Effects of 2,4-Dinitrophenol and Other Metabolic Inhibitors on The

(CANCER RESEARCH 40, 3669-3673. October 1980] 0008-5472/80-0040-OOOOS02.00 Effects of 2,4-Dinitrophenol and Other Metabolic Inhibitors on the Bidirectional Carrier Fluxes, Net Transport, and Intracellular Binding of Methotrexate in Ehrlich Ascites Tumor Cells1

David W. Fry, J. Courtland White, and I. David Goldman2

Department ol Medicine, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298

ABSTRACT edly influenced by (a) the concentration of the inhibitor, (b) whether measurements are taken during influx, net transport, 2,4-Dinitrophenol (DNP), an uncoupler of oxidative phos- or the steady state, (c) whether total intracellular or freely phorylation, has been frequently used to evaluate the effects exchangeable MTX is determined, and (d) whether or not of energy depletion on methotrexate (MTX) transport. The glucose or other energy-producing substrates are present. results from these studies, however, have shown a multiplicity of effects that suggest a more complicated interaction with the MTX transport system than adenosine 5'-triphosphate deple INTRODUCTION tion alone. Accordingly, studies were undertaken to compare Membrane transport of MTX3 is a complex process. While the effects of DNP with a variety of other metabolic inhibitors this system conforms in many respects to other carrier-me on influx, efflux, net uptake, and intracellular binding of MTX in diated processes with the demonstration of temperature and Ehrlich ascites tumor cells. Low concentrations of DNP (0.1 sulfhydryl dependence (10), heteroexchange diffusion (9, 25), mw) inhibited efflux and increased the intracellular steady-state and inhibition by structural analogs (8, 10), some properties of concentration of MTX, both of which were totally reversed by the transport system are poorly understood. For instance, influx glucose. These alterations were similar to those caused by and net transport are highly sensitive to changes in the anionic inhibitors of the electron transport system (azide, rotenone, (8, 13) and cationic (5) composition of the extracellular com antimycin A, cyanide, and oligomycin) and anaerobic glycolysis partment. Further, metabolic poisons enhance, rather than (2-deoxyglucose) and are compatible with inhibition of an en inhibit, influx and net transport of MTX in some systems, ergy-dependent exit pump. As the concentration of DNP was presumably related to inhibition of an energy-dependent exit increased, influx was competitively inhibited with a KÂ¡of 336 pump (7). The effects of DNP on MTX transport are of particular UM. Inhibition was instantaneous and was reversed by removal interest. This agent has been commonly used as an uncoupler of DNP but not by addition of glucose. The inhibition of MTX of oxidative phosphorylation to evaluate the effects of ATP influx by DNP was different from the effects of other metabolic depletion on MTX transport. However, a wide variety of effects inhibitors which consistantly stimulated influx, an effect totally of DNP on MTX uptake have been reported, ranging from reversed by glucose. Likewise, as DNP concentrations were inhibition (15, 22), to stimulation (11 ), and to no effect at all (4, increased, inhibition of efflux was enhanced but was only 16). Although some discrepancies may be explained by differ partially reversed by glucose, which suggests that at high ences in cell lines, the data suggest that the effects of DNP concentrations of DNP inhibition was due to both energy de may be more complex than energy depletion alone and that pletion and an additional mechanism not associated with en this agent may interact in more than one way with the MTX ergy metabolism. DNP, as well as all other metabolic inhibitors, transport system. Accordingly, studies were undertaken to increased the apparent nonexchangeable fraction of MTX; further clarify the effects of DNP on MTX transport. The results evidently, this is due to energy depletion, since this effect was indicate that, whereas DNP produces changes similar to other totally reversed by glucose. The additional nonexchangeable metabolic inhibitors, there is a second effect that appears to MTX remained unchanged over at least 2 hr of incubation and be based upon a direct interaction between DNP and the MTX was not diminished by repeated changes of the extracellular carrier or its microenvironment which inhibits the bidirectional fluid; this suggests that it was tightly bound. Dicumarol, another flows of this antifolate. These observations provide useful in uncoupler of oxidative phosphorylation, produced effects sim formation on (a) the application of DNP as an agent to evaluate ilar to those of DNP; however, arsenale had little effect on MTX the energetics of MTX transport, (D) the nature of the bidirec transport, which suggests that these alterations were not due tional flows of MTX across the Ehrlich ascites tumor membrane, to uncoupling alone. The results indicate that although most and (c) the different effects of energy inhibitors on the unidi metabolic inhibitors affect MTX transport by inhibition of energy rectional fluxes and net transport of MTX in this cell system. metabolism, DNP affects the system in at least two ways: (a) by energy depletion similar to that of other metabolic inhibitors; and (Â£>)byan apparent interaction with the carrier at the cell MATERIALS AND METHODS membrane which inhibits bidirectional fluxes. The results may Chemicals. [3',5',9-3H]MTX was obtained from Moravek resolve some of the variation in the literature on the effects of (City of Industry, Calif.) and purified by DEAE-cellulose chro- DNP on MTX transport by showing that observations are mark matography (10). [carboxy-14C]lnulin was obtained from New

1 Supported in part by Grants AM-07150 and CA-16906 from the NIH. 2 To whom requests for reprints should be addressed. ' The abbreviations used are: MTX, methotrexate (4-amino-N'Â°-methylptero- Received December 3, 1979; accepted July 14, 1980. lyglutamic acid); DNP. 2,4-dinitrophenol

OCTOBER 1980 3669

England Nuclear (Boston, Mass.). Antimycin A, sodium arse Table 1 nale, 2-deoxyglucose, dicumarol, oligomycin, and rotenone Effects of DNP and other metabolic inhibitors on MTX influx were from Sigma Chemical Company (St. Louis, Mo.). Sodium Cells were exposed to 2-deoxyglucose for 10 min, or to other inhibitors for 5 min before MTX. Influx was measured over 100 sec. All values were compared azide and DNP were from Fisher Chemical Company (Fair with the control. Lawn, N. J.), potassium cyanide was from Allied Chemical InhibitorDNP of controlinflux96.1 (Morristown, N. J.), and d/-5-formyltetrahydrofolate was from "73.4 Â± 2.3a- (0.1mM)DNP Â±3.1C48.1 Lederle Laboratories (Pearl River, N. Y.). (0.25mM)DNP Â±1.8o31 Cells, Media, and Incubation Techniques. Ehrlich ascites (0.5mM)DNP (1.0mM)DNP .5 Â±3.6d16.4 tumor cells were grown in male CF-1 male (Sprague-Dawley, Â±0.33d87.9 mM)Glucose(1 0 mM) + glucose (10 Â±2.7C87.0 Madison, Wis.) and passed weekly by i.p. inoculation of 0.2 ml mM)Arsenale(10.0 Â±1.9C38.5 mM)Dicumarol(1.0 of undiluted ascitic fluid. Cells were suspended in buffer com +5.1"196.3 /IM)Rotenone(50.0 Â±4.2d83.2 posed of 136 mM NaCI, 4.4 mM KCI, 16 mw NaHCO3, 1.1 mM /JM)Rotenone(1 .0 mM)Antimycin(1 .0 fiM) + glucose (to Â±12.16178.1 KHjPCv 1.0 mM MgCI?, and 1.9 mM CaCI?. The pH was Â±12.1Â°175.9Â± (IM)PotassiumA (0.5 maintained at 7.4 by passing warmed and humidified 95% O2/ 7.9d150.0Â± mM)Oligomycincyanide (1 .0 4.6"149.3Â± 5% CO? over the cell suspension. The suspension was stirred fig/ml)Azide (0.3 5.2d80.0 mM)Azide(10.0 by a Teflon paddle in specifically designed flasks inserted into Â±1.9C134.8 a 37Â°water bath. Unidirectional fluxes, net uptake, and intra- mM)2-Deoxyglucose(10.0 mM) + glucose (10 (50.0 mM)n33332844524441044% Â± 2.8o cellular binding of [3H]MTX were measured as previously de 1Mean Â±S.E. of n experiments performed on separate dates. scribed (7, 10). Transport fluxes were stopped by injection of * p>0.2 (paired f test). the cell suspension into 10 volumes of 0Â°0.85% NaCI solution cp<0.01 (paired (test). a p < 0.001 (paired ( test). (pH 7.4). The cell fraction was separated by centrifugaron (2000 x g for 30 to 60 sec) and washed twice with the 0Â° 0.85% NaCI solution. The washed pellet was aspirated into the tip of a Pasteur pipet, extruded onto a polyethylene tare, and dried overnight at 70Â°. The dried pellets were weighed on a Cahn electrobalance (Cahn Instruments, Paramount, Calif.), placed in a scintillation vial, and dissolved in 0.2 ml of 1 N KOH for 1 hr at 70Â°.The digest was neutralized with 0.2 ml of 1 N HCI, and 3 ml of Ready-Solv (Beckman Instruments, Inc., Irvine, Calif.) were added to the scintillation vial. Radioactivity was determined in a Beckman LS-230 scintillation spectrometer, 025mM DNP and counting efficiencies were determined using [3H]- or CONTROL [14C]toluene internal standards. Determination of Intracellular Water and Chloride Distri bution Ratio. Intracellular water was determined from the dif ference between the wet weight and the dry weight of a cell 50 pellet, less the [14C]inulin space as described in detail else where (2, 10). The chloride distribution ratio was measured as [MTX]e an indication of changes in membrane potential (2, 10). Chart 1. Double reciprocal plot of extracellular MTX concentration, [MTX]., versus influx at different concentrations of DNP. Influx was measured over 100 sec, and lines were plotted by the method of least squares. RESULTS

Effect of DNP on MTX Influx. Unlike a variety of other Dicumarol, another uncoupler of oxidative phosphorylation metabolic poisons that deplete cell ATP and stimulate MTX which possesses some structural similarity to DNP, was also a influx, DNP had no effect at 0.1 mM but inhibited influx as the potent inhibitor of MTX influx. However, arsenale, which is level of DNP was increased (Table 1). Inhibition occurred structurally unrelated to DNP but still exhibits uncoupling activ instantaneously upon exposure of cells to DNP, was reversed ity, caused only slight inhibition (Table 1). Phenol and dinitro- when the cells were washed free of the inhibitor, and was benzene (analogs of DNP which lack the nitro and the hydroxyl competitive with a KÂ¡of336 UM (Chart 1). This is in contrast to groups, respectively) had no effect on MTX influx (Table 1). other metabolic inhibitors that increased the Vmax,decreased Effects of DNP on MTX Efflux. Intracellular MTX consists of the Km, and exhibited a small lag phase before producing an exchangeable fraction which rapidly leaves the cells and a maximal effects. Glucose alone produced a small but significant fraction that is tightly bound and is not significantly different inhibition of influx and reversed the stimulatory effects of azide from the dihydrofolate reductase binding capacity (3). Cells and rotenone (Table 1), as well as of antimycin A, cyanide, were brought to a steady state with 2 JUMMTX, washed twice oligomycin, and 2-deoxyglucose (not shown), that was appar at 0Â°, and resuspended in MTX-free buffer with or without ently related to restoration of cell ATP by anaerobic glycolysis. DNP, as indicated in Chart 2. DNP (0.1 mM) inhibited efflux of The inhibition of MTX influx by DNP, however, does not appear exchangeable MTX. Further, DNP increased the nonexchange- to be related to energy depletion, since addition of 10 mM able fraction, an effect that was not diminished by repeated glucose, which repletes cellular ATP in the presence of DNP washing of the cells in MTX-free buffer and continued incuba (14), did not reverse its effect on MTX influx. Indeed, the tion for up to 120 min. The inhibition of MTX efflux by 0.1 mM inhibitory effects of glucose and DNP were additive (Table 1). DNP was reversed by glucose, which also restored the non-

3670 CANCER RESEARCH VOL. 40

7 control cells exceeded the expected equilibrium ratio by 3.29- fold. Addition of 0.1 or 0.25 mM DNP increased the distribution 6- ratio to 9.88- and 11.8-fold, respectively, over the equilibrium value. If DNP concentrations were increased to 0.5 mM and higher, the distribution ratio declined. Indeed, the electrochem ÃŽMâ€¢D ical potential for MTX in the presence of 1 mM DNP was less O> B 4 - than the control, although exchangeable levels are difficult to quantitate under these conditions because of the very slow exit rate and the high nonexchangeable component. The effects of low concentrations of DNP were similar to those observed with many other metabolic inhibitors. Table 3 shows a 12- to 20- fold augmentation of the MTX distribution ratio in the presence of a variety of inhibitors. Glucose alone decreased the ratio and reversed the effects of all metabolic inhibitors.

DISCUSSION 0 15 30 45 60 75 90 MINUTES Previous studies have suggested an energy-dependent exit Chart 2. Effects of DNP or DNP plus glucose on net efflux of MTX. Cells were incubated at 37Â° for 30 min with 2 JIM MTX, washed twice in 0Â°buffer, and pump for MTX, based upon inhibition of efflux and stimulation resuspended into 40 ml of MTX-free buffer containing: â€¢.no additions; O, 0.1 of uphill transport of MTX in cells by metabolic poisons (8, 11). ITIMDNP; A, 0.1 mM DNP plus 10 mM glucose; â€¢1.0 rriM DNP; and A, 1.0 mw DNP, an uncoupler of oxidative phosphorylation, is a commonly DNP plus 10 mM glucose. used inhibitor of energy metabolism which depletes cellular ATP. While some of the effects of this agent on MTX transport exchangeable fraction to the dihydrofolate reducÃase binding level (Chart 3), which suggests that these effects were due 20- completely to energy depletion. The effects of 0.1 mM DNP on I8 - MTX efflux were similar to those observed with azide, cyanide, â€¢ rotenone, antimycin A, and 2-deoxyglucose, all of which re 025 mM DNP duced the efflux rate constant, increased the nonexchangeable fraction, and were totally reversed by glucose (Table 2). If the 0 5 mM ONP DNP concentration was increased to 1 mM, efflux was further 0 I mM ONP inhibited and the major portion of intracellular MTX was non- IO - exchangeable (Chart 3). However, addition of glucose only 8 - partially reversed inhibition of efflux by this level of DNP (Chart * I 0 mM DNP 3), which suggests that at high concentrations of DNP, efflux 6- is inhibited, in part, both by energy depletion and by an energy- 4 - independent mechanism. Table 2 indicates efflux rate con 2- stants for exchangeable MTX and the nonexchangeable MTX levels in the presence of 0.1 and 1 mM DNP and a variety of 0 other metabolic inhibitors. O 45 60 75 90 105 120 Effects of DNP on Net MTX Transport. When cells at the MINUTES steady state with 1 /Â¿MMTX were exposed to low concentra Chart 3. Effect of DNP on net uptake of MTX. Cells were incubated at 37Â°in the presence of 1 /IM MTX. At 33 min. portions of the media were exposed to the tions of DNP, net uptake of MTX was stimulated and the cells indicated concentrations of DNP. achieved a higher steady-state level (Chart 3). As DNP concen trations increased from 0.1 to 0.25 mM, net accumulation Table 2 increased. However, at high DNP concentrations (0.5 to 1.0 Effects of metabolic inhibitors on the efflux rate constant for exchangeable mM), the steady-state levels of MTX declined toward the con MTX and the nonexchangeable MTX level Cells were incubated for 30 min with 2 /Â¿MMTX. washed twice in 0Â°buffer, trol. The increase in net accumulation in the presence of 0.1 to and resuspended into 40 ml MTX-free buffer at 37Â°with or without inhibitor.

1.0 mM DNP were totally abolished by 10 mM glucose (Chart rate con stant3(min 4). Hence, while glucose did not alter DNP inhibition of influx bleMTX and only partially reversed inhibition of efflux by 1 mM DNP, it tration0. ')0.171 level2.46 Â±0.012b0.070 abolished entirely DNP effects at the steady state. Table 3 ControlDNPDNPAzideCyanide Â±0.1153.21 indicates the steady-state distribution ratio for MTX (the ratio 1mM1.0 Â±0.0110.032 Â±0.2365.56 mM10.0 Â±0.0110.073 Â±0.4974.24 of the exchangeable MTX concentration in the intracellular mM1.0 Â±0.0070.070 Â±0.3703.33 water, [MTX]Â¡,to the extracellular MTX concentration, [MTX]e) mw Â±0.031 Â±0.464 Rotenone 4 1.0 fiM 0.050 Â±0.029 5.03 Â±0.629 in control cells and with varying concentrations of DNP and Antimycin A 3 0.5 /AM 0.054 Â±0.022 5.35 Â±0.782 other inhibitors with or without glucose. The measured MTX 2-Deoxyglucosen134674 3Concen50.0 mMEfflux0.089 Â±0.036Nonexchangea3.39 Â±0.454 concentration gradient was compared to the expected concen The rate constant is determined as the slope obtained from a semilogarithmic tration gradient for an equilibrating system for a bivalent aniÃ³n, plot of exchangeable MTX as a function of time after resuspension into MTX-free buffer. Exchangeable MTX is determined from the net MTX level in the cell less based on the membrane potential as estimated from the chlo the nonexchangeable fraction. ride distribution ratio. The measured [MTX]Â¡/[MTX]e ratio in Mean Â±S.E. of n experiments performed on separate days.

OCTOBER 1980 3671

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1980 American Association for Cancer Research. D. W. Fry et al. are similar to those of other metabolic inhibitors, other effects creased, efflux is further inhibited and there is only partial are different. Low levels of DNP, sufficient to slow efflux and reversal by glucose, which indicates that high concentrations enhance net MIX transport, do not alter MIX influx. Higher of DNP not only inhibit efflux by energy depletion but also by concentrations of DNP reduce MIX influx in sharp contrast to an energy-independent mechanism. Finally, while low concen influx stimulation observed with other metabolic inhibitors. trations of DNP stimulate accumulation of exchangeable MIX While the effects of other inhibitors of oxidative phosphorylation at the steady state, an effect observed with other inhibitors that on MIX influx are reversed by glucose, inhibition by high is presumably related to inhibition of an exit pump, as the DNP concentrations of DNP is not affected, which suggests a mech level is increased, stimulation of net MIX transport is reduced anism independent of alterations in energy metabolism. This is and ultimately net accumulation may be inhibited. further supported by the observation that arsenale, another These data thus indicate an interaction between DNP and uncoupler of oxidative phosphorylation, does not alter MIX the MTX-tetrahydrofolate cofactor carrier that results in a influx. Likewise, at low levels of DNP, inhibition of efflux is marked reduction in the bidirectional flows of this antifolate in completely reversed by glucose, but, as DNP levels are in- addition to effects based upon inhibition of energy metabolism. This is not surprising, in view of the complex interactions 1 between DNP and mammalian cells. Mitchell (17, 18) sug gested that agents such as DNP uncouple phosphorylation 12- from electron transport by forming lipid-soluble proton donor- acceptor systems in the membrane which facilitate proton transport, thus short-circuiting the proton potential. Indeed, it was shown that DNP greatly increased the conductance of lipid membranes (1 ), due almost entirely to proton transport (12). These effects are produced not only in the mitochondrial mem brane but in the plasma membrane as well. Thus, DNP has 6 been reported to abolish proton gradients across the cell membrane (20, 21 ) and to cause hyperpolarization or depolar 4- ization, depending on the pH (14). DNP has also been reported to directly inhibit a variety of membrane phenomena such as (a) aniÃ³n transport in erythrocytes (19), (to) swelling in mito chondria (23), and (c) adenosine triphosphatase activities (24). In view of this and the observation that inhibition of MTX 60 75 90 transport by these compounds is instantaneous, reversible, MINUTES and demonstrates competitive kinetics, it is likely that this agent Chart 4. Effect of DNP or DNP plus glucose on net uptake of MIX. Cells were incubated at 37Â°in the presence of 1 nu MIX. At 33 min, portions of the media interacts with the MTX carrier directly at the cell membrane. were exposed to: â€¢.no additions; â€¢0.1 mM DNP; O, 0.1 HIM DNP plus 10 HIM DNP and dicumarol contain substituted phenols with an acidic glucose; A, 1.0 mM DNP; and A. 1.0 mM DNP plus 10 mM glucose. hydroxyl group (pK ~ 4). The ionization of this hydroxyl group

Table 3 Effects of DNP and other metabolic inhibitors on the MTX electrochemical potential

pected0 ured/ [MTX1/ ex InhibitorControlDNP (/il/mg)3.753.453.763.503.553.47Â±Â±Â±Â±Â±Â±0.096e0.0970.0600.0810.1320.134[ci-yicii."0.5020.4750.5440.4100.5210.493Â±Â±Â±Â±Â±Â±0.0300.0520.0310.0070.0080.013Ex[MTX).0.2520.2260.2950.1680.2720.243Measured[MTX],/[MTX],0.8282.492.972.450.5443.630.4262.110.6973.864.950.468Â±pected3.299.88'11.799.72'2.4112.31.69'12.62.76'14.219.6'1.93 0.066Â± (0.1mM)DNP(0.25mM)DNP 0.389Â± 0.194Â± (0.5mM)DNP 0.323Â± (1.0mM)AzidedO.O 0.127Â± mM)AzidedO.O 0.395Â± +glucose mM) 0.081Â± mM)2-Deoxyglucose(50.0(10 0.320Â± mM)2-Deoxyglucose(50.0 0.051Â± glucose(10.0mM) + mM)Antimycin /IM)RotenoneA (0.5 0.674Â± .0/iM)Glucose(1 0.037Â± (10.0 mM)[HOH1/DW" 0.072n1432378333226Meas (HOHl/DW, intracellular water/dry weight. 6 (CI ],/[CI I,, ratio of intracellular to extracellular chloride concentration. c Ratio of intracellular to extracellular MTX concentration for an equilibrating system calculated from the Nernst equation. The chloride distribution ratio was used as a measure of membrane potential and MTX was assumed to be a bivalent aniÃ³n at pH 7.4. d Experimentally measured ratio of intracellular to extracellular MTX concentration. Intracellular MTX was based on the net intracellular MTX level at the steady state in the presence of 1 /IM MTX less the nonexchangeable fraction for each condition. 8 Mean Â±S.E. of n experiments. ' Values were determined using the expected [MTXl/JMTX], for the control.

3672 CANCER RESEARCH VOL. 40

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1980 American Association for Cancer Research. Effects of DNP on MIX Transport appears to be an important factor in the inhibitory properties of 3. Cohen. M., Bender, R. A., Donehower, R.. Myers. C. E.. and Chabner. B. A. these compounds, since unsubstituted phenol (pK =. 9.89) or Reversibility of high-affinity binding of methotrexate in L1210 murine leu kemia cells. Cancer Res.. 38 2866-2870, 1978. dinitrobenzene (no hydroxyl group) does not inhibit MTX influx. 4. Divekar. A. Y., Vaidya. N. R.. and Braganca. B. M. Active transport of Since DNP and dicumarol are anions at physiological pH, aminopterin in Yoshida sarcoma cells. Biochim. Biophys. Acta, 135: 927- 936. 1967. inhibition may be due, in part, to the general sensitivity of MTX 5. Fry, D. W.. and Lawrence. W. Evidence for bidirectional fluxes of methotrex influx to anionic compounds (8, 13). However, this inhibition is ate in Ehrlich ascites tumor cells by the same carrier. Proc. Am. Assoc. Cancer Res., 27. 21, 1980. different from that of other organic anions in that it retards the 6. Fyfe. M. J., and Goldman, I. D. Characteristics of the vincristine-induced bidirectional flows of MTX, while other organic anions tested augmentation of methotrexate uptake in Ehrlich ascites tumor cells. J. Biol. markedly inhibit influx but have no effect on efflux (13). Chem., 248. 5067-5073. 1973. 7. Goldman. I. D. Transport energetics of the folie acid analogue, methotrexate, DNP, as well as other metabolic inhibitors, increase the in L1210 leukemia cells: enhanced accumulation by metabolic inhibitors. J. apparent nonexchangeable fraction of MTX (Chart 2; Table 2). Biol. Chem.. 244: 3779-3785. 1969. This fraction appears to represent tight binding rather than 8. Goldman, I. D. The characteristics of the membrane transport of amethopterin and the naturally occurring folates. Ann. N. Y. Acad. Sci.. 786. 400- slow efflux of exchangeable MTX, since (a) this component 422. 1971. persists over several hr of observation and is not diminished by 9. Goldman, I. D. A model system for the study of heteroexchange diffusion: multiple changes of the extracellular fluid, (o) the quantity of methotrexate-folate interactions in L1210 leukemia and Ehrlich ascites tumor cells. Biochim. Biophys. Acta, 233. 624-634, 1971. MTX that leaves the cells in the presence of metabolic inhibitors 10. Goldman, l. D., Lichtenstein. N. S., and Oliverio. V. T. Carrier-mediated over an extended period of time does not correspond to the transport of the folie acid analogue, methotrexate. in the L1210 leukemia amount predicted from the rate constant if all MTX were ex cell. J. Biol. Chem., 243. 5007-5017, 1968 11. Hakala, M. T. On the nature of the permeability of Sarcoma 180 cells to changeable, and (c) efflux of MTX in the presence of metabolic amethopterin in vitro. Biochim. Biophys. Acta. 702. 210-225, 1965. inhibitors does not conform to a single exponential unless 12. Hopfer, U., Lehninger, A. L.. and Thompson, T. E. Protonic conductance across phospholipid bilayer membranes induced by uncoupling agents for corrected for the increased binding. The nature of this addi oxidative phosphorylation. Proc. Nati. Acad. Sei. U. S. A.. 59 484-490. tional nonexchangeable fraction is unclear and under further 1968. investigation. 13. Jennette. J. C., and Goldman, I. D. Inhibition of the membrane transport of folates by anions retained in uremia. J. Lab. Clin. Med., 86. 834-843. 1975. The multiple effects of DNP on the MTX transport system 14. Johnstone. R. M. The hyperpolarizing and depolarizing effects of 2.4-dini- reported in this paper may clarify discrepancies in the effects trophenol on Ehrlich cells Biochim. Biophys. Acta. 5Ã•2. 550-556, 1978. 15. Kessel. D., and Hall, T. C. Amethopterin transport in Ehrlich ascites carci of this agent in different studies and dissimilarities between this noma and L1210 cells Cancer Res.. 27. 1530-1543. 1967. and other metabolic inhibitors. Hence, in evaluating the effects 16. Kessel. D.. Hall, T. C.. and Roberts. D. Modes of uptake of methotrexate by of DNP on MTX transport, the results obtained will be markedly normal and leukemic leukocytes in vitro and their relation to drug response. Cancer Res.. 28. 564-570. 1968. influenced by the experimental conditions in which the meas 17. Mitchell. P. Coupling of phosphorylation to electron and hydrogen transfer urements are made, such as (a) concentration of the inhibitor, by a chemi-osmotic type of mechanism. Nature (Lond.). (9). 144-148. (o) whether the measurement is taken during influx, net trans 1961. 18. Mitchell, P. Chemi-osmotic coupling in oxidative and photosynthetic phos port, or at the steady state, (c) whether total intracellular MTX phorylation Biol. Rev. Camb Philos. Soc.. 41: 445-502. 1966. or freely exchangeable MTX is determined, and (d) whether or 19. Omachi. A. Sulfate transport in human red cells: inhibition by some uncou- not glucose or other energy-producing substrates are present. plers of oxidative phosphorylation. Science (Wash. D. C.), 745 1449-1450. 1964. 20. Poole, D. T. The effect of uncoupling agents on the pH gradient across the plasma membrane of the Ehrlich ascites tumor cell. Biochem. Biophys. Res. Commun. 32. 403-407, 1968. ACKNOWLEDGMENTS 21. Poole, D. T.. Butler, T. C., and Williams. M. E. The effects of nigericin, valinomycin. and 2.4-dinitrophenol on intracellular pH. glycolysis. and K' The excellent technical assistance of Linda Anderson is gratefully acknowl concentration of Ehrlich ascites tumor cells. Biochim. Biophys. Acta. 266 edged. 463-470. 1972. 22. Rubin, R.. Owens, E., and Rail, D. Transport of methotrexate by the choroid plexus. Cancer Res., 28. 689-694, 1968 23. Tapley. D. F. The effect of thyroxine and other substances on the swelling REFERENCES of isolated rat liver mitochondria. J. Biol. Chem.. 222. 325-339. 1956. 24. Yamada, Y., and Watanabe. S. Competitive and uncompetitive effects of 1. Bielawski, J., Thompson. T. E., and Lehninger. A L. The effect of 2.4- 2,4-dinitrophenol on ATPase activities of rabbit skeletal actomyosin and dinitrophenol on the electrical resistance of phospholipid bilayer membrane. myosin. J. Biochem. (Tokyo), 82. 893-900. 1977. Biochem. Biophys. Res. Commun., 24 948-954. 1966. 25. Yang, C. H.. Peterson. H. F.. Sirotnak, F. M.. and Chello. P. L. Folate analog 2. Bobzien, W. F.. III. and Goldman. I. D. The mechanism of folate transport in transport by plasma membrane vesicles isolated from L1210 leukemia cells. rabbit reticulocytes. J. Clin. Invest., 51: 1688-1696. 1972. J. Biol. Chem., 254. 1402-1407, 1979.

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1980 American Association for Cancer Research. Effects of 2,4-Dinitrophenol and Other Metabolic Inhibitors on the Bidirectional Carrier Fluxes, Net Transport, and Intracellular Binding of Methotrexate in Ehrlich Ascites Tumor Cells

David W. Fry, J. Courtland White and I. David Goldman

Cancer Res 1980;40:3669-3673.

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