Europ. J. Cancer Vol. 11, pp. 845-854. Pergamon Press 1975. Printed in Great Britain

Changes of Molecular Properties Associated with the Development of Resistance Against Methotrexate in Human Lymphoblastoid Cells* D. NIETHAMMER and R. C. JACKSON," Department of Paediatrics, University of Ulm, Germany and Department of Applied Biochemistry, Institute of Cancer Research, Sutton, Surrey, United Kingdom Abstract--WI-L2 human lymphoblastoid cells were exposed, in culture, to progressively increasing concentrations of methotrexate. By this procedure a series of sublines was obtained with varying degrees of resistance to this drug. Membrane transport of folates and antifolates and the activities of folate dependent enzymes were studied in these sublines. Three mechanisms of drug resistance were observed. As in many previous studies, elevated cellular activities of dihydrofolate redactase werefound; in some sublines the activity increased over 200-fold. A detailed study was made of the electrophoretic pattern of dihydrofolate redactase in the resistant cells, and the increase in activity was found to be of two kinds; initially electrophoretic form I increased, whereas in cells with the highest enzyme activities predominantly form II was found. The other enzymes tested, thymidine kinase, thymidylate synthetase, serine hydroxymethyltransferase, lO-formyltetrahydrofolate synthetase and 5,10-methylenetetrahydrofolate dehydrogenase, did not change greatly during the course of development of resistance. Serine hydroxymethyltransferase and thymidylate synthetase were both inhibited by methotrexate but these inhibitions were weak. The contributions made by these secondary sites of action to the cytotoxicity of the drug are discussed. A relatively high frequency of mutants was found with impaired membrane transport of methotrexate ; these mutants appeared rather late in the course of development of resistance, and in all these cases enzyme and transport mutations occurred together. As in LI210 mouse leukaemia cells, 5-methyltetrahydrofolate, 5-formyltetrahydrofolate and methotrexate shared a common pathway for uptake into the cell; in contrast to L1210 cells, wefound the predominant change in the Vmaz, rather than the K,,, of the transport system of resistant mutants. It seems that resistance involving impaired methotrexate transport during treatment of human malignant disease may be a comparatively frequent phenomenon, and may occur together with elevation of dihydrofolate reductase activity. Accepted 9 September 1975. *Experimental work in this paper was supported by grants from Deutsche Forschungsgemeinschaft (SFB 112) and from the Ludwig Institute for Cancer Research (London Branch). ~'Present address: Laboratory for Experimental Oncology Indiana University School of Medicine, Indianapolis 46202, U.S.A. ~:Abbreviations used: MTX, methotrexate; FH 2, dihydrofolate; FH4, tetrahydrofolate.

INTRODUCTION THE DEVELOPMENT of resistance to methotrexate (MTX)~ has been studied in numerous experimental tumours. The known mechanisms of acquired resistance involve either an elevated level of the target enzyme, dihydrofolate (FH2) reductase [1-8], increased activity of thymidylate synthetase [9, 10], impaired

845

846

D. Niethammer and R. C. Jackson

membrane transport of the drug [7, 8, 11-15], or utilisation of the "salvage pathway" for biosynthesis of thymidylate [16]. Our own studies with L1210 mouse leukaemia cells [17] and experiments by others [14] have shown that defects of cellular uptake of M T X tend to appear early in the course of development of resistance; these mutants are then outgrown by cells containing high dihydrofolate reductase levels [17, 18]. The relationship of the multiple electrophoretic forms of dihydrofolate reductase has been studied in detail in Lactobacillus casei [19]. Preliminary studies with L1210 cells have explored the relation of these different forms to the increased total reductase activity found in MTX-resistant cells [17, 20, 21]. As resistance developed, electrophoretic form I increased first, but the highly resistant cells which emerged later contained predominantly form II of the enzyme. Corresponding studies have not been reported with human cells. A complete understanding of resistance of human cells to M T X will also require a fuller knowledge of the molecular properties of folate transport systems in the membrane of human cells. Kessel, Hall and Roberts [22] demonstrated that permeability to M T X of human leukaemic leucocytes was correlated with clinical response to the drug. At low drug concentrations M T X entered the cells by a saturable, temperature-sensitive process of facilitated diffusion [22], but at extracellular concentrations greater than 2 x 10 .5 M a significant amount of the drug could enter the cells by passive diffusion [23]. Much greater concentrations than this may be achieved during high-dose M T X infusions. Experiments with transport mutant L1210 cells [17] indicated that ability to transport 5-methyltetrahydrofolate was lost in parallel with M T X transport activity, but that folic acid transport ,.'as unimpaired; the Vma x for uptake of M T X was similar to that of the drug-sensitive line, whereas K , increased by a factor of 3-5. The present report describes analogous studies with human cells, and considers the relevance of the results to the treatment of human malignant disease. MATERIAL AND METHODS aH-MTX, 5-methyl-(14C)-tetrahydrofolate and aH-folic acid were obtained from the Radiochemical Centre, Amersham. Their purity, as monitored by thin layer chromatography, was about 98%. Unlabelled M T X was a product of Lederle, enzyme substrates and cofactors were from Boehringer, and reagent

chemicals were obtained from the usual commercial sources, analytical grades being used where available.

Cell culture The W1-L2 human lymphoblastoid cell line was originated in 1968 from the spleen of a patient with spherocytic anaemia [24]. The cells were maintained in continuous suspension culture using R P M I 1640 medium, supplemented with 10% foetal calf serum and antibiotics. Cells were subcultured every second day into Falcon plastic tissue culture flasks, using an inoculum of 6-8 x 104 cells/ml. The generation time under these conditions was about 14 hr and the cultures reached a final density of about 1.5 x 106 cells/ml. The dose-response curves were obtained by adding the required concentrations of M T X to triplicate cultures at zero time, and counting the cultures in a haemocytometer after 48 hr. What is measured under these conditions is thus inhibition of cell growth, not cell lysis or decrease in viability. Resistant sublines were obtained by growing sensitive W1-L2 cells in the continuous presence of M T X . The initial concentration used was 2 x 10-8 M. Under these conditions more than 70% of the cells died. After 48 hr the surviving cells were transferred to fresh drugcontaining media. Over the course of 5-10 culture generations the cell growth rate increased and eventually no dead cells were seen. When the cell growth rate was almost back to normal the drug concentrations was doubled and the procedure was repeated. Five parallel lines were maintained in this way, and the total duration from step 1 to step 9 was about four months. The studies here described are based on the final two stages in the development of resistance of each line (steps 8 and 9). Terminology is as follows: WS indicates the original drug-sensitive line, and W R the resistant lines; the first number indicates the stage of resistance, and the second refers to the cell line (e.g. WR9-3 is line 3 at the 9th stage of resistance). The WR8-1ines were maintained in the presence of 1.6x 10 -6 M M T X , and the WR9lines in the presence of 3.2 x 10- 6 M M T X . Transport experiments Cells were collected during logarithmic growth and resuspended in phosphate-buffered saline [25] pH 7.2 at 37 ° at a concentration of about 5 × 106 cells per ml. Radioactive substrate was added at zero time and 2 ml aliquots were withdrawn at the indicated times and were blown into 9 ml of ice-cold physiological saline which stopped transport immediately

The Developmentof Resistance Against Methotrexatein Human LymphoblastoidCells [23]. During the course of the experiments the pH of the medium did not change, no cell lysis was seen, and the integrity of the membrane, as monitored by trypan blue, remained unimpaired. The pellet was washed twice in ice-cold saline and was digested overnight in 1 ml Protosol (New England Nuclear). After adding 10 ml of scintillation counting fluid ( 5 g PPO and 0.3g dimethylPOPOP/l of toluene) the samples were poured into plastic vials and counted in a refrigerated scintillation counter. Counting efficiences under these conditions were 40-5% for tritium and 70% for 14C. Extracellular volume was determined by the use of x4C.inulin ' which is not taken up by the cells. Radioactivity already present at zero time was probably due to a rapid nonspecific adsorption to the outside of the cell membrane [23]. Data are calculated as nmoles of folate compound taken up per 109 cells (approx. 0.92 g wet weight).

847

of resistance showed wide variation. The most resistant line obtained, WR9-4, was capable of growth at 70% of the normal rate at the highest M T X concentration tested, 2.5 x 10-5 M. Dihydrofolate reductase was measured in cell-free preparations of each cell line. All ten resistant sublines showed elevations of this

'~

WR 8

%.

WR 9

~-------.~

60 50

,,

40 30

2o

~o..

'°

Enzyme assays All enzyme assays were performed at 30 ° on cell free preparations. For this purpose cells were disrupted with a Branson sonifier (position 6.20 sec) in ice-cold 0.05 M Tris-chloride buffer pH 7.5, and the lysate was centrifuged at 25000 x g for 20 rain to remove particulate material. Dihydrofolate reductase was measured according to the method of Matthews and Huennekens [26], thymidylate synthetase by the method .of Wahba and Friedkin [27], thymidine kinase by the method of Voytek and co-workers [28], serine hydroxymethyltransferase by the method of Scrimgeour and Huennekens [29], 5-10-methylene tetrahydrofolate dehydrogenase according to Ramasastri and Blakley [30] and 10-formyltetrahydrofolate synthetase as described by Rabinowitz and Pricer [31]. Polyacrylamide gel electrophoresis was carried out by the procedure of Davis [32], and gels were stained for dihydrofolate reductase activity as described by Dunlap and coworkers [19], and scanned in a gel scanner attached to a Gilford model 2400-S spectrophotometer. Affinity chromatography was performed by the method of Whiteley and coworkers [33].

111=4

.

lo-e

Dose-response curves for the various resistant lines and for the sensitive line are shown in Fig. 1 and Fig. 2 respectively. Though the five lines of each stage of resistance were obtained in the same way, and kept at the same maintenance concentration of drug, their actual degree

MTX

CM'-]

.

10i-5

I

I

3 '10-5

Fig. 1. Dose-response curves of the ten resistant sublines, measured in culture. Ordinate: cell count after 4 8 hr growth in presence of drug as percent of uninhibited control; Abscissa: M T X concentration (log scale). 100 90 80 70 60 50

x

x

40 30

20

e-

O~ 8

"6 67 ae 5

3

RESULTS

~

¸

ii

i

I

10-9

10-8

MTX

Fig. 2.

10-7

I"M'-I

Dose-response curve for the drug-sensitive W1-L2 cells. Details as for Fig. 1.

848

D. Niethammer and R. C. Jackson

enzyme activity ranging from 20-fold to over 200-fold relative to the sensitive line (Table 1). Figure 3 shows scans of polyacrylamide gels

E=26

E=

a A~

X )

WR9-4

0 0 (,0 I.< >I--

WR9-1 (E=23, x)

Z u.I 0

O I--O_

o

A

WR9-1 \

( after ch alfinily 0 a 0g aphy

R f --->

Fig. 3. Optical density scans of polyacrylamide gel electrophorograms stained for dihydrofolate reductase activity. The numbers under the peaks indicate the percentage of each form. E = elevation of total dihydrofolate reductase activity (relative to WS) for each line.

stained for dihydrofolate reductase activity; enzyme from the sensitive line and three of the resistant lines (showing varying degrees of elevation of the activity) was examined in this way. The major peak at Rf = 0.24 is electrophoretic form I, and the double peak at Rf = 0.39 and 0.44 corresponds to electrophoretic form II; (although this peak is a doublet, both components are termed "Forms I I " in order to conform with the terminology used by previous workers). This electrophoretic pattern corresponds closely to that previously described for mouse L1210 cells and human leukaemic leucocytes [17, 18, 34, 35]. It should be noted that the band widths shown are much wider than on the original gels, because of diffusion during the activity staining process. The two minor peaks at Rr = 0.17 and ,0.61 developed also in the presence of 10-4 M M T X , suggesting that these bands represent NADPH oxidase activity. In the sensitive line form II predominated with over 60% of the total activity. In line WR9-3, with a 26-fold elevation of enzyme activity the increase has been predominantly in form I, now 63% of the total activity. In line WR9-4 (total elevation 126-fold) the further increase was apparently in Form II, and the proportions of the two forms are now close to those of the parent line. In WR9-1, with a total dihydrofolate reductase elevation of 234-fold, the increase in form II again accounted for most of the extra enzyme. It is noteworthy that in this highly resistant line Form II is no longer a doublet, but consists entirely of material with Rr = 0.39. Following affinity chromatographyof the dihydrofolate reductase from crude extracts of WR9-1 cells all the activity appeared as Form I (Fig. 3). Since recovery from the column was 70%, and 66% of the original activity before chromatography was Form II, it is clear

Table 1. Properties of some MTX-resistant W1-L2 sublines

Dihydrofolate reductase Line WS WR WR WR WR WR WR WR WR WR WR

8--1 8-2 8-3 8-4 8-5 9-1 9-2 9-3 9-4 9-5

IDso [/~M]

Resistance - fold

0.013 2.2 3.2 3-0 3-8 2.8 6-8 6.9 11.0 > 16.0 6.1

(1) 170 240 230 290 220 520 530 840 > 1230 440

IU/10 9

cells*

0.22 48.1 17.9 4.5 21.7 30-6 50.7 4-9 5.6 27-3 14.9

elevation - fold (1) 222 83 21 100 141 234 23 26 126 69

*Values q u o t e d are m e a n s for d u p l i c a t e or triplicate d e t e r m i n a t i o n s .

Transport % of control* (100) 108.3 93.1 19.7 78.5 93-8 107.4 27.3 11.2 10-2 76.5

The Development of Resistance Against Methotrexate in Human Lymphoblastoid Cells that at least 50% of the original Form II enzyme was converted to Form I during purification. The molecular relationship and biological significance of the electrophoretic forms of dihydrofolate reductase are discussed later. Table 2 records the activities of five other folate enzymes in the sensitive line and three of the resistant sublines. No large changes were detected; activity of thymidylate synthetase was slightly decreased in all three lines, and thymidine kinase showed increases of 21-60%. The biological significance of these small changes in cellular thymidine kinase is difficult to assess; no thymidine was present in the culture medium, but it is possible that traces of thymidine were present in the serum used to supplement the medium. The activities of serine hydroxymethyltransferase and 5.10methylenetetrahydrofolate dehydrogenase were high, indicating a considerable potential for utilizing serine as a source of one-carbon groups for purine biosynthesis; the activity of 10form~,ltetrahydrofolate synthetase was rather lower, suggesting that formate may be a less important one-carbon group source in these cells. In the W1-L2 cell dihydrofolate reducTable 2.

tase is the only component known to interact strongly with M T X ; the Ki value for the W1L2 reductase with M T X has been estimated to be 7.3 x 10 -12 M (Jackson, Hart and Harrap, unpublished results). Table 3 summarizes the inhibition of some other enzymes of folate metabolism by M T X in the sensitive cell line. Thymidylate synthetase and serine hydroxymethyltransferase from W1-L2 cells were also inhibited, though much more weakly, by MTX. Transport of M T X was measured in the sensitive line and in all resistant sublines. Figure 4 shows the progress curves for cellular uptake of 3H-MTX with time. Only one of the sublines of stage 8 (WR8-3) showed significant impairment of transport capacity, the other sublines at this stage having approximately normal rates. At the final stage 9 all but one of the cell lines (WR9-1) showed significant impairment of transport. Whereas drug uptake in the parental line ceased after 20 min, at which time the rates of uptake and efflux had become equal, such a steady state was not attained with the mutant lines, during the time of the experiments. The amount of bound M T X present in the cell at the steady state is

Enzyme activities in W1-L2 cell sublines

Thymidylate synthetase Thymidine kinase 10-Formyltetrahydrofolate synthetase Serine hydroxymethyl transferase 5,10-Methylenetetrahydrofolate dehydrogenase

WS

WR9-I

WR9-3

WR9-4

0.031 0.056

0.023 0.068

0.029 0-070

0.021 0.090

0.14

0"22

0" 15

0" 12

1.14

1.64

1.50

1.10

0-49

0.39

0-55

0-55

Activities are given as I.U./109 cells, when 1 I.U. = pmole ofsubstrate converted per minute at 30 °.

Table 3.

Inhibition of some enzymes of folate metabolism in W1-L2 cells

Enzyme Dihydrofolate reductase Thymidylate synthetase I 0-formyltetrahydrofolate synthetase Serine hydroxymethyltransferase 5,10-methylenetetrahydrofolate dehydrogenase

849

Activity I.U./109 cells

Concentration of M T X required to give 50% inhibition

0.22 0.02

3.1 x 10-9M* 2"2 × 10- SM

0.14

- I"

1.14

3"2 x I0- 3M

0.49

-- "[" I

*Enzyme conen, used in this experiment was 1.5 x 10-9M. 1"No detectable inhibition by M T X at 10-SM.

850

D. Niethammer and R. C. Jackson

determined by the number of binding sites, and clearly the increased amount of dihydrofolate reductase in the resistant lines may account for the increased amount of M T X 3,0-

// 1

2,5 u)

\ 2,0

2,5

4

r-

z LLI

....

1,5

S

hU '~

1,0

0,5-

~/,

/ /

MTX:

10

20

[1,6.1()6M]

30

40

50

TIME, min 3,0-

y 5

/

2,5 -

\

u~

(:D 0

2,0-

z UJ

1,5-

1,0-

4 0,5"

WR9 1-5

3

] 10

20

30

40

50

TIME, min

Fig. 4. Uptake of M T X by W1-L2 sublines. Extracellular concentration of M T X was 1 ltM. (a) Sublines WR8-1 to WR8-5, (b) Sublines WR9-1 to WR9-5. ]

taken up by the cells. The transport mutants are very stable, since their properties have not changed over six months continuous growth in the presence of MTX. Figure 5 shows the uptake curves for MTX, folic acid and 5-methyltetrahydrofolate in the transport mutant WR9-4 compared with the sensitive line. The uptake of 5-methylFH4 is impaired to the same extent as that of M T X whereas the influx of folic acid is identical in both lines, p-Choromercurisulphonate (20/~M) blocked the influx of M T X and 5-methylFH4 completely in both lines, but did not change the rate of uptake of folic acid. Table 4 summarizes some of the kinetic data for the MTX transport system obtained with the parental W1-L2 line, and with WR9-4. 5-FormylFH4 (folinic acid) competitively inhibited uptake of MTX, suggesting that it may enter the cell by the same route. In the mutant line WR9-4, the Km for M T X of the transport system was not significantly altered from the normal value, but the Vm,x was decreased by a factor of more than seventeen. Finally, Table 1 summarizes relative rates of M T X transport, when the extracellular M T X concentration was 1 #M, for all the sublines, together with the dihydrofolate reductase activities. It is clear that the greatest drug resistance is associated with an effective transport mutation in combination with large amounts of dihydrofolate reductase Form II (WR9-4). Rather lesser degrees of drug resistance are associated with either the possession of very large amounts of dihydrofolate reductase, Form II, but normal transport properties (WR8-1, WR9-1) or with an effective transport mutation, but lower activities of dihydrofolate reductase, and that preponderantly Form I (WR9-3). It is interesting to note that in two populations (2 and 5) the increase in drug resistance from stage 8 to stage 9 which was accompanied by a reduction in transport activity was also associated with reduction in the dihydrofolate reductase activity. This is probably the consequence of a change in cell population as cells containing the transport mutation but modest activities of the reductase outgrow cells with very high reductase levels but normal drug transport. By contrast, the development of the most resistant cells of all is probably the result of the simultaneous aquisition of the two mutations by the same cell.

DISCUSSION The biochemistry and pharmacology of M T X have been studied in great detail in many

The Development of Resistance Against Methotrexate in Human Lymphoblastoid Cells

MTX

[S-MeFH4

1"0

Fig. 5.

2~

3"0

4"0

1"0

851

FA

TIME,rain 2"0

gO

4~

lb

I

2"0

'

3~0

'

4'0

Uptake of M T X (1 aM), 5-methyltetrahydrofolate(0-8 aM) andfolio acid (5 I~M) in the sensitive cell line (©) and the resistantsubline WR9-4 (O).

experimental systems and a huge literature exists; however, a number of questions remain as topics of current debate. Some such questions are (i) Are the conclusions drawn from studies with mouse L1210 cells, which indicate that two membrane transport systems exist for folate compounds, also applicable to human cells? (ii) What is the significance of the multiple electrophoretic forms of dihydrofolate reductase? (iii) How important is the direct inhibition of thymidylate synthetase by M T X ? The experiments described in this paper are attempts to answer these questions within the context of our experimental model of human lymphoblastoid cells grown in culture. The first of these questions alone admits of a conclusive answer. From the results here outlined it is clear that 5-methyltetrahydrofolate and M T X share a common carrier, but that folic acid enters the cell by a separate mechanism. This is suggested by the simultaneous and quantitatively similar loss of MTX- and 5methylFH4-transporting activity in the drug resistant sublines, in which, however, folic acid uptake remains unimpaired. The effect of p-chloromercurisulphonate mimics the transport mutation: M T X and 5-methylFH, transport activity are again lost together, and folic acid transport is again unaffected. The kinetic results suggest that the M T X transport pathway may also be shared by 5-formylFH4. With regard to the development of MTXresistance by virtue of changes in membrane permeability, three aspects of the present study should be emphasized. Firstly although previous kinetic studies of transport mutants (with mouse L1210 cells) have shown increases in the Km of the transport protein for MTX, with very little change in the transport V=a,, the present results with subline WR9-4 indicate the reverse: an appreciable decrease was found

in Vm,z. This is of clinical significance, since the effect of a K=-type transport mutation may be largely overcome by increasing the extracellular drug concentration, but the effect of a V=az-type transport mutation cannot be reversed in this way. Secondly, the frequency and stability of the transport mutations in these human lines must be stressed. Studies with mouse L1210 cells [14, 17] have suggested that transport mutations occur rather early in the selection process, that they are less frequent than the high dihydrofolate reductase mutants, and that they tend to be overgrown or replaced by the latter type of MTX-resistant cell. By contrast, our results with the W1-L2 sublines indicate that transport mutations are in this case rather frequent, and very stable. This may be partly explained by the continued effectiveness of the V=ax-type of transport mutation at high drug concentrations, in contrast to the Kin-type transport mutations found in L1210 cells. Thirdly, the fact that human lymphoblasts have a pathway for folic acid uptake separate from that for M T X suggests the desirability of having antifolate drugs which enter the cell by the folic acid pathway, and which would thus circumvent MTX-resistance resulting from transport mutations. The molecular relationship of the different electrophoretic forms ofdihydrofolate reductase in higher organisms is not yet entirely clear. In Lactobacillus casei, Form II consists simply of the apoenzyme, Form I, to which is bound an equimolar amount of NADPH [19]. The situation in mammalian cells is more complex: Form II appears on the polyacrylamide gels as a doublet: attempts to interconvert F o r m I into Form II, or vice versa, (using enzyme from mouse L1210 cells) by incubation with NADPH or FH 2 respectively did result in some interconversion, but this was slow and i n c o m '

852

D. Niethammer and R. C. Jackson

plete, unlike the situation in L. casei, (R. C. Jackson, unpublished work) and furthermore, experiments with the cell line L1210/R6, a mutant line containing an 80-fold elevation of dihydrofolate reductase, largely Form II, seemed to indicate that the cells did not contain enough NADPH for all the dihydrofolate reductase to exist as holoenzyme [17]. Moreover, the enzyme from L1210/R6 appeared to have slightly different kinetic properties from the normal L1210 dihydrofolate reductase [17]. The present study has indicated that only Form I is observed after purification of the enzyme by affinity chromatography, and the total recovery of activity is sufficiently gxeat that at least half of the Form II must be converted to Form I on the affinity column. The only explanation consistent with all the observations is that "Form I I " consists of two entities, one of which is Form I plus an equimolal amount of NADPH, and the other of which is a structural modification, and that this latter modification is not recovered under the conditions of affinity chromatography. The change in electrophoretic pattern of dihydrofolate reductase as M T X resistance develops appears to be a consistent one. Earlier work with LI210 cells, both in vivo [20] and in vitro [17] has shown a rapid increase in Form I, followed, as higher degrees of drug resistance develop, by a rise in Form II. The present study, although not following changes in the electrophoretic pattern with time, has shown that in those sublines with moderate increases in total dihydrofolate reductase activity this increase is mainly in Form I, but that the highly resistant sublines showing the greatest total reductase activity have predominantly Form II. Two suggestions may be advanced for the biological significance of Form II in highly MTX-resistant cells. One suggestion is that the drug is preferentially bound by Form II, thus leaving Form I to function normally; evidence to support this suggestion has been obtained with L1210 cells, where the Form II enzyme from the highly resistant clone L1210/R7A has a K i for M T X of 2.0 x10-12 M, compared to 5.3 x 10 -12 M for the normal Form I L1210 enzyme [36]. An alternative suggestion is that Form II has a longer cellular half-life, which confers a selective advantage on the cell in the presence of M T X ; this possibility is supported by direct measurements of enzyme half-lives in both L. casei and L 1210 cells [21 ]. Finally, the present study has attempted briefly to consider the importance to human ceils of sites of action of M T X other than dihydrofolate reductase. The great activity of

serine hydroxymethyltransferase, and the feebleness of its inhibition by M T X make it most unlikely that this enzyme ever becomes rate-limiting in the presence of MTX. For thymidylate synthetase, the situation is much harder to assess: the inhibition by MTX, although two orders of magnitude stronger than that for serine hydroxymethyltransferase, is many orders of magnitude weaker than for dihydrofolate reductase; on the other hand the Vm,~ of thymidylate synthetase is much lower than that of dihydrofolate reductase. McBurney and Whitmore [37] have recently reported that hamster cells in the presence of growth-inhibitory concentrations of M T X showed no accumulation of FH2, and thus suggested that thymidylate synthetase may be rate-limiting in these conditions. As yet, no measurements have been made of FH 2 in MTX-inhibited W1-L2 cells, but the known kinetic parameters of the folate enzymes in these cells make possible a tentative prediction of the behaviour of the system. The intracellular concentration of dihydrofolate reductase is about 0.19/~M (given that mol. wt. = 21,000 and specific activity = 89 I.U./mg). To reduce the potential steady-state flux through the pathway by 50% (to half the Vma~of the rate-limiting enzyme) will require inhibiting dihydrofolate reductase by 95.45%. From the Straus-Goldstein equation [38] we may calculate that this will require a total cellular M T X concentration of 0"226 #M (given Ks = 7.3 pM, K,, for FH2 = 0.13vM, and cellular FH2 = 3 7 v M ; this makes the pessimistic assumption that 80% of total cellular folate accumulates as FH 2: if this is not true, slightly less M T X will be required). At this level of total MTX, the free M T X concentration will be 0.044 vM [38]. At the normal cellular level of 5,10-methyleneFH, (about 6/~M) this low concentration of free M T X will cause negligible inhibition of thymidylate synthetase. However, in the highly resistant subline WR9-1 the situation is rather different; the reductase activity is 234-fold higher, and to make it rate-limiting it must be inhibited 99.96%. Consider a total intracellular M T X level of 6 0 # M : this would give 99-68% inhibition of dihydrofolate reductase (not enough to make it rate-limiting). Under these conditions the free M T X concentration would be 15.5 vM. This would give appreciable inhibition ofthymidylate synthetase. We conclude that although a direct interaction of M T X with thymidylate synthetase is probably not a major determinant of cytotoxicity in the parental W1-L2 cell line, it may become important in those resistant sublines

The Development of Resistance Against Methotrexate in Human Lymphoblastoid Cells with greatly elevated dihydrofolate reductase activity. The validity of these conclusions leans on the assumption that the kinetic parameters for the polyglutamyl forms which constitute a major proportion of cellular folate coenzymes are similar to those measured for the monoglutamyl folates. Some authors [39, 40] have stressed the importance of free cellular M T X as a determinant of toxicity of this drug. The above calculations indicate that even in the sensitive W1-L2 line, where inhibition of dihydrofolate reductase is probably the major mode of action, the extensive degree of inhibi-

853

tion required inevitably entails measurable concentrations of free drug. In summary, the response of human lymphoblastoid cells to M T X is reassuringly similar, in general, to that of the useful murine model systems, although a few interesting differences exist. Years of research have made M T X one of the most completely documented of all antitumour drugs, but the last word has yet to be written about this interesting compound. Aclmowledgements--The authors are grateful for the skilled technical assistance of Miss C. Bullmann and Mr. G. A. Taylor.

REFERENCES I. J. R. BERTINO, D. R. DONOHUE, B. W. GA~RIO, R. SILBER, A. A. LENTY, M. M A Y E R and F. M. HUENNEKENS, Increased level ofdihydrofolate reductase in leucocytes of patients treated with amethopterin Nature (Lord.) 193, 140 (1962). 2. S. FUTTER-MANand M. SILVERMAN, The inactivation of folio acid by liver. J. biol. Chem. 224, 31 (1957). 3. M . J . OSBORN, M. FmSEMANand F. M. HUENI~KENS, Inhibition of dihydrofolio reductase by aminopterin and amethopterin. Proc. Soc. exp. Biol. (N.Y.) 97, 429 (1958). 4. D. ROBERTS,I. WODmSKYand T. C. HALL, Studies on folio reductase III. The level of enzyme activity and response to methotrexate of transplantable mouse tumours. CancerRes. 25, 1899 (1965). 5. J . P . P~RKmS, B. L. HILLCOATandJ. R. BERTINO,Dihydrofolate reductase from a resistant subline of the L1210 lymphoma. J. biol. Chem. 242, 4771 (1967). 6. J.L. BIEDLER,A. W. SCHmSCrmRand D. J. HUTCHISON,Selection of chromosomal variant in amethopterin-resistant sublines of leukaemia L1210 with increased levels ofdihydrofolate reductase. J. nat. CancerInst. 31, 575 (1963). 7. G. A. FISCHER, Increased levels of folic acid reductase as a mechanism of resistance to amethopterin in leukemic cells. Biochern. Pharrnacol. 7, 75 (1961). 8. U. D. K. MISRA, S. R. HUMPHREYS,M. FRIEDKIN,A. GOLDIN and E. J. CRAWFORD, Increased dihydrofolate reductase activity as a possible basis of drug resistance in leukaemia. Nature (Lord.) 189, 39 (1961). 9. D. ROBERTS, T. C. HALL and D. ROSENTI-IAL, Coordinated changes in biochemical patterns: the effect of cytosine arabinoside and methotrexate on leucocytes from patients with acute granulocytic leukaemia. CancerRes. 29, 571 0969). 10. W. WILMANNS,Effects of amethopterin treatment on thymidylate synthesis in human leucocytes and bone marrow cells. Ann. N.Y. Acad. So/. 186, 365 (1971). 1 I. J. R. BERTINO, The mechanism of action of the folate antagonists in man. Cancer Res. 23, 1286 (1963). 12. G.A. FISCHER,Defective transport ofamethopterin (methotrexate) as a mechanism of resistance to the antimetabolite in L5178Y cells. Biochem. Pharmacol. U , 1233 (1962). 13. D. K~SSEL, T. C. HALL, D. ROBERTSand I. WODINSKY,Uptake as a determinant ofmethotrexate response in mouse leukaemias. Science 150, 752 (1965). 14. F.M. SIROTNAK,S. KURITAand D. J. HUTCmSON,On the nature of a transport alteration determining resistance to amethopterin in the L1210 leukaemia. Cancer Res. ~.8, 75 (1968). 15. W. C. WERKn~ISER, L. W. LAW, R. A. ROOSA and C. A. NmnoL, Further evidence that selective uptake modifies cellular response to 4-amino-folate antagonists. Proc. Am. Assoc. CancerRes. 4, 71 (1963). 16. M. T. HAKALA, S. F. ZAKREZEWSgJ and C. A. NICHOL, Relation of folic reductase to amethopterin resistance in cultured mammalian cells. J. biol. Chem. 236, 962 (1961).

854

D. Niethammer and R. C. Jackson 17. 18.

19. 20. 21. 22. 23. 24. 25. 26. 27. 28.

29. 30. 31. 32. 33.

34. 35.

36. 37. 38. 39. 40.

R. C. JACKSON,D. NIETHAMMERand F. M. HUENNEKENS,Enzymic and transport mechanisms of amethopterin resistance in L1210 mouse leukaemia cells. Cancer Biochem. Biophys., in press. F . M . HUENNEKENS,J. I. RADER, V. NEEF, F. OTTINO, R. C. JACKSONand D. NIETHAMMER,Folate antagonists: transport and target site in leukemic cells. In: Erythrocytes, Thrombocytes, Leukocytes. (Edited by E. GERLACH,K. MOSER, E. DEUTSCHand W. WILMANNS)p. 496, Thieme, Stuttgart (1973). R.B. DUNLAP,L. E. GUNDERSENand F. M. HUENNEKENS,Interconversion of the multiple forms of dihydrofolate reductase from amethopterin-resistant LactobaciUus casei. Biochem. Biophys. Res. Commun. 42, 772 (1971). N . G . L . HARDING, M. F. MARTELLIand F. M. HUENNEKENS,Amethopterininduced changes in the multiple forms of dihydrofolate reductase from L1210 cells. Arch. Biochem. Biophys. 137~ 295 (1970). R . C . JACKSONand F. M. HUENNEKENS,Turnover of dihydrofolate reductase ill rapidly-dividing cells. Arch. Biochem. Biophys. 1549 192 (1973). D. KESSEL,T. C. HALL and D. ROBERTS, Modes of uptake of methotrexate by normal and leukaemic human leucocytes in vitro and their relation to drug response. CancerRes. 28~ 564 (1968). I . D . GOLDMAN,The characteristics of the membrane transport of amethopterin and the naturally-occurring folates. Ann. N.Y. Acad. Sci. 186~400 (1971). J. A. LEVY, M. VmOLAINEN and V. DEFENDI, Human lymphoblastoid lines from lymph node and spleen. Cancer22~ 517 (I 968). R. DULBECCOand M. VOOR, Plaque formation and isolation of pure lines with poliomyelitis viruses, or. exp. Med. ~ 167 (1954). C. K. MATHEWS and F. M. HUENNEKENS, Further studies on dihydrofolic reductase, or. biol. Chem. 238~ 3436 (1963). A . J . WAHBA and M. FRIEDKIN,Direct spectrophotometric evidence for the oxidation of tetrahydrofolate during the enzymatic synthesis of thymidylate. J. biol. Chem. 236~ PC. 11 (1961). P. VOYTEK,P. K. CHANOand W. M. PRUSOFF,Purification of deoxythymidine kinase by preparative disc gel electrophoresis and the effects of various halogenated nucleoside triphosphates on its enzymatic activity. J. biol. Chem. 2469 1432 (1971). K. G. SCRIMCEOUR and F. M. HUENNEKENS, Dihydrofolic reductase. In: Methods in Enzymology. (Edited by S. P. COLOWICKand N. O. KAPLAN) Vol. VI, p. 364. Academic Press, London (1963). R. V. RAMASASTRIand R. L. BLAKLEY,5,10-Methylenetetrahydrofolate dehydrogenase from baker's yeast. J. biol. Chem. 237~ 1982 (1962). J. C. RAmNOWITZand W. E. PRICER, Formyltetrahydrofolate synthetase. In: Methods in Enzymology. (Edited by S. P. COLOWmKand N. O. KAPLAN) Vol. VI, p. 375. Academic Press, London (1963). B.J. DAws, Disc electrophoresis II. Method and application to human serum protein. Ann. N.Y. Acad. Sci. 121,404 (1964). J. M. WHITELEY,R. C. JACKSON,G. P. MELL,J. H. DRAIS and F. M. HUENNEKENS, Folate antagonists covalently linked to carbohydrates: synthesis, properties and use in the purification of dihydrofolate reductases. Arch. Biochem. Biophys. 150~ 15 (1972). G.P. MELL, M. F. MARTELLI,J. KIRCHNERand F. M. HUENNEKENS,Multiple forms ofdihydrofolate reductase. Biochem. Biophys. Res. Commun. 33~ 74 (1968). F . M . HUENNEKENS, G. P. MELL, N. G. L. HARDINO, L. E. GUNDERSENand J. H. FREISHEIM,Structure and catalytic mechanism ofdihydrofolate reductase. In: Chemistry andBiology of Pteridines (Edited by K. IWAI, M. AKINO, M. GOTO and Y. IWANAMI)p. 329 International Academic, Tokyo (1970). L.I. HART, R. C. JACKSONand K. R. HARRAP (unpublished). M . W . McBURNEY and G. F. WmTMORE, Mechanism of growth inhibition by methotrexate. CancerResearch 35, 586 (1975). O . H . STRAUSand A. GOLDSTEm, Zone behaviour of enzymes, J. gen. Physiol. 26, 559 (1943). J. C. WHITE and I. D. GOLDMAN,Free intracellular methotrexate is a critical factor in the inhibition ofdihydrofolate reduction in Ehrlich ascites tumour cells in vitro. Proc. Am. Assoc. CancerRes. 16~ 45 (1975). F . M . SmOTNAKand R. C. DONSBACH,The intracellular concentration dependence of antifolate inhibition of DNA synthesis in L1210 leukaemia cells. CancerRes. 3,t~ 3332 (1974).