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Methotrexate resistance in L5178Y mouse leukemia cells Dedhar, Shoukat 1982

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M E T H O T R E X A T E R E S I S T A N C E IN L5178Y MOUSE L E U K E M I A C E L L S B y S H O U K A T D E D H A R B . S c . U n i v e r s i t y of A b e r d e e n , 1975 A T H E S I S S U B M I T T E D IN P A R T I A L F U L F I L M E N T OF T H E R E Q U I R E M E N T S FOR T H E D E G R E E OF M A S T E R OF S C I E N C E In T H E F A C U L T Y OF G R A D U A T E S T U D I E S (Depar tment of Patho logy ) We accept t h i s t h e s i s as confo rming to the r e q u i r e d s t a n d a r d T H E U N I V E R S I T Y OF B R I T I S H C O L U M B I A © S H O U K A T D E D H A R , 1982. In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head of my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood t h a t c o p y i n g or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department of PfirTHOl^O^ V  The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 Date J l P L - / g/.ST7 / 7 & 2-. ii A B S T R A C T Methotrexate, a folic acid antagonist, has been used in the clinical treatment of a wide variety of malignant neoplasms for over 20 years, either as a single agent or in combination with other antineoplastic agents. It is a cell cycle specific inhibitor and kills cells only in the S phase of growth. MTX is a potent inhibitor of the enzyme dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP + oxidoreductase, EC 1.5.1.3.)/ which catalyses the NADPH dependent reduction of dihydrofolic acid and folic acid to tetrahydrofolic acid: the metabolically active coenzyme form of folic acid essential in the biosynthesis of dTMP from dUMP by thymidylate synthetase. Inhibition of DHFR therefore leads to the inhibition of DNA synthesis and cell death. Methotrexate has many favourable properties; for instance, it interacts directly with intracellular sites without the need for prior metabolic transformation. It can be administered in large doses because toxicity to normal cells can be minimized by the administration of folinic acid ( N 5 formyl tetrahydrofolic acid) shortly after the administration of MTX. However, the effectiveness of MTX is inevitably compromised by the emergence of dru g resistance, which can be either intrinsic, i.e. the tumour cells are resistant to MTX at the outset, or the tumour cells acquire resistance after exposure to MTX. An understanding of the mechanisms of resistance to MTX is therefore v e r y important if treatment with this potent antineoplastic agent is to be improved. Three mechanisms of resistance to MTX have been determined from studies with experimental tumour systems: impaired uptake of MTX; increased levels of dihydrofolate reductase; and appearance of altered i i i d i h y d r o f o l a t e reduc tase wi th a lower a f f i n i t y fo r M T X . Impaired u p t a k e of M T X and inc reased levels of O H F R can both t h e o r e t i c a l l y be overcome b y s u s t a i n i n g i n c r e a s e d concent ra t ions of f r e e i n t r a c e l l u l a r M T X . T h i s can be ach ieved b y e x p o s i n g the ce l ls to h i g h e r concent ra t ions of M T X , and many chemotherapeut ic regimens now use ' h i g h - d o s e ' M T X wh ich _3 can ach ieve plasma concent ra t ions of M T X as h i g h as 10 M . H o w e v e r , r e s i s t a n c e to M T X is s t i l l a major c l in i ca l problem and the use of ' h i g h -dose' M T X has not s i g n i f i c a n t l y i n c r e a s e d the t h e r a p e u t i c index of M T X t r e a t m e n t . A p p e a r a n c e of D H F R with a lower a f f i n i t y f o r M T X s u g g e s t s as an a l t e r n a t i v e the s y n t h e s i s of an agent wh ich would be a potent i n h i b i t o r of the a l te red e n z y m e , and r e q u i r e s the deta i led c h a r a c t e r i z a t i o n of the p r o p e r t i e s of t h i s e n z y m e . If the a l te red enzyme re ta ins some a f f i n i t y f o r M T X , the admin is t ra t ion of M T X and the more potent agent would r e s u l t in bet te r g rowth i n h i b i t i o n of the r e s i s t a n t t u m o u r . In t h i s t h e s i s , a mouse leukemia cel l l ine ( L5178Y) g r o w n in s u s p e n s i o n c u l t u r e was used to isolate two M T X - r e s i s t a n t cel l l ines and these were used to s t u d y the mechanisms leading to M T X r e s i s t a n c e . B o t h r e s i s t a n t cel l l ines e x h i b i t e d impaired M T X u p t a k e when -6 _4 exposed to 10 M M T X b u t not when exposed to 10 M M T X , B o t h l ines also had e levated D H F R leve ls (7 to 9 f o l d ) . A v a r i a n t form of D H F R p r e s e n t in small amounts in both cel l l ines was isolated by M T X -sepharose a f f i n i t y c h r o m a t o g r a p h y . T h e a l te red D H F R d i f f e r e d f rom the major form of reduc tase p r e s e n t in these cel ls in i ts m a r k e d l y lower a f f i n i t y (100,000 f o l d ) fo r M T X . T h e two forms of the enzyme were iv purified from the most resistant cell line and their properties compared. They were found to differ moderately in their Km for substrates, however, the Ki of MTX differed by a factor of 100,000 for the two forms. In addition there were marked differences in their heat stability, isoelectric points and sensitivity to p-chloromercuriphenyl-sulphonate, and a minor difference in their molecular weights. It is concluded that the presence of a highly resistant form of DHFR in these cell lines represents an important mechanism in conferring a high degree of resistance to these cells. The importance of this form of DHFR in MTX resistance is discussed in relation to impaired transport and elevated DHFR levels. Experiments to determine the amino acid sequence of the altered enzyme are underway and once determined should facilitate the synthesis of specific inhibitors of its activity. V T A B L E OF CONTENTS Page A B S T R A C T ii LIST OF T A B L E S vii LIST OF FIGURES viii LIST OF ABBREVIATIONS xi ACKNOWLEDGEMENTS xiii INTRODUCTION 1 1. Cytotoxic mechanism of action of methotrexate 1 2. Properties of dihydrofolate reductase 3 3. Mechanisms of resistance to methotrexate 10 (1) Increased levels of dihydrofolate reductase. 12 (2) Impaired transport of methotrexate. 14 (3) Altered dihydrofolate reductases with lowered affinity for methotrexate. 18 MATERIALS AND METHODS 23 1. Chemicals 23 2. Cell culture 23 3. Procedures for establishing methotrexate-resistant cells 24 4. Measurement of the initial rate Of entry and the intra-cellular steady-state concentrations of methotrexate 24 5. Preparation of extracts 25 6. Assay for folate reductase activity 26 7. High pressure liquid chromatography of reduced and oxidized folates 27 8. Procedures for the isolation and purification of folate reductase 27 (a) MTX-sepharose affinity chromatography 28 v i ( b ) Sephadex G -100 c h r o m a t o g r a p h y 28 ( c ) C h r o m a t o g r a p h y of Form 2 fo late reduc tase on D E A E A f f i g e l B l u e 29 9 . Gel E l e c t r o p h o r e s i s 30 10. Isoelectr ic f o c u s i n g 31 R E S U L T S 32 1 . G r o w t h p r o p e r t i e s of the M T X - s e n s i t i v e and M T X -r e s i s t a n t L5178Y mouse leukemic c e l l s . 32 2 . P r o p e r t i e s of M T X u p t a k e into L 5 1 7 8 Y ( S ) and ( R 3 ) c e l l s . 38 3 . Ev idence of the p resence of two forms of fo late reduc tase in M T X - r e s i s t a n t L5178Y c e l l s . 44 4 . P u r i f i c a t i o n of Forms 1 and 2 of D H F R f rom L 5 1 7 8 Y ( R 4 ) c e l l s . (a ) A f f i n i t y c h r o m a t o g r a p h y 53 ( b ) Sephadex G -100 c h r o m a t o g r a p h y 56 5 . Molecu lar we ight determinat ion 56 P r o p e r t i e s of the M T X - s e n s i t i v e (Form 1) and M T X -i n s e n s i t i v e (Form 2) fo late r e d u c t a s e s . (a) pH optima 67 ( b ) Heat inac t i va t ion 67 (c ) E f fec t of p C M S on enzyme ac t i v i t i es 67 ( d ) K i n e t i c p r o p e r t i e s 72 (e) E l e c t r o p h o r e t i c p r o p e r t i e s 79 D I S C U S S I O N A N D C O N C L U S I O N S 85 R E F E R E N C E S 103 VII LIST OF TABLES T a b l e 1 . P h y s i c a l and k ine t i c p r o p e r t i e s of known d i h y d r o f o l a t e r e d u c t a s e s . Page T a b l e 2 . P r o p e r t i e s of L5178Y ( S ) , ( R 3 ) and ( R 4 ) c e l l s . 37 T a b l e 3 . P r o p e r t i e s of 3 H - M T X i n f l u x into L 5 1 7 8 Y ( S ) ce l ls when exposed to 10 M M T X . 42 T a b l e 4 . P r o p e r t i e s of 3 H - M T X i n f l u x into L5178Y(S ) and -4 ( R 3 ) ce l l s when exposed to 10 M M T X . 43 T a b l e 5 . P u r i f i c a t i o n of Form I and Form 2 fo late reduc tases f rom L5178Y ( R 4 ) c e l l s . 59 T a b l e 6 . Summary of the k ine t i c p r o p e r t i e s of Form 1 and Form 2 fo late r e d u c t a s e s . 75 VIII LIST OF FIGURES Figure 1. Structural formulae of methotrexate and folic acid. Figure 2. Summary of pathways of 'one-carbon' transfer reactions. Figure 3. Diagrammatic illustration of a carrier transport model to account for the unidirectional influx of MTX which occurs prior to saturation of the dihydrofolate reductase binding sites. Figure 4. Growth curve of L5178Y(S) cells. Figure 5. Effect of MTX on proliferation of L5178Y(S), ( R 3 ) and ( R 4 ) cells. 3 Figure 6. Time course of uptake of H-MTX by L5178Y(S) and ( R 3 ) cells. Figure 7. Methotrexate-sepharose affinity chromatography of cellular lysates from A. L5178Y(S) cells B. L5178Y ( R 3 ) cells C. L5178Y ( R 4 ) cells. Figure 8. High pressure liquid chromatography of various folate compounds and the reaction products of folate reductase assays. A. HPLC of a standard mixture of folates. B. HPLC of the reaction products of folate reductase activity in L5178Y(S) lysate. C. HPLC of the reaction products of Form 2 folate reductase activity. Figure 9. Inhibition of folate reductase activity from MTX-sensitive and MTX-resistant cells by MTX. Figure 10. Gel filtration on Sephadex G-100 of folate reductase Forms 1 and 2 from L5178Y ( R 4 ) cells. A. Form 1 B. Form 2. Figure 11. Photograph of SDS-polyacrylamide disc gel electrophoresis stained with Coomassie blue A. Form 2 folate reductase from R 4 cells B. Form 1 folate reductase from R 4 cells C. Molecular weight standards. IX Page Figure 12. Photograph of SDS-polyacrylamide slab gel electrophoresis stained by the silver staining method. Electrophoresis of Form 2 folate reductase at various stages of purification. 64 Figure 13. Chromatography of Form 2 folate reductase on DEAE Affigel Blue. 66 Figure 14. Effect of temperature on the enzyme activities of Form 1 and Form 2 folate reductases A. Effect of heat on the enzyme activities of L5178Y(S) and ( R 4 ) lysates. B. Effect of heat on the enzyme activities of partially purified Form 1 and Form 2 folate reductases. 69 Figure 15. Effect of pCMS on the enzyme activities of Form 1 and Form 2 folate reductases. 71 Figure 16. Double reciprocal plots of initial velocity patterns. A. Km for folic acid of Form 2 folate reductase B. Km for folic acid of Form 1 folate reductase C. Km for NADPH of Form 1 folate reductase D. Km for NADPH of Form 2 folate reductase. 74 Figure 17. Dixon plots for the determination of MTX dissociation constants. A. L5178Y(S) folate reductase 77 B. L5178Y(R 3) Form 2 folate reductase 78 C. L5178Y(R 4) Form 2 folate reductase. 78 Figure 18. Electrophoresis of Form 1 and Form 2 folate reductases on 7.5% non-denaturing polyacrylamide gels. A. Stained for enzyme activity in the absence of MTX. B. Stained for enzyme activity in the presence of MTX. 81 Figure 19. Photograph of Giemsa-trypsin-stained metaphase chromosomes from: A. L5178Y (S) cells. 83 B. L5178Y ( R 4 ) cells. 84 Schematic representation of the effect of MTX on folate metabolism in MTX-sensitive and MTX-resistant L5178Y cells. xi LIST OF ABBREVIATIONS AH Sepharose 4B: Aminohexyl sepharose 4B. DEAE: Diethylaminoethyl. DHFR: Dihydrofolate reductase or folate reductase depending on whether dihydrofolic acid or folic acid is used as substrate. DNA: Deoxyribonucleic acid. dTMP: Deoxythymidine monophosphate. dUMP: Deoxyuridine monophosphate. E. Coli: Escherischia Coli. EDC: 1 -Ethyl-3(3-Dimethyl-aminopropyl)-carbodiimide hydrochloride. HnRNA: Heterogeneousnuclear RNA. HPLC: High pressure liquid chromatography. HSR: Homogeneous staining region. KHz: Kilohertz. L. Casei: Lactobacillus Casei. mRNA: Messenger RNA. MTT: 3(4,5-dimethylthyazolyl-2)-2,5-diphenyltetrazolium bromide. MTX: Methotrexate. NADP: Nicotinamide adenine dinucleotide phosphate. NADPH: Nicotinamide adenine dinucleotide phosphate, reduced form. pCMB: Para-chloromercuribenzoate. pCMS: Para-chloromercuriphenylsulphonate. Poly(A): Polyadenylate. Q 1 0 : Change in reaction velocity for every 10° difference in the temperature of reaction. XII RNA: Ribonucleic acid. SDS: Sodium dodecyl sulphate. TEMED: N, N, N 1, N'-Tetramethylethylenediamine. U.V.: Ultraviolet. Vmax: Maximum velocity of reaction. xi I i ACKNOWLEDGEMENTS I would like to express my gratitude : To my research supervisor, Dr. James Goldie, for his support, advice and valuable guidance throughout the course of this investigation; To Dr. Godolphin, Dr. Beer and Dr, Pontifex, members of my graduate committee, for their interest and helpful suggestions, and to Dr. Pierce for his assistance and kind support; To Dr. Krystal for stimulating discussions and advice; To Daria Hartley and Dierdre FitzGibbons for expert technical assistance and for keeping the cells alive; To Linda Wood for typing the manuscript, and the Department of Medical illustrations at the Cancer Control Agency for their help with the illustrations; 7 And finally to my wife, Carole, for her support, patience, and diligent proofreading. T h i s work was partially supported by a grant to Dr. Goldie from the National Cancer Institute of Canada. INTRODUCTION 1. C Y T O T O X I C MECHANISM OF ACTION OF M ETHOTREXATE. In the early 1940's, nutritional studies designed to discover the cause of certain forms of anaemia indicated the lack of an important factor in the diets of subjects exhibiting such a disorder, (Pfiffner et al_, 1943). Th i s factor was isolated and its chemical and physical properties characterized. It was shown to be a pteridine derivative and was called "Folic acid," (Latin, folium, leaf). The structure of folic acid was elucidated in 1946, (Angier et al, 1946) and was soon followed by the synthesis and subsequent investigation of the biological activity of a number of potent antagonists. The three most widely studied and clinically useful folate antagonists were found to be aminopterin, methotrexate (MTX), and 3', 5' dichloro-MTX. These compounds are all 4-amlno analogs of folic acid and are powerful inhibitors of the enzyme dihydrofolate reductase (DHFR). Of these compounds MTX is the most potent inhibitor of DHFR and has been in clinical use as an antineoplastic agent since the early 1950's. The structural formulae of folic acid and MTX are shown in Fig. 1. Dihydrofolate reductase carries out the NADPH dependent reduction of folic acid and dihydrofolic acid to tetrahydrofolic acid, (Futterman, 1957; Osborn et al_, 1958; Peters et a[, 1959). Tetrahydrofolic acid is the metabolically active coenzyme form of folic acid, and functions as a carrier of "one carbon" units in the biosynthesis of the purine r i n g , thymidylate, methionine, serine and histidine (Huennekens, 1963; Huennekens et a[, OH M^C\ JX^ 9 10 r=\ ft H N ^ ^ C ^ 5 ^ c - C H 2 - N - ( \ /VC-N-C-CHRCH2-COOH H a N ^ / ^ ^ ^ C 0 0 H Pteroic Acid P - Amino Glutamic Acid Benzoic Acid FOLIC ACID (Pteroylglutamic Acid) NH 9 ' 2 O 9 10 f = \ H H N ^ 4 C s^ c -CH-N - ^ x ^ - C - N - C - C H j - C H g - C O O H H 2 N ^ ^ 8 ^ H C H 3 COOH METHOTREXATE (2, 4 diamino N10Methyl Pteroylglutamic Acid) 3 1968; B l a k e l e y , 1969; Rader and H u e n n e k e n s , 1973) . A summary of these v a r i o u s pathways is shown in F i g . 2 . A lack of th i s coenzyme, t h e r e f o r e , caused e i ther b y d i e t a r y d e f i c i e n c y or the admin is t ra t ion of a fo late a n t a g o n i s t r e s u l t s in decreased s y n t h e s i s of p u r i n e s , t h y m i d y l a t e and p r o t e i n s . A l t h o u g h t h e r e have been r e p o r t s i n d i c a t i n g tha t in c e r t a i n t i s s u e s a defect in p u r i n e s y n t h e s i s is r e s p o n s i b l e fo r the e f fec ts of ant i fo la tes ( e . g . M T X ) , (Go ld thwai t et a l , 1952; S a r t o r e l l i et a[, 1958) , a number of s t u d i e s showed t h a t an i n h i b i t i o n of t h y m i d y l a t e s y n t h e s i s is the p r i m a r y event lead ing to cel l death a f te r e x p o s u r e to M T X . ( T o t t e r et a l , 1955: B o r s a et a l , 1969; C a p i z z i et a[, 1971) . T h i s t y p e of cel l death is ca l led " T h y m i n e l e s s " death due to the decrease in the i n t r a c e l l u l a r pools of t h y m i d y l a t e . M T X has been shown to be most potent d u r i n g the S phase of the cel l c y c l e , ( H r y n i u k et a[, 1969; B r u c e , 1970) . In add i t ion to be ing a v e r y s t r o n g i n h i b i t o r of D H F R , M T X has also been f o u n d to be a weak , compet i t i ve i n h i b i t o r of t h y m i d y l a t e s y n t h e t a s e , ( B o r s a and Whitmore, 1969) . S i n c e the e a r l y s t u d i e s on the mechanism of act ion of M T X , i t has become i n c r e a s i n g l y a p p a r e n t tha t the p r i m a r y i n t r a c e l l u l a r t a r g e t of M T X is d i h y d r o f o l a t e r e d u c t a s e , and the s u b s e q u e n t decrease in the leve ls of t e t r a h y d r o f o l i c ac id coenzymes , (espec ia l l y methy lene t e t r a h y d r o f o l i c ac id ) leads to a decrease in i n t r a c e l l u l a r t h y m i d y l a t e pools and to decreased and unba lanced D N A s y n t h e s i s . 2 . P R O P E R T I E S OF D I H Y D R O F O L A T E R E D U C T A S E A l l known d i h y d r o f o l a t e reduc tases r e a d i l y reduce 7 , 8 - d i h y d r o f o l a t e [EQ. (1 ) ] a n d , more s l o w l y , fo l i c ac id [EQ . ( 2 ) ] , to t e t r a h y d r o f o l a t e ( B l a k e l e y , 1969; H u e n n e k e n s et a l , 1971) . 4 F I G U R E 2 A summary of pathways illustrating the central role of dihydrofolate reductase in 'one-carbon' transfer reactions leading to the biosynthesis of purines, pyrimidines and amino acids. 5 FA 2 N A D P H ^ ^ A- NADPH >8< 2NADP--+ Serine "•NADP Methionine + FH 4 Homocysteine >nine + F Pyridoxal Phosphate Ns-Methyl AD dUMP NADH / I© N s, N10-Methylene FH 4 I VXFH j h A D P V d T M P - D N A Glycine FH 4 + HCO; H » N 1 ° -Formyl _ „N s , N10Methenyl FH*——-Purines FH 4 Purines (7) Dihydrofolate Reductase (5) Serine Hydroxymethyl Transferase ©.Thymidy la te Synthetase (4) Cyclohydrolase (5) FH 4 Formylase 6 [EQ . (1 ) ] 7 , 8 - D i h y d r o f o l a t e + N A D P H + H + -> 5 , 6 , 7 , 8 -t e t r a h y d r o f o l a t e + N A D P + [EQ . (2) ] Folate + 2 N A D P H + 2 H + -> 5 , 6 , 7 , 8 - t e t r a h y d r o f o l a t e + 2 N A D P + Most of the known d i h y d r o f o l a t e reduc tases are s t r o n g l y i n h i b i t e d b y M T X ; ( W e r k h e i s e r , 1961; B e r t i n o et a l , 1964) the i n h i b i t i o n is pH d e p e n d e n t and s to ich iometr ic at ac id p H , and compet i t ive w i th d i h y d r o f o l i c a c i d , at phys io log ia l p H ; the repor ted Ki va lues be ing _9 a p p r o x i m a t e l y 10 M or lower , ( H u e n n e k e n s , 1968a; B l a k e l e y , 1969) . Except ions to t h i s o b s e r v a t i o n have been r e p o r t e d h o w e v e r , and wi l l be d i s c u s s e d more f u l l y in the sect ion on mechanisms of res i s tance to M T X . D i h y d r o f o l a t e reduc tases have been p u r i f i e d and c h a r a c t e r i z e d f rom a v a r i e t y of s o u r c e s , i n c l u d i n g severa l normal and mal ignant cel l l i n e s . While all s h a r e one common p r o p e r t y , i . e . , c a t a l y s i s of the react ions in E Q . (1) and ( 2 ) , these enzymes e x h i b i t c o n s i d e r a b l e d i v e r s i t y wi th respec t to both s t r u c t u r a l and k ine t i c p a r a m e t e r s . T h e s e p r o p e r t i e s are summarized in Tab le 1 . T h e molecular we ights can be g r o u p e d into f o u r genera l c a t e g o r i e s , w i th the D H F R s f rom mammalian sources (w i th the except ion of calf t h y m u s enzyme-MWt . 33,500) h a v i n g molecular we ights r a n g i n g f rom 20,000 to 2 3 , 0 0 0 . Most of the mammalian reduc tases also e x h i b i t doub le pH op t ima , b u t t h e r e is c o n s i d e r a b l e v a r i a t i o n in the t u r n o v e r numbers and the Km va lues f o r the s u b s t r a t e s , d i h y d r o f o l a t e and N A D P H . T A B L E I Properties Of Some Dihydrofolate Reductases. Km (M) SOURCE MWt. -T-SAc. pH of Assay NADPH Dihydrofolate Beef Liver 20,000 1.09 6.25 1.5x10" 5 6x10" 6 21,100 103 6.0 -7 5x1O - 8 7 Pig Liver 20,000 7.2 3.1x10 ' Chicken Liver 22,000 15 7.4 1.8x10";? 1.2x10"' Calf Thymus 33,500 16.3 6.0 3.3x10 2.3x10 Ehlrich „ ~-6 Ascites Cells 20,200 1.0 7.5 5.6x10 ° „-7 L1210 Cells (R) 20,000 44.5 7.0 5.0x10" b 4x10 in mice „ „-6 L1210 Cells (R) 20,000 23 7.0 3.8x10 in culture 1.7x10" 6 -7 Sarcoma 180 21,000 7.2 3x10 cells (R) -4 Baby Hamster 23,000 4.7 7.0 1.6x10 N kidney cells (R) * Specific activity is expressed in umoles/min/Mg protein, a. Resistant to folate antagonist. From Huennekens et al, (1976). 8 T h e amino ac id sequences of d i h y d r o f o l a t e reduc tases f rom S t r e p t o c o c c u s faecium ( G l e i s n e r et a[, 1974) , E s c h e r i c h i a col i ( B e n n e t et a[, 1977; Stone et a[, 1977) , Lac tobac i l lus casei ( B i t a r , K . G . et a l , 1977) , c h i c k e n l i v e r ( K u m a r et a[, 1980) , L1210 mouse leukemia ce l ls (Stone et a[, 1979) and p o r c i n e l i v e r (Smith et a[, 1979) , have been determined so f a r . C o n s i d e r a b l e homology , espec ia l l y in the f i r s t N - t e r m i n a l 3 0 - 3 5 amino ac id r e s i d u e s , e x i s t s between the above mentioned mammalian reductases ( F r e i s h e i m et a[, 1979) . X - r a y c r y s t a l l o g r a p h i c s t u d i e s of the bacter ia l enzymes have ident i f i ed some of the amino ac ids i n v o l v e d in the b i n d i n g of the s u b s t r a t e s ( d i h y d r o f o l i c ac id o r fo l i c ac id and N A D P H ) and M T X (Mathews et a j , 1978) , and most of these are c o n s e r v e d in all the reduc tases sequenced so f a r . Seve ra l of the mammalian d i h y d r o f o l a t e reduc tases have two c y s t e i n e r e s i d u e s per molecule , one b u r i e d and one a c c e s s i b l e . Modi f i cat ion of the access ib le res idue b y o r g a n i c mercur ia l compounds s u c h as p C M S , p C M B , or m e t h y l m e r c u r i c h y d r o x i d e (MeHgOH) r e s u l t s , in some i n s t a n c e s , to an inc rease in c a t a l y t i c a c t i v i t y of the enzyme ( P e r k i n s and B e r t i n o , 1966; Kaufman 1964, 1966; F re ishe im et a l , 1979; Gold ie et al_, 1981) . H o w e v e r , bacter ia l reduc tases are i n h i b i t e d b y these compounds (Warwick and F r e i s h e i m , 1975) . T h e v a r i o u s d i h y d r o f o l a t e reduc tases also d i f f e r wi th r e s p e c t to t h e i r pH p r o f i l e s , e . g . , the reduc tase f rom c h i c k e n l i v e r c e l l s , d e s c r i b e d b y Mathews and H u e n n e k e n s , (1963) has a doub le pH optimum (wi th h i g h e r a c t i v i t y at ac id ic pH v a l u e s ) ; tha t f rom Lactobac i l lus L e u h r a n i i , a s ing le pH optimum (Kesse l and R o b e r t s , 1965) ; and tha t 9 f rom human l e u k o c y t e s , a s ing le but v e r y b road optimum o v e r a wide pH range ( B e r t i n o et a[, 1970. ) K i n e t i c data f o r numerous d i h y d r o f o l a t e reduc tases are ava i lab le and the Km va lues fo r the s u b s t r a t e s , d i h y d r o f o l i c ac id and N A D P H are l i s t e d " in T a b l e 1 . A n a l y s e s of k inet i c data have revea led tha t both the L1210 mouse leukemia (McCo l lough et a[, 1971) , and E. Co l i ( B u r c h a l l , 1970) enzymes u t i l i ze a random t y p e mechan ism, i . e . e i the r d i h y d r o f o l a t e (or fo l i c a c i d ) , or N A D P H may b i n d f i r s t . K i va lues fo r _9 M T X of most D H F R s are of the o r d e r of 10 M or lower . A v e r y i n t e r e s t i n g f e a t u r e of d i h y d r o f o l a t e reduc tases is t h e i r a b i l i t y to e x i s t in mul t ip le forms tha t can be separated e l e c t r o p h o r e t i c a l l y or c h r o m a t o g r a p h i c a l l y . E l e c t r o p h o r e s i s of p a r t i a l l y p u r i f i e d p r e p a r a t i o n s f rom c h i c k e n l i v e r and L1210 mouse leukemia ce l ls on ce l lu lose acetate membranes , fo l lowed b y s t a i n i n g fo r enzyme a c t i v i t y , revea led the p resence of at least 2 forms of reduc tases (Mell et a l , 1968) . F u r t h e r w o r k wi th the enzyme f rom M T X - r e s i s t a n t L. Casei showed tha t the 2 forms of reductases were re lated b y the p resence or absence of one e q u i v a l e n t of N A D P H , wh ich appeared to be t i g h t l y bound in L. Casei but o n l y weak ly bound in the c h i c k e n l i v e r e n z y m e , ( D u n l a p et a[, 1971) . Some of the m u l t i p l i c i t y o b s e r v e d wi th d i h y d r o f o l a t e reduc tase is however due to the e x p r e s s i o n of genet i ca l l y d i f f e r e n t forms of the enzyme b y both p r o c a r y o t i c and e u c a r y o t i c c e l l s . S . Faecuim v a r . d u r a n s A conta ins two r e d u c t a s e s ; one is s p e c i f i c fo r d i h y d r o f o l a t e whereas the o ther can u t i l i ze both d i h y d r o f o l a t e and fo late as s u b s t r a t e 10 ( N i x o n et a j , 1968; A l b r e c h t et a[, 1969) . It has been shown that t h e r e are severa l d i s t i n c t t y p e s of E. Col i wh ich conta in ext rachromosomal R - f a c t o r s and p r o d u c e both the normal t r imethopr im (a D H F R i n h i b i t o r ) s e n s i t i v e chromosomal d i h y d r o f o l a t e reduc tase and novel t r imethopr im r e s i s t a n t R - f a c t o r reduc tase ( S k o l d et a[ , 1974; Pat tscha l l et a[, 1977) . In a d d i t i o n , E. Co l i RT500 has two genet i ca l l y i n d e p e n d e n t i soenzymes , as revea led b y t h e i r d i f f e r e n c e s in p r i m a r y s t r u c t u r e ( B a c c a n a r i et al_, 1979) . Gen et i ca l l y d i f f e r e n t forms of d i h y d r o f o l a t e reduc tases have also been shown to be p r e s e n t in mammalian c e l l s , h o w e v e r , the appearance of these forms is u s u a l l y assoc iated w i th r e s i s t a n c e to M T X . In a d d i t i o n , the d i f f e r e n t forms t e n d to have d i f f e r e n t b i n d i n g capac i t ies f o r M T X wi th the r e s i s t a n t ce l l s p r o d u c i n g reduc tases wi th r e d u c e d a f f i n i t i e s f o r M T X ( A l b r e c h t et a|_, 1972; F l in to f f et a j , 1976, 1980; Go ld ie et a l , 1980, 1981; Melera . et a[ , 1980; Haber et a|, 1981) . 3 . M E C H A N I S M S OF R E S I S T A N C E T O M T X Methot rexate is one of the most usefu l an t ineop las t i c agents p r e s e n t l y a v a i l a b l e , h a v i n g a b road spec t rum of ant i tumour a c t i v i t y . It has been f o u n d c l i n i c a l l y usefu l aga ins t a v a r i e t y of c a n c e r s , e i ther as a s ing le agent o r in combinat ion wi th o ther ant ineop las t i c d r u g s . B e i n g a cel l c y c l e s p e c i f i c i n h i b i t o r , k i l l i n g ce l ls o n l y in the S phase of g r o w t h , i t is most potent aga ins t tumours wi th large g rowth f r a c t i o n s and s h o r t generat ion times and least potent aga ins t tumours w i th a small g r o w t h f r a c t i o n and long genera t ion t imes . 11 MTX interacts directly at intracellular sites without the need for prior metabolic transformation. It can be administered in large doses because toxicity to normal cells can be reversed by the administration of folinic acid ( N 5 formyl tetrahydrofolic acid), the so called "Folinic acid rescue." In spite of these favourable properties, the clinical usefulness of MTX is limited inevitably by drug resistance: either intrinsic resistance, or acquired resistance after exposure of the tumour to MTX. From numerous studies on experimental tumour systems, ithree mechanisms of acquired resistance have been determined: (1) Increased levels of dihydrofolate reductase (Fischer, 1961; Chang and Littlefield (1976); Schimke et al, 1978; Niethammer and Jackson, 1975; Dolnick et al, 1979; and Goldie et al, 1980). (2) Impaired transport of MTX (Fischer, 1962; Harrap et a[, 1971; Jackson et a]_, 1975). (3) Generation of altered dihydrofolate reductase with a lower affinity (increased Ki) for MTX (Jackson et al, 1976; Flintoff et al, 1976, 1980; Gupta et al, 1977; Goldie et a[, 1980, 1981; Haber et al, 1981). Other possible mechanisms of resistance have been reported in the literature and include increased levels of thymidylate synthetase (Maley and Maley, 1971) and the induction of the thymidine kinase salvage pathway (Wilmanns, 1971); however, the latter mechanism depends upon the presence of relatively high levels of exogenous thymidine not usually encountered under physiological conditions. 12 Each of the mechanisms of resistance will now be considered in more detail: (1) Increased levels of dihydrofolate reductase. T h i s phenomenon was f i r s t described by Hakala et a[, (1961) who found that cultured Sarcoma 180 cells gradually became resistant to MTX upon exposure to the drug, and at the same time dihydrofolate reductase activity increased proportionally with the degree of resistance. The enzyme from the resistant cells (elevated up to 154 fold) did not differ from the enzyme from sensitive cells in terms of Km for the substrates or turnover number per MTX binding site, and it was deduced that resistance in this case resulted from the production of a large excess of normal DHFR which immobilized all of the intracellular MTX, leaving enough free enzyme to ca r r y on its normal functions. Similar observations were made by Fischer, (1961) in MTX-resistant L5178Y cells. Since that time many other cell lines resistant to MTX exhibited this phenomenon. However, the mechanism for the increased levels of DHFR were not clear until 1972 when Nakamura and Uttlefield demonstrated by immunologic and inhibitor titration studies that this elevation in DHFR activity was due to an increase in the reductase protein resulting from an increase in the relative rate of DHFR synthesis. T h i s was substantiated later and was found to be due to an alteration in the regulatory mechanism controlling the rate of synthesis of the enzyme (Hainggi and Utt l e f i e l d , 1976; Alt et al, 1976). The increased rate of synthesis of DHFR was shown to be related to elevated quantities of DHFR mRNA which was capable of synthesizing DHFR in a cell-free system derived from wheat germ (Chang and Uttl e f i e l d , 1976; Kellems et al, 1976). Some of the MTX-resistant cell 13 lines have up to 300 fold elevations in the amount of DHFR (representing up to 6% of the total soluble protein in these cells) , as compared with the parent sensitive cells. The simultaneous increase in the mRNA for the enzyme facilitated the in vitro synthesis of the corresponding complimentary DNA (cDNA), and utilizing this probe Alt et al, (1978) and Schimke et a[, (1978) were able to demonstrate selective gene multiplication leading to an increase in the number of dihydrofolate reductase genes. This amplification of the dihydrofolate reductase genes in stably MTX-resistant cell populations has been found to be associated with a long homogeneously staining chromosomal region (usually on chromosome 2) upon Trypsin-Giemsa banding analysis of metaphase chromosomes. Non banding (homogeneous) chromosomal regions in cells producing high dihydrofolate reductase levels was f i r s t demonstrated by Biedler et a[, in 1974. Similar findings have been reported since in MTX-resistant sarcoma S-180 cells (Numberg et a[, 1978), in MTX-resistant L5178Y cells (Dolnick et a[, 1979; Berenson et a[, 1981) and in MTX-resistant mouse lymphoma EL4 cells and mouse melanoma P619 cells (Bostock and Tyler-Smith, 1981). In all of these cases resistance was shown to be stable i.e. MTX resistance and DHFR gene copy number were stable when cells were grown in the absence of selection pressure (i.e. MTX). MTX resistant cell lines exhibiting unstable amplification of DHFR gene sequences (loss of resistance upon removal of selection pressure and simultaneous loss of amplified DHFR DNA sequences) have been found to be associated with small, paired chromosomal elements denoted "double minute chromosomes." (Kaufman et a[, 1979; Bostock and Tyler-Smith, 1981). 14 The mechanisms by which the amplified DHFR sequences become incorporated into normal chromosomes or appear as "double minute chromosomes" involve extensive chromosomal rearrangement with many intermediate chromosomal forms leading, either to stable, or unstable amplification (Tyler-Smith and Alderson, 1981; Bostock and Tyler-Smith, 1981; Tyler-Smith and Bostock, 1981), the details of which are beyond the scope of this thesis. The appearance of amplified DHFR gene sequences in MTX-resistant mouse S-180 cells has led to the elucidation of the structure and organization of mouse DHFR gene (Nunberg et a[, 1980). The gene contains a minimum of five intervening sequences and spans a minimum of 42 kilobase pairs on the genome. Four of the intervening sequences occur within the protein-coding region of the gene, and one intervening sequence occurs within the 5' untranslated region. Such a large amount of intervening (untranslated) sequences has not been found in any other gene analyzed to date, and its significance is not clear. The active mRNA was found to be only 1600 nucleotides long and contained a 3' poly(A) t a i l . 2) Impaired Transport of Methotrexate. A high-affinity transport system for MTX has been established in a number of mammalian cells which conform to a carrier model that MTX shares with N 5-methyl tetrahydrofolate and N 5-formyl tetrahydrofolate (Goldman, 1971; Bender, 1975). The mechanism for active influx of MTX into cells is illustrated in Figure 3. The relationship between the extracellular MTX concentration and the unidirectional influx of MTX adheres to Michaelis-Menten kinetics, and 5-methyl tetrahydrofolate and 5-formyl tetrahydrofolate are competitive inhibitors of this process. 15 FIGURE 3 A carrier transport, model to account for the undirectional influx of MTX which occurs prior to the saturation of dihydrofolate reductase binding sites. Membrane carrier is denoted by 'C. The dissociation constant for the MTX-carrier reactions at the outer and inner cell membrane are denoted as k-i/kx and k - 3 / k 3 respectively. Adapted from Goldman (1971). 16 EXTRACELLULAR MEMBRANE MTX MTX MTX MTX+ MTX MTX MTX MTX MTX MTX k-4 k-1 4 k4 M k3 k2 |MTX-C^MTX-C k-2 INTRACELLULAR MTX Binds to Dihydrofolate Reductase 17 In f lux is h i g h l y tempera tu re dependent and pH dependent and the c a r r i e r - M T X in te rac t ion invo l ves s u l p h y d r y l g r o u p s , s ince i n f l u x is s t r o n g l y i n h i b i t e d by p - h y d r o x y mercur ibenzoate (Goldman et a[ , 1968; S ino tnak et a[, 1968) and p - c h l o r o m e r c u r i p h e n y l su lphonate ( p C M S ) ( R a d e r et a[ , 1974) . Plasma membrane -assoc ia ted p r o t e i n components a p p e a r i n g to p lay a role in M T X t r a n s p o r t in L1210 lymphoma ce l ls have been i d e n t i f i e d and p a r t i a l l y c h a r a c t e r i z e d (McCormick et §|, 1979) . T h e s t e a d y - s t a t e i n t r a c e l l u l a r level of M T X not on ly depends on the rate of i n f l u x of M T X , but also on the concent ra t ion of DHFR and on the rate of e f f l u x of M T X . T h e lat ter has been shown to be i n h i b i t e d b y metabol ic i n h i b i t o r s (sodium az ide and d i n i t r o p h e n o l ) , and t h u s e x p o s u r e of ce l ls to M T X in the p resence of metabol ic i n h i b i t o r s r e s u l t s in a net inc rease in the i n t r a c e l l u l a r s teady state level ( H a k a l a , 1965; Go ld man , 1969) . Dembo and S i r o t n a k (1976) have p r o v i d e d data f o r the h y p o t h e s i s tha t i n f l u x and e f f l u x of M T X t a k e place v ia d i f f e r e n t c a r r i e r s t h u s a c c o u n t i n g fo r the fac t tha t i n f l u x and e f f l u x of M T X are d i f f e r e n t i a l l y a f fected b y metabol ic i n h i b i t o r s . Impaired membrane t r a n s p o r t of M T X as a mechanism of c e l l u l a r r e s i s t a n c e to t h i s agent was f i r s t o b s e r v e d in a s t r a i n of L5178Y leukemia ce l ls in v i t r o , b y F i s c h e r (1962) . L a t e r , the re la t ionsh ip between i n f l u x of M T X in v i t r o and c y t o t o x i c i t y in v i v o was c o r r e l a t e d f o r a v a r i e t y of mur ine leukemias ( K e s s e l et al_, 1965) . It has been shown that f r e e i n t r a c e l l u l a r M T X in excess of the D H F R - b i n d i n g capac i t y is needed to s u p p r e s s c e l l u l a r d i h y d r o f o l a t e r e d u c t i o n (White and G o l d m a n , 1976) . When f r e e i n t r a c e l l u l a r M T X is low b u t d i h y d r o f o l a t e is h i g h , d isp lacement of bound M T X b y d i h y d r o f o l a t e p l a y s an impor tant role in r e v e r s i n g the t o x i c e f fects of M T X (Whi te , 18 1979) . T h e amount of f r e e i n t r a c e l l u l a r M T X is re lated to i ts i n f l u x k i n e t i c s . Impaired t r a n s p o r t in M T X - r e s i s t a n t human l ymphoblas to id cel l l ine was a t t r i b u t e d to a marked decrease in the i n f l u x Vmax f o r the M T X , c a r r i e r system (Niethammer and J a c k s o n , 1975) , and impai red t r a n s p o r t in r e s i s t a n t l ines of L1210 mouse leukemia ce l ls has been re lated to an inc rease in the i n f l u x Km ( J a c k s o n et a[, 1975; S i r o n t r a k et a[, 1968) . H i l l et a[, (1979; 1982) , have shown that whi le the d i f f e r e n c e in M T X t r a n s p o r t in s e n s i t i v e and r e s i s t a n t L5178Y cel ls is p r o f o u n d at low c o n c e n t r a t i o n s , t h i s d i f f e r e n c e is d imin i shed as the e x t r a c e l l u l a r concent ra t ions a re i n c r e a s e d . H e n c e , when the e x t r a c e l l u l a r M T X level is s u f f i c i e n t l y h i g h , r e s i s t a n c e to t h i s agent shou ld be overcome. 3) A l t e r e d d i h y d r o f o l a t e reduc tases wi th lowered a f f i n i t y f o r M T X . A s mentioned e a r l i e r , genet i ca l l y d i f f e r e n t forms of d i h y d r o f o l a t e reduc tases have been shown to be p r e s e n t in mammalian c e l l s . T h e s e v a r i a n t forms of DHFR are most of ten assoc iated wi th r e s i s t a n c e to M T X and the enzymes u s u a l l y have a lower a f f i n i t y ( h i g h e r K i ) f o r M T X than the D H F R s f rom the p a r e n t M T X - s e n s i t i v e c e l l s . I n t r i n s i c r e s i s t a n c e to M T X of c u l t u r e d mammalian ce l ls has been c o r r e l a t e d w i th the K i ' s of d i h y d r o f o l a t e reduc tases f o r M T X ( H a r r a p et al_, 1971; J a c k s o n et a|, 1976) . In the s t u d y b y H a r r a p et a[, the cel l M n e s t Y o s h i d a asc i tes s a r c o m a , mouse leukemia L1210 and mouse leukemia L5178Y, were eva luated wi th r e s p e c t to s e n s i t i v i t y to M T X (dose of M T X r e q u i r e d to reduce the s u r v i v i n g f r a c t i o n of ce l ls to less than 10%). Y o s h i d a ce l ls were 10 fo ld more r e s i s t a n t than L5178Y ce l ls wh ich were 10 fo ld more r e s i s t a n t t h a n L1210 c e l l s . T h e K i ' s of D H F R s fo r M T X 19 r f rom these t h r e e cel l l ines fo l lowed t h i s same o r d e r , i . e . the Y o s h i d a DHFR had a h i g h e r Ki ( lower a f f i n i t y ) f o r M T X than L5178Y DHFR which had a h i g h e r K i t h a n L1210 D H F R . A l l t h r e e ce l l l ines t r a n s p o r t e d the d r u g at comparable rates and the s p e c i f i c a c t i v i t i e s of d i h y d r o f o l a t e reduc tases were also comparab le . It appeared t h e r e f o r e that in these t h r e e cell l ines d i f f e r e n c e s in M T X s e n s i t i v i t y cou ld be d i r e c t l y c o r r e l a t e d wi th the K is df d i h y d r o f o l a t e reduc tases f o r M T X . T h e s e r e s u l t s were conf i rmed b y J a c k s o n et a[, (1976) who c a r r i e d out s imi lar s tud ies wi th more s t r i n g e n t ana lyses of the Ki v a l u e s . Human melanoma ce l ls have been r e p o r t e d to be i n t r i n s i c a l l y r e s i s t a n t to M T X ( K u f e et a[, 1980) , and a l though the r e s i s t a n c e in t h i s case was a t t r i b u t e d to e levated i n t r a c e l l u l a r leve ls of D H F R , the p resence of D H F R wi th a lower a f f i n i t y of M T X could not be e l iminated because of the method used fo r the assay fo r D H F R a c t i v i t y ( 3 H - M T X l igand b i n d i n g a s s a y ) . P r o l i f e r a t i n g normal r a b b i t epidermal cel ls have also been f o u n d to be i n t r i n s i c a l l y r e s i s t a n t to M T X ( H a r p e r and F l a x m a n , 1981) , a l though the mechanism of r e s i s t a n c e in t h i s ins tance was not c l e a r . Mammalian c e l l s , i n i t i a l l y h i g h l y s e n s i t i v e to M T X , a c q u i r e res i s tance to the d r u g upon e x p o s u r e to i n c r e a s i n g concent ra t ions of M T X . A l b r e c h t et a l , (1972) r e p o r t e d the s y n t h e s i s of a l te red D H F R in two M T X - r e s i s t a n t l ines of C h i n e s e hamster c e l l s . Ki va lues f o r M T X of the D H F R f rom these ce l ls were not d e t e r m i n e d ; h o w e v e r , whereas the enzyme f rom the paren t M T X - s e n s i t i v e ce l ls i n te rac ted s to ich iomet r i ca l l y wi th M T X , the i n h i b i t i o n of D H F R f rom the r e s i s t a n t ce l ls was r e v e r s i b l e , r e f l e c t i n g weak i n t e r a c t i o n . In a d d i t i o n , some o ther p r o p e r t i e s of the enzyme were d i f f e r e n t f rom those of D H F R f rom s e n s i t i v e ce l ls in terms of tempera tu re s e n s i t i v i t y , pH op t ima , and the e f fec t of N A D P H on the i n h i b i t i o n of D H F R a c t i v i t y . 20 J a c k s o n and Niethatnmer (1977) d e s c r i b e d the p r o p e r t i e s of an a l te red d i h y d r o f o l a t e reduc tase f rom M T X - r e s i s t a n t human _6 l ymphoblasto id c e l l s . T h e s e cel ls were r e s i s t a n t to 10 M M T X ; the Km f o r d i h y d r o f o l a t e was 1 8 - f o l d h i g h e r than tha t of the D H F R f rom the p a r e n t l i n e , and the a f f i n i t y f o r M T X was 5 0 - f o l d lower . T h e " l o w - a f f i n i t y " D H F R d i f f e r e d in i t ' s t e m p e r a t u r e s e n s i t i v i t y (more heat lab i le ) f rom the p a r e n t DHFR but both forms of the enzyme had s imi lar molecular we ights ( 2 2 , 5 0 0 ) . In add i t ion to the s t r u c t u r a l a l te ra t ion of the DHFR f rom M T X - r e s i s t a n t c e l l s , the total a c t i v i t y of the enzyme had i n c r e a s e d g r e a t l y ove r that in the pa ren t ce l ls (230 fo ld h i g h e r ) , p r o b a b l y b y the mechanism of gene a m p l i f i c a t i o n . F l in to f f et a[, (1976) demonst rated the p resence of normal amounts of an a l te red D H F R wi th d ec r eased a f f i n i t y f o r M T X f rom M T X - r e s i s t a n t C h i n e s e hamster o v a r y c e l l s . Se lect ion fo r i n c r e a s e d res i s tance p r o d u c e d cel ls wh ich appeared to possess inc reased a c t i v i t y of a l te red enzyme ( G u p t a et a[ , 1977) . T h e mutant enzymes (6 to 8 fo ld more r e s i s t a n t to i n h i b i t i o n b y M T X than wi ld t y p e enzyme) and the wi ld t y p e enzyme also demonst rated small d i f f e r e n c e s in pH op t ima , Km fo r fo late and heat s t a b i l i t y . F u r t h e r c h a r a c t e r i z a t i o n of the wi ld t y p e and a l te red reduc tases b y two -d imens iona l gel e l e c t r o p h o r e s i s fa i led to reveal any c h a r g e d i f f e r e n c e s between the two f o r m s . Most r e c e n t l y , Haber et a[, (1981) demonst rated the p r e s e n c e of e levated levels of a l te red D H F R wi th a lower a f f i n i t y f o r M T X in h i g h l y M T X - r e s i s t a n t 3T6 mouse embryo f i b r o b l a s t s . T h i s enzyme e x h i b i t e d a 2 7 0 - f o l d reduc t ion in b i n d i n g a f f i n i t y fo r M T X . In add i t ion the Km f o r d i h y d r o f o l i c ac id was inc reased 3 - f o l d , the pH optimum was d i f f e r e n t a n d , a l though the molecular we ight was s imi lar to the enzyme f rom p a r e n t c e l l s , the a l te red enzyme demonst rated a s i g n i f i c a n t bas ic s h i f t in e l e c t r o p h o r e t i c m i g r a t i o n . 21 Ample evidence therefore exists for the presence of altered forms of DHFRs in MTX-resistant cells. Although the alterations are associated with decreased affinity for MTX in all of these cases, the degree of loss of affinity varies considerably, as do the alterations in other properties. It therefore appears that in as much as amino acid substitution at various points in the dihydrofolate reductase molecule can effect MTX-binding (Matthews et a[, 1977), a variety of altered enzymes which differ in physical and kinetic properties can be expected. Selection with stepwise increases in concentrations of MTX initially appear to yield MTX-resistant cells expressing wild type DHFR expressed at high levels, however, growth at high MTX concentrations result in the prevalence of cells expressing elevated levels of DHFR with reduced affinity for MTX (Haber et a[, 1981). It has been noted by Biedler et a[, (1972), that the correlation between DHFR activity and the level of drug resistance varied for different sublines of Chinese hamster cells grown in increasing inhibitor concentrations. The cells containing lower levels of DHFR than expected for their degree of resistance were found to contain an enzyme with reduced affinity for MTX (Albrecht et a[, 1972). MTX-resistant cells exhibiting two of the three mechanisms of resistance have been isolated. For example cells may have impaired uptake of MTX as well as elevated levels of DHFR, or elevation of DHFR is also accompanied by an alteration in the structure of the enzyme leading to decreased affinity for MTX. The present investigation was undertaken with the aim of studying these three mechanisms furt h e r and to assess the importance of each in imparting a high degree of resistance to the cell. In addition, if an 22 altered form of DHFR with a lower affinity for MTX was present in MTX-resistant cells, isolation, purification and characterization of its physical and chemical properties could be carried out and compared with the enzyme from MTX-sensitive cells i.e. one with a high affinity for MTX. These experiments would lead to an insight into the nature of the alterations leading to reduced affinity, and would facilitate the synthesis of more potent inhibitors of its activity. 23 METHODS AND MATERIALS 1. Chemicals [ 2 1 4 C ] Folic acid (55mCi/m mole), [3,5,9 3H] MTX, [3,5,7,9 3H] Folic acid(29Ci/m mole) and PCS liquid scintillation fluid were purchased from Amersham Corp. ,Oakville,Ontario. Fisher's medium for leukemic cells of mice,horse serum (dialyzed and undialyzed),glutamine and penicillin/streptomycin were all obtained from Grand Island Biological Co.,Grand Island N.Y. Dihydrofolic acid, NADPH, MTT, AH-Sepharose-4B, EDC, pCMS, 5-methyltetrahydrofolic acid, p-aminobenzoic acid, p-aminobenzoyl glutamate, tetrahydrofolic acid were purchased from Sigma Chemicals, St.Lo.,Miss. N 5-Formylfolic acid was purchased from ICN pharmaceuticals Inc., Plainview, N.Y. Methotrexate was purchased from Lederle,Montreal. Acrylamide, Methylenebisacrylamide, ammonium persulphate, TEMED, SDS, DEAE Affigel blue were purchased from Bio-Rad Laboratories, Richmond, California. Sephadex G100 was obtained from Pharmacia (Canada)lnc.,Dorval, Quebec. 2. Cell culture: L5178Y mouse leukemia cells were initially passaged in the peritonium of BDF^ mice. One week after the innoculation the cells were removed and were grown at 37°C as suspension cultures in plastic flasks or tubes (Corning) under an atmosphere of 5% C O p in air. The 24 growth medium was Fisher's medium supplemented with 10% dialyzed horse serum, 1% glutamine and 100U/ml penicillin - 100ul/ml streptomycin (Grand Island Biological Co., Grand Island, New Y o r k ) . Cell stocks were diluted every one or two days with fresh medium to maintain the cultures in exponential growth. Under these conditions the doubling time for all three cell lines was approximately 12 hours. Total cell numbers were determined by counting on a Model Coulter Counter (Coulter Electronics, Hialeah, Fla.)-3. Procedures for establishing MTX-resistant cells. Two stable MTX-resistant cell lines were developed from the parent L5178Y cells, designated L5178Y(S). The properties of these cells are summarised in Table 2. The two resistant sublines were designated L5178Y(R 3) and L5178Y(R 4). The resistant sublines were obtained by subculturing the cells in progressively increasing sublethal concentrations of MTX until the desired level of resistance had been achieved. Both resistant lines were maintained in MTX containing -5 -3 medium; the R 3 cell line grew in 10 M MTX and R 4 cells in 10 M MTX. R 4 cells were derived from R 3 cells. 4. Measurement of the initial rate of entry and the intracellular steady-state concentrations of MTX. L5178Y lymphoblasts in exponential growth at a density of 5-7 x 5 10 cells/ml were dispensed in 2 ml aliquots into stoppered tubes. MTX-resistant cells were washed twice with MTX-free Fisher's medium without serum and allowed to grow for 2-3 days in drug-free media, with serum, prior to experimentation. 25 R a d i o l a b e l e d M T X was added to the s h a k i n g cel l s u s p e n s i o n s at _6 _4 37°C to a f ina l concent ra t ion of 10 M or 10 M . A t v a r i o u s t i m e s , t u b e s were removed f rom the water b a t h , p l u n g e d into ice and 8 ml i c e - c o l d 0.9% sodium c h l o r i d e so lut ion was added to each t u b e and the ce l ls immediately removed f rom s u s p e n s i o n b y c e n t r i f u g i n g at 350 g . f o r 6 m i n . at 4°C . T h e ce l ls were washed twice f u r t h e r wi th i c e - c o l d 0 .9% NaCI s o l u t i o n . T h e r e s u l t i n g cel l pe l le ts were s o l u b i l i z e d in 0 . 5 ml IN NaOH at 37°C fo r 12 h o u r s . 0 .25 ml of the r e s u l t i n g so lu t ions were added to 10 ml P C S l i q u i d sc in t i l l a t ion f l u i d . 50ul 6N HCI was added to each s c i n t i l l a t i o n cockta i l to neut ra l i ze the a lka l i and 0.7ml d i s t i l l e d water was added to obta in a homogeneous, c lear s c i n t i l l a t i o n c o c k t a i l . R a d i o a c t i v i t y was determined u s i n g a Sear le Mark III l i q u i d sc in t i l l a t ion s p e c t r o m e t e r . E f f i c i e n c y of c o u n t i n g was 40% fo r H . Init ial rate of i n f l u x ( e n t r y ) s t u d i e s were c a r r i e d out b y s t o p p i n g the react ion at 2 m i n . In t race l lu la r s teady state concent ra t ions were determined b y s t o p p i n g the react ion at 30 m i n . 5 . P r e p a r a t i o n of E x t r a c t s . g A p p r o x i m a t e l y 10 ce l ls in logar i thmic g rowth were removed f rom s u s p e n s i o n by c e n t r i f u g i n g at 250 g . at 4°C fo r 6 m i n . ( P r i o r to p r e c i p i t a t i o n of cel l e x t r a c t s , R 3 and R 4 ce l ls were g rown in M T X - f r e e medium at 37°C f o r 24 h o u r s to remove all e n z y m e - b o u n d M T X ) . T h e ce l ls were washed twice in i c e - c o l d P B S ( 0 . 1 5 M N a H g P C y H ^ O ; 0 . 1 5 M N a 2 H P 0 4 pH 7 . 2 in 0 .9% NaCI) and then r e s u s p e n d e d in 4 - 6 ml i c e - c o l d 0 .05 M T r i s - H C I pH 7 . 5 . T h e ce l ls were d i s r u p t e d b y son ica t ing at 20 26 KHz for 30 seconds at 4°C (Branson Sonifier, cell disrupter 350, Branson Sonic Power Co., Connecticut). The lysate was then centrifuged at 100,000 g. for one hour at 4°C in a Beckman L5 Ultracentrifuge. The supernatant was termed 'crude lysate'. 6. Assay for Folate Reductase Activity: Folate reductase activity was measured using the radiolabeled folic acid method of Uttlefield (1969). The 100 ul incubation mixture consisted of 20 ul 0.01 M potassium acetate buffer, pH 5.0; 30 ul 3.33 mM NADPH; 30 ul 330uM [2- 1 4C] Folic acid (S. Ac. :55mCi/mmol), or [3',5',7,9-3H] Folic acid (S. Ac. :29Ci/mmol) and 20ul enzyme preparation. Inhibitors (10 ul ) were incubated with the enzyme preparation for 5 min. at room temperature prior to starting the reaction with labelled folic acid. After incubation at 37°C for 20 minutes, the reaction mixture was chilled on ice and 20ul 0.027 M unlabelled folic acid was added. Unreduced folic acid was precipitated by the addition of 20 ul 0.17 M zinc sulphate and 5 ul glacial acetic acid. After 10 minutes in ice the mixture was centrifuged at 1,000 g. for 15 minutes at 4°C. 50 ul of the supernatant fraction was added to 10 ml PCS scintillation cocktail and counted in a Searle Mark III liquid scintillation spectrometer. All assays were corrected for a blank reaction mixture lacking enzyme which showed less than 2% of the added radioactivity. 1 unit of activity is defined as that amount of enzyme reducing 1 nmole of folic acid in 20 minutes per ml. Protein was measured using the Bio-Rad protein assay (Bio-Rad Laboratories, Chemical Division, Technical Bulletin 1051, April 1977), which is based on the observation that the absorbance maximum of Coomassie Brilliant Blue G-250 dye shifts from 465nm to 595nm when binding to protein occurs. 27 7. High pressure liquid chromatography of reduced and oxidized  folates. The products of the radioactive folate reductase assay were determined by HPLC. The method used was a modification of Stout et al_, (1976), for the separation of substituted pteroyl monoglutamates and pteroyl oligo- -glutamates. Tetrahydrofolates, dihydrofolate and unreduced folic acid were separated by anion exchange chromatography on a Model 7000 B liquid chromatograph (Micromeretics, Nocross, Ga., U.S.A.) using a Reeve-Angel partisil 10 SAX column 25 x 4.6mm i.d. (Whatman, Clifton, N.J., U.S.A.). The eluent (0.16M NaCI solution containing 4mM NaNH^HPO^ pH6.5) was formed by mixing distilled water (primary reservoir) and 2M NaCI containing 0.05M NaNH^HPC^ pH6.5 (secondary reservoir.). The run was carried out isocratically with the secondary reservoir set at 8%. The column temperature and flow rate were maintained at 29°C and 2.0 ml/min respectivley. Before the chromatographic run, the column was equilibriated with 0.6M NaCI obtained by the use of a 30% setting for the secondary resevoir. Standard solutions of reduced and oxidized folates were detected in the column effluent by their U.V. absorbance at 254 nm., using a chromonitor 785 flow spectrophotometric detector (Micromeretics). Fractions of the column effluent (1ml.) were also collected automatically into scintillation vials on a fraction collector. PCS scintillation fluid (10ml.) was added to each vial and the radioactivity counted on a Mark III liquid scintillation spectrometer. 3 14 Radiopurities of HFolic acid and CFolic acid were determined by HPLC on an anion exchange column as described above. Both were found to be 98% pure with the main impurities being para-aminobenzoic acid. 28 8 . P r o c e d u r e s f o r the isolat ion and p u r i f i c a t i o n of fo late r e d u c t a s e . (a ) . M T X - S e p h a r o s e A f f i n i t y C h r o m a t o g r a p h y : A H - s e p h a r o s e - 4B (5 g . ) was swol len and washed wi th 1 l i te r 0 . 5 IVl NaCI on a s i n t e r e d g lass f i l t e r ( G 3 ) . T h e gel was then washed wi th 500 ml of d i s t i l l e d water (made pH 4 . 5 wi th 1 N H C I ) . T h e gel was r e s u s p e n d e d in 25 ml d i s t i l l e d water (pH 4 . 5 ) ; M T X (10 mg f o r S c e l l s ; and 50 mg fo r R 3 and R 4 c e l l s ) and EDC (88 mg and 440 mg r e s p e c t i v e l y ) were added to the gel and i n c u b a t e d f o r 16 h o u r s at room t e m p e r a t u r e on a mult i p u r p o s e r o t a t o r . T h e M T X sepharose was then washed wi th 0 .05 M T r i s - H C I , pH 8 . 0 ; 2 M KCI and p o u r e d into a 1 cm x 8 cm g lass c o l u m n . T h e p a c k e d column was then e q u i l i b r i a t e d wi th 200 ml 0 . 0 5 M T r i s - H C I pH - 5 7 . 5 c o n t a i n i n g 10 M N A D P H . ' C r u d e lysate ' (4 ml) was then app l ied to the column and c h r o m a t o g r a p h y was c a r r i e d out u s i n g 20 ml 0 .05 M T r i s - H C I pH 7 . 5 ; 1 0 _ 5 M N A D P H ( B u f f e r 1 ) , fo l lowed b y 30 mi 0 . 2 M T r i s - g l y c i n e b u f f e r pH 9 . 5 ; 2 M K C I , 1 0 _ 5 M N A D P H ( B u f f e r 2) and the fo late reduc tase was then e lu ted wi th 0 . 2 M T r i s - g l y c i n e pH 9 . 5 , 2 M KCI and 0 . 5 mM d i h y d r o f o l i c ac id ( B u f f e r 3 ) . F rac t ions (3 ml) were co l lected automat ica l ly on a G i l son micro f r a c t i o n a t o r and scanned f o r p ro te in ( U . V . a b s o r b a n c e at 280 nM) and fo late r e d u c t a s e a c t i v i t y . ( b ) . S e p h a d e x G -100 C h r o m a t o g r a p h y : T h e f r a c t i o n s c o n t a i n i n g fo late r e d u c t a s e a c t i v i t y in the B u f f e r 1 wash of the M T X - s e p h a r o s e a f f i n i t y column ( F o r m 2) were p o o l e d , d i a l y z e d aga ins t 4mM T r i s - H C I pH ^ 7 - 5 , l y o p h i l i z e d and d i s s o l v e d in 3 ml of d i s t i l l e d w a t e r . A l i q u o t s ( 1 . 5 - 2 . 0 ml) were app l ied to Sephadex G -100 columns ( 1 . 6 x 81 cm) and e lut ion was c a r r i e d out wi th 50 mM T r i s - H C I (pH 7 . 5 ) at a f low rate of 24 m l / h r . F rac t ions (6 ml each) were co l lected and assayed fo r p ro te in ( O . D . 280 nm) and fo late reduc tase a c t i v i t y . T h e ac t i ve f r a c t i o n s were 29 pooled (total volume - 18 ml), dialyzed extensively against 2.8 mM Tris-HCI (pH 7.5), lyophilized and dissolved in 1 ml distilled water. The fractions containing folate reductase activity in the Buffer 3 wash of the MTX-sepharose affinity column (Form 1) were dialyzed extensively against 50 m M Tris-HCI (pH 7.5) to remove all of the dihydrofolic acid. The active fractions were then pooled, dialyzed against 5 mM Tris-HCI (pH 7.5), lyophilized and dissolved in 2 ml of distilled water. Aliquots (2 ml) were applied to Sephadex G-100 columns and chromatographed as described above for Form 2 activity. ( c ) . Chromatography of Form 2 folate reductase on DEAE Affigel blue: DEAE Affigel blue is a Crosslin ked agarose which has both Cibacron blue and diethylaminoethyl groups covalently linked to it. Form 2 folate reductase from R 4 cells, after fractionation through MTX-Sepharose affinity column and gel filtration through G100 Sephadex was applied to a DEAE Affigel blue column (1.5x5.4 cm.).Prior to equilibriation of the column the gel was washed with 50ml 0.1M acetic acid, pH 3.0 containing 1.4M NaCI and 40% isopropanol. The column was then equilibriated with at least 5 bed volumes of starting buffer which was 50mM T r i s - H C L pH 7.5. Approximately 6.0ml (2.0mg) of the enzyme preparation from G100 sephadex in 50mM T r i s - H C L , pH 7.5 was applied , and eluted with 5 bed volumes of the starting buffer. The column was then eluted with a step gradient from 1.0M NaCI to 2.5M NaCI in starting buffer. 30 Gel Electrophoresis: Samples (50-75 ul) containing 25 ug protein were electrophoresed on 7.5% polyacrylamide gels (acrylamide: methylenebisacrylamide, 29.2 : 0.8) according to the procedure of Huennekens et a[, (1971). The running buffer was 25 mM Tris-glycine (pH 8.3) and electrophoresis was carried out for 1.5 hr. at 4°C using a current of 2.2 mA per tube. The gels were stained for dihydrofolate -5 reductase activity in the presence and absence of MTX (10 M), as described by Huennekens et a[, (1971). Electrophoresis in the presence of sodium dodecyl sulphate (SDS) was carried out using the procedure of Laemmli, (1970) . Samples (75ul) containing 25-50 ug protein for detection with Coomassie blue, or 2 to 4ug for detection by the silver staining technique, were electrophoresed using a running buffer consisting of 0.025 M Tris, 0.192 M glycine, 0.1% SDS (pH 8.3). After electrophoresis for 4hr. at 4°C and 2.2 mA per tube, the gels were either stained in 0.2% Coomassie blue 'R1 in 50% trichloroacetic acid for 2h at room temperature and destained by diffusion in 7% acetic acid, 10% isopropanol; or were stained by a modification of the silver staining technique (Switzer et a[, 1979), described below. Silver staining of 13% SDS-PAGE slab gels was carried out as follows: The gel was removed from the electrophoresis apparatus and fixed for 2 periods of 30 min.in 25% methanol/10% acetic acid. The gel was then rinsed in distilled water (3x20min.), followed by fixation in 10% gluteraldehyde solution for 30 min.; it was then rinsed free of fixative with distilled water (3x10 min.) and left overnight in the same. The gel was next placed in diamine solution (0.36% NaOH (46ml); concentrated NH^OH (3.1ml); 20%(w/v) AgNOg (9ml) and 20% ethanol (165ml)) for 15min, rinsed with distilled water (3x10 min.) 31 and then placed in the developing solution containing 100ml. absolute ethanol, 1% citric acid (5ml.), 3.7% formaldehyde (5ml.) and 890 ml. distilled water. The bands developed almost immediately and the reaction was stopped by rinsing the gel with several washes of distilled water, once the desired intensity of the bands had been achieved. The dark background and the surface deposits of silver were removed as per Switzer et al, (1979). Isoelectric focusing: Isoelectric focusing was carried out in 130 x 2.5 mm glass tubes as described by O'Farrell, (1975). Gels contained a final concentration of 2% Ampholines (LKB), 1.5% pH range 5 to 7 and 0.5% pH range 3.5 to 10. Following electrophoresis for 12 hr. at 400 volts the gels were extruded into 50% methanol, 10% acetic acid containing 0.03% Coomassie Brilliant Blue ("R" grade, Sigma) for 2 hr. Gels were then destained overnight against 7% acetic acid, 5% methanol. To determine the pH gradient, gels were sliced into 1 cm sections and incubated in sealed vials with 5 ml of degassed distilled water. After 1 hr. of shaking at 23°C the pH was measured using an Orion Research pH meter. 32 RESULTS 1. Growth properties of the MTX-sensitive and MTX-resistant L5178Y mouse leukemic cells. A typical growth curve for L5178Y(S) cells is shown in Fig.4. The growth of these cells could be divided into 3 phases: a lag phase, followed by an exponential phase and finally a stationary phase. An initial lag phase was always present at low cell concentrations ( i . e . less than 50,000 cells/ml). T h i s was followed by an exponential phase characterized by a doubling time of 11 to 14 hours. Cell growth began to slow down at a cell concentration of greater than 700,000 cells/ml g with zero growth at cell concentrations of 10 /ml. Growth could be stimulated with the addition of fresh medium containing serum. Routine cultures were maintained at a cell concentration of 2 to 6 x 10^ cells/ml, i.e. in the exponential phase. Upon exposure of the S cells to MTX during the selection of MTX-resistant cells, the doubling time slowed down considerably; however, the cells were kept exposed to the drug in culture medium until the cell number doubled every 11 to 14 hours. At this point the selection process of resistant cells to that particular concentration of MTX was regarded to be essentially complete. Th i s was substantiated by determining the I-D.^Q ( i . e . the dose of MTX required to inhibit cell proliferation by 50% of control) for MTX of these cells. The inhibition of L5178Y(S), (R3), and ( R 4 ) cells with increasing concentrations of MTX is shown in Fig.5., and the I -D.^Q values are listed in Table 2. Thus, L5178Y(R 3) cells were approximately 1000 fold, and R 4 cells 180,000 fold more resistant to MTX than the parent L5178Y(S) cells. 33 FIGURE 4 A typical growth curve of L5178Y(S) cells under the conditions described in the text. The figure shows these cells growing with a doubling time of 12 hours. Once established, R 3 and R 4 cells exhibited similar growth profiles and doubling times. Cell numbers were determined by counting on a Model Z b x Coulter Counter. 3 4 35 FIGURE 5 Determination of I.D.^Q values (i.e. concentration of MTX required to inhibit proliferation of cells by 50% of control) for MTX-sensitive and MTX-resistant cells. -4 Cells, at a concentration of approximately 5x10 cells/ml were allowed to grow under standard conditions in increasing concentrations of MTX for 24 hours. Cell numbers were counted at this time and the results plotted as % increase in cell number of control against MTX concentration. Each point is the mean of three separate experiments. L5178Y(S) O ; L5178Y(R 3) • ; L5178Y(R 4) • . 36 [MTX] M T A B L E 11 Properties of L5178Y MTX-Sensitive and MTX Resistant Cell Lines. Cell-Line I.D. 5 0 (MTX) Folate Reductase % Folate Reductase S.Ac. (units/Mg Protein) Activity Represented By Low-Affinity ' Variant. Intracellular Steady State Level (nmoles/10 9 cells) of MTX at 'Low' and 'High' Extracellular MTX. 10" 6M 10" 4M L5178Y(S) 2.8x10" 8M L5178Y(R 3) 2.6x10"5M L5178Y(R 4) 5.0x10 °M 1.0 7.0 8.6 0% 2% 8% 1.3 0.3 32.3 30.0 38 2. Properties of MTX uptake into L5178Y(S) and (R.Q cells MTX transport into L1210 murine leukemia and Ehrlich ascites tumour cells is compatible with a carrier transport model (Lichtenstein et a[, 1968; Goldman et a[, 1971; Mckormick et a[, 1979), and evidence exists that the mode of transport in L5178Y cells is similarly carrier mediated (Harrap et a[, 1971). The sequence of events when carrier interacts with MTX during the influx process can be accounted for by a series of four reversible reactions as illustrated in Fig.3. The time course of uptake of MTX can therefore be divided into three phases. During the initial phase, the carrier from which MTX has dissociated at the inner cell membrane reorients towards the cell exterior in the unloaded condition. During this phase , the net uptake of MTX is equal to it's unidirectional influx velocity, and the uptake is a linear function of time. Following saturation of all the intracellular binding sites (mainly dihydrofolate reductase), free intracellular MTX appears and can interact with the carrier at the inner ceil membrane to produce a unidirectional efflux which leads to a declining net uptake until a steady state is reached, when these bidirectional fluxes are equal. These three phases are illustrated for the uptake of MTX into L5178Y(S) cells in Fig.6 (a).: a linear phase during the first 5min. of -6 exposure to 10 M MTX, followed by a declining uptake phase and then a plateau phase (steady state phase) after 20 min. exposure. Under these conditions the Km for influx was 9.1uM and Vmax was 0.4nmoles/ 9 _g min/10 cells. In contrast, MTX (10 M) uptake into R3 cells was greatly impaired, achieving 23% of the intracellular steady state MTX concentration of S cells. Uptake of MTX into I_5178Y(S) cells was found to be temperature dependent (Qin=3.0) .and the influx was strongly 39 FIGURE 6 The time course of uptake of 3H-MTX into L5178Y(S) and ( R 3 ) cells. A. Cells were exposed to 10 M MTX for various time periods (from 0 to 40 min.) and the intracellular levels of 3H-MTX determined as described in the text. L5178Y (S) ® ; L5178Y(R 3) p -4 B. Cells were exposed to 10 M MTX and treated as described above. L5178Y(S) • ; L5178Y(R 3) o . In both cases the experiment was carried three times with identical results. 40 A 4 1.4 1 o 111111 1 1 i 1 1 r 1 r 012345 10 15 20 25 30 35 40 45 TIME (min.) 41 inhibited by p-chloromercuriphenylsulphonate (pCMS) (Table 3.), suggesting the involvement of sulphydryl groups in the carrier-MTX interaction. The effect of 5-methyl tetrahydrbfolate and folinic acid (5-formyl tetrahydrofolate) on MTX influx and on the intracellular steady-state concentration of the drug was also determined. (Table 3). It was noted that both of these folate compounds reduced the level of the steady-state concentration of MTX, though this was much less than the inhibition seen on the initial rate of influx (Table 3). The above studies could not be carried out on the R 3 cells since the influx of MTX was sp slow and the extent of drug uptake so limited that accurate determinations were not possible. Next, the extent of MTX uptake with time in the two cell lines was determined when the extracellular concentration of drug was increased -4 to 10 M. ( F i g 6 b . ) . It is now seen that significant amounts of drug entered both cell lines. At all points the amount of MTX associated with both cell types was nearly identical, with no significant difference observed in either the initial rate of entry of MTX or in the intra-cellular concentrations achieved at steady-state levels after a 30 min. -4 incubation. At this 10 MTX concentration not only are higher intracellular levels achieved, but there is also a considerable quantity of drug associated with the cells at zero time, which is consistent with earlier reports and is considered to represent the component bound to the cell surface (Goldman, 1975; Hill et a[, 1979). When either S or R 3 -4 cells were exposed to 10 M MTX for either 2 or 30 mins. at 37°C, drug uptake was not significantly influenced by the presence of 70 uM pCMS, -4 -4 10 M methyl tetrahydrofolate or 10 M folinic acid (Table 4). T A B L E III P roper t ies of H - M T X Inf lux into L5178Y(S) Ce l l s at 10 M E x t r a c e l l u l a r M T X . Initial rate of i n f l u x * % Inhib i t ion of S t e a d y - S t a t e Inhib i t ion of (nmoles/mn/10 ce l l s ) M T X on ly ( C o n t r o l ) M T X + pCMS (70uM) M T X + fi 5 - C H 3 F H 4 (10 M) M T X - + Fol in ic acid ( 1 0 " b M ) 0.256 ± 0.16 0.029 ± 0.001 0.063 ± 0.005 0.105 ± 0.012 in i t ia l rate C o n c e n t r a t i o n * * S t e a d y - S t a t e g (nmoles/10 ce l l s ) 89% 75% 60% 1.58 ± 0 .36 0.41 ± 0 .084 1.06 ± 0 .10 1.21 ± 0 .07 74% 33% 23% •6„ 3 *S cel ls were exposed to 10 M H - M T X fo r 2 m i n . in the p resence or absence of the above compounds . **S cel ls were exposed to 10 M H - M T X fo r 30 m i n . in the p resence o r absence of the above compounds . Each point rep resents the mean of 3 va lues ± s t a n d a r d e r r o r . T A B L E IV P roper t ies of 3 H - M T X Inf lux into L5178Y(S) and ( R 3 ) Ce l l s at 1 0 _ 4 M E x t r a c e l l u l a r M T X . M T X - s e n s i t i v e cel ls M T X - r e s i s t a n t ce l ls M T X on ly (Contro l )*** M T X + pCMS (70uM) M T X + . 5 - C H 3 F H 4 (10 M) M T X . + Fol in ic ac id (10 M) % Inhibi t ion of % Inhib i t ion of Initial rate of S t e a d y - S t a t e i n f l u x * Concent rat ion** 0% 11% 0% 0% 10% 10% % Inh ib i t ion % Inh ib i t ion of Init ial rate of S t e a d y - S t a t e i n f l u x * Concent ra t ion** 6% 0% 7% 15% 5% 15% *Cel ls were exposed to 10~ M H - M T X fo r 2 m i n . in the p resence of the above c o m p o u n d s . - 4 3 **Cells were exposed to 10 M H - M T X fo r 30 m i n . in the p r e s e n c e of the above c o m p o u n d s . g ***lnit ia l rate of i n f l u x in nmoles/min/10 cel ls in cont ro l cel ls was 3 . 9 ±3 0 . 4 and 3 . 6 ± 0 . 4 fo r S and R cel ls r e s p e c t i v e l y . S t e a d y - S t a t e concent ra t ions in nmoles/10 cel ls in cont ro l ce l l s were 3 2 . 3 ± 1.2 and 29 .5 ± 1 . 3 fo r S and R cel ls r e s p e c t i v e l y (see F i g u r e 3 ) . Each point rep resents the mean of 3 est imat ions . 44 These data therefore suggest that at high extracellular drug concentration the impairment in drug uptake in MTX-resistant cells can be overcome, but that under these conditions the mechanism by which MTX enters the cells (both the sensitive and the resistant variants) may differ from the apparent active transport process demonstrated at -6 the lower drug concentration.of 10 M in the L5178Y(S) cells. 3 .Evidence for the presence of two forms of Folate reductase  in MTX resistant L5178Y cells. Folate reductase activity in the lysates of S, R 3 and R 4 cells was assayed as described in the Methods and Materials section. The specific activities (in Units/Mg protein) of DHFR in these cell lines are shown in Table 2. Both resistant cell lines had elevated levels of DHFR, being 7fold higher in the R 3 cells and 8.6-fold higher in the R 4 cells, as compared to the level in the parent S cells. A f f i n i t y chromatography of cellular lysate from L5178Y(S) cells on MTX-sepharose affinity column resulted in the binding of the total DHFR activity applied.This is apparent from Fig.7a. where DHFR activity is absent in the buffer 1 and buffer 2 elutions, which resulted in the elution of large amounts of protein.(solid lineFig.7a.). DHFR activity was only eluted with buffer 3 which contained the substrate dihydrofolic acid. In contrast, chromatography of R 3 lysate resulted in the elution of a DHFR peak in buffer 1 in addition to the bound activity eluted with buffer 3 (Fig.7b.). The activity eluted with buffer 1 therefore represented a portion of the total DHFR activity which did not bind to the affinity column. 45 FIGURE 7 Methotrexate-sepharose a f f i n i t y chromatography of lysates from L5178Y(S), (R 3) and (R 4) c e l l s . C e l l u l a r lysates (tynl.) were applied to MTX-sepharose a f f i n i t y columns and eluted stepwise with the following b u f f e r s : Buffer 1: 0.05M T r i s - C l , pH7.5; 10~5M NADPH. Buffer 2: 0.2M T r i s - G l y c i n e , pH9.5 i n 2M KC1; I O - 5 NADPH. Buffer 3: 0.2M T r i s - G l y c i n e , pH9.5 i n 2M KC1; 5mM d i h y d r o f o l i c acid. 3.5-ml f r a c t i o n s were c o l l e c t e d and p r o t e i n was estimated by absorbance at 280nM. Fractions eluted with Buffer 3 were dialyzed at 4°C for 18 h. against two changes of 2 l i t e r s of 0.05M T r i s - C l , pH7.5, before assaying a l l f r a c t i o n s f o r enzyme a c t i v i t y O » A28O p r o f i l e ; 9 , enzyme a c t i v i t y ( u n i t s ) . A, c e l l u l a r lysate from L5178Y(S) c e l l s ; B, c e l l u l a r l y s a t e from L5178Y(R 3) c e l l s ; and C, c e l l u l a r lysate from L5178Y(R 4) c e l l s . 46 A T 1 1 1 1 1 r FRACTION NUMBER 47 • BufferQ- • Buffer®. Buffer (3 )— 12 16 20 24 28 32 36 Fraction number C T 1 1 1 r FRACTION NUMBER 48 Chromatography of L5178Y(R 4) lysate showed a similar profile of protein and DHFR activity elution (Fig.7c.) as R 3 lysate; however, the proportion of unbound activity in buffer 1 was greater than that seen in R 3 cells. The question now arose as to whether the unbound activities eluting with buffer 1 upon chromatography of R 3 and R 4 lysates represented a different form of the enzyme with a lower affinity for MTX, or 'spill-over' of the bound enzyme due to the saturation of binding sites on the column. Another possibility could be that the activity observed in buffer 1 elution was artifactual resulting from insufficient precipitation of the unreduced radiolabeled folic acid. To answer these questions, fractions with DHFR activity in buffer 1 elution were pooled, concentrated and assayed in the absence of NADPH. No enzyme activity was detected. In order to show that the products of this NADPH dependent enzymatic reaction was indeed tetrahydrofolic acid, the supernatant of the radioassay was subjected to HPLC on an anion exchange column (see Methods and Materials). Fig.8a. shows the separation of a standard mixture of N^ formyl tetrahydrofolic acid, N^ methyl tetrahydrofolic acid, tetrahydrofolic acid, p-aminobenzoylglutamate, p-aminobenzoic acid, dihydrofolic acid and folic acid Fig.8b. represents the radioactive profile of the reaction products of the MTX sensitive DHFR present in the cell lysate from L5178Y(S) cells. Three radioactive peaks are evident with two of them corresponding to the retention times of the tetrahydrofolate cofactors. These two peaks were therefore tentatively identified as one or more forms of tetrahydrofolate. 49 FIGURE 8 Separation of folate compounds by high pressure liquid chromatography on an anion exchange column. Experimental details are given in the text. A, Chromatography of a standard mixture of folate compounds. B, Chromatography of the reaction products of folate reductase activity in L5178Y(S) lysate. C, Chromatography of the reaction products of Form 2 folate reductase activity. 3 9 H-CPM; O.D.2S4 profile of separation of a standard mixture of folate compounds. DU 0.5 H 0.4 H E c CVJ 0.3 Q Cl 0.2 H 0.H P-AMINO BENZOYL GLUTAMATE 5- FORM YL - TE TR AH YDROFOLIC ACID/ TETRAHYDROFOLIC ACID 5-METHYL-TETRAHYDROFOLIC ACID DIHYDROFOLIC ACID FOLIC ACID 2 4 6 8 10 12 14 TIME (MIN) 51 52 CM I o 0_ o I 2 4 -22 -20 t 18 • I] 16 - II 14 - 11 12 - ;"j 10 -8 - 1 1 I i 6 -1 1 1 1 I i 4 -2 -f 1 l 1 V 0 -| • A ^x t • Y O A 0.5 h0.3 r-0.2 r-0.1 0 2. 4 6 8 10 12 14 TIME (Min.) O d cn 53 Fig. 8c. shows the radioactive profile of the products of the DHFR enzyme activity eluted in buffer 1 upon chromatography of R 3 lysate. Four radioactive peaks were present this time with two of these peaks corresponding to the retention times of tetrahydrofolate cofactors. It was concluded therefore that this latter activity represented DHFR capable of generating tetrahydrofolic acid from folic acid. However the problem of 'spill-over' still remained. The unbound activities from both R 3 and R 4 cells were therefore assayed in the presence of increasing concentrations of MTX to determine the sensitivity of these enzymes to MTX. This is shown in Fig.9. where the percentage inhibition of the enzyme of control is plotted against increasing MTX concentration. Included for comparison is the sensitivity of DHFR's derived from S cells, and the affinity column bound reductase from R 4 cells. It can be ~6 seen that both sensitive enzymes were 100% inhibited by 10 M MTX. In contrast the unbound DHFR from R 3 cells was only inhibited 50% by 10" 3M MTX and that from the R 4 cells was inhibited less than 40% by 10" 3M MTX. It was therefore concluded that R 3 and R 4 cells have in addition to MTX sensitive DHFR (affinity column bound activity) a variant form of DHFR characterized by marked insensitvity to MTX and inability to bind to MTX-sepharose affinity column. Hereafter, the DHFR activity from R 3 and R 4 cells which bound to the affinity columns was called Form 1, and the activity which did not bind to the columns was called Form 2. 4. Purification of Forms 1 and 2 of DHFR from L5178Y ( R 4 ) cells. (a). A f f i n i t y chromatography: As described earlier, affinity chromatography of the R 4 cell lysate on MTX-sepharose columns resulted in the complete separation of the two forms of reductase. T h i s 54 FIGURE 9 Inhibition of folate reductase activity from MTX-sensitive and MTX-resistant cells by MTX. Enzyme preparations were incubated with NADPH (1mM) and MTX in reaction buffer (0.01M potassium acetate, pH5.0) at room temperature for 5 min. The enzyme reaction was then started by the addition of 2- 1 4C folic acid (100uM).® L5178Y(S) lysate; • L5178Y(R 4) Form 1 DHFR; • L5178Y(R 3) Form 2 DHFR; o L5178Y(R 4) Form 2 DHFR. DO METHOTREXATE CONCENTRATION (M) 56 procedure also achieved substantial purification of both forms of the enzyme (see later and Table 5). ( b ) . Sephadex G-100 chromatography: Further purification of the two dihydrofolate reductases from the R 4 cells was carried out by means of gel filtration through Sephadex G-100. Both forms eluted in a position corresponding to an apparent molecular weight of approximately 20,000 ( F i g . 10a) and b ) ) . Similar results were obtained in low and high salt concentrations. The extent of purification obtained at each stage is shown in Table 5. The total enzyme activity applied to the affinity column was 155.1 nmoles/20 min. As indicated in Table 5, 8.3% of this activity was found to be due to Form 2 dihydrofolate reductase, determined by integrating the areas under Peaks I and II following MTX-sepharose affinity chromatography (Fig 7C). On the basis of this assumption the purification of Form 1 dihydrofolate reductase after affinity chromatography was 211 and that of Form 2 reductase was 31.5. Chromatography of Form I through sephadex G-100 resulted in a total purification of 1767 fold. Some high molecular weight proteins apparently bind to methotrexate-sepharose affinity columns and are eluted with Form 1 dihydrofolate reductase (Kaufman and Pierce, (1971)). These proteins are eluted in the void volume when applied to G-100 sephadex (Fig 10a). A 741 fold overall purification was achieved for the Form 2 reductase after Sephadex G-100 chromatography. Molecular weight determination: Form 1 (from affinity column) and Form 2 (from Sephadex G-100) enzymes were electrophoresed in the presence of sodium dodecyl sulphate as described in the Methods Materials section. These gels were stained with Coomassie blue and 57 FIGURE 10 Gel filtration on Sephadex G-100 of folate reductase Forms 1 and 2 from L5178Y(R4) cells. Samples (1.5 to 2.0ml) were applied to a sephadex G-100 column (1.6x81cm) and elution was carried out with 50mM Tris-HCI, pH7.5, at a flow rate of 24ml./hr. 6ml. fractions were collected and assayed for protein (absorbance at 280nM) and folate reductase activity , the A 28o profile; S , enzyme activity (units). A. Fractions eluted with Buffer 3 from methotrexate-sepharose affinity column and containing reductase activity (Form 1) were pooled, dialyzed at 4°C for 14hr. against 5mM Tris-HCI, pH7.5, lyophilized, dissolved in 2ml. of water and applied to the column. B. Fractions eluted with Buffer 1 from methotrexate-sepharose affinity column and containing reductase activity (Form 2) were pooled, dialyzed at 4°C for 14hr. against 4mM Tris-HCI, pH 7.5, lyophilized, dissolved in 3ml. of water and applied to the column. 58 A 1 1 1 1 1 1 1 1 r Vo 67,000 43,000 25,000 13,700 FRACTION NUMBER B 12 16 20 24 FRACTION NUMBER 28 32 36 T A B L E V Partial Purification of Dihydrofolate Reductases from L5178Y ( R 4 ) Cells. Total Activity Protein Specific Activity Purification Yield Form I Cell lysate Af f i n i t y column Sephadex G-100 nmol/20 min 142 b 114 106 M9 48,000 180 ^20 nmol/20 min/ug 0.0030 0.63 5.30 -fold 1 210 1770 % 100 88 81.5 Form 11 Cell lysate Af f i n i t y column Sephadex G-100 12.9 b 12.7 4.0 48,000 1,500 ^20 0.00027 0.0085 0.2 1 31.5 740 100 98 32 a T h e s e values were based on the assumption that Forms I and II contribute 91.7 and 8.3%, respectively, of the total activity in the crude lysate. These values were calculated on the assumptions that Form II contributes 8.3% of the total activity in the crude lysate. This percentage was determined by intergrating the areas under Peaks I and II following methotrexate-Sepharose affinity chromatography ( F i g . 1B). Thi s value was furt h e r substantiated by the heat stability studies performed on the L5178Y ( R 4 ) crude lysate in which 70% of the total enzyme activity corresponds to the heat-stable Form II dihydrofolate reductase (see Fig. 5). Since the Vmax of Form II enzyme is approximately 9.0-fold lower than Form I enzyme (Table II), the actual concentration of Form II enzyme in L5178Y R 4 cells is approximately 38% of the total dihydrofolate reductase concentration. 60 single protein bands were observed for both reductases corresponding 'to apparent molecular weights of 18,500 (Form 1) and 20,500 (Form 2). Molecular weight standards were electrophoresed simultaneously ( F i g . 11). Because of the unusual nature of Form 2 DHFR in terms of it's very low affinity for MTX, it was desirable to be able to determine the amino acid sequence of this enzyme. In order to do this the enzyme had to be purified to homogeneity. Recently a highly sensitive silver staining techinique for the visualization of proteins in polyacrylamide gels has been reported in the literature (Switzer et a[, 1979). Th i s techinique has been used to reveal proteins not visible with Coomassie blue; a modification of the method was therefore used to determine the homogeneity of Form 2 reductase after filtration through G-100 sephadex. As shown in Fig.12.(lane 2), the main band corresponds to DHFR (M.Wt 20000), however, three or four minor bands, not previously observed upon staining with Coomassie blue, were present. Further purification was therefore essential and was carried out by chromatography on a DEAE affigel blue column as described in the Methods and Materials section. The enzyme bound tightly to the column and was eluted with 1.5M NaCI in 50mM T r i s - C l pH 7.5 ( F i g . 13.) The fractions under this peak were pooled and concentrated by ultrafiltration through a 10,000 M.Wt. filter (Amicon). Polyacrylamide gel electrophoresis in the presence of SDS followed by silver staining now revealed an essentially homogeneous enzyme corresponding to a M.Wt. of 20,000 with one minor contaminating band of lower M.Wt. (Fig.12.-lane 4). Isoelectric f o c u s i n g — I s o e l e c t r i c focusing on pH gradients of 3.5 to 9.5 resulted in single peaks corresponding to pi values of 8.4 for Form 1 and 6.0 for Form 2. 61 FIGURE 11 Molecular weights of folate reductase Forms 1 and 2 from L5178Y(R4) cells in polyacrylamide-sodium dodecyl sulphate gels. Samples (75ul) containing 25-50ug of protein were applied to 10% gels, prepared as described by Leammli (1970), and electrophoresed at 4°C for 4 hr. at 2.2mA/tube. The gels were stained in 0.2% Coomassie blue R. in 50% trichloroacetic acid and destained in 7% ocetic acid, 10% isopropyl alcohol. In Gel A, enzymatically active fractions from sephadex G-100 chromatography of Form 2 reductase were pooled, dialyzed at 4°C for 14hr. against 2 liters of 3mM Tris-HCI, pH7.5, lyophilized, and dissolved in 1ml. of water. Gel B, Form 1 reductase, obtained from sephadex G-100 chromatography as described above. Gel C, molecular weight standards (Bio-Rad Laboratories, Mississaauga, Ontario). From top to bottom: phosporylase b, 94,000; bovine serum albumin, 68,000; ovalbumin, 43,000; carbonic anhydrase, 30,000; soyabean trypsin inhibitor, 21,000; lysozyme, 14,300. o A B C 63 FIGURE 12 Polyacrylamide-sodium dodecyl sulphate gel electrophoresis of Form 2 folate reductase from L5178Y(R 4) cells. Samples (50ul) containing not more than 6ug of protein were applied to a 13% slab gel described by Laemmli (1970), and electrophoresed at 10°C for 4 to 5 hr. at 30mA total. The gel was stained by the silver staining technique described in detail in the text. The gel was photographed immediately after developement. Lane 1: Molecular weight standards (described in Fig. 11); Lane 2: Form 2 folate reductase preparation from sephadex G-100; Lane 4: Form 2 folate reductase after subsequent chromatography on DEAE affigel blue. Lane 3: No protein applied. The two bands appearing in all lanes are artifacts of electrophoresis and staining since they appear in the blank lane (lane 3) as well. 64 M * 65 FIGURE 13 Chromatography of Form 2 folate reductase on DEAE affigel blue. Form 2 folate reductase activity from three sephadex G-100 columns was pooled and applied to the column. The column (1.5x8cm) was eluted step-wise with the buffers indicated and folate reductase activity was determined in each fraction by measuring the rate of decrease in absorbance at 340nm (i. e . oxidation of NADPH to NADP) in the presence of dihydrofolic acid. 66 0.05 M TRIS-CL ,pH7.5 0.05M TRIS-CL pH7.5 0.05 M TRIS pH7.5 1.0M NaCI 0.008 0.05 M TRIS -CL pH 7.5 2.5 M NaCI 8 12 15 19 23 27 31 35 39 Fraction number 67 P r o p e r t i e s of the M T X - s e n s i t i v e (Form 1) and M T X - i n s e n s i t i v e  (Form 2) folate reductases o a) pH O p t i m a : T h e pH optimum fo r the Form 1 reductase was f o u n d to be 4 . 0 , whereas that fo r Form 2 was between 4 . 5 and 5 . 0 (data not s h o w n ) . These f i n d i n g s are cons i s ten t wi th p r e v i o u s r e p o r t s (Smith et a[, 1979) in tha t when fo l i c ac id is used as a s u b s t r a t e , a c t i v i t y is l imited to the ac id ic p H . b ) Heat Inac t i va t ion : T h e heat s t a b i l i t y of the two forms of d i h y d r o f o l a t e reductase p r e s e n t in the R 4 ce l ls was compared to tha t of the reduc tase p r e s e n t in the l ysate of the parent sens i t i ve S c e l l s . Incubat ion of the L5178Y(S ) l ysate at 60°C, resu l ted in total inac t i va t ion of fo late reduc tase a c t i v i t y in 10 min ( F i g . 1 4 ) . Incubat ion of the L 5 1 7 8 Y ( R 4 ) l ysate at 60°C resu l ted in inac t i va t ion of 92% of the a c t i v i t y ; h o w e v e r , the remain ing 8% was stable fo r up to 34 min of incubat ion at t h i s tempera tu re ( F i g . 1 4 ) . P a r t i a l l y p u r i f i e d Form 1 and Form 2 reduc tases were incubated at 60°C f o r 30 m i n . Whereas Form 1 lost 98% of i ts a c t i v i t y w i th in 10 min (and 100% by 30 m i n ) , Form 2 was completely s table f o r the en t i re incubat ion per iod ( F i g s . 1 4 ) . c ) E f fect of p a r a - c h l o r o m e r c u r i p h e n y l s u l p h o n a t e on fo late reduc tase a c t i v i t i e s : T h e two forms of reduc tase responded v e r y d i f f e r e n t l y to the o r g a n i c m e r c u r i a l , p C M S . P a r t i a l l y p u r i f i e d enzyme p r e p a r a t i o n s - 9 were incubated with v a r i o u s concent ra t ions of p C M S , r a n g i n g f rom 10 - 4 to 10 M, fo r 5min at room t e m p e r a t u r e , p r i o r to a s s a y i n g fo r enzyme a c t i v i t y . F i g . 15 . shows the ef fect of i n c r e a s i n g concent ra t ions of pCMS on the a c t i v i t y of. the two forms of d i h y d r o f o l a t e r e d u c t a s e . Form 68 FIGURE 14 Effect of temperature on the enzyme activities of the two folate reductases from L5178Y(R 4) cells. Enzyme preparations (50ul) were incubated at 60°C for various time periods and cooled in ice prior to assaying for enzyme activity using the assay described in the text. A. Effect of heat on the enzyme activities of l_5178Y(S) ( • ) and L5178Y(R 4) ( o ) cell lysates. B. Effect of heat on the enzyme activities of partially purified Form 1 ( o ) and Form 2 ( o ) folate reductases. 70 F I G U R E 15 Ef fect of p C M S on the enzyme a c t i v i t i e s of the two fo late reduc tases f rom L 5 1 7 8 Y ( R 4 ) c e l l s . Samples (20ul) c o n t a i n i n g 50ug of p ro te in were i n c u b a t e d w i th v a r i o u s concent ra t ions of p C M S at 25°C f o r 5 m i n . p r i o r to a s s a y i n g fo r fo late reduc tase a c t i v i t y o , e f fec t of p C M S on the enzyme a c t i v i t y of p a r t i a l l y p u r i f i e d Form 1 fo late r e d u c t a s e ; , e f fec t of p C M S on the enzyme a c t i v i t y of p a r t i a l l y p u r i f i e d Form 2 fo late r e d u c t a s e . V 0 , enzyme a c t i v i t y in the absence of p C M S ; V , enzyme a c t i v i t y a f te r e x p o s u r e to p C M S . 71 T 1 1 I I I 11 I 1 1 1—I l l l l l 1 1 1 I l l l l l 1 1 1 I I I I II 1 1 1 I I I I I I 1 0 9 1 0 8 1 0 7 1 0 6 1 0 5 1 0 " (pCMS) M 72 2 was f o u n d to be v e r y s e n s i t i v e to low concent ra t ions of p C M S , the a c t i v i t y almost d o u b l i n g in the p resence of 10 ^ - 1 0 p C M S . H o w e v e r , the a c t i v i t y d r o p p e d to below that obta ined in the absence of p C M S , at - 4 h i g h e r c o n c e n t r a t i o n s (10 M ) . Form 1 appeared to be ac t i va ted v e r y s l i g h t l y b y p C M S , the a c t i v i t y i n c r e a s i n g wi th i n c r e a s i n g concent ra t ions -4 of p C M S s u c h tha t at 10 p C M S it was approx imate l y 1.1 x the level ob ta ined in the absence of p C M S . d ) K i n e t i c p r o p e r t i e s : D issoc iat ion cons tants f o r fo l i c ac id and N A D P H wi th both forms of the enzyme were determined f rom L i n e w e a v e r - B u r k doub le rec ip roca l p lots ( L i n e w e a v e r and B u r k , 1934) , ( F i g . 16 a - d ) . In each case one s u b s t r a t e was v a r i e d f rom 10 - 100 uM whi le the o ther was he ld c o n s t a n t . A summary of the cons tants f rom a f i t of data to the equat ion is shown in Tab le 6 . T h e Km fo r N A D P H is - 5 approx imate l y 4 x 10 M f o r both f o r m s . H o w e v e r , the Km fo r fo l i c ac id of Form 2 (1.66 x 10~ 4 M) is approx imate l y 2 . 5 fo ld h i g h e r t h a n tha t -5 of Form 1 ( 6 . 8 x 10 M ) . M o r e o v e r , the Vmax fo r Form 2 wi th e i ther s u b s t r a t e is s u b s t a n t i a l l y less t h a n the Vmax f o r Form 1 . T h e Ki f o r M T X of D H F R f rom S ce l ls as well as the two forms of reduc tase p r e s e n t in R 4 ce l ls were determined in p a r t i a l l y p u r i f i e d p r e p a r a t i o n s wi th the use of D ixon plots ( D i x o n , 1955) , ( F i g 1 7 . a , b , and c . ) ( A p p e n d i x I ) . T h e i n h i b i t o r d i ssoc ia t ion cons tants ( K i ) a re also summarized in T a b l e 6 . T h e Ki f o r M T X of the L 5 1 7 8 Y ( S ) reduc tase - 9 was approx imate l y 2 . 6 x 1 0 M . Form 1 enzyme f rom R 4 ce l ls had K i fo r _o M T X of 2 . 0 x 1 0 M , a 1 0 - f o l d a p p a r e n t i n c r e a s e . T h e s e va lues are o n l y approx imate because of the t i g h t b i n d i n g na tu re of M T X to s e n s i t i v e d i h y d r o f o l a t e reduc tases and also the h i g h e r molar concent ra t ion of the 73 FIGURE 16 Double reciprocal plots of initial velocity patterns with folic acid or NADPH as the varied substrates. A, determination of Km for folic acid of Form 2 folate reductase. The NADPH concentration was kept constant at 1mM. B, determination of Km for folic acid of Form 1 folate reductase. C, determination of Km for NADPH of Form 1 folate reductase. The folic acid concentration was kept constant at 100uM. D, determination of Km for NADPH of Form 2 folate reductase. In all cases, the enzyme concentration was 0.7mg/ml. Points were fitted with a straight line estimated by least squares linear regression analysis. B I s > z Q P U 2 0.10 E S 0.08 < 8 0.06 E Q. 0 0 .04 -U l s , 0 . 0 2 -Km -0-02 0 0.02 0.O4 0.06 0.08 0.10 RECIPROCAL OF SUBSTRATE CONCENTRATION ("4") M M FOLIC ACID 75 TABLE VI A Summary of the Kinetic Properties of DHFRs From L5178Y(S) and (R 4) Cells. DIHYDROFOLATE Vmax Km Km Ki REDUCTASE (units) (Folic Acid) (NADPH) (MTX) Molar Molar Molar S 12.3 1.72 x 10"5 - 2.6 x 10 R 4 83.3 6.8 x 10"5 5.0 x 10"5 2.0 x 10 'High Aff R. 10.0 1.7 x 10"4 2.7 x 10"5 7.5 x 10 4 'Low Aff 76 FIGURE 17 Dixon plots of the receprocal of initial velocity of reaction against MTX concentration at two substrate (folic acid) concentrations. Two lines with different slopes are obtained and intersect at a point to the left of the ordinate axis. T h i s point lies at a value of -Ki (see appendix I). Enzyme assays were carried out as described in the text. The NADPH concentration was kept constant at 1mM in all cases. A, L5178Y(S) folate reductase. B, Form 2 folate reductase from L5178Y(R 3) cells. C, Form 2 folate reductase from L5178Y(R 4) cells. Points were fitted with a straight line estimated by least squares linear regression analysis. 77 METHOTREXATE CONCENTRATION (M x 10"9) 78 B 79 enzyme in R 4 c e l l s . T h e Ki fo r M T X of Form 2 enzyme ( 7 . 5 x 1 0 ^M) was approx imate l y 2x10^ fo ld h i g h e r than Form 1 reductase and ind icates a v e r y low ( b u t detectab le ) a f f i n i t y f o r M T X . e) E l e c t r o p h o r e t i c p r o p e r t i e s : E lec t rophores i s of the two forms of folate reduc tase on n o n - d e n a t u r i n g po l yac ry lamide gels demonst rated a marked d i f f e r e n c e in e l e c t r o p h o r e t i c mobi l i t y ( F i g . 1 8 A ) . T h e gels were s ta ined fo r a c t i v i t y as d e s c r i b e d by Huennekens et a[, (1971) . Both forms of the enzyme were also incubated wi th 10 ^M M T X in the p resence of N A D P H at 4°C fo r 30 m i n . , then e lec t rophoresed as above and s ta ined f o r a c t i v i t y in the p resence of 10 M M T X f o r 15 m i n . F ig 1 8 ( B ) shows the format ion of a complex between Form 1 and M T X , as ind ica ted by an inc rease in the v a l u e . In c o n t r a s t , t h e r e is no change in the R^ of Form 2 d i h y d r o f o l a t e r e d u c t a s e , i n d i c a t i n g the lack of complex format ion between M T X and the enzyme. S t a i n i n g the gels - 6 f o r enzyme a c t i v i t y in the p resence of 10 M M T X resu l ted in a band of v e r y low i n t e n s i t y fo r Form 1 . However , the i n t e n s i t y of the Form 2 enzyme remained the same in the p resence and absence of M T X ( F i g 1 8 B ) . A n inc rease in R^ wi th t e r n a r y complex format ion between D H F R s , N A D P H and M T X has been demonst rated before by severa l w o r k e r s (Neef and H u e n n e k e n s , 1975; G u n d e r s e n et a[, 1972) . 80 FIGURE 18 Electrophoresis of dihydrofolate reductases Form 1 and 2 on 7.5% non-denaturing polyacrylamide gels. Samples (50-75ul) containing 25ug protein were electrophoresed on 7.5% polyacrylamide gels in 25mM T r i s - g l y c i n e , ph8.3, at 2.2mA/tube for 1.5h. at 4°C. The gels were stained for dihydrofolate reductase activity by placing the gels in a freshly prepared solution containing NADPH, dihydrofolic acid and 3(4,5-dimethylthyazoly(-2)-2, 5-diphenyltetra-zoluim bromide in 50mM T r i s - C I , pH 8.0. After staining the gels were washed with distilled water, followed by 5% acetic acid and then photographed. A. Gel A: Form 1 reductase; Gel B: Form 2 reductase. B. Samples were incubated with 10 methotrexate for 30 min. at 4°C before electrophoresis. The gels were -6 stained for activity in the presence of 10 M MTX. Gel A: Form 2 reductase; Gel B: Form 1 reductase. Z O l > 3 0 O - S Z O l > 3 J O — K 61 81 91 81 » l Cl 21 11 01 • • 1111111 l l l l l l l l i Inn ii|[mi l i i i i l t i n " " ' l i i l lllllltl! 82 FIGURE 19 Giemsa-trypsin-stained metaphase chromosomes. Cells 4 (3x10 cells/5ml) were incubated for 72hr. at 37°C in Fisher's meduim containing 10% horse serum. Colcemid was added to a final concentration of 16ng/ml. and after 90 min. at 37°C cells were centrifuged at 300g. for 3 min. and resuspended in 5ml. of 75mM KCI (37°C) for 8 min. Cells were centrifuged as above and resuspended gently in 3ml. methanol: acetic acid (3:1 V/V), centrifuged as before and resuspended in 5ml. of the same solution. The cell suspension was then dropped on wet slides and allowed to air dry. Slides were stored at room temperature for 1 week, heated for 16hr. at 56°C, incubated in 25mM potassium phosphate (pH 6.8: 56°C) for 8 min., and then stained with Giemsa-Trypsin. Slides were rinsed in distilled water, air dried, and examined by light microscopy. A, L5178Y(S); B, L5178Y(R4). The homogeneous staining region on one of the chromosomes of R4 cells is indicated by the arrow. 83 84 85 D I S C U S S I O N The effectiveness of MTX as a cytotoxic agent depends on its potency as an inhibitor of dihydrofolate reductase. In cells resistant to MTX the inhibition of the DHFR activity may not be complete. Th i s may be due to insufficient intracellular levels of MTX because of reduced uptake; increased levels of DHFR resulting in the immobilization of all of the intracellular MTX leaving free DHFR; or, the appearance of altered DHFR with reduced affinity for MTX resulting in incomplete inhibition of DHFR activity. In the MTX-resistant cells examined in this thesis all three mechanisms were found to be present to some extent. L5178Y(S) cells, growing rapidly with a population doubling time of 11 to 14 hours are ver y sensitive to MTX ( F i g . 5). The '•D.gQ of -8 these cells was 2.8x10 M MTX, with total inhibition of cell proliferation -ft occuring at 10 M MTX. L5178Y ( R 3 ) cells derived from S cells by growing them in media containing progressively increasing sublethal -9 concentrations of MTX (starting from 10 M), had an ' - D . ^ Q of 2.5x10 ^M. This constitutes a 1000-fold increase in resistance over the S cells. R 3 cells also had a population doubling time of 11 to 14 hours. The properties of S and R 3 cells were studied in terms of MTX influx and the amount and type(s) of dihydrofolate reductase. Tables 2 and 3 show that whilst uptake of MTX into S cells at "low" (10 _ 6M) extracellular concentration was significantly inhibited by pCMS, 5-methyl tetrahydrofolate and 5-formyl tetrahydrofolate, uptake into S -4 and R 3 cells at "high" . (10 M) extracellular concentration was not influenced by these compounds. At "low" extracellular MTX concentrations drug uptake in S cells appears to occur via the carrier 86 mediated transport process demonstrated in other cell types (Harrap et a[, 197.1; Hill et a[, 1979), and is defective in the R 3 cells, resulting in considerable lower uptake of MTX ( F i g . 6a). In contrast, at "high" MTX concentrations, both drug-sensitive and resistant lines took up equivalent amounts of MTX ( F i g . 6b). In addition, the lack of competition for uptake between MTX and 5-methyl tetrahydrofolate or 5-formyl tetrahydrofolate, strongly suggest that most of the MTX was not taken up via the physiological carrier that accounts for transport at "low" extracellular concentrations. The nature of this different process is not known; however, the marked reduction in the sensitivity of uptake to pCMS indicates that sulphyhdryl groups are probably not involved in contrast to the uptake at "low" MTX concentrations which is inhibited by pCMS. The resistant cells were markedly less susceptible to MTX cytotoxicity despite the fact that potentially cytocidal intracellular concentrations of drug were achieved. Therefore other factors must be contributing to the resistance of these cells. L5178Y(R 4) cells were derived from R 3 cells by exposing them to -3 increasing concentrations of MTX, up to 10 M. R 4 cells had an l-D-gg of between 5x10 - 3M and 10 _ 2M MTX, representing a 200,000-fold increase in resistance. Despite being extremely resistant to MTX, these cells demonstrated identical drug uptake properties to R 3 cells (described above). It would therefore appear that the transport barrier is not a significant mechanism of resistance in these two highly MTX-resistant cell lines. Folate reductase activity, assayed by the radiolabeled folic acid method, was next determined in the cellular lysates of L5178Y(S), ( R 3 ) 87 and ( R 4 ) cells. The specific activities are listed in Table II. Folate reductase activity in R 3 cells was 7-fold higher than in S cells and the activity in R 4 cells was approximately 9-fold higher. As explained in the 'Introduction' of this thesis, many MTX-resistant cell lines have elevated levels of folate reductase, some as much as 300-fold higher than the level in the corresponding wild-type cells. This elevation in DHFR level has been associated with the specific amplification of the DHFR gene (Alt et al, 1978; Schimke et a[, 1978). In addition, the amplification has been correlated with the appearance of a homogeneously staining chromosomal region (HSR) upon Trypsin-Giemsa banding analysis of metaphase chromosomes (Numberg et a[, 1978; Dolnick et al_, 1979; Berenson et a[, 1981). It was of interest therefore to see if the moderate elevation in DHFR activity in R 4 cells was associated with an HSR region. Fig. 19(b) shows just such a region in one of the chromosomes of R 4 cells, and is not present in any of the chromosomes of S cells ( F i g . 19(a)). R 4 cells grown in the absence of MTX for over 100 generations retained the 9-fold elevation in the DHFR level and their resistance to MTX. This observation substantiates the finding that stably resistant cells with DHFR gene amplification are associated with an HSR region on the chromosomes whereas unstably resistant cells with gene amplification are associated with 'double minute 1 chromosomes which are lost, together with the elevated levels of DHFR, upon removal of the selection pressure (Kaufman et a[, 1979). R 3 and R 4 cells, in addition to the elevation of DHFR activity also contained two forms of the enzyme. The principal form (Form 1) in R 3 and R 4 cells exhibited high affinity for MTX and bound tightly to the MTX-sepharose affinity column. The second form (Form 2) present in 88 smaller amounts in both cell lines, failed to bind to the MTX-affinity column and exhibited markedly lower affinity for MTX (see Table VI). In the parent sensitive cells there were no detectable amounts of the Form 2 type of enzyme ( F i g . 7). The Form 2 DHFR in the resistant cells retained its ability to reduce folic acid, suggesting capability of physiological function in the intact cell. R 4 cell-line, the most resistant subline, had higher levels of Form 2 DHFR and this enzyme had also a higher Ki (lower affinity) for MTX than the Form 2 reductase from R 3 cells. Therefore a distinct correlation can be made between the amount of 'low-affinity' (Form 2) enzyme present, its Ki for MTX and the degree of resistance of the cells (Table VI). Purification of the two forms of DHFR was carried out from the R 4 cell line since this subline contained the highest levels of both forms of the enzyme. It was carried out by affinity chromatography on MTX-sepharose columns, followed by gel filtration on sephadex G-100. These two steps afforded 1760-fold purification of Form 1 (high affinity) DHFR and 740-fold purification for Form 2 (low affinity) DHFR. Further purification of the Form 2 enzyme had to be carried out on DEAE-Affigel blue columns to achieve homogeneity as determined by SDS polyacrylamide gel electrophoresis and staining for protein with the ver y sensitive silver staining method (see Results Section). The apparent molecular weights of the two forms of the enzyme differ by 2000, both being in the range of previously reported molecular weights for mammalian dihydrofolate reductases (^20,000) (Bertino et a|, 1965;' Perkins et a[, 1967; Neef and Huennekens, 1975; Smith et a[, 1979). The apparent molecular weights determined by gel-filtration through sephadex G-100 ( F i g . 10) were consistent with 89 those determined by SDS polyacrylamide gel electrophoresis (Fig. 11 and 12). The two forms of the enzyme had different isoelectric points(pl). Form 1 had a pi of 8.4 and agrees well with the pis of DHFRs exhibiting high affinities for MTX (Flintoff et a[, 1976; Kaufman and Kemerer, 1977; and Smith et a[, 1979). The isolectric point of Form 2 enzyme was 6.0 and represents a considerable acidic shift. These results suggest that the alteration in the structure of DHFR (Form 2) resulting in a huge reduction in affinity for MTX is probably associated not only with changes in the amino acids normally responsible for the tight binding of MTX but also in the amino acids elsewhere in the molecule. This difference in the overall charge and the affinity for MTX (and therefore the ability to form a stable ternary complex with MTX and NADPH) was demonstrated by carrying out electrophoresis on 7.5% polyarylamide gels under non-denaturing conditions (i.e. in the absence of SDS). Gels were stained for enzyme activity by placing them in a freshly prepared solution containing NADPH, dihydrofolic acid and 3(4,5-dimethylthyazolyl-2)-2, 5-diphenyltetrazolium bromide in 50mM Tris-CI, pH 8.0. DHFR activity was indicated by a purple zone of reduced tetrazoluim. Fig. 18 (a and b) shows the differential migration of the two forms of DHFR under these conditions. Staining for enzyme -6 activity in the presence of MTX (10 M) diminished the intensity of the colour due to the activity of Form 1 but not Form 2 reductase (Fig. 18). Polyacrylamide gel electrophoresis has been used by Gundersen et a_[, 1972 and Neef and Huennekens, 1975 to visualize complex formation between DHFRs and their substrates and inhibitors. These investigations have shown an increase in the R^  value of enzyme-NADPH-MTX complex. Upon pre-incubation of the two forms of 90 DHFRs with NADPH and MTX (10 M) for 30 min. at 4°C, an increase in the R^ value of Form 1 was observed indicating the formation of a ternary complex ( F i g . 18). However, the R f value of Form 2 enzyme (low-affinity enzyme) remained the same and indicated clearly the inability of this enzyme to bind MTX ( F i g . 18). This was a 'visual' confirmation of the differences in the MTX binding capacities of the two forms of DHFR. Dihydrofolate reductase activity can be stimulated by organic mercurial compounds such as pCMB and pCMS (Kaufman, 1964; Perkins and Bertino, 1964). The latter authors reported that whereas pCMB stimulated the activity of DHFR from Ehrlich ascites cells 3-4-fold when dihydrofolic acid was used as the substrate, no stimulation of activity occured when folic acid was used as the substrate. in this present study, only a slight increase in activity of Form 1 enzyme occured upon incubation with pCMS (upto 10~ 4M) ( F i g . 15), whereas Form 2 activity -7 -5 was stimulated almost 2-fold by pCMS (10 to 10 M) using folic acid as substrate ( F i g . 15). Further increasing the pCMS concentration to -4 10 M was inhibitory to the enzyme activity. An observation similar to this one was reported b y Blumenthal and Greenberg (1970), who showed that the DHFR activity from a MTX-resistant mouse lymphoma cell line was stimulated by low concentrations of pCMB, but was inactivated by -4 higher concentrations (3x10 M). In contrast, the activity of DHFR from their MTX-sensitive cell line was only stimulated slightly. The differential response of the two forms of DHFR, reported in this thesis, to pCMS would suggest the presence of cysteine residues at different positions in the two enzymes, Form 2 having cysteine groups which are 91 more readily accessible to pCMS; the interaction of pCMS with these cysteine groups then causing the enzyme to undergo a conformational change resulting in a more rapid reduction of folic acid. The marked difference observed in the stability of the two enzymes to heat ( F i g . 14) suggests different tertiary structures and together with the differences in the overall charge and the differential effect of pCMS on enzyme activity suggests substantial alteration in the amino acid sequence of Form 2 reductase from that of Form 1 reductase. Enzyme kinetic studies of the two enzymes showed that while the Ki for MTX was very different for the two forms, the Km for folic acid and NADPH (Table 6) were relatively similar. In order to explain this phenomenon one has to speculate that although MTX and folic acid (or dihydrofolic acid) compete for the active site at physiological pH, they do not bind to the active site in the same way; since, it appears that the binding site for folic acid is relatively unaltered, whereas that for MTX is very different, perhaps due to the substitution of one or several amino acids crucial to the tight binding of MTX. In this regard, Hood and Roberts (1978) and Charlton et al_, (1979) have shown by x-ray diffraction and stereochemical studies, respectively, that folic acid and dihydrofolic acid bind to DHFR in the same orientation, whereas MTX binds to the enzyme-NADPH ( complex in the reverse orientation. The Vmax of Form 2 DHFR was lower than that of Form 1 by a factor of 9.0, so that alterations leading to decreased affinity for MTX also led to diminished enzyme activity. Such a reduction in the Vmax has recently been reported by Haber et a[, (1981) for an altered DHFR with a 250-fold decrease in affinity for MTX in MTX-resistant 3T6 mouse fibroblasts. 92 In light of the data discussed so far, three points need to be raised. F i r s t l y , whether exposure of sensitive cells to MTX induces mechanisms which lead to resistance or whether variant cell types expressing the mechanisms of resistance appear during normal growth and are selected for upon MTX exposure; secondly, what are the molecular mechanisms for the phenotypic expression of altered dihydrofolate reductases; and t h i r d l y , does the presence of altered DHFRs with substantially lower affinities for MTX constitute an important mechanism of resistance, and if so what types of compounds may be more potent than MTX in inhibiting their activity. The question of whether MTX induces mutations leading to resistance ( i . e . by epigenetic processes), or whether variant cell types expressing the mechanisms of resistance appear during normal growth as a result of spontaneous mutations ( i . e . genetic processes) is still under considerable debate. These phenomena apply to drug resistance in general in both procaryotic and eucaryotic systems. Substantial evidence exists for the latter process, i.e. drug resistant phenotypes arise spontaneously and with a definite frequency (L u r i a and DelbrCick, 1943; Law, 1952; Brockman, 1974; Poche et al_, 1975; Ling et al_, 1975). Utilizing the 'fluctuation test' Luria and DelbrCick (1943) were able to show the occurence of this process for bacterial populations resistant to viruses. Law (1952) utilized the same test to show that spontaneous mutation and selection constitute the mechanism by which MTX-resistant L1210 mouse leukemic cells develop in vivo. Siminovich (1976) has suggested that a similiar process exists for the appearance of resistant cells to many of the currently used chemotherapeutic agents, although an epigenetic process may be a factor in the case of some of these 93 agents. Goldie and Coldman (1979) have recently developed a mathematical model which relates drug sensitivity of tumours to their spontaneous mutation rate towards phenotypic drug resistance. Analysis of this model indicated that the probability of the appearance of drug resistant phenotypes increases with the mutation rate. The answer to the second question can at best be very speculative at the present time because the study of gene amplification and altered dihydrofolate reductases is very recent and any significant understanding of molecular mechanisms must await further work. Whether alteration in the structure of dihydrofolate reductase occurs as a result of errors during translation or transcription; or whether separate genes coding for different forms of DHFRs exist, remains an open question. It is unlikely that errors during translation would lead to such drastically altered species of enzymes, and in addition any mutations leading to errors in the translation of proteins would probably be lethal to the cells. Errors during the transcription of DHFR gene, or during the processing of mRNA from HnRNA, may be possible. The dihydrofolate reductase gene from a MTX-resistant mouse cell line has been shown to be 42 kilobase pairs long and to contain a minimum of five intervening sequences (four of which are in the protein coding region) (Nunberg et aj, 1980). If such a large amount of intervening sequence is a common property of all mouse DHFR genes then clearly, errors in the transcriptional process, due to either, point mutations in the genetic sequences of 'start' and 'stop' codons (resulting in the expression of 'noncoding' sequences), or due to errors in the processing of mRNA after transcription, could lead to the expression of mRNA coding for altered DHFRs. The existence of separate genes coding for altered DHFRs is also a distinct possibility. 94 Perhaps such genes are repressed in MTX-sensitive cells, and, those few cells that express these genes due to some mutational event, are selected for upon exposure to increasing concentrations of MTX. In the case of L5178Y R 4 cells which express two distinct species of enzymes, it is difficult to visualize how the expression of two such different proteins can take place from a single gene. Rather, it is very likely that two genes, one coding for Form 1 and one for Form 2 enzyme exist in these cells and both are expressed. In this regard multiple functional mRNA species have been shown to be present in MTX-resistant cells (Dolnick and Bertino, 1981; Setzer et a]_, 1980). The expression of multiple mRNAs for a single enzyme is rather unique, and if it is a common property of DHFR expression, one can speculate that at least some of these mRNAs code for more than one species of enzyme. Therefore, in resistant cells which express a single species of altered enzyme with a moderate decrease in affinity for MTX (Haber et a[, 1981), errors in the transcription of 'normal' DHFR (i.e. one usually coding for high affinity DHFR) or errors in subsequent processing of mRNA, probably constitute the mechanism; whereas the expression in the same cell, of low affinity DHFR of the Form 2 type together with high affinity DHFR probably results from the expression of two separate genes. It is entirely possible that the MTX-resistant cell populations such as R3 and R 4 are not homogeneous, but composed of several cell types expressing different mechanisms of resistance. Therefore, the 'low-affinity' and 'high-affinity' DHFRs in these cell lines may be expressed by two different cells. The only certain way for establishing 95 this fact is to localize DHFR activity in these cells by histochemical means, in the presence and absence of MTX. Although a histochemical technique for the detection of DHFR has been reported (Tzortzatou and Hayhoe, 1974), it is fraught with problems relating to non-specific background staining, and also difficulty in quantitation, and therefore is not very accurate. The frequency of spontaneous mutation to an altered dihydrofolate -9 reductase has been calculated as 10 with the number of mutants present in an uncloned population estimated at 2-10x10 7 (Flintoff et aj_, 1976; Flintoff et al, 1976). Most cell lines (including the ones described in this thesis) which have been treated with stepwise increasing concentrations of MTX have yielded resistant cells containing wild type dihydrofolate reductase expressed at high levels (Raunio and Hakala, 1967; Nakamura and Uttlefield, 1972; Hanggi and Uttlef i e l d , 1976; Alt et al, 1976). Whereas the highly MTX-resistant L5178Y cells described in this thesis expressed elevated levels of DHFR, a proportion of which constituted an enzyme species with drastically reduced affinity for MTX, the study of other cells resistant to progressively increasing MTX concentrations (Albrecht et al_, 1972; Biedler et al, 1972; Haber et al, 1981), has shown that growth at high drug concentrations can result in the prevalence of cells expressing elevated levels of a DHFR with reduced affinity for MTX. The question remains as to whether the mutational event leading to the expression of altered reductase occured in the parental diploid L5178Y(S) population, and cells having a genotype for this alteration were selected for upon exposure to MTX, or, whether it occured in cells already containing amplified DHFR genes i.e. the expression of altered DHFR is secondary 96 to gene amplification. Some cells are found to be intrinsically resistant to MTX (Vanden Berg et al, 1981; Kufe et al, 1980) and if such cells are found to express normal amounts of DHFR with a low affinity for MTX then this would mean that mutations leading to altered DHFR with reduced affinity can occur in the absence of an increase in gene dosage. Flintoff et a|, (1976 and 1980) treated Chinese hamster ovary cells with a single high dose of MTX. Resistant cells were found to express normal levels of altered DHFR with sevenfold reduction in affinity for MTX. Therefore exposure of cells to high concentrations of MTX may select for cells with altered reductases in the absence of gene amplification. However, it is also conceivable that extended periods of growth in the presence of MTX selection, of cells with an increased number of target genes would increase the probability of the expression of an altered DHFR gene. Regardless of the mechanism for the expression of altered reductases with lower affinity for MTX, considerable evidence exists for the presence of these forms in MTX-resistant cells. In the highly resistant L5178Y ( R 3 ) and ( R 4 ) cells described in this thesis, altered DHFR with a very low affinity for MTX exists together with a moderate elevation in the total DHFR activity, and transport defect for Mow' extracellular concentration of MTX. The significance of this altered form of DHFR in relation to elevation in DHFR activity and impaired drug transport, needs to be established. From the experiments carried out on the uptake of MTX by R 3 and R 4 cells at 'low' and 'high' extracellular MTX concentrations, it would appear that growth of these cells in high concentrations of drug allows the achievment of potentially cytocidal intracellular drug levels. Despite this fact the cells were still not susceptible to MTX cytotoxicity. The appearance of cells expressing impaired uptake of 97 'low' (10 M) extracellular concentrations of MTX maybe an early event in the stepwise selection of resistant mutants. These cells, resistant to low concentrations of the drug, were probably so by virtue of impaired uptake alone and did not have elevated levels of DHFR or 'low-affinity' DHFR. With the exposure of these cells to increasing concentrations of MTX, cytocidal levels of intracellular MTX would be achieved and this new selection pressure would select for cells also expressing elevated levels of DHFR and/or 'low affinity' DHFR. Although the most resistant cell line (L5178Y R 4) had a 9-fold elevation in DHFR level over the wild type S cells, 8% of this was represented by the 'low-affinity' (Form 2) enzyme. Since the Vmax of the Form 2 enzyme was approximately 9-fold lower than that of Form 1 ('high-affinity') enzyme, the actual concentration of Form 2 DHFR in R 4 was approximately 38% of the total DHFR concentration (Table 5). Therefore the net elevation in DHFR activity with high affinity for MTX was only 5.6 fold over that in S cells. Clearly, an elevation of approximately 6-fold in the 'high affinity' DHFR activity of R 4 cells could not alone account for the 200,000 fold increase in resistance of these cells to MTX. The amount of DHFR represented by the 'low-affinity' enzyme in R 4 cells is therefore 3.4 fold higher than the total DHFR in S cells. It has been estimated (Jackson and Harrap, 1973; White and Goldman, 1976) that in murine tumour cells no more than 5% of the DHFR activity is required to generate sufficient tetrahydrofolate cofactors to maintain cell viability. Low affinity DHFR activity in excess of this amount is present in R 4 cells and, as shown in Fig. 9, at a concentration of MTX 98 w h e r e b y i n h i b i t i o n of the h i g h a f f i n i t y D H F R is complete , t h e r e is no s i g n i f i c a n t e f fect on the ac t i v i t i es of low a f f i n i t y DHFR (Form 2) form R 3 o r R 4 c e l l s . F i g . 20 schemat ica l l y i l l u s t r a t e s how a moderate e levat ion in h i g h a f f i n i t y D H F R , a long wi th the p resence of s i g n i f i c a n t levels of low a f f i n i t y DHFR f u n c t i o n together in i m p a r t i n g a h igh degree of res i s tance to M T X in cell t y p e s such as L5178Y ( R 4 ) . In c o n c l u s i o n , the p r e s e n t i nves t iga t ion has shown that ext reme r e s i s t a n c e to M T X in L5178Y mouse leukemia ce l ls is assoc iated wi th the e x p r e s s i o n of an a l te red t a r g e t enzyme ( D H F R ) with a v e r y low a f f i n i t y f o r M T X . A l t h o u g h these ce l ls e x h i b i t e d impaired u p t a k e of ' low' - 6 concent ra t ions (10 M) of M T X ; when exposed to ' h i g h ' concent ra t ions - 4 (10 M) of the d r u g , e q u i v a l e n t i n t r a c e l l u l a r s teady state levels were ach ieved in both s e n s i t i v e and r e s i s t a n t c e l l s . T h e moderate ( 9 - f o l d ) e levat ion in the DHFR concent ra t ion cannot account fo r the approx imate l y 200,000 fo ld inc rease in res i s tance of these c e l l s . T h e s e r e s u l t s agree wi th those r e p o r t e d by B i e d l e r et a[, (1972) and A l b e r c h t et a[, (1972) who noted tha t the co r re la t ion between DHFR a c t i v i t y and the level of d r u g res i s tance v a r i e d fo r d i f f e r e n t s u b l i n e s of C h i n e s e hamster ce l ls g r o w i n g in i n c r e a s i n g i n h i b i t o r c o n c e n t r a t i o n s , and that the ce l ls c o n t a i n i n g lower levels of DHFR than expected fo r t h e i r degree of r e s i s t a n c e were f o u n d to conta in an enzyme wi th reduced a f f i n i t y f o r M T X . T h e appearance of a l te red D H F R s in ext reme res i s tance to M T X is qu i te common (Haber et a[, 1981) , a l though the f i n d i n g of t h i s i n v e s t i g a t i o n , i . e . the p resence of s i g n i f i c a n t amounts of a DHFR wi th v e r y low a f f i n i t y f o r M T X together wi th a DHFR wi th h i g h a f f i n i t y fo r M T X , is u n i q u e . 99 The mechanisms of resistance to MTX in the clinical situation are far from clear. If dihydrofolate reductases with very low affinities for MTX are expressed in human MTX-resistant tumour cells J_Q vivo, then compounds with significant inhibitory effects towards these types of enzymes should prove very useful in overcoming resistance to MTX. If, in addition, these variants are expressed together with normal DHFRs with high affinity for MTX, then the administration of high-doses of MTX together with compounds capable of inhibiting the low affinity DHFRs (either simultaneously or in some kind of sequence) should result in better growth inhibition of the resistant tumours. The synthesis of compounds capable of inhibiting altered DHFRs must await the elucidation of the amino acid sequence as well as the secondary and tertiary structures of these enzymes. Amino acid sequencing and x-ray crystallographic data on the 3-dimensionai structure of DHFRs from E. Coli and chicken liver (Matthews et al_, 1977; Volz et al, 1982) have shown that MTX is bound in a cavity, 15A° deep. The presence of an amino group on position 4 (see Fig. 1) of MTX increases the basicity of the pteridine ring by about three pK units resulting in the protonation of the ring at the nitrogen on position 1. Thi s protonated nitrogen interacts strongly with the side chain of an aspartate residue (in E. Col i ) . This interaction apparently enhances the binding of MTX relative to folic acid and dihydrofolic acid, which do not have the amino group on position 4 ( F i g . 1). It can therefore be visualized that the substitution of a less polar amino acid for the aspartate residue, or the substitution of a highly basic amino acid (such as arginine) close to the aspartate residue would result in a substantial decrease in the binding affinity of MTX to the altered DHFR. The latter of the two processes has recently been described by 100 Baccanari et al, (1981) for an altered form of E. Coli. DHFR with a lower affinity for MTX. Although altered DHFRs reported so far have different kinetic and physical properties, it is very likely that only a limited number of changes in the amino acids responsible for the binding of MTX at the active site, lead to a decreased affinity for i t . Once these changes have been established, compounds capable of binding to this altered active site can be synthesized. The present investigation has therefore demonstrated the existence of a biochemical target which can be exploited by chemotherapy in overcoming the heretofore unresolved problem of resistance to MTX. 101 FIGURE 20 Schematic representation of the effect of MTX on folate metabolism in MTX-sensitive and MTX-resistant L5178Y cells. A. MTX-sensitive cells: MTX is actively taken up by the cells and binds strongly to the single species of 'high-affinity' folate reductase present. Inhibition of the enzyme results in the depletion of tetrahydrofolate pools with the consequent decrease in dTMP levels, leading to unbalanced and decreased DNA synthesis. The tendency of the increase in dihydrofolic acid levels (due to the inhibition of folate reductase) to displace MTX from the enzyme can be overcome by sustaining free intracellular MTX. B. MTX-resistant cells: When exposed to 'high' -4 concentrations (10 M) MTX, the drug enters the cell and binds strongly to the elevated levels of 'high-affinity' folate reductase ( HA), inactivating it and leaving v e r y little free intracellular MTX. The 'low-affinity' folate reductase ( L A ) (with a considerable higher Ki for MTX) present in these cells will continue to be functional at the free intracellular concentrations of MTX now present. The uninhibited activity of the Mow-affinity 1 enzyme ensure the synthesis of sufficient amounts of tetrahydrofolate cofactors and thus of dTMP. 102 A MTX-SENSITIVE CELL B MTX-RESISTANT CELL 103 REFERENCES 1. Albrecht, A., Pearce, F.R., Suling, W.J., and Hutchison, D.J.: Folate reductase and specific dihydrofolate reductase of the amethopterin-sensitive streptococcus faecium var. durans, Biochemistry, 8, 960-967, 1969. 2. 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Wilmanns, W.: Effects of methotrexate treatment on thymidylate synthesis in human leukocytes and bone marrow cells, Ann. N.Y. Acad. S c i . , 186, 365-371, 1971. 114 APPENDIX I A graphical method of obtaining values for Ki in the case of competitive and non-competitive inhibitors was described by Dixon in 1953. With a competitive inhibitor the effect of varying independently bot|h [S] and [I] has to be determined in order to obtain Ki. However, if / v i (where vi is the initial reaction velocity) is plotted against [I], keeping [S] constant, a straight line is obtained, and if this is done at two different substrate concentrations, [ S x ] and [ S 2 ] , the resultant lines will intersect at a point to the left of the ordinate axis (as in Fig. 17). T h i s point lies at a value of - Ki which can thus be read directly from the graph. This may be proved as follows: E + S — l i 1 ES k + I E + P; E + l v = ? E I . k " i where S is the substrate, E the enzyme, I the inhibitor, and P the product of the reaction. It is assumed that El does not undergo furth e r reaction. Inhibitor concentration is denoted by [I] and the dissociation constant of enzyme-inhibitor complex by Ki. For equilibrium conditions: (i.e k+x « k- x) The rate equation is v = k + 2 [ES] and the conservation equation [Eo] = [E] + [ES] + [El] 115 also Ks = [E] [S] •IES] Ki = [E] [I] [E l ] Hence [Eo] = K s [ E S ] + [ES] + K s [ E S ] [I] [S] [S] Ki T , k+o [Eo ] T h u s v = — — - . — For s teady state cond i t ions the re la t ionsh ip i s : v = k + g [Eo] 1 [s] u K i ; where Km = k + 2 + k - -\ k + I R e a r r a n g i n g E q . 2 in the form 1 = Km (1 + K i ) 1 + 1 V k+ 2 [Eo] IS] k+ 2 [Eo] and r e p l a c i n g k + 2 [Eo] by V as fo l lows : 1 = £ * < i + in ) 1 + 1 Vi V V l " - L ^ J [S] V Ki T h e equat ion r e s p r e s e n t s each l ine and at the point of i n t e r s e c t i o n ' /v i and [I] wi l l be the same fo r both l ines as also wi l l be V . C o n s e q u e n t l y , -I , [I] N Km , [I] , Km ( 1 + K i } [ S J - ( 1 + K i } [ S 2 ] T h i s can be t r u e on ly if e i the r [Sx] = [ S 2 ] or if [I] the fo rmer is not t r u e , [I] = - K i . = - K i , and s ince 116 From Dawes (1972). T h i s method of determining values for Ki is not ve r y accurate for 'tight-binding' inhibitors i.e. inhibitors which interact stoichiometrically with the enzyme. Therefore Ki values of MTX obtained for the dihydrofolate reductase from MTX-sensitive cells are only estimates and not strictly accurate. However, the DHFRs from the MTX-resistant cells exhibit higher I .D.^Q values for MTX and therefore MTX does not interact stoichiometrically with these enzymes, and the Ki values obtained for these DHFRs are probably quite accurate as determined by Dixon plot. PUBLICATIONS: 1. B . S c . (HONS) Thesis , University of Aberdeen, Scotland: The substructure of Herpes Simplex Virus I DNA molecules (1975). A B S T R A C T S : 1. Gudauskas, G . A . , Hartely, D . , Dedhar', S. and Goldie, J . H . : Computer assisted data acquisition and processing of flow microfluorometric data. Proc. 7th Canadian Medical and Biological Engineering Conference (1978). 2. Goldie, J . H . , Hartley, D . , Gudauskas, G . A . and Dedhar, S.: A methotrexate insensitive form of folate reductase present in methotrexate-resistant L5178Y mouse leukemia cells. 7th Annual Proceedings of the American Association of Cancer Research, No. 60, 16, (1979). A R T I C L E S : 1. T i d d , D . M . and Dedhar, S.: Specific and sensitive combined high performance liquid chromatographic-flow fluorometric assay for intracellular 6-Thioguanine nucleotide metabolites of 6-Mercaptopurine and 6-Thioguanine. J . Of • Chromatography 145, 237-246, (1978). 2. Goldie, J . H . , Krystal , G . , Hartley, D . , Gudauskas, G . A . and Dedhar, S . : A methotrexate insensitive variant of folate reductase present in two lines of methotrexate-resistant L5178Y cells. European J . of Cancer, 16, 1539-1546, (1980). 3. Goldie, J . H . , Dedhar, S. and Krystal , G . : Properties of a methotrexate-insensitive variant of dihydrofolate reductase derived from methotrexate-resistant L5178Y cells. J . Biological Chemistry, 256, 11629-11635, (1981). 4. Hill, Bridget T . , Dedhar, S. and Goldie, J . H . ' : Evidence that at "High" extracellular methotrexate concentrations the transport barrier is unlikely to be an important mechanism of drug resistance. Biochemical Pharmacology, 31(2), 263-266, (1982)^ ':!• t 5. Inhibition by folic acid antagonists of a methotrexate-insensitive dihydrofolate reductase from methotrexate-resistant L5178Y cells (In Preparation). SDII/D/82.03.05 

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