In the experiment described inChart 2, the concentration of MTX in the medium was 0.6

@M.The normal cells exhibited a rapid association withMTX which persisted for about 15 mm, followed by a longer

period of several hr during which the drug accumulated. Athigher levels of extracellular MTX (2 to 25 SM), a similarprolonged period of uptake lasting 4 to 6 hr was observed,but the rapid influx noted at the lower concentrations (Chart2) was not as clearly defined. The linear phase of uptakewas concentration dependent and saturable. Steady statekinetics were approached by 16 hr, after which little furtheruptake of MTX was observed.

Neither H35A1nor H35RbIlexhibited the sustained uptakeof MTX shown by the normal cells (Chart 2). At a mediaconcentration of 0.6 @M,a very slow uptake was observedthat reached saturation within 2 hr, increasing only slightly

Ui

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HOURS IN CULTUREChart 1. Growth curve for H35 and H35RI cells. Cells were inoculated and

cultured, and growth curves were conducted as described in Materials andMethods.â€•Triplicate plates were removed at the indicated time points, andthe average values are presented. MTX was added to the media at 72 hr(arrow) with the H35 cells and was included throughout the culture cyclewith the H35R1cells.

MARCH 1979 737



2 4 6

Intracellular concentration of MTXin H35and H35R1cellsEightcultures each of H35 and H35RI cells were grown for104hr

at which time the indicated amount of MTX was added, andtheincubationwas continued for 16 hr. At that time, the cellswerewashed

and digested as described in â€œMaterialsandMethods.â€•Enzymicassays for MTX (39) were conducted on thepooledmaterial

from the 8 cultures. Parallel duplicate plates were evaluatedforcellularproteintocalculateintracellularvolume(â€˜â€˜Matenials and Methodsâ€•).Cell

MTX(â€”)

MTXCell (ii.M) FreeBoundH35

0.03@ 0.251.00.306.60.9H35RI

0.3â€• 0 0.7

J. Galivan

H35 cells (0.03 MM), the amount of free MTX slightly exceeded that which was tightly bound. On increasing themedia level to 0.3 @M,a similar level of bound MTX wasfound , but the level of free MTX was greatly increased . TheH35RI cells contained only slightly less bound MTX but nodetectable free MTX at the 0.3 @Mbevel. This result wasconfirmed by using radioactive MTX and by subjecting analiquot of the cell extract to Sephadex G-25 gel filtrationwhere all the radioactivity migrated with the protein in the

0.5

I/MTX, ,uM

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INCUBATION(hr)

Chart 2. Uptake of MTX by H35, H35R1, and H35RIII cells in culture.Uptake was measured as described in â€œMaterialsand Methods.â€•Intracellularvolume was based upon a value of 0.00315 mI/mg cell protein, and [3',5',9-3HIMTx,i .4 x 10'dpm/nmol,wasaddedto themediaata finalconcentration of0.6 @.cM.

after that time, and the resulting intracellular concentrationwas approximately equal to that of the media. At mediaconcentrations between 2 and 25 p.M, there was a rapidinflux of MTX which was usually complete within 30 mm,and no further uptake was observed over a 4-hr period. Theresultant cellular concentration of MTX was always lessthan the media concentration. The H35R cells did notexhibit the prolonged phase of transport shown by the H35cells at concentrations as high as 200 p.M. Both MTXresistance and reduced transport appear to be stable alterations in the H35A1 cells since growth in the absence ofMTX for 85 generations results in a normal growth curve in

the presence of 0.3 @MMTX and uptake phenomena identical to those depicted in Chart 2.

The effect of temperature on the uptake of MTX in thenormal and resistant cells was examined. Reducing theincubationtemperatureto0Â°preventedtheextendedlinearuptake of MTX normally observed with H35 cells (Chart 2),but had little effect on the initial, rapid uptake. A similarreductionintemperaturedidnotdiminish,and ifanything,slightly increased uptake of MTX by H35A1cells.

Saturability of the Transport System In H35 Cells. Thedependence of the transport rate of MTX in H35 cells wasmeasured as a function of its concentration in the medium.The rate was measured by taking duplicate measures ofuptake of 30, 60, 90, and 120 mm which was linear at allconcentrations tested. This experiment showed that transport system was saturated at approximately 18 @Mand thatlittle increase in the rate of uptake was observed at higherlevels (Chart 3). The Vm@was 0.32 @moI/min/litercell waterwhich corresponds to 1.1 nmol/min/g protein. The appanent K@for the accumulation of tnitium from [3',S',g-'H]MTXis5.2 Â±1.4 @M(S.E.).

Intracellular Concentration of MTX in H35 and H35RCellsat SteadyState. The amountof MTXaccumulatedbythe normal and resistant cells at the steady state wasanalyzed by measuring the bevels of tnitiated MTX in cellextracts and also by the use of purified dihydrofolatereductase (Table 2). At the lowest level that is toxic to the

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Chart 3. Kinetics of MTX uptake by H35 cells. Uptake of MTX wasmeasured as described in â€˜Materialsand Methods,â€•and the concentrationin the medium was varied between 1 and 25 @cM[3',5',9.3H)MTX, 2 x 10@dpm/nmol. Rates of uptake were determined by measuring duplicate cultures at 30, 60, 90, and 120 mm, a portion of the curve which was linear at allconcentrations of MTX measured.

Table2

a These experiments were also conducted using radioactive

MTX(3 x 10@dpm/nmol) to measureintracellular MTXand Sephadex G-25gel filtration to distinguish free MTXfrom bound. The H35cells ([MTXL@@= 0.03 @.a@i;ext, extracellular had concentration of0.35 @Mfree MTX and 1 .1 p@ibound MTX. The H35R1 cells ([MTX]ext= 0.3 @M) had 0.56 p.M bound MTX and no detectable free MTX.

CANCERRESEARCHVOL. 39738



. No addition

A +50,uMMTX0 + 50 jiM 5-CH3lfPteGIu(--)

0 2 4 0@

INCUBATION (hr)

Chart 5. The effect of 5-CH,I-tI PteGIu and PteGIu on the efflux and influxof MTX in H35 cells. A, efflux. The cultures were allowed to reach steadystate (16-hr incubation) with 10 @cM[3',5',9-3HJMTX, 1 X 1O@dpm/nmol, inthe medium. The medium was removed and replaced with 2 ml of PteGIufree Swims 5-77 containing the indicated additions. Retained intracellular[3',5',9-'H)MTX was measured as a function of time at 37â€•by the sameprocedures used to measure uptake of MTX described in â€œMaterialsandMethods.â€•B, Influx. Uptake was measured as described in â€œMaterialsandMethodsâ€•with 2 @LM[3',5',9-'HJMTX, 3 x 1O@dpm/nmol, in the medium andadditions as indicated.

studies on the efflux of MTX from the H35 cells. Studieswith L1210 cells, which have an active transport system forMTX, have shown that the intracellular to extracellularconcentration ratio does not generally exceed one (23, 56),and the maximum intracellular concentration achievable is5.59 @tM(23). Secondly, efflux of MTX from L1210 cells hasbeen shown to be very rapid (23, 54, 56). Similar studieswithH35 cellssuggesteda greatlyimpairedabilityfortheintracellular MTX to be released into the medium. Cellsequilibrated with MTX for 18 hr and placed in media lackingMTX demonstrated a very slow efflux which is unaffectedby either 5-CH)-H4PteGlu or unlabeled MTX (Chart 5). Bycontrast, 5-CH3-H4PteGbumarkedly inhibited MTX uptake(Chart 58), and a 5-fold excess of PteGlu had no effect onMTX uptake which has been observed previously with MTXtransport in cultured mammalian cell systems (28). Thesedata taken together strongly suggested that MTX was converted to a less permeable form once inside the cell.Binding to intracellular proteins was an unlikely possibilitybecausethedatainTable2 suggestedthatallintracellularMTX in excess of that which approximated the concentration of dihydnofobate reductase was free in the cell extract.

Sephadex G-15 was used to examine the cellular contentsafter exposing the H35 cells to 0.03, 1.0, and 10 @M

â€˜? radioactive MTX (Chart 6). At 1 .0 and 10 @M extracellular

6 MTX, higher-molecular-weight forms of MTX predominated@ in the elution pattern. In both cases, the amount of unme

@( tabolized MTX stayed relatively constant throughout the 24-

@%@% hr time course while the pobyglutamates increased in

@ amount. In addition, the majority of the radioactivity shiftedc@< from Fractions 10 to 12 to the peak at Fractions 6 to 9

during the course of the experiment. Since the earliestebuting peak (Fractions 6 to 9) corresponded to MTX(G3)and Fractions 10 to 12 corresponded to MTX(G2), this shiftsuggested the further addition of glutamate residues. At0.03 @MMTX, which is the minimal toxic level (Chart 1),unaltered MTX predominated at 1 and 4 hr, whereas 90% ofthe MTX at 24 hr was present as high-molecular-weightderivatives. When H35A cells were analyzed by the sameprocedure, it was found after 24 hr that a similar distributionof MTX metabolites was observed with the peak Fractions 6

200


excluded fraction. The amount of bound MTX in theseexperiments approximated the intracellular concentrationof dihydrofolate neductase which was found to be 0.8 and0.9 @.tMwhen measured by titration with MTX (54) in 2independent measurements. These results suggest that thebound MTX observed in Table 2 corresponds to that whichis associated with dihydrofolate neductase.

In all the studies conducted, the intracellular bevelof MTXdetermined with [â€˜H]MTXagreed well with the amountmeasured with dihydrofolate reductase. This result mdicates that the MTX was not degraded. The media from theresistant cell cultures was also examined by enzyme assayand by thin-layer chromatography (â€œMaterialsand Methodsâ€•)and confirmed that resistance was not due to breakdown of the drug. The concentration of tnitiated MTX insidethe H35 cells at steady state varied with respect to theconcentration of MTX in the media (Chart 4) and wassaturable. The maximum intracellular concentration accumulated was 115 p.Mwhich was at a media concentration of50 @MMTX on greater. Above that concentration, no higherlevels of MTX were found in the cell. These data also showthat the capacity to accumulate MTX from the media increased as the concentration of MTX in the media decreased. The data described in Charts 2 through 4 arecharacteristic of an uphill active transport system (20).However, this interpretation is complicated by the extensivemetabolism of MTX in these cells to be described below.

Metabolism of MTX by H35 and H35R Cells. By measuring the intracellular concentration of MTX with radioactivityand dihydrofolate neductase, the identitiy of radioactivematerialas a stoichiometnicinhibitorofdihydrofolatereductase is assured. However, the enzyme assay cannotdistinguish between MTX and its polyglutamate derivativessince the batten compounds are also potent inhibitors ofdihydrofolate reductase (32, 60). The possibility that suchcompounds were present was suggested by the high intracellular levels of MTX at steady state (Chart 4) and by

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MTX (pM)

Chart 4. Cell MTX concentration as a function of medium MTX concentration at steady state. The indicated levels of [3',5',9-3H)MTx, 1 x 10 dpm/nmol were added to the medium, incubated for 18 hr, and the intracellularMTX measured as described in â€œMaterialsand Methods.â€•Duplicate plateswere measured for each point. The results are expressed as either theconcentration (pM) of total intracellular MTX or as ratio of intracellular MT@to extracellular MTX (MTX,/MTX0) as a function of the medium concentration.

MARCH 1979 739



39%hr

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J. Galivan

to 9 predominating in media containing 50 @MMTX (Chart7). However, at a media concentration of 0.3 @tM,thepredominant intracellular species was MTX. In this study,the intracellular level of MTX plus MTX polyglutamates didnot exceed 0.5 @Meven at 24 hr, and of this, approximately70% was unaltered MTX. A similar predominance of unaltered MTX was observed with normal cells in 0.03 @MMTXat 1 and 4 hr when the intracellular concentration of MTXdid not greatly exceed the concentration of dihydrofolatereductase.

Although the coincidence of these peaks with MTX(G))and MTX(G2) suggests that polyglutamates are beingformed, it does not prove their existence. The fact that allof the radioactive MTX within the cell acts as a stoichiometnc inhibitor of L. casei dihydrofolate reductase, eventhough as much as 98% is converted to the higher-moleculan-weight derivatives, supports this contention. Samplesfrom Fractions 6 to 9 and 10 to 12 were treated withpancreatic conjugase, and all the radioactivity comesponded to MTX when subjected to Sephadex G-15 gelfiltration. The conjugase-treated samples were also subjected to thin-layer chromatography (â€œMaterialsand Methodsâ€•)and were identical to authentic MTX. The actualnumber of glutamate residues in the earliest fractionsremains in doubt since higher-molecular-weight derivativesof MTX may not be resolved from MTX(G:i) in Sephadex G15gelchromatography(60).

0.3,@uM MTX

0.5

z0I-0a:U-,.â€˜%â€˜

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b

10 30 0 30 0 30

FRACTION

Chart 7. Chromatography of intracellular MTX from H35RI cells. Theexperimental procedures are identical to those used in Chart 6. The specificactivity of [3',5',9-3H]MTX was 1 x 10@dpm/nmol (50 @M)and 6 x 1O@dpm/nmol (0.3 SM).

DISCUSSION

Transport of MTX in a number of cell lines and especiallyL1210 cells (reviewed in Refs. 20 and 28) is an active,energy- and temperature-dependent, saturable process.Transport processes in tumor cells of hepatic origin havenot been widely investigated. The data reported here mdicate that transport in H35 hepatoma cells in culture hasseveral properties which are shared by L1210 cells. Theoverall process of accumulation of radiolabeled MTX ispreventedby a reductionin temperatureto 0Â°and issaturablewitha K1of 5.2 @Mand a Vmaxof protein,1.1nmob/min/g. These values are in the same range as arethose reported for other cell lines (23, 56). The capacity ofthe cells to accumulate tnitium is also saturable. Althoughthese properties are consistent with an active transportsystem, the extensive intracellular conversion of MTX topolyglutamate derivatives in H35 cells during the â€˜â€˜concentrativeâ€•phase of uptake complicates thisinterpretation.Thus, it is possible that the kinetics of uptake and effect oftemperature reduction on uptake reflect the rates of conversion to po!yglutamate derivatives and not transbocationacross the cell membrane. In spite of this qualification, thegeneral similarity of H35 cell transport of MTX to thatobserved in L1210 cells makes it possible that the transportsystems are not greatly different (Charts 2 and 3). Otherexperimental observations suggest that this is the case.Uptake is completely inhibited by 5-CH3-H4PteGIu but isrelatively insensitive to PteGlu as is the L1210 transportsystem (28). The inhibition by 5CH:iH4PteGlu is stronglysuggestive of a carrier-mediated system. Sodium azide hasbeen shown toenhance therateofinfluxand steadystatedistribution of MTX in L1210 cells (19). This appears to be

0.03@iM MTX

z0Iâ€”U

a:U.

a-0

b

20

l0

020 020 020

FRACTION

Chart 6. Chromatography of intracellular MTX from H35 cells. The isolation of intracellular MTX and its products and Sephadex G-15 gel chromatography are described in the â€œMaterialsand Methods.â€•MTx(G,) eluted atFractions 6 to 9, MTX(G,) eluted at Fractions 10 to 13, and MTX eluted atFraction 25. The concentration in the media was 0.03, 1.0, and 10 @.cM[3'.S' ,9-3H)MTX, 6, 4, and 1 x 10Â°dpm/nmol, respectively. The percentageof conversion of MTX to its higher-molecular-weight derivatives is indicatedwithin each graph. Each experiment utilized the pooled washes of 4 culturedishes, and identical duplicate plates were used to calculate the intracellularvolume.

CANCER RESEARCH VOL. 39740




due to suppression of the efflux component of the transportsystem. The rate of uptake of MTX by H35 cells was alsostimulated by sodium azide,3 possibly by a similar mechanism. The major difference between the H35 and L1210systems appears to be the duration of linear uptake of MTXand high intracellular levels of MTX achieved in the hepatoma cells, the basis for which may be the extensiveconversion of MTX to its pobyglutamates in the H35 cells.Since these derivatives are believed to permeate the cellmembrane much less readily than does MTX (43, 58, 60),their formation would then result in higher retention of totalMTX within the H35 cells. A direct analysis of the transportsystem in H35 cells awaits the elucidation of conditionswhich inhibit MTX metabolism without affecting transport.

The extent to which MTX is converted to its polyglutamates has been discovered only recently and is cruciallyimportant in understanding the pharmacology of the antifolate. In the tissues that MTX polyglutamate synthesis hasbeen studied, MTX glutamate has predominated except inL1210 cells in vivo where MTX(G2) is the major species (60).Some tissues such as small intestine and thymus do notappear to convert MTX to polyglutamate derivatives (61).H35 cells appear to carry out a more rapid and extensiveconversion of MTX than that reported in other systems.When the intracellular concentration of MTX was in excessof 1 to 2 @M,the conversion to polyglutamates was 90% ormore of the total cell MTX in normal and resistant cells.Comparable figures with other cells are human fibrobbasts,55% (50); Li 210 cells in vivo , 56% (60); liver, 67% (61); andkidney, 28% (61). Primary cell cultures of rat hepatocyteshave also been examined, and at a media concentration of10 @MMTX, 85% conversion to MTX(G2) and MTX(G3) wasfound after a 24-hr incubation.3 In the H35 cells, the predominant species of MTX at steady state coincides withMTX(G)) Ofl Sephadex G-i5 gel filtration although this peakelutes in the void volume and higher molecular species maybe present. Previously only MTX glutamate and MTX(G2)have been observed (50, 60, 61). The reason for the presence of more glutamate residues on MTX derivatives in H35cells awaits a more detailed examination of the enzyme(s)involved in this process.

Titration of dihydrofolate reductase with MTX, MTX(G2),MTX(G3), and the 2 major peaks (Fractions 6 to 9 and 10 to13) isolated from H35 cells by Sephadex 0-15 chromatography was conducted using the enzyme from MTX-resistantL. casei (18) and also that from extracts of H35 cells. In allcases examined the inhibition curves were identical, mdicating that the polyglutamates are equally effective as MTXininactivatingdihydrofolatereductasefromthesesources.These results confirm similar studies previously conductedby Jacobs et a!. (32) and Whitehead (60).

H35 cells are among the more susceptible cell lines withregard to MTX toxicity (59). At the minimal level of MTX thatis toxic to H35 cells (0.03 MM), only a small amount of freeMTX was observed within the cell, which is consistent withthe hypothesis that MTX must be present in excess of thebevel of dihydnofolate reductase to inhibit cell growth (21,22, 31, 55). At a 10-fold higher concentration of MTX (0.3MM), the H35 cells had a level of free MTX which was

3 J. Galivan, unpublished observations.

elevated seven-fold over the amount bound and approximately 20-fold oven the media concentration (Table 2). Freeand bound MTX in this context is more accurately definedas a combination of MTX and its polyglutamates. The factthatthe polyglutamatesinhibitdihydrofolatereductaseaseffectively as does MTX suggests that they will be distnibuted in both the free and bound species. By contrast, H35AIcells accumulated no detectable free MTX or its polyglutamates when cultured in 0.3 @MMTX, a concentration whichdoes not inhibit cell growth. It is presumed that the intracellular bound MTX is associated with d ihydnofolate reductase since the concentration of bound MTX in severalstudies (0.56 to 1.0 @tM;Table 2) corresponded to theconcentration of dihydrofolate red uctase.

At all concentrations of MTX tested, the H35AI cellsshowed a reduced capacity to allow MTX access to theintracellular compartment. The data shown in Table 2 andChart 3 suggest that this is related to an alteration in thetransport system. MTX pobyglutamate formation does notappear to be impaired in resistant cells since efficientconversion of MTX is observed when intracellular levels areadequate (Chart 7). In addition, the rate of MTX pobyglutamate synthesis was identical over a 20-hr period whenmeasured in H35 and H35RI cell extracts.3 Two changes inthe properties of the H35RI cell further suggested an alteration in the membranes of the resistant cells. These cellsare much more sensitive to trypsinization, and reducedtemperatures (O@)than were normal cells and detached fromthe culture dishes rapidly under either of these conditions. Itmay be tentatively proposed that the resistant cells differprimarily by preventing the accumulation of MTX within thecell and that it is due to an alteration in the membraneproperties. The MTX that enters the resistant cells may doso by diffusion since the rate of uptake is not altered byreduction of the temperature to@ and 5-CH3-H4PteGludoes not inhibit uptake as it does with normal cells.

The presence of MTX polyglutamates in this study extends the cell types in which they have been found andindicates that they are far more prevalent than originallythought. This has several implications regarding the chemothenapeutic activity of MTX, some of which have beendiscussed by Rosenblatt et a!. (50) and Whitehead et a!. (60,61). Chief among these would be a potentially elevatedtoxicity of MTX in cells which convert MTX to its pobyglutamates due to a longer half-life within the cells and a greaterpossibility of inhibition of other folate requiring enzymes. Alikely second site is thymidylate synthetase which hasalready been postulated as a site of action of MTX (8, 11,21 ) and is known to have a much higher affinity for polyglutamate analogs of ptenidines than for their monoglutamates(16, 17, 35).

ACKNOWLEDGMENTS

The author wishes to thank Joyce Becker and Dr. van Potter of theMcArdle Laboratories, University of Wisconsin, Madison, Wis., for supplyingstocks of H35 cells and for their helpful edvice in maintaining the cultures,Dr. Charles M. Baugh, Department of Biochemistry, University of SouthAlabama, Mobile, Ala., for generously providing the MTX polyglutamate andpteroylpolyglutamate standards, and Dr. Gladys Maley for preparing theLactobadllluscase! dihydrofolate reductase. The helpful suggestionsof Dr.Frank Maley in the preparation of the paper and the excellent technicalassistance of Pat Fox and Zenia Nimec are deeply appreciated.

MARCH 1979 741



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1979;39:735-743. Cancer Res John Galivan Resistant Cultured Rat Hepatoma CellsTransport and Metabolism of Methotrexate in Normal and

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