Polymorphism of dihydrofolate reductase from a methotrexate-resistant subline of L1210 cells

POLYMORPHISM OF DIHYDROFOLATE REDUCTASE FROM A METHOTREXATERESISTANT SUBLINE OF L1210 CELLS T. H. DUFFY, S. B. BECKMAN*, J. K. SATOt, H. NAGAE$, K. S. VITOLS and F. M. HUENNEKENS Division of Biochemistry, Department of Basic and Clinical Research, Scripps Clinic and Research Foundation, La Jolla, California 92037

INTRODUCTION:

METHOTREXATE

RESISTANCE

IN

CANCER C H E M O T H E R A P Y Methotrexate (MTX), the 4-amino-10-methyl analog of folic acid that is used extensively in cancer chemotherapy, is taken into cells by an active transport system whose primary substrate is 5-methyltetrahydrofolate (reviewed in (1)). Subsequent polyglutamylation of the drug enhances its intracellular retention (2). The target for M T X is dihydrofolate reductase (EC 1.5.1.3), and inhibition of this enzyme prevents thymidylate synthesis and leads to cell death (3). Resistance to the drug, which ultimately limits its clinical usefulness, has been attributed (4) to several mechanisms: (a) decreased uptake of M T X due to a reduced amount of the membrane-associated transport protein; (b) decreased uptake of M T X due to alterations in the transport protein that result in a lower affinity for the drug; (c) decreased polyglutamylation of MTX; (d) metabolic conversion of M T X to the 7-hydroxy derivative; (e) increased amount of dihydrofolate reductase; and (f) alterations in dihydrofolate reductase that result in a lower affinity for the drug. T u m o r cells propagated in vitro provide convenient model systems for studying the mode of action of MTX and mechanisms of resistance. A cloned subline (R6) of L1210 murine leukemia cells, 240-fold resistant to MTX, was obtained by culturing cells on gradually increasing concentrations of the drug (5) and is maintained in the presence of 10-6 M MTX. Dihydrofolate reductase, which is elevated ca. 80-fold in these cells, was purified to homogeneity, as judged by S D S - P A G E (6). M T X inhibition studies, however, indicated that the preparation consisted of at least two components, varying in affinity for the drug (7). Polyacrylamide gel electrophoresis (PAGE) or isoelectric *Present address: University of California, Irvine, California. tVisiting Investigator. Permanent address: Division of Hematology/Oncology, Childrens Hospital of Los Angeles, Los Angles, California. SPresent address: Department of Pediatrics, University of Nagoya, Japan.

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T.H. DUFFY, et

al.

focusing (IEF) resolved the preparation into two bands (8). The present investigation was undertaken to provide additional information about the polymorphism of the L1210/R6 dihydrofolate reductase and its relationship to MTX resistance.

E L E C T R O P H O R E T I C ANALYSIS OF L1210/R6 D I H Y D R O F O L A T E REDUCTASE The enzyme preparation, isolated as described previously (8), exhibited a single, sharp band when subjected to S D S - P A G E (Fig. 1, left); the molecular mass was estimated to be 21,500 daltons. When examined under the nondenaturing conditions of P A G E at pH 8.3 (Fig. 1, middle), two proteinstaining bands were observed. On duplicate gels (not shown), both bands stained for catalytic activity (11); inclusion of M T X in the staining medium prevented this response. The minor band and the principal band (designated 1 and 2,) had R~- values of 1.0 and 0.55, respectively. Band 1, which had the greater net charge under the conditions of this experiment, could be observed only on gels in which the dye marker was omitted. Isoelectric focusing provided pI values of 7.4 and 8.2 for bands 1 and 2, respectively (Fig. 1, right). Densitometric analysis of gels stained for protein and activity (8) revealed that 1 comprised about 10% of the total protein and at least 20% of the total activity; the specific activity of 1, therefore, was ca. 2fold higher than that of 2. When these bands were examined further, the material in 1 had the higher affinity for MTX, was more stable at 37 °, and appeared to be less activatable by KC1 (8). Subsequent observations, however, have indicated that band 2 was not homogeneous. It was rather diffuse, did not stain uniformly, and was only partially inactivated by prolonged exposure to 37 °. By narrowing the pH range in the I E F procedure, it was possible to obtain a further resolution of 2 (Fig. 2). Several components, staining for both protein and enzymatic activity, appeared to be present. Additional resolution was achieved when the enzyme was denatured with urea and subjected to IEF (Fig. 3). Under these conditions, four major bands and two minor bands were observed. Experiments are currently in progress to ascertain which of these denatured bands have arisen from 2 and which from 1. For comparative purposes, dihydrofolate reductase partially purified from parental (MTX-sensitive) El210 cells was analyzed by IEF (data not shown). Two bands, staining for protein and enzymatic activity, were observed. The major and minor protein bands (representing 40 and 60% of the activity, respectively) had pI values of 8,2 and 8.5. The relationship of the 8.2 band to its counterpart in the R6 enzyme is not yet known. A series of monoclonal antibodies directed against the R6 enzyme, which have been developed

DIHYDROFOLATE REDUCTASE POLYMORPHISM

5

recently in this laboratory, may aid in detecting epitopes common to the parental and R6 dihydrofolate reductases.

ENZYME-SUBSTRATE AND ENZYME-INHIBITOR COMPLEXES

Dihydrofolate reductases form stable binary and ternary complexes with substrates and inhibitors (6), and the electrophoretic behavior of these complexes provides information about their structures. Various complexes of the major band of the R6 enzyme were readily visualized by P A G E (Fig. 4). The slight, but significant, increase in the mobility of the band when it was complexed with dihydrofolate or MTX results from the two additional negative charges provided by the ligand. In contrast, the N A D P H complex had a greater mobility than would be expected from the charges contributed by the nucleotide. In this latter instance, it appears that complexation of the protein has caused a conformational change, exposing more negatively charged groups to the environment. Addition of MTX to the enzymeN A D P H complex produced an additional increase in mobility. The enzyme complexes were also examined by IEF (Fig. 5). Under these conditions, binary and ternary MTX complexes of the minor band were seen, along with those of one or more components of the major band. Changes in pI values produced by these complexations were greater than expected from the charges contributed by the ligands and must be the result, therefore, of conformational alterations. Two of the monoclonal antibodies to the R6 enzyme (see above) showed a slightly reduced ELISA response to the E - M T X complex. More pronounced losses of ELISA activity were observed with M T X - E - N A D P H , confirming the IEF results, which had indicated that significant conformational changes accompanied formation of the ternary complex.

CONFORMATIONAL

CHANGES OF DIHYDROFOLATE REDUCTASES

The relatively large difference in isoelectric points (7.4 vs. 8.2) of bands 1 and 2 suggests that it should be possible to separate components of the R6 enzyme on a preparative scale via ion-exchange chromatography. This may be complicated, however, by the apparent interconvertibility of certain of these components. When bands 1 and 2 were excised, eluted, and re-run on IEF, 2 gave rise mainly to 2 along with a small amount (ca. 10%) of 1; band 1, alternatively, produced 2 and no detectable amount of 1 (data not shown). Although the forms were stable during PAGE or IEF, interconversion between 1 and one of the components of 2 apparently occurred when the separate entities were allowed to stand in solution between successive IEF

6

T.H. DUFFY, et al.

p r o c e d u r e s . C o n d i t i o n s accelerating or r e t a r d i n g this i n t e r c o n v e r s i o n are c u r r e n t l y u n d e r investigation. T h e o c c u r r e n c e o f i n t e r c o n v e r s i o n w o u l d suggest that c o n f o r m a t i o n a l l y different forms o f the same p o l y p e p t i d e are involved in the relevant c o m p o n e n t . This possibility is consistent with the k n o w n ability o f d i h y d r o f o l a t e r e d u c t a s e s to u n d e r g o " a c t i v a t i o n " , a process believed to be due to c o n f o r m a t i o n a l changes (14). E u k a r y o t i c d i h y d r o f o l a t e reductases d i s p l a y several-fold increases in catalytic activity when t r e a t e d with salts, c h a o t r o p i c agents (e.g., u r e a o r g u a n i d i n i u m h y d r o c h l o r i d e ) o r sulfhydryl reagents (e.g., mercurials, 5 , 5 ' - d i t h i o b i s ( 2 - n i t r o b e n z o i c acid) ( D T N B ) , I2, o r t e t r a t h i o n a t e ) . R e s p o n s e s o f several r e p r e s e n t a t i v e d i h y d r o f o l a t e reductases to these agents are s u m m a r i z e d in T a b l e 1. M a m m a l i a n a n d avian reductases have a single Cys residue l o c a t e d n e a r the N - t e r m i n a l end (22), which is the target for the v a r i o u s SH-agents. A c t i v a t i o n p r o b a b l y involves a c o m m o n m e c h a n i s m , since the effects o f different agents are not additive. The a c t i v a t e d enzymes are m o r e susceptible to heat d e n a t u r a t i o n a n d proteolysis t h a n their n o n - a c t i v a t e d c o u n t e r p a r t s , suggesting that a c t i v a t i o n is a c c o m p a n i e d by c o n v e r s i o n to a m o r e o p e n c o n f o r m a t i o n .

TABLE 1. ACTIVATION OF DIHYDROFOLATE REDUCTASES BY VARIOUS AGENTS Enzyme L1210 Chicken liver Beefliver E. coli L. casei

Salts

H+

Chaotropes

SH-Agents

Ref.

+ 0 + 0 0

+ + + 0 0

+ + + 0 0

+ + + 0

15-17 18-21 22-24 25,26 27

Activation (+); No effect (0); Inhibition (-).

A C T I V A T I O N OF THE L I 2 1 0 / R 6 ENZYME BY DTNB: E F F E C T ON MTX I N H I B I T I O N Previous studies have s h o w n t h a t 1 has a higher specific activity t h a n 2 a n d that it does n o t r e s p o n d to a c t i v a t i o n (8). These o b s e r v a t i o n s suggested that 1 might a l r e a d y exist in an " a c t i v a t e d " but stable c o n f o r m a t i o n . Since 1 has a high affinity for M T X , it seemed w o r t h w h i l e to explore the possibility that t r e a t m e n t o f the enzyme with v a r i o u s a c t i v a t i o n p r o c e d u r e s m i g h t i m p r o v e the affinity o f 2 for the drug. Kinetic studies o f the enzyme (7) h a d i n d i c a t e d that t r e a t m e n t o f the enzyme with D T N B , f o l l o w e d by d i t h i o t h r e i t o l ( D T T ) , might achieve this objective. In the current investigation, the enzyme was t r e a t e d only with D T N B . U n d e r these c o n d i t i o n s , b o t h 1 a n d 2 were c o n v e r t e d into mixed disulfides c o n t a i n i n g t h i o n i t r o b e n z o i c acid (TNB), which could be

pl 9.5 q=m,

W

-.-,--- pl 3.5 E

E- FH 2

E-MTX

E-NADPH

E..MTX "NADPH

FIG. 5. Isoelectric focusing of substrate and inhibitor complexes of enzyme. Complexes were formed by incubation (30 min at 4 °) of 12.2 #g of enzyme with a 5-fold excess of the indicated substrate a n d / o r inhibitor. Gels were focused on 0.5% agarose using pH 3-10 ampholytes (LKB) and stained for protein with Coomassie Blue as described in the legend to Fig. 1.

8

1". H. DUFFY, et aL

upon close examination, can be detected with the aid of electrophoresis and specific activity staining methods. This naturally-occurring polymorphism can result from: (a) genetically distinct proteins; (b) conformationally different forms of the same protein; or (c) the presence or absence of enzymebound N A D P H (14, 27). Deamidation of glutamine and asparagine residues, which could occur naturally within the cell or artificially during preparation of the cell extract, is possibly a further source of multiple forms. During purification, minor forms may be lost and additional forms created (e.g., folate or dihydrofolate complexes produced when these ligands are used to displace the enzyme from MTX-affinity columns (discussed in (28)). The existence of these multiple forms raises several questions. Do they have different functions in the cell? Are they genetic, conformational or artifactual variants of the prototype enzyme? If they are genetic variants, does each cell contain the individual genes for all forms, or is the population a mixture of cells, each containing the gene for one form? In this latter situation, maintenance of the cellular microheterogeneity requires that no form should have an adverse effect upon the replication rate. A change in the amount or properties ofdihydrofolate reductase is the most commonly encountered mechanism of M T X resistance. An increased amount of enzyme is due to gene amplification and an elevated level of m R N A (29); it is not clear, however, whether genes for all minor forms are similarly affected. Amplified genes for dihydrofolate reductase may be located either on extrachromosomal, self-replicating elements (termed "double minute chromosomes") or on elongated chromosomes (in "homogeneously-staining regions"). Examples of MTX-resistant cells in which the increased dihydrofolate reductase activity is provided entirely by increased amounts of unmodified parental enzyme have been reviewed by Morandi and Attardi (30). H u m a n cell lines showing this mechanism of resistance have been described by Cheng (31). The complexity of this problem is illustrated, however, by the work of Melera et al. (32), who have shown that independently-derived MTX-resistant sublines of Chinese hamster lung cells over-produce two isoelectric forms of either a 20 K or a 21 K dihydrofolate reductase; both enzymes are also present in the parental cells. There are several well-studied examples of altered dihydrofolate reductases that are characterized by a reduced affinity for the drug (8, 33-36). It is not yet clear whether the higher-affinity counterparts (present in the MTX-sensitive, parental cells) also coexist with the mutant enzymes in the resistant cells. The low-affinity enzymes are found in large amounts, but apart from being less sensitive to MTX, they do not appear to have uniformly common characteristics. Most, but not all, show higher pI values than the parental enzymes, and the specific activities are usually lower. The simplest alteration leading to a mutant enzyme would be a change in a single amino acid. In the enzymes from MTX-resistant E. coli (36) and 3T6

DIHYDROFOLATE REDUCTASE POLYMORPHISM cells (37), substitution of an arginine for a leucine has been shown to be the extent of the mutation, and the reduced affinity for M T X has been attributed to neutralization of the carboxylate group of a neighboring aspartate residue (believed to interact with N ~of M T X (38)) by the positively charged arginine. It should be noted that a single amino acid substitution could also allow the enzyme to assume a new conformation that is less favorable for M T X binding. Other mutant enzymes, however, differ by more than one amino acid from their MTX-sensitive counterparts, and in such cases the mutational sequence is more difficult to envision. Resistance to M T X is an exceedingly complex problem, both in terms of the number of possible mechanisms and the present lack of understanding about how these mechanisms become operative. There is wide variation in the manner by which parental cells (either in the laboratory or in the patient) are challenged by MTX, and the cloning procedures applied to an MTX-resistant population may arbitrarily select only a limited number of examples of the resistance mechanisms. The L 1210/R6 subline used in the present study arose from an extended series of M T X challenges and may have undergone multiple mutations. For example, it shows defective transport of M T X (L. Pope, unpublished results), as well as several altered forms of the enzyme that are apparently absent in the parental strain. Thus, it is difficult to assess the degree to which any given MTX-resistant subline is "representative". But sublines containing altered enzymes with reduced affinity for M T X are valuable as sources of these proteins. Three-dimensional structures of the latter, when available, may provide guidance for the synthesis of new analogs of M T X capable of high-affinity interaction with the modified binding site. In addition, further studies on the in vitro conversion of low-affinity to highaffinity forms (cf. Fig. 7) may offer clues for achieving the same objective in vivo using physiologically acceptable procedures.

SUMMARY Dihydrofolate reductase, purified to homogeneity (as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis), from a subline of L 1210 murine leukemia cells resistant to 10 -6 M methotrexate, was resolved into two principal forms (1 and 2) by polyacrylamide gel electrophoresis at p H 8.3 or isoelectric focusing. In the latter procedure, these forms had pI values of 7.4 and 8.2, respectively; both stained for protein and catalytic activity. F o r m ! appears to be a single component, comprising ca. 10% of the total protein and at least 20% of the total catalytic activity. It is also more sensitive to inhibition by MTX, more heat-stable, and less susceptible to activation than form 2. Multiple components of 2 were observed by narrowing the p H range in isoelectric focusing, and further resolution was achieved by urea denaturaAER-B~

10

T.H. DUFFY, et al.

tion. S u b s t r at e and i n h ib it o r c o m p l e x e s o f 1 and 2, differentiated by p o l y a c r y l a m i d e gel el e c t r o p h o r e s i s or isoelectric focusing, p r o v i d e d i n f o r m a tion a b o u t the ability o f the enzyme to u n d e r g o c o n f o r m a t i o n a l changes. I n t e r c o n v e r s i o n o f 1 with one o f the c o m p o n e n t s o f 2 m ay also involve c o n f o r m a t i o n a l isomerism. These c o n c l u s i o n s are consistent with the wellk n o w n ability o f e u k a r y o t i c d i h y d r o f o l a t e reductases to exhibit increased catalytic activity ( a t t r i b u t e d to t r a n s f o r m a t i o n s to m o r e open c o n f o r m a t i o n s ) when tr eat ed with salts, c h a o t r o p e s , or c y s t e i n e - m o d i f y i n g agents. T r e a t m e n t o f the L 1210/R6 e n z y m e p r e p a r a t i o n with one o f these activating agents, 5,5'd i t h i o b i s ( 2 - n i t r o b e n z o i c acid), d e r i v a ti z e d both 1 and 2 (changing their pl values to 7.3 and 6.9, respectively) and altered the enzyme such that s t o i c h i o m e t r i c inhibition for M T X was observed.

ACKNOWLEDGMENTS This p a p e r is p u b l i c a t i o n n u m b e r BCR-3696 f r o m the Research Institute o f Scripps Clinic. T h e w o r k was s u p p o r t e d by grants f r o m the N a t i o n a l Institutes o f H e a l t h (CA06522) a n d the A m e r i c a n C a n c e r Society (CH-31). T. H. D u f f y is the recipient o f a P o s t d o c t o r a l F e l l o w s h i p (PF-2493) f r o m the A m e r i c a n C a n c e r Society, an d J. K. Sato is the recipient o f a P h y s i c i a n - I n v e s t i g a t o r D e v e l o p m e n t A w a r d (CA00964) f r o m the N a t i o n a l C a n c e r Institute.

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