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UBC Theses and Dissertations

Studies on the expression of normal and structurally altered dihydrofolate reductase in mouse and human… Dedhar, Shoukat 1984

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S T U D I E S O N T H E E X P R E S S I O N O F N O R M A L A N D S T R U C T U R A L L Y A L T E R E D D l H Y D R O F O L A T E R E D U C T A S E IN M O U S E A N D H U M A N M E T H O T R E X A T E - R E S I S T A N T T U M O U R 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 M . S c , 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 , 1982. 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 L M E N T O F 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 O F D O C T O R O F P H I L O S O P H Y in T H E F A C U L T Y O F G R A D U A T E S T U D I E S Department of Pathology We accept th is thes is as conforming to the r e q u i r e d s t a n d a p d . T H E U N I V E R S I T Y / O F B R I T I S H C O L U M B I A DECEMBER 1984 © S H O U K A T D E D H A R , 1984 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date T ^ v /7^S" 79) II A B S T R A C T T h e ac t i v i t y of d ihyd ro fo la te r e d u c t a s e , a key enzyme in the de novo b iosyn thes i s of t h y m i d y l a t e , p u r i n e s , and some amino a c i d s , had p r e v i o u s l y been found to be inc reased in a me tho t rexa te - res i s tan t mouse leukemia (L5178Y) cel l l ine as compared to the ac t i v i t y in the parenta l me tho t rexa te -sens i t i ve cel l l i ne . T h e inc reased a c t i v i t y was composed of two forms of the enzyme, one of w h i c h , form 2 , was h i g h l y i nsens i t i ve to inh ib i t ion by metho t rexa te . Bo th forms were p u r i f i e d to near homogenei ty and us i ng the ant ibod ies p repa red aga ins t them, it cou ld be demonst ra ted tha t the two forms are an t igen ica l l y d i s t i n c t . T h e inc reased d ihyd ro fo la te reduc tase ac t i v i t y p resen t in the methot rexate res i s tan t ce l ls resu l ted f rom an o v e r p r o d u c t i o n of both forms of the enzyme due to the p resence of abundan t mRNA cod ing f o r these enzymes . A n inc rease in the d ihyd ro fo la te reduc tase gene copy number cou ld be demon-s t ra ted in the res i s tan t c e l l s . mRNA cod ing fo r form 1 and form 2 enzymes was g rea t l y en r i ched by polysome immunoprec ip i ta t ion and compl imentary DNA ( c D N A ) was s y n t h e s i z e d in v i t r o f rom these en r i ched mRNA molecu les. Ev idence was found fo r the p resence of me tho t rexa te - i nsens i t i ve forms of d i hyd ro f l a t e reduc tase in the b las t ce l ls of th ree out of e igh t acute myelogenous leukemia pa t i en t s , and in two (d i s t i nc t f rom the above) of the e igh t pat ients the ac t i v i t y was s i gn i f i can t l y i n c r e a s e d . In con t ras t to the o v e r p r o d u c t i o n of d ihyd ro fo la te reduc tase p ro te in in the me tho t rexa te - res i s tan t mouse cel l l i ne , i nc reased enzyme ac t i v i t y in a me tho t rexa te - res i s tan t human p romye locy t i c leukemia ( H L - 6 0 ) cell l ine cou ld not be co r re la ted wi th an inc rease in I l l the enzyme p r o t e i n . F u r t h e r m o r e , the amounts of d i hyd ro fo l a te reduc tase mRNA and gene -dosages were s imi lar in the parenta l metho-t r e x a t e - s e n s i t i v e and me tho t rexa te - res i s tan t c e l l s . T h e enzyme f rom the res i s tan t ce l ls d i f f e red s i g n i f i c a n t l y in some of i ts phys i ca l and k ine t ic p rope r t i es f rom tha t p resen t in the parenta l c e l l s . A n inc rease in d ihyd ro fo la te reduc tase ac t i v i t y r esu l t i ng f rom a modi f icat ion of the enzyme ra the r than gene ampl i f icat ion has not to date been repor ted in the l i t e ra tu re and may p resen t a novel mechanism of res is tance to metho t rexa te . IV T A B L E OF C O N T E N T S P A G E A B S T R A C T II L IST OF T A B L E S VI L IST OF F I G U R E S VII G L O S S A R Y X A C K N O W L E D G E M E N T S XIII C H A P T E R 1: I N T R O D U C T I O N 1 O r i g i n of the D r u g Res is tan t Pheno type 2 Mechanism of Ac t i on of Metho t rexa te 12 Res is tance to Methot rexa te 19 B iochemis t r y and Molecu lar Genet ics of D ihyd ro fo la te Reduc tase 30 T h e Scope of th i s T h e s i s 43 Re fe rences 51 C H A P T E R 2: O V E R P R O D U C T I O N OF TWO A N T I G E N I C A L L Y D I S T I N C T FORMS OF D I H Y D R O F O L A T E R E D U C T A S E A N D G E N E A M P L I F I C A T I O N IN A H I G H L Y M E T H O T R E X A T E R E S I S T A N T M O U S E L E U K E M I A C E L L L I N E . 62 In t roduc t ion 62 Mater ia ls and Methods 63 Resu l t s 75 D i scuss ion 103 Refe rences 110 C H A P T E R 3 : P O L Y S O M E I M M U N O P R E C I P I T A T I O N OF L5178Y ( R 4 ) F O R M 1 A N D F O R M 2 D I H Y D R O F O L A T E R E D U C T A S E mRNA A N D S Y N T H E S I S OF C O M P L E M E N T A R Y D N A ( c D N A ) . 115 In t roduc t ion 115 Mater ia ls and Methods 116 Resu l t s and D i scuss ion 123 Refe rences 136 V P A G E C H A P T E R 4: H E T E R O G E N E I T Y IN T H E S P E C I F I C A C T I V I T Y A N D M E T H O T R E X A T E - S E N S I T I V I T Y OF D I H Y D R O F O L A T E R E D U C T A S E F R O M B L A S T C E L L S OF A C U T E M Y E L O G E N O U S L E U K E M I A P A T I E N T S . 138 In t roduct ion 138 Mater ia ls and Methods 139 Resu l t s 141 D iscuss ion 158 Refe rences 163 C H A P T E R 5 Sect ion 1: I N C R E A S E D D I H Y D R O F O L A T E R E D U C T A S E A C T I V I T Y IN A M T X - R E S I S T A NT H U M A N P R O M Y E L O C Y T I C L E U K E M I A ( H L - 6 0 ) C E L L L I N E . L A C K OF C O R R E L A T I O N B E T W E E N I N C R E A S E D A C T I V I T Y A N D O V E R P R O D U C T I O N . 165 In t roduc t ion 165 Mater ia ls and Methods 166 Resu l t s 171 D i scuss ion 189 References 196 Sect ion 2: P R O P E R T I E S OF A N A C T I V A T E D F O R M OF D I H Y D R O F O L A T E R E D U C T A S E F R O M M E T H O -T R E X A T E - R E S I S T A N T H L - 6 0 C E L L S . 198 In t roduc t ion 198 Mater ia ls and Methods 199 Resu l t s and D iscuss ion 201 References 213 A P P E N D I X I: I N H I B I T I O N OF T H E M E T H O T R E X A T E I N S E N S I T I V E ( F O R M 2) D I H Y D R O F O L A T E R E D U C T A S E FROM L5178Y ( R 4 ) C E L L S B Y S U B S T I T U T E D T R I A Z I N E S A N D Q U I N A Z O L I N E S . 214 Mater ia ls and Methods 215 Resu l t s and D iscuss ion 215 Refe rences 225 S U M M A R Y A N D C O N C L U S I O N S 226 VI LIST OF TA B LES PAGE CHAPTER 1 Table 1-1 Table 1-2 Some properties of vertebrate and bacterial dihydrofolate reductases. Restriction enzyme analysis of human and mouse DHFR gene. 33 36 CHAPTER 2 Table 2-1 Restriction endonuclease fragment lengths hybridizing to 3 2 P - D H F R cDNA. 98 CHAPTER 4 Table 4-1 Activities and MTX-inhibitory properties of DHFR activity present in blast cell lysates from AML patients, and normal white blood cell lysates. 145 CHAPTER 5 Section 1 i Table 5-1 Table 5-2 Table 5-3 Synthesis of DHFR. MTX-sepharose affinity chromatography of DHFR from HL-60 (S) and (R 4 ) cells. Kinetic properties. 190 202 209 APPENDIX 1 Table 1 Table 2 Table 3 Quinazolines (Type 1). Quinazolines (Type 2). Substituted diaminotriazines. 218 220 221 VII LIST OF FIGURES PAGE CHAPTER 1 Figure 1-1 Fluctuation test. 5 Figure 1-2 Structural formulae of tetrahydrofolate and MTX polyglutamates. 15 Figure 1-3 Mechanism of action of MTX. 17 Figure 1-4 Diagramatic representation of the deletion of the diploid locus of Chinese hamster DHFR. 40 Figure 1-5 Schematic representation of the effect of MTX on Folate metabolism in MTX-sensitive and -resistant L5178Y cells. 45 Figure 1-6 (A) Overall schematics of the determination of the reasons for increased DHFR activity in L5178Y (R 4 ) cells. 49 (B) Strategy for the enrichment of mRNAs coding for form 1 and form 2 DHFRs. 49 Figure 1-7 Map of double stranded cDNA clone (pDHFR II). 50 CHAPTER 2 Figure 2-1 SDS-PAGE of form 2 DHFR preparations. 77 Figure 2-2 Titration of anti-DHFR antibodies. 81 Figure 2-3 Competition assay illustrating antigenic diversity between form 1 and 2 DHFRs. 83 Figure 2-4 SDS-PAGE of 3 H-leucine labeled proteins and partially purified 3H-labeled preparations of form 1 and form 2 DHFR from L5178Y (R 4 ) cells. 87 Figure 2-5 mRNA directed synthesis of DHFR in vitro. 89 Figure 2-6 Immunoprecipitation of in vitro translated DHFR by antibodies of two forms of DHFR. 91 Figure 2-7 Northern blot analysis of total L5178Y (S) and (R 4 ) poly A + RNA. 94 Figure 2-8 Southern blot analysis of L5178Y (S) and (R 4 ) genomic DNA. 97 VIII PAGE Figure 2-9 Dot blot analysis of genomic DNA from L5178Y (S) and (R 4 ) cells. 100 Figure 2-10 Giemsa-trypsin stained metaphase chromosome spread of an L5178Y (R 4 ) cell. 102 CHAPTER 3 Figure 3-1 SDS-PAGE of in vitro translation of total poly-somal and immunoprecipitated polysomal mRNA. 125 Figure 3-2 SDS-PAGE of in vitro translation of preadsorbed, immunoprecipitated polysomal mRNA. 128 Figure 3-3 SDS-PAGE of in vitro translation of "non-precipitated" polysomal mRNA. 131 Figure 3-4 Northern blot analysis of total polysomal and immunoprecipitated polysomal mRNA from L5178Y (R 4 ) cells. 134 CHAPTER 4 Figure 4-1 Stability of DHFR activity in AML blast cell lysates. 143 Figure 4-2 Inhibition of DHFR activity by MTX in blast cell lysates of AML patients. 147 Figure 4-3 Inhibition of DHFR activity by MTX in blast cell lysates of AML patients. 149 Figure 4-4 Inhibition of dihydrofolate reductase activity by MTX in white blood cell lysates of normal healthy individuals. 152 Figure 4-5 Non-denaturing PAGE of cell lysate from AML patient D.Ro. on 7.5% acrylamide gels. 155 Figure 4-6 Scan of non-denaturing PAGE of cell lysate from patient J . N . 157 Figure 4-7 Non-denaturing PAGE of blast cell lysate from patient A . P. 160 IX PAGE CHAPTER 5 Section 1 Figure 5-1 Figure 5-2 Figure 5-3 Figure 5-4 Figure 5-5 Figure 5-6 Figure 5-7 Figure 5-8 Section 2 Figure 5-9 Figure 5-10 Figure 5-11 Time course for the development of MTX-resistance in HL-60 and HeLa cells. 173 Southern blot analysis of HeLa (S) and ( R 4 ) genomic DNA. 175 DHFR activity in cellular lysates. 177 Estimation of DHFR gene dosage. 180 DHFR mRNA content in MTX-sensitive and -resistant HL-60 cells. 183 SDS pol^acrylamide gel electrophoresis of lysates from HL-60 (S) and (R 4 ) cells. 186 Immune dot-blot analysis. 188 Autoradiograph of SDS polyacrylamide gel electrophoresis of 3 5S-methionine-labeled cellular proteins. 192 Heat stability of HL-60 (S) and (R 4 ) DHFR activities in cell lysates. 205 Comparison of the heat stability of DHFR activity in cell lysates and partially purified preparations. 208 Effect of pCMS on the DHFR activity from HL-60 (S) and (R 4 ) cells in the presence and absence of KCI. 211 APPENDIX 1 Figure 1 Structural formula of MTX and core structures of quinazoline and triazine compounds. 217 X GLOSSARY Codon: a group of three nucleotides that codes for an amino acid. Complementary DNA (cDNA): DNA that is complimentary to messenger RNA; used for cloning or as a specific probe in hybridization studies. Dot-blot analysis: obviates the need for fractionating DNA or RNA and can act as a rapid screening method for detecting specific sequences from a complex mixture; DNA or RNA is applied as a dot directly onto nitrocellulose filter and allowed to hybridize with a specific 3 2 P-labeled DNA probe. Exon: portion of DNA that codes for the final mRNA. Genome: all the genes of an organism or individual. Intervening sequence: a portion of DNA that is transcribed but does not appear in the final mRNA transcript. Intron: Intervening sequence in DNA. Nick translation: procedure for labeling DNA in vitro using DNA polymerase I. XI N o r t h e r n - b l o t a n a l y s i s : method of t r a n s f e r r i n g R N A f ragments separa ted by e lec t rophores i s to a n i t roce l lu lose o r o ther t y p e s of t rea ted p a p e r s . The R N A can then be v i s u a l i z e d by h y b r i d i z a t i o n wi th a 3 2 P - l a b e l e d D N A p r o b e . P lasmid : ex t rachromosomal . autonomously r e p l i c a t i n g , c i r c u l a r D N A segment . Po l yadeny la t i on : n o n t r a n s c r i p t i v e add i t ion of po ly ( A ) ( po l yadeny la te ) to the 3' end of euca ryo t i c R N A . Promote r : a D N A sequence at wh ich R N A po lymerase b i n d s , and then in i t ia tes t r a n s c r i p t i o n . Pseudogene : a sequence tha t looks l ike a gene bu t does not f unc t i on as one . It may have appeared in the genome by r e v e r s e t r a n s c r i p t i o n of a mature m R N A . Res t r i c t i on endonuc lease : s i t e - s p e c i f i c e n d o d e o x y r i b o n u c l e a s e : c leavage is s e q u e n c e - s p e c i f i c ; both s t r a n d s are c l e a v e d ; usua l l y isolated f rom b a c t e r i a . S o u t h e r n blot a n a l y s i s : method of t r a n s f e r r i n g DNA f ragments that have been separa ted b y gel e lec t rophores i s to a n i t roce l lu lose f i l t e r such that re la t i ve pos i t ions of the DNA f ragments are ma in ta ined ; the DNA can then be v i sua l i zed by h y b r i d i z a t i o n wi th a 3 2 P - l a b e l e d D N A p r o b e . XII Termina t ion sequence : a DNA sequence at the end of a t r a n s c r i p -t ional un i t tha t s igna ls the end of t r a n s c r i p t i o n . T r a n s f o r m a t i o n : the i n t roduc t i on of an exogeneous D N A p repa ra t i on into a c e l l . T r a n s l a t i o n ( in v i t r o ) : the p rocess in wh ich the gene t i c code conta ined in the nuc leot ide sequences of m R N A d i r e c t s the o r d e r of amino ac ids in the format ion of pep t ide (can be c a r r i e d out in v i t r o u n d e r the r i gh t c o n d i t i o n s ) . X I I I A C K N O W L E D G E M E N T S I w ish to e x p r e s s my s ince re a p p r e c i a t i o n : to my s u p e r v i s o r s D r . J . H . Gold ie ( H e a d , D i v i s i on of Medica l O n c o l o g y , C a n c e r Con t ro l A g e n c y of B . C . ) and D r . Godo lph in (Depar tment of Pa tho logy ) fo r t he i r en thus ias t i c s u p p o r t , and c r i t i ca l gu idance d u r i n g the cou rse of t h i s p ro jec t ; to members of my a d v i s o r y committee, D r . Ross M a c G i l l i v r a y (Depar tment of B i o c h e m i s t r y ) , D r . Paul Renn ie (Depar tment of C a n c e r E n d o c r i n o l o g y , C a n c e r Con t ro l A g e n c y of B . C . ) , D r . C . T . Bee r (Depar tment of B i o c h e m i s t r y ) and D r . R. Pearce (Depar tment of Pa tho logy ) fo r t he i r help and encouragement ; to D r . Mar t i n Gi les (Depar tment of C a n c e r E n d o c r i n o l o g y , C a n c e r Con t ro l A g e n c y of B . C . ) fo r i nva luab le adv ice and s t imu la t ing d i s c u s s i o n s ; to Dar ia Ha r t l ey and D e i r d r e F i t z - G i b b o n s fo r e x p e r t techn ica l a s s i s t a n c e ; to members of the Medical I l l us t ra t ions Depar tment , C a n c e r Con t ro l A g e n c y of B . C . f o r t he i r ass is tance in the p repara t i on of the i l l u s t r a t i o n s ; to L inda Wood fo r t i r e less sec re ta r ia l ass is tance d u r i n g the p repa ra t i on of t h i s t h e s i s ; a n d , f i na l l y to the C a n c e r Con t ro l A g e n c y of B . C . fo r t he i r s u p p o r t . T h i s work was pa r t i a l l y suppo r t ed by a g r a n t to D r . Gold ie f rom the Nat ional C a n c e r Inst i tu te of C a n a d a . T h i s thes i s is ded ica ted to C a r o l e , my w i fe , f o r her who lehear ted s u p p o r t at all t imes , fo r neve r doub t i ng that I would f i n i s h i t , and fo r be ing the re in t imes of n e e d . 1 . C H A P T E R 1 I N T R O D U C T I O N " T h e prob lem of res i s tance in neop las t ic c e l l s , as in m ic ro -o r g a n i s m s , wi l l remain a most impor tant pe rpe tua l th rea t to the success fu l use of t he rapeu t i c agen ts " ( L a w , 1956) ( 1 ) . Chemothe rapy is p l ay i ng an i n c r e a s i n g l y impor tant role in the t reatment of pat ients wi th cancer and p r e s e n t l y cu res t housands of pat ients wi th a v a r i e t y of d isseminated ma l ignanc ies . However , it is apparen t that the g rea t major i ty of mal ignanc ies tha t are t rea ted wi th chemotherapeut ic d r u g s u l t imate ly become r e f r a c t o r y to the t rea tment . Overcoming d r u g - r e s i s t a n c e is t he re fo re of paramount impor tance in the e f fec t i ve management of ma l i gnancy . T h e l is t of c l i n i ca l l y e f f i cac ious an t ineop las t i c agents ava i lab le to the p resen t day medical onco log is t is a long one and con t inues to g row at an i n c r e a s i n g pace as ou r u n d e r s t a n d i n g of the mal ignant pheno type becomes c l e a r e r . T h e c l i n i ca l l y usefu l chemotherapeut ic d r u g c lasses inc lude the a l ky l a t i ng a g e n t s , p la t i num-con ta in ing compounds , fo late an tagon i s t s , p u r i n e and py r im id i ne nuc leos ide ant imetabo l i tes , and the an th racyc l i ne and b leomycin an t i b i o t i c s . A l l of these d r u g s are ' cy to tox i c ' and i n te r fe re wi th some aspect of DNA s y n t h e s i s , t hus k i l l i ng ce l ls wi th a p ro l i f e ra t i ve c a p a c i t y . In add i t ion to these cy to tox i c d r u g s , novel t he rapeu t i c agents may inc lude membrane t a r g e t s , r a d i o s e n s i t i z e r s , and noncy to tox i c approaches des igned to p r e v e n t metas tases, i nduce terminal d i f fe ren t ia t ion of mal ignant c e l l s , i nh ib i t tumour ang iogenes is and s u p p r e s s c a r c i n o -g e n e s i s . - 2 -T h i s thes is is conce rned spec i f i ca l l y w i th the molecular p r o p e r -t ies of ce l ls wh ich are res i s tan t to a w ide ly used folate an tagon i s t , methot rexate ( M T X ) . In pa r t i cu l a r the thes i s is conce rned wi th an examinat ion of the biochemical and molecular p rope r t i es of the enzyme d ihyd ro fo la te reduc tase ( the i n t race l l u l a r t a rge t f o r M T X ) f rom M T X -res is tan t cel l l ines as well as f rom cel ls of c l in ica l neoplasms wh ich are r e f r a c t o r y to M T X . T h e ob jec t ives of these s tud ies are to not on ly enhance ou r knowledge of d ihyd ro fo la te r e d u c t a s e , an enzyme int imate ly assoc ia ted wi th the p rocess of cel l d i v i s i o n , bu t also to u n d e r s t a n d the t ypes of changes that t h i s enzyme can unde rgo in M T X - r e s i s t a n c e c e l l s , wi th the ul t imate aim of overcoming res is tance to t h i s o therw ise h i g h l y e f fec t i ve an t ineop las t i c agen t . O r i g i n of the D r u g - R e s i s t a n t Pheno type Res is tance to v i r t u a l l y all the cy to tox i c d r u g s can be demon-s t ra ted in exper imenta l s y s t e m s , and c l i n i ca l l y even the most r espons i ve tumours even tua l l y become r e f r a c t o r y to any one o r a combinat ion of these cy to tox i c d r u g s . What is the o r i g i n of these res i s tan t ce l ls? Do they a r i se spon taneous ly due to random mutat ional even ts o c c u r r i n g w i th in the cel l popu la t i on , wi th some of the resu l t i ng va r i an t ce l ls hav ing the p rope r t i es of be ing res i s tan t to the chemical agen t ; o r do they ar ise by an ep igenet i c mechan ism, i . e . the se lec t ing agent in the env i ronment somehow induces a h e r e d i t a r y adaptat ion in a p ropo r t i on of the cel l popu la t ion r e n d e r i n g them res i s tan t to the d r u g . A cons ide rab le amount of ev idence ex i s t s in s u p p o r t of the former mechan ism, i . e . a genet ic bas is fo r the o r i g i n of the d r u g res i s tan t c e l l s . T h e ev idence fo r th i s mechanism wi l l be l - 3 -rev iewed here b r i e f l y and spec i f i c po in ts wi l l be rev iewed in more detai l in the subsequen t c h a p t e r s . F luc tua t ion A n a l y s i s In 1943 L u r i a and D e l b r u c k (2) used the f l uc tua t i on tes t to show that the appearance of b a c t e r i o p h a g e - r e s i s t a n t bac te r ia was a random process and not d i r ec ted by the phage themse lves . T h e p r i n c i p l e of the f l uc tua t i on tes t is based on the suppos i t i on tha t if pheno typ i c v a r i a n t s are a r i s i n g spon taneous ly and randomly then the re wi l l be a cons ide rab le f l uc tua t ion in the numbers of such pheno types in a la rge sample of mul t ip le rep l ica te c u l t u r e s ( F i g . 1 -1 ) . T h e f l uc tua t ion tes t has s ince been used to demonst ra te the genet ic o r i g i n of d r u g r e s i s -tant pheno types in v i t r o and in v i v o ( 3 - 6 ) . A l t h o u g h a pos i t i ve resu l t in a f l uc tua t ion tes t is i n d i r e c t , and not de f i n i t i ve proof of a gene t i c o r i g i n , it is cons i s ten t wi th such an o r i g i n . In recent yea rs s tud ies in molecular genet ics have p r o v i d e d data wh ich is to ta l ly c o n s i s t e n t , and p rov i des d i r ec t ev idence fo r a genet ic o r i g i n of d r u g r e s i s t a n c e . Gene Ampl i f i ca t ion and K a r y o t y p i c C h a n g e s  Assoc ia ted With D r u g Res is tance T h e phenomenon of se lec t i ve gene ampl i f icat ion was f i r s t obse rved in 1978 as a mechanism fo r res is tance to the fo late an tago-n is t and an t ineop las t i c agen t , M T X ( 7 , 8 ) . S ince tha t t ime, i n c r e a s i n g numbers of examples of gene ampl i f icat ion in mammalian ce l ls have been r e p o r t e d , i n c l u d i n g ampl i f icat ion of genes cod ing f o r spec i f i c p ro te ins in o ther d r u g - r e s i s t a n t ce l ls ( 9 -11 ,126 ) . In the case of - 4 -F I G U R E 1-1 F luc tua t ion T e s t . Ce l l s are subc loned f rom a la rge paren t s tock c u l t u r e and equal numbers of cel ls are then t r a n s f e r r e d f rom each of the subc loned popula t ions into second cu l t u re sys tems con ta in ing the se lec t ing agen t . U n d e r these cond i t ions t he re is a l a r g e , s ta t i s t i ca l l y s i g n i f i -cant va r ia t i on or " f l u c t u a t i o n " in the number of res i s tan t co lonies per t u b e . If the se lec t ing agent was i n d u c i n g the res i s tance almost equal numbers of res i s tan t co lonies would be expec ted per t u b e . Howeve r , if r es i s tan t ce l ls had been genera ted by random spontaneous mutat ions and the d r u g was s imply se lec t ing fo r res i s tan t c e l l s , then a la rge va r ia t i on in the number of res i s tan t co lonies would be expec ted per t u b e . T h e lat ter mechanism has been shown to opera te f o r most d r u g s . 5 FLUCTUATION TEST Parent —Population Growth of Subclones/ Drug Resistant Mutants - 6 -res is tance to M T X the genes ampl i f ied are those cod ing fo r the enzyme d ihyd ro fo la te reduc tase ( D H F R ) , the p r ima ry i n t r ace l l u l a r t a rge t fo r M T X . T h e ex t ra gene copies resu l t in marked ly e levated i n t race l l u l a r levels of D H F R . Res is tance to M T X is t he re fo re due to the p resence in the v a r i a n t ce l ls of su f f i c i en t f ree enzyme to genera te enough te t rahyd ro fo la te fo r ce l l u la r s y n t h e t i c p r o c e s s e s . T h e r e s i s -tan t pheno type and ampl i f ied genes can be e i ther s tab le o r u n s t a b l e , i . e . in some cel l l i n e s , in the absence of se lect ion p r e s s u r e ( M T X ) , the res is tance and ampl i f ied D H F R genes are s tab le , whereas in o ther cel l l ines the ampl i f ied genes are uns tab le and both the genes and M T X res is tance can be lost r a p i d l y . T h e ampl i f ied gene sequences mani fest themselves as cy togene t i c abnormal i t ies such as homo-geneous ly s ta in ing reg ions ( H S R ) on one of the two homologous chromosomes e i ther at the s i te of the nonampl i f ied gene (12,13) o r on o ther chromosomes (113) . H S R s are expanded reg ions of a chromo-some wh ich s ta in homogeneously ra the r than g i v i n g a s t r i a ted pa t te rn upon t r y p s i n - g i e m s a band s t a i n i n g . T h e y were f i r s t d e s c r i b e d by B ied le r and S p e n g l e r (14) in M T X - r e s i s t a n t Ch inese hamster lung cel l l ines wh ich had inc reased D H F R a c t i v i t y . T h e HSR chromosomes are usua l l y assoc ia ted wi th the s tab le M T X - r e s i s t a n t pheno type ( 1 5 ) , a l though excep t ions have been repor ted (16 ) . In the uns tab l y ampl i f ied s ta te , DHFR genes are f ound on smal l , of ten pa i red ext rachromosomal e lements , ca l led doub le minutes ( D M s ) . T h e s e elements lack cent romeres and the re fo re can be p ropo r t i oned unequa l l y into d a u g h t e r cel ls at mitosis and hence can be lost r a p i d l y (13 ) . - 7 -T h e D H F R encoded by ampl i f ied genes may be s imi lar to tha t p resen t in the M T X - s e n s i t i v e cel ls or it may be a l t e r e d , r esu l t i ng in a lower a f f i n i t y fo r M T X (17 ,18 ) . The gene ampl i f icat ion assoc ia ted wi th the e x p r e s s i o n of a l te red D H F R ' s may be s tab le ( 1 9 ) , o r uns tab le wi th the genes local ized on DMs (20 ) . In one of the cases (17) the gene cod ing f o r a l te red DHFR has been c loned and sequenced (21 ) . In th is case the a l te red enzyme va r i es f rom i ts normal c o u n t e r p a r t by a s ing le amino ac id s u b s t i t u t i o n , wh ich resu l t s in the enzyme h a v i n g a lower a f f i n i t y fo r M T X and thus i nc reas ing the degree of M T X -r e s i s t a n c e . T h i s then p rov i des s t r o n g ev idence tha t d r u g res is tance can resu l t f rom small changes in the genome. R e c e n t l y , D H F R gene ampl i f icat ion has been d i r e c t l y demon-s t ra ted in leukemic ce l ls of pat ients t rea ted wi th methot rexate ( 2 2 , 2 3 ) , and the assoc ia ted cy togene t i c changes ( i . e . DMs and H S R s ) have also been repor ted in pa t ien ts t rea ted wi th M T X for o ther mal ignancies (24 ) . In a d d i t i o n , the M T X - s e n s i t i v i t y of D H F R f rom pat ients wi th acute myelogenous leukemia has been found to be ex t reme ly heterogeneous (25) (see C h a p t e r 4 ) . S ince d i f f e rences in the s e n s i t i v i t y to M T X re f lec ts the th ree -d imens iona l s t r u c t u r e and the re fo re the amino-ac id sequence of D H F R , th i s he te rogene i t y demonst ra tes a h igh degree of s t r u c t u r a l po lymorph ism at the D H F R locus . T h e r e f o r e , gene ampl i f icat ion and mutat ions are not r es t r i c t ed to ce l ls in c u l t u r e bu t ac tua l l y do take place in pa t i en t s . P le io t rop ic D r u g Res is tance K a r y o t y p i c changes and gene ampl i f icat ion are not r es t r i c t ed to M T X - r e s i s t a n c e . K a r y o t y p i c changes such as the p resence of DMs - 8 -and H S R s have been found in ce l ls e x h i b i t i n g mu l t i d rug res i s tance (26) wh ich is a complex p le io t rop ic pheno type of c r o s s - r e s i s t a n c e to unre la ted compounds (27 ) . Ce l l s res i s tan t to v i n c r i s t i n e (9) or co lch ic ine e x h i b i t c r o s s - r e s i s t a n c e to o ther un re la ted d r u g s such as p u r o m y c i n , d a u n o r u b i c i n and a d r i a m y c i n . Such mutants a r i se appa ren t l y f rom a s ing le genet ic event lead ing to a l te red d r u g permeab i l i t y (27 ) . T h e s e ce l ls have been shown to o v e r p r o d u c e a h igh molecular we ight plasma membrane g l ycop ro te i n (gp 150) ( 2 6 , 2 7 ) . In the case of v i n c r i s t i n e res is tance a low molecular we igh t cy toso l i c p ro te in (V19) (26) has been shown to be o v e r p r o d u c e d . T h e f unc t i ons of these o v e r p r o d u c e d p ro te ins however s t i l l remain o b s c u r e . Recen t l y ad r iamyc in and co lch ic ine res i s tan t C h i n e s e hamster ce l ls have been shown to conta in ampl i f ied D N A f r a g m e n t s , some of wh ich are ampl i f ied in both i ndependen t l y d e r i v e d cel l l ines (28 ) . These resu l t s s u g g e s t tha t the D N A sequences wh ich are ampl i f ied in common in m u l t i - d r u g - r e s i s t a n t cel l l ines i nc lude g e n e ( s ) respons ib le fo r a common mechanism of m u l t i - d r u g res i s tance in these c e l l s . T h e phenomenon of m u l t i - d r u g res is tance is a f r e q u e n t c l in ica l f i n d i n g bu t whether gene ampl i f icat ion con t r i bu tes to i t is not k n o w n . Once the ampl i f ied genes in m u l t i - d r u g res i s tan t human cel l l ines have been c loned it shou ld be eas ier to tes t f o r ampl i f icat ion of these genes in tumour b i ops ies . T h e p le io t rop ic pheno type appears to show all the mani festat ions of a phenomenon a r i s i n g f rom a s ing le genet ic mutat ion i . e . 1) i ndependen t c lones se lected f o r co lch ic ine res is tance in a s ing le s tep d i s p l a y p le io t ropy ( 2 9 ) , 2) the f r e q u e n c y of appearance of s u c h c lones is s imi lar to o ther d r u g - r e s i s t a n t pheno types ( 2 9 ) , 3) r e v e r -- 9 -tan t ce l ls isolated in a s ing le s tep e x p r e s s a concomi t tant r eve rs i on of p le io t ropy ( 3 0 ) , 4) h y b r i d ce l ls d i s p l a y i n g dominance fo r co lch ic ine res is tance are also dominant fo r p le io t ropy ( 3 1 ) , 5) D N A mediated t r a n s f e r of co lch ic ine res is tance also resu l t s in the p le io t rop ic pheno type (32 ) . C e l l : Ce l l H y b r i d i z a t i o n S tud ies T h e techn ique of c e l l : cel l h y b r i d format ion has been used fo r s t u d y i n g the e x p r e s s i o n of the d r u g - r e s i s t a n t pheno type in a cel l con ta in ing o ther genomes s p e c i f y i n g d i f f e ren t t r a i t s . T h e dominant or r ecess i ve na tu re of the res i s tan t pheno type can be determined wh ich -in t u r n can be used to i den t i f y d i f f e ren t mechansims of r e s i s t a n c e . Chromosome loss o c c u r s in h y b r i d ce l ls espec ia l l y wi th c rosses between ce l ls of d i f f e ren t s p e c i e s . T h e s e reduced h y b r i d s have been used to map the determinants of d r u g - r e s i s t a n t pheno types of spec i f i c chromosomes and th i s t y p e of ana l ys i s has p r o v i d e d s t r o n g ev idence fo r a chromosomal o r i g i n and a gene t i c bas is fo r d r u g r e s i s t a n c e . In p r i n c i p l e , a recess i ve mutat ion would not be e x p r e s s e d at h igh enough f r equenc ies in d ip lo id ce l ls where two o r more mutat ions at the same locus would be r e q u i r e d fo r the e x p r e s s i o n of the pheno-t y p e . It has been shown that the major i ty of pheno types res i s tan t to chemotherapeut ic d r u g s in cu l t u re ce l ls are dominant (29) and t h e r e -fo re in t h e o r y can be e x p r e s s e d in po lyp lo id ce l ls as a resu l t of a s ing le muta t ion . D r u g Res is tance Mediated by Gene T r a n s f e r Pe rhaps the most c o n v i n c i n g ev idence fo r a genet ic bas is fo r - 10 -d r u g res is tance comes f rom the recent advances in mammalian cel l genet ics i n v o l v i n g chromosome and DNA-med ia ted gene t r a n s f e r . D N A f rom d r u g - r e s i s t a n t ce l ls (32) o r c loned D N A c a r r y i n g the d r u g -res i s tan t marke r (33-35) have been used to t r ans fo rm d r u g sens i t i ve ce l ls to d r u g res i s t ance . T h e t rans fo rmed ce l ls can be shown to d i sp lay s tab le and her i tab le spec i f i c d r u g r e s i s t a n c e . T h i s t echn ique has been used to demonst ra te the genet ic bas is of d r u g - r e s i s t a n c e fo r a number of well cha rac te r i zed sys tems (29) and unequ i voca l l y demonst ra tes the genet ic bas is f o r d r u g - r e s i s t a n c e . T h e genet ic changes i nvo l ved in the genera t ion of d r u g - r e s i s t a n t pheno types p r o b a b l y i nc lude mutat ions (base s u b s t i t u t i o n s ) , de le t i ons , t r anspos i t i on of genet ic e lements , chromosomal r e a r r a n g e m e n t s , t rans loca t ions and gene amp l i f i ca t ion . T h e resu l t of these changes may be a l t e r e d , d im in ished or i nc reased gene p r o d u c t s . One reason as to why tumour cel ls seem to deve lop h igh levels of d r u g res i s tance so read i l y whi le the normal cel l renewal sys tems of the body do not (54) may be assoc ia ted wi th the fac t tha t mal ignant cel l popu la t ions are cha rac te r i zed by 'genet ic i ns tab i l i t y ' r esu l t i ng in the above ment ioned genet ic c h a n g e s . A l t h o u g h normal cel l renewal sys tems such as the ce l ls of the u p p e r r e s p i r a t o r y , d i ges t i ve t r a c t and bone marrow ce l ls e x h i b i t v a r y i n g degrees of s e n s i t i v i t y to the an t ineop las t i c a g e n t s , as do mal ignant c e l l s , th i s degree of s e n s i t i v i t y or res is tance appears to be re la t i ve l y s tab le and does not change o v e r the per iod of the t reatment (54 ) . It has been pos tu la ted by C a i r n s (55) that w i th in a normal cel l renewal sys tem the ' immortal ' D N A s t r a n d in the stem cel l l ine is conse rved w i th in the p r o g e n y of the stem cel l tha t re ta ins stem cel l c a p a c i t y . T h e newly s y n t h e s i z e d D N A - 11 -s t r a n d is passed on to the d i f f e ren t i a t i ng pool of c e l l s . In the ma l ig -nant pheno type h o w e v e r , t h i s d i f fe ren t ia l re tent ion of the immortal s t r and in the stem cel l is lost and hence the newly s y n t h e s i z e d s t r a n d , toge ther wi th any mutat ions r e s u l t i n g f rom c o p y i n g e r r o r s , would also be re ta ined in the stem cel l popu la t i on . T h u s the ma l ig -nant stem cel l would be much more l i ke ly to p r o g r e s s i v e l y accumulate s u c c e s s i v e mutat ional e r r o r s . Genet ic i ns tab i l i t y seems to unde r l i e the phenomenon of tumour p r o g r e s s i o n (56) i . e . the p r o g r e s s i v e acqu is i t i on of g rea te r tumor cel l he te rogene i t y , lead ing to , among o ther p r o p e r t i e s , i nc reased metastat ic potent ia l and d r u g r e s i s t a n c e . T h e mal ignant state and the capac i t y to genera te a b road range of d r u g res i s tan t pheno types the re fo re appear to be in t imate ly r e l a ted . In summary , the ev idence in s u p p o r t of a genet ic o r i g i n f o r the d r u g - r e s i s t a n t pheno type i n c l u d e : the pheno type is spon taneous ly genera ted wi th a rate equ iva len t to the mutat ion rates in normal -5 -7 popu la t ions ( t y p i c a l l y 10 to 10 fo r s tab l y d r u g res i s tan t pheno-t y p e s ) ; i t is s tab l y i nhe r i t ed in the absence of se lec t i on ; the f r e q u e n c y of the appearance can be i nc reased wi th known mutagens ; chromosomal local izat ion of the de terminant is assoc ia ted wi th the d r u g - r e s i s t a n t t r a i t ; demonst ra t ion of an a l te red gene p r o d u c t ; and an a l te red gene can be demonst ra ted at the D N A l eve l . T h e t h e o r y tha t d r u g - r e s i s t a n t pheno types a r i se spon taneous ly and wi th a de f in i te f r e q u e n c y has been used by Gold ie and Coldman (124) to deve lop a mathematical model fo r re la t ing the d r u g s e n s i t i v i t y of tumours to t he i r spontaneous mutat ion ra te . T h e model p red i c t s tha t the p ropor t i on and abso lu te numbers of res i s tan t ce l ls wi l l i nc rease wi th time and that the f rac t i on of res i s tan t ce l ls w i th tumour - 12 -colonies of the same s ize wil l v a r y depend ing on whe ther the mutat ion was an ea r l y or late e v e n t . F u r t h e r m o r e , the appearance of a res is tan t pheno type inc reases wi th mutat ion rate and tha t t he re would be a sudden dec l ine in the expec ta t ion of c u r e as cel l numbers i nc rease . T h e model has v e r y impor tant impl icat ions in the schedu l i ng of d r u g s d u r i n g chemotherapy and on the t iming of ad juvan t chemothe rapy . T h i s thes is focuses on one wide ly used chemotherapeut ic agen t , •methot rexate , and on i ts p r ima ry i n t race l l u l a r t a rge t d ihyd ro fo la te r e d u c t a s e . T h e res t of th i s i n t roduc t i on wi l l be devo ted to a rev iew of the l i t e ra tu re c o n c e r n i n g the mechanism of act ion and mechanisms of res is tance to M T X , and the b iochemis t ry and molecular gene t i cs of d i hyd ro fo la te r e d u c t a s e . Mechanism of Ac t i on of Methot rexa te Metho t rexa te ( M T X ) is the most w ide ly used ant imetabol i te in cancer chemotherapy and is used most of ten in the t reatment of mal ignanc ies such as acute l ymphocy t i c leukemia , n o n - H o d g k i n ' s lymphoma, os teosarcoma, cho r i oca rc inoma, head and neck c a n c e r , and b reas t cance r ( 36 ) . It is also used in the t reatment of severe pso r ias i s (37) and as an immunosuppress i ve agent a f ter bone marrow t ransp lan ta t i on (38 ) . M T X is a c lose s t r u c t u r a l analogue of fo l i c ac id and the re fo re is a fo late an tagon is t . T h e folate v i tamins are essent ia l co fac to rs that c a r r y o n e - c a r b o n g r o u p s wh ich are r e q u i r e d fo r the b i osyn thes i s of ce r ta in amino a c i d s , p u r i n e s and t hym idy l i c a c i d , the la t ter be ing essent ia l f o r D N A s y n t h e s i s and cel l d i v i s i o n . T h e folate co fac to rs - 13 -i n c l u d i n g M T X all sha re the common s t r u c t u r a l f ea tu res shown in F i g . 1-2. T h e s t r u c t u r e cons is t s of a m u l t i - r i n g p te r i d i ne g roup l inked to para -aminobenzo ic ac id wh ich l i nks a terminal g lu tamic ac id r e s i d u e . T h e fo late co fac tors must be reduced to t he i r t e t r a h y d r o form at pos i t ions 5 ,6 ,7 and 8 of the p te r i d ine r i n g fo r a c t i v i t y . T h e enzyme respons ib le fo r c a r r y i n g out th is v e r y impor tant f unc t i on is d i h y d r o -folate reduc tase ( D H F R ) , wh ich is po ten t ly i nh ib i ted by M T X . Of the many forms of ant i fo la tes ava i l ab le , M T X , the 4-amino, N 1 0 methyl analogue of fo l i c ac id ( F i g . 1-2) is the most w ide ly used in cancer chemothe rapy . T h e mechanism of act ion of M T X is i l l us t ra ted in F i g . 1-3. M T X en te rs cel ls v ia a well cha rac te r i zed (39,40) ac t i ve t r a n s p o r t sys tem used by c i r c u l a t i n g fo lates N 5 - m e t h y l t e t r a h y d r o -fo late and N 5 - f o r m y l t e t r ahyd ro fo l a t e . A t h i ghe r concen t ra t ions of M T X (g rea te r than 20 pM) a second less well cha rac te r i zed e n t r y mechanism is used ( 4 1 , 4 2 ) . T h i s mechanism p r o b a b l y i nvo l ves d i f f us ion and accounts f o r the ab i l i t y of the ' t r a n s p o r t - r e s i s t a n t ' ce l ls ( d i s c u s s e d la ter ) to take up M T X at h igh ex t r ace l l u l a r concen t ra t i ons of M T X (42 ) . Upon en te r i ng the ce l ls M T X inh ib i t s cy toso l i c D H F R to -10 wh ich it b i nds wi th v e r y h igh a f f i n i t y ( K i of ^10 M ) . Inh ib i t ion of D H F R resu l t s in the dep le t ion of i n t r ace l l u l a r t e t rahyd ro fo la te pools and thus an i nd i r ec t i nh ib i t i on of t hymidy la te syn the tase wh ich 5-10 requ i res N me thy lene - te t rahyd ro fo la te as a co fac tor in the s y n t h e s i s of d T M P f rom dUMP ( F i g . 1-3 A ) . For complete inh ib i t i on of D H F R , ' f ree ' ( u n b o u n d ) i n t race l l u l a r M T X is r e q u i r e d s ince inh ib i t ion of D H F R leads to an accumulat ion of d i h y d r o f o l i c ac id wh ich at h igh concen t ra t ions tends to compete wi th M T X fo r b i n d i n g to D H F R ( F i g . 1-3 B ) ( 43 ,44 ) . - 14 -F I G U R E 1-2 S t r u c t u r a l formula of T e t r a h y d r o f o l a t e ( A ) and Metho t rexa te ( B ) po lyg lu tamates . In ( A ) o n e - c a r b o n g roups ( R ) are t r a n s p o r t e d on n i t rogen 5 or 10 or b o t h . 15 OH H , N ^ -R r CH, N O COOH O II I II C —NH —CH —CH ? —CH 2—C COOH O I II NH—CH—CH5—CHj — C • OH j n NH, HjN^^N-^^N-^ CH3 O COOH O CH— CH, —CHj^C COOH O I I II NH—CH— CH ?—CH ? —C -4-OH J n Reridine Ring P-Aminobenzoic Acid Glutamyl Residues - 16 -F I G U R E 1-3 Mechanism of Ac t i on of Me tho t rexa te . M T X denotes metho t rexa te , DHFR d ihyd ro fo la te r e d u c t a s e , T S thymidy la te s y n t h e t a s e , F H 4 t e t r a h y d r o f o l a t e , F H 2 d i h y d r o f o l a t e , G iu g l u tamy l , d T M P t h y m i d y l a t e , dUMP d e o x u r i d y l a t e . B r o k e n l ines ind icate enzyme i n h i b i t i o n . In B : React ion 1. is ca ta lyzed by d U T P a s e . 2. by D N A dependen t DNA po lymerase 3. by u rac i l D N A g l y c o s y l a s e 17 N 5 -Methyl -FH 4 MTX INTRACELLULAR — - N 1 0 -Formyl -FH 4 (G lu n ) Purine Synthesis N 5" 1 010-Methenyl-FH 4(Glu n) \ N 8 - 1 0 10 -Methy lene-FH 4 (Glu n ) F H 4 (Glun) T S DHFR MTX F H 2 (GlUn) MTX (GlUn ) dUMP dTMP I dTTP J DNA B MTX - 18 -T h e consequence of e f fec t i ve inh ib i t i on of D H F R is a dep le t ion in the i n t race l l u l a r d T M P pools and an inc rease in the dUMP poo ls . T h e dUMP is phospho ry la ted to d U T P and it has recen t l y been shown (45) -5 tha t the e f fec t i ve rat io of i n t race l l u l a r d U T P / d T T P changes f rom <10 --] to >10 . T h i s t remendous inc rease in d U T P levels resu l t s in i n -co rpora t ion of u rac i l into D N A , desp i te the p resence of d U T P a s e ac t i v i t y wh ich conve r t s d U T P back to dUMP ( F i g . 1-3 B ) . U rac i l i nco rpo ra ted into D N A is removed by an exc i s ion - r e p a i r pa thway in i t ia ted by u r a c i l - D N A g l y c o s y l a s e (46 ) . Howeve r , Gou l ian et a[, (45) have shown that as long as the s y n t h e s i s of d T M P is i nh ib i t ed at tempts to f i l l in the gaps left in the cou rse of e x c i s i o n - r e p a i r at s i tes of u rac i l removal may resu l t in ex tens i ve degrada t ion of D N A , and may resu l t in i nco rpo ra t i on of add i t iona l dUMP into the repa i r p a t c h . T h i s cyc le of dUMP remova l , re inco rpo ra t i on and removal resu l t s in ex tens i ve degrada t ion of D N A , and may con t r i bu te s i g n f i -can t l y to the cy to tox i c i t y of M T X , in add i t ion to the b lock in DNA s y n t h e s i s due to the lack of t hymidy la te and p u r i n e s . A recent f i n d i n g , and of cons ide rab le pharmacologica l i n t e r e s t , has been tha t l ike the phys io log i c fo la tes , M T X is e x t e n s i v e l y metabol ized in tnace l lu la r l y to po lyg lu tamate d e r i v a t i v e s wh ich conta in mul t ip le g lutamate g r o u p s l i nked by gamma-pept ide b o n d s . T h e s e were f i r s t o b s e r v e d in red ce l ls (47) and have now been iden t i f i ed in v a r i o u s mur ine and human t i s sues ( 4 8 , 4 9 ) . T h e M T X - p o l y g l u t a m a t e s have at least equal a f f i n i t y f o r D H F R (50) but appear to be d i s -sociated f rom D H F R at a s lower rate than M T X , and the re fo re are po tent ia l l y less r e v e r s i b l e i nh ib i t o r s than the paren t d r u g . Thie po lyg lu tamates are also able to remain w i th in the cel l in the absence - 19 -of ex t r ace l l u l a r d r u g ( 5 1 , 5 2 ) , in con t ras t to M T X , wh ich r a p i d l y leaves the ce l ls a f ter ex t r ace l l u l a r d r u g d i s a p p e a r s . In a d d i t i o n , the re tent ion is i n f l uenced by cha in l e n g t h : i . e . the longer cha in leng ths (3 to 4) are re ta ined i n t r ace l l u l a r l y fo r longer per iods than the compounds wi th sho r t e r cha in l eng ths . Longer re tent ion has been assoc ia ted wi th p ro longed inh ib i t i on of D H F R and ex tended c y t o -t o x i c i t y . Recent exper iments have shown that in add i t ion to i nh ib i t i ng D H F R , the add i t ion of one g lu tamyl res idue to M T X t rans fo rms the d r u g into, a more potent i nh ib i t o r of t hym idy la te syn the tase (53 ) . T h e s e data show that g lu tamyla t ion of M T X is impor tant to the cy to tox i c e f fects of M T X . S ince the length of the po lyg lu tamate cha in inc reases wi th the dose of M T X g i ven (57 ) , the po lyg lu tamate format ion has obv ious bea r ing on the c l in ica l use of M T X . Res is tance to Methot rexa te T h e c l in i ca l use fu lness of M T X is l imited u l t imate ly by the emergence of ce l ls w i th in the tumour wh ich are res i s tan t to M T X . T h e mechansims of res i s tance to M T X have been i n t ens i ve l y i nves t i ga ted and f rom numerous s tud ies wi th animal tumour ce l ls and human cel l l ines th ree major mechanisms of res i s tance have been r e c o g n i z e d : a) dec reased up take of M T X ; b) i nc reased level of the d r u g ta rge t enzyme d ihyd ro fo la te reduc tase ( D H F R ) ; c ) dec reased a f f i n i t y of D H F R fo r M T X . In add i t ion to these th ree mechan isms, dec reased format ion of methot rexate po lyg lu tamates and decreased ac t i v i t y of t hym idy la te syn the tase may also p lay a role in some cases of M T X r e s i s t a n c e . - 20 -Decreased Up take of M T X Tumour ce l ls exposed to M T X in v i t r o may become res i s tan t because of impairment of the ac t i ve up take sys tem (41,57) t h r o u g h a decrease in the a f f i n i t y of the c a r r i e r f o r M T X . T h i s t y p e of mechanism has not been c lea r l y documented in pat ients wi th res i s tance to M T X . T h e role of impaired up take of M T X in M T X - r e s i s t a n c e is becoming c lea re r as more M T X - r e s i s t a n t cel l l ines are c h a r a c t e r i z e d . F l in to f f et a l , (58 ,59) have been able to isolate s tab le mutants possess ing d i f f e ren t mechanisms of res is tance by u s i n g c lonal se lect ions in s i n g l e - s t e p d r u g t rea tments of Ch inese hamster o v a r y ce l ls in c u l t u r e . One of the mutants was res i s tan t to M T X by v i r t u e of impai red M T X u p t a k e . C e l l : cel l h y b r i d i z a t i o n s tud ies revea led that th i s t ype of mutant was recess i ve whereas those e x h i b i t i n g inc reased D H F R levels and a l te red DHFR were dominant . T h e y found tha t the impai red up take mutants d i sp l ayed i nc reased s e n s i t i v i t y to t h ree c lasses of d i am inopy r im id ines , low molecular we igh t l ypoph i l i c i nh ib i t o r s of D H F R . T h i s co l la tera l s e n s i t i v i t y of up take mutants to l i p i d - so lub le ant i fo la tes has been o b s e r v e d by o ther w o r k e r s and these compounds are be ing deve loped fo r c l in i ca l use ( 6 0 , 6 1 ) . A s desc r i bed ea r l i e r ' u p t a k e - r e s i s t a n t ' ce l ls are able to take up M T X at h igh ex t r ace l l u l a r concen t ra t ions by v i r t u e of a second d r u g e n t r y mechanism wh ich p r o b a b l y i nvo l ves d i f f us ion ( 4 1 , 4 2 ) . In a h i g h l y M T X - r e s i s t a n t L5178Y mouse leukemia cel l l i ne , the o b s e r v e d M T X -up take b a r r i e r cou ld be overcome by i ncuba t i ng the ce l ls at 100 fo ld h i g h e r e x t r a c e l l u l a r M T X concen t ra t ions (42 ) . Desp i te t h i s o b s e r -va t ion the ce l ls remained res i s tan t to M T X and the re fo re o ther mechanisms such as inc reased D H F R leve ls and e x p r e s s i o n of a low - 21 -M T X - a f f i n i t y D H F R may p lay more impor tant ro les in h i gh degrees of M T X - r e s i s t a n c e . Indeed , a recent repo r t on the deve lopment of M T X - r e s i s t a n c e in a human squamous cel l carc inoma of the head and neck in c u l t u r e (62) shows that a s tep -w i se inc rease in M T X c o n c e n -t ra t ion resu l t s in i t ia l l y in va r i an t s wh ich are res i s tan t to low c o n c e n -t ra t ions of M T X due to impai red up take of M T X . However when these ce l ls are exposed to i n c r e a s i n g l y h i ghe r concen t ra t i ons of M T X , inc reased levels of D H F R p lay a more impor tant role in r e s i s t a n c e , and f i n a l l y , the v e r y h i g h l y M T X - r e s i s t a n t ce l ls may e x p r e s s a low M T X - a f f i n i t y form of D H F R . In add i t ion the h i g h l y res i s tan t ce l ls no longer e x h i b i t impai red i n f l ux of M T X . T h e s e data show tha t impai red up take of M T X does not p lay a major role in moderate to h igh degrees of M T X - r e s i s t a n c e and tha t it can be overcome by e i ther i nc reas ing the dose of M T X o r by admin i -s t e r i ng more l ip id so lub le folate an tagon i s t s . Increased Leve ls of D H F R T h i s phenomenon was f i r s t d e s c r i b e d by Hakala et a[, (63) who found tha t c u l t u r e d sarcoma 180 ce l ls g r a d u a l l y became res i s tan t to M T X upon e x p o s u r e to the d r u g , and at the same time D H F R ac t i v i t y i nc reased p ropor t i ona te l y wi th the degree of r es i s t ance . T h e enzyme from the res i s tan t ce l ls (e levated up to 154 fo ld ) had s imi lar p r o p e r -t ies to the enzyme from the sens i t i ve ce l ls and it was deduced that res is tance in t h i s case resu l ted f rom the p roduc t i on of a la rge excess of normal D H F R wh ich immobl i l ized all of the i n t race l l u l a r M T X , leav ing enough f ree enzyme to c a r r y on i ts normal f u n c t i o n s . S imi la r obse rva t i ons were made by F i she r (64) in M T X - r e s i s t a n t L5178Y c e l l s , - 22 -and s ince tha t time many o ther cel l l ines res i s tan t to M T X have been shown to exh ib i t t h i s phenomenon (59 ,65 ) . Howeve r , the mechanism fo r the i nc reased levels of D H F R were not c lear unt i l 1972 when Nakamura and L i t t le f ie ld (66) demonst ra ted b y immunologic and inh ib i to r t i t ra t i on s tud ies tha t t h i s e levat ion in D H F R ac t i v i t y was due to an inc rease in the reduc tase p ro te in r e s u l t i n g f rom an inc rease in the re la t i ve rate of D H F R s y n t h e s i s . T h i s was subs tan t i a ted later and was found to be due to an a l te ra t ion in the r egu la to r y mechanism con t ro l l i ng the rate of s y n t h e s i s of the enzyme ( 6 7 , 6 8 ) . T h e i nc reased rate of s y n t h e s i s was shown to be re la ted to e levated quan t i t i es of D H F R mRNA wh ich was capable of s y n t h e s i z i n g D H F R in a ce l l - f r ee p ro te in s y n t h e s i z i n g system ( 6 9 , 7 0 ) . Some of the M T X - r e s i s t a n t cel l l ines have up to 300 fo ld inc reases in the amount of D H F R ( r e p r e -sen t ing up to 6% of the total so lub le pro te in in these c e l l s ) , as compared to the sens i t i ve c e l l s . T h e h igh levels of D H F R mRNA p resen t in these res i s tan t cel ls fac i l i ta ted the in v i t r o s y n t h e s i s (7) and subsequen t c lon ing (71) of the D H F R compl imentary D N A ( c D N A ) . U t i l i z i ng th i s p robe A l t et a]_, (7) were able to demonst ra te tha t the inc reased levels of D H F R mRNA resu l ted f rom a se lec t ive gene mu l t i -p l ica t ion leading to an inc rease in the number of D H F R g e n e s . T h i s phenomenon is known as gene ampl i f i ca t ion . A s d e s c r i b e d ear l i e r in th i s chap te r D H F R gene ampl i f icat ion (and the assoc ia ted M T X - r e s i s t a n c e ) can be s tab le , wi th res i s tance be ing assoc ia ted wi th a long homogeneously s ta in ing chromosomal reg ion ( H S R ) on one (or more) chromosomes, uns tab le wi th res is tance be ing assoc ia ted wi th sma l l , pa i red chromosomal elements ca l led 'doub le minute ' (DM) chromosomes. In s i tu h y b r i d i z a t i o n s tud ies wi th - 23 -1 1 2 5 - l a b e l e d D H F R c D N A chromosome sp reads has shown tha t p r e f e r -ent ia l h y b r i d i z a t i o n takes place at the s i te of H S R s and on DMs ind i ca t i ng that the D H F R gene is indeed local ized at these s i tes (78 ) . Johns ton et EH, (72) have shown tha t D H F R gene ampl i f icat ion can o c c u r spon taneous ly and in the absence of se lect ion p r e s s u r e . In Ch inese hamster o v a r y ce l ls the f r e q u e n c y of a 2- fo ld inc rease in -3 gene copy number was found to be of the o r d e r of 1x10 / c e l l genera t ion in sens i t i ve c e l l s . T h i s is an i n c r e d i b l y h igh f r e q u e n c y and pe rhaps exp la ins the ease wi th wh ich ce l ls o v e r p r o d u c i n g D H F R can be obta ined in the p resence of M T X . T h e f r e q u e n c y wi th wh ich ce l ls become res i s tan t to M T X can be i nc reased dramat ica l l y by v a r i o u s t reatments of ce l ls p r i o r to M T X se lec t i on . P r i o r t reatment of ce l ls wi th M T X enhances the f r e q u e n c y of M T X res i s tance a p p r o x i -matley 10 fo ld (73,74) and inc lus ion of the tumour p romotor , 12 -0 - te t radecanoy lpho rbo l -13 -ace ta te f u r t h e r enhances the f r e q u e n c y of M T X res i s tance and gene amp l i f i ca t ion . Inh ib i t ion of D N A rep l i ca t ion wi th h y d r o x y u r e a , an i nh ib i t o r of r i bonuc leo t ide reduc tase also enhances the subsequen t emergence of M T X - r e s i s t a n t co lonies (75 ) . T h u s , t rea tment of ce l ls w i th agents tha t a l ter D N A rep l i ca t ion or D N A s t r u c t u r e may ac tua l l y fac i l i ta te gene ampl i f icat ion and the emergence of r e s i s t a n c e . R e c e n t l y , Mar ian i and Sch imke (76) have shown tha t gene ampl i f icat ion can o c c u r in a s ing le cel l cyc le in C h i n e s e hamster o v a r y c e l l s . T h e y showed tha t the D H F R gene is rep l i ca ted d u r i n g the f i r s t 2 h r . of the S phase and tha t if DNA s y n t h e s i s is t r a n s i e n t l y i nh ib i t ed s h o r t l y a f ter S phase has commenced, t h e n , in a s ing le cel l c y c l e , ce l ls w i th inc reased D H F R enzyme levels and D H F R genes can - 24 -be p r o d u c e d . From these uns tab le v a r i a n t c e l l s , colonies emerge wi th enhanced M T X res is tance and D H F R gene copy number . T h e mechanism appears to be one in wh ich upon resumpt ion of D N A s y n t h e s i s fo l lowing removal of the i n h i b i t o r , v i r t u a l l y all the D N A rep l i ca ted p r i o r to i n h i b i t i o n , i n c l u d i n g the D H F R g e n e , is r e r e p l i c a t e d . T h e in i t ia l event t he re fo re is the re rep l i ca t i on of a la rge amount of genome. U n d e r se lect ion ( i . e . p resence of M T X ) , gene rea r rangemen t , recombinat ion and loss of nonse lec ted D N A sequences o c c u r , r esu l t i ng in apparen t spec i f i c gene amp l i f i ca t ion . T h i s new data f a v o u r s the ' d i sp ropor t i ona te rep l i ca t i on ' model of gene ampl i f icat ion p roposed by Sch imke (77) in wh ich the re can o c c u r more than a s ing le in i t ia t ion of rep l i ca t ion on a por t ion of a chromosome w i th in a s ing le S phase of the cel l c y c l e . T h e mul t ip le in i t ia t ions lead to the genera t ion of f ree s t r ands of D N A wi th in a rep l i ca t ion ' b u b b l e ' . T h e loose ends of the newly rep l i ca ted D N A can unde rgo recombina t ion , gene ra t i ng ex t rachromosomal , c i r c u l a r chromosomal s t r u c t u r e s (not un l i ke D M s ) . T h e c i r c u l a r DNA s t r u c t u r e can f u r t h e r unde rgo ro l l i ng c i r c l e rep l i ca t ion fo l lowed b y recombinat ion wi th chromosomal D N A to genera te a tandem head- to - ta i l repeat of the ampl i f ied genes (not un l i ke H S R s ) . A l t h o u g h in some cases the enzyme coded by the ampl i f ied genes is i nd i s t i ngu i shab le f rom the enzyme s y n t h e s i z e d by the w i l d - t y p e g e n e s , in o the rs the ampl i f ied genes code fo r a l te red D H F R s , some of them wi th a reduced a f f i n i t y fo r M T X ( 1 7 , 1 9 , 6 5 ) . D H F R s wi th a reduced a f f i n i t y fo r M T X rep resen t the t h i r d mechanism of res i s tance to M T X and are d i s c u s s e d below. - 25 -Decreased A f f i n i t y of D H F R fo r M T X Exper imenta l tumour ce l ls res i s tan t to M T X may conta in D H F R wi th decreased a f f i n i t y fo r M T X . T h i s mechanism has been demon-s t ra ted in va r i ous mouse (17,79-81) as well as human cel l l ines ( 8 1 ) . In these s y s t e m s , res is tance can be co r re la ted wi th dec reased M T X a f f i n i t y of D H F R , in ce l ls not p r e v i o u s l y exposed to M T X ( i n t r i n s i c r e s i s t a n c e ) , o r in those wh ich were in i t ia l l y sens i t i ve to M T X bu t acqu i r ed res i s tance wi th p ro longed e x p o s u r e to M T X ( a c q u i r e d r e s i s t a n c e ) . I n t r i ns i c res is tance to M T X of c u l t u r e d mammalian ce l ls has been co r re la ted wi th the K i ' s of d ihyd ro fo la te reduc tases f o r M T X ( 8 2 , 8 3 ) . In the s t u d y by Ha r rap et a[, (82) the cel l l i nes : Y o s h i d a asc i tes sa rcoma, mouse leukemia L1210 and mouse leukemia L5178Y, were eva lua ted wi th respec 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 rac t i on of ce l ls to less than 10%). Y o s h i d a ce l ls were 10 fo ld more res i s tan t than L5178Y ce l ls wh ich were 10 fo ld more res i s tan t than L1210 c e l l s . T h e K i ' s of D H F R s fo r M T X from these th ree cel l l ines fo l lowed th i s same o r d e r , i . e . the Y o s h i d a D H F R had a h i ghe r K i ( lower a f f i n i t y ) f o r M T X than L5178Y D H F R wh ich had a h i g h e r Ki than L1210 D H F R . A l l t h ree cel l l ines t r a n s p o r t e d the d r u g at comparable rates and the spec i f i c ac t i v i t i es of d i h y d r o -fo late reduc tases were also comparab le . It appeared t he re fo re that in these th ree cel l l ines d i f f e rences 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 co r re la ted wi th the K is of d ihyd ro fo la te reduc tases f o r M T X . These resu l t s were conf i rmed by Jackson et a[, (83) 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 repor ted to be i n t r i n s i c a l l y res i s tan t to M T X - 26 -(84) and a l though the res is tance in t h i s case was a t t r i bu ted to e levated i n t race 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 cou ld not be el iminated because of the method used fo r the assay f o r D H F R ac 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 ) . D H F R a c t i v i t y in the b las t cel l l ysa tes of 8 pat ients w i th A M L (acute myelogenous leukemia) v a r i e d w ide ly in t he i r s e n s i t i v i t y to i nh ib i t i on by M T X ( C h a p t e r 5 ) . A M L does not usua l l y respond well to M T X and the p resence of i nsens i t i ve D H F R s in the b las t ce l ls of some pat ien ts sugges t s tha t th i s may be the u n d e r l y i n g mechanism of c l in ica l i n t r i n s i c r e s i s t a n c e . Mammalian c e l l s , i n i t i a l l y h i g h l y sens i t i ve to M T X , acqu i re r e s i s -tance to the d r u g upon e x p o s u r e to i nc reas ing concen t ra t ions of M T X . A l b r e c h t et al_, (85) repor ted 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 fo r M T X of the D H F R from these ce l ls were not de te rm ined ; h o w e v e r , whereas the enzyme from 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 i ch io -met r ica l ly wi th M T X , the inh ib i t i on of D H F R from the res i s tan t ce l ls was r e v e r s i b l e , re f l ec t i ng 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 rope r t i es of the enzyme were d i f f e ren t f rom those of D H F R from sens i t i ve 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 ef fect of N A D P H on the inh ib i t i on of D H F R a c t i v i t y . Jackson and Niethammer (81) d e s c r i b e d the p rope r t i es of an a l te red d ihyd ro fo la te reduc tase f rom M T X - r e s i s t a n t human lympho--6 b las to id c e l l s . T h e s e ce l ls were res i s tan t to 10 M M T X ; the Km fo r d ihyd ro fo la te was 18- fo ld h i ghe r than tha t of the D H F R from the paren t l i ne , and the a f f i n i t y fo r M T X was 50- fo ld lower . T h e " l ow -a f f i n i t y " D H F R d i f f e red in i t ' s tempera tu re s e n s i t i v i t y (more heat - 27 -lab i le) f rom the paren t D H F R bu t both forms of the enzyme had s imi lar molecular we ights (22 ,500 ) . In add i t ion to the s t r u c t u r a l a l tera t ion of the D H F R f rom M T X - r e s i s t a n t c e l l s , the total ac t i v i t y of the enzyme had inc reased g rea t l y o v e r tha t in the paren 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 amp l i f i ca t ion . F l in to f f et aH, (58) demonst ra ted the p resence of normal amounts of an a l te red D H F R wi th dec reased a f f i n i t y fo r M T X f rom M T X -res i s tan t C h i n e s e hamster o v a r y ce l ls se lected by a s ing le s tep se lect ion p r o c e s s . E x p o s u r e of these ce l ls to i n c r e a s i n g c o n c e n -t ra t ions of M T X resu l ted in the se lect ion of ce l ls wi th i nc reased a c t i v i t y of a l te red enzyme (86 ) . T h e mutant enzymes (6 to 8 fo ld more res is tan t to inh ib i t i on by M T X than wi ld t y p e enzyme) and the wi ld t y p e enzyme also demonst ra ted small d i f f e rences in pH op t ima, Km fo r folate and heat s t a b i l i t y . F u r t h e r cha rac te r i za t i on of the wi ld t y p e and a l te red reduc tases by two-d imens iona l gel e lec t ropho res i s fa i led to reveal any cha rge d i f f e rences between the two fo rms . Haber et a[, (17) demonst ra ted the p resence 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 exh ib i t ed a 270- fo ld reduc t ion in b i nd ing a f f i n i t y f o r M T X . In add i t ion the Km fo r d i h y -d ro fo l i c ac id was inc reased 3 - f o l d , the pH optimum was d i f f e ren t a n d , a l though the molecular we igh t was s imi lar to the enzyme f rom pa ren t c e l l s , the a l te red enzyme demonst ra ted a s i gn i f i can t bas ic sh i f t in e lec t rophore t i c m ig ra t i on . In all of the above examples the ex ten t of decrease in the a f f i n i t y of M T X fo r D H F R is smal l . D H F R s wi th la rge decreases (of the o r d e r of 10,000 to 100,000 fo ld ) in M T X - a f f i n i t i e s have also been - 28 -repor ted ( 3 4 , 7 9 , 8 7 ) . Gold ie et a[, (79) d e s c r i b e d a v a r i a n t form of D H F R ( the sub jec t of C h a p t e r s 2 and 3) p resen t in h i g h l y M T X -res i s tan t L5178Y ce l ls wh ich d i f f e red in i ts a f f i n i t y fo r M T X f rom the D H F R in the sens i t i ve ce l ls by a fac to r of 100,000. T h i s a l te red form also d i f f e red in o ther p rope r t i es (18) s u c h as a l te red e lec t rophore t i c mob i l i t y , heat s t a b i l i t y , and ac t i va t ion by o rgan i c mercur ia l compounds . DNA f rom a cel l l ine e x p r e s s i n g a h i g h l y M T X - r e s i s t a n t D H F R has been used to t r ans fo rm mouse bone marrow to M T X - r e s i s t a n c e (87 ) . T h i s enzyme was p resen t in a M T X - r e s i s t a n t mouse f i b r o b l a s t cel l l i ne , 3 T 6 R 1 , and was found to be >10,000 fo ld more res i s tan t to inh ib i t ion by M T X than the enzyme f rom the M T X - s e n s i t i v e c e l l s . In almost all of these examples the a l te red enzyme is e i ther i tse l f e x p r e s s e d at h i ghe r leve ls than the D H F R in the sens i t i ve ce l ls ( 1 7 , 8 1 , 8 7 ) , o r is e x p r e s s e d in cel l popu la t ions wh ich also e x p r e s s e levated levels of D H F R wi th no rma l , h igh a f f i n i t y fo r M T X (79 ) . T h e common p r o p e r t y of all these ce l ls is that t hey were se lec ted f o r M T X - r e s i s t a n c e by a s tep -w i se inc rease in M T X c o n c e n t r a t i o n . T h i s form of se lect ion usua l l y leads to D H F R gene amp l i f i ca t ion , and a l te red D H F R s may be genera ted as a consequence of gene amp l i f i -cat ion e . g . due to mutat ions genera ted d u r i n g the r e - rep l i ca t i on cyc l e desc r i bed e a r l i e r . In the one case (58) in wh ich se lect ion was c a r r i e d out in a s ing le s t e p , a normal amount of a l te red D H F R was e x p r e s s e d . T h e mechanism fo r the genera t ion of a l te red D H F R s may be : a s t r u c t u r a l mutat ion in the D H F R cod ing sequence ( r e s u l t i n g f rom base s u b s t i t u t i o n s , de le t ions or even sequence r e a r r a n g e m e n t s ) ; o r , e x p r e s s i o n of a va r i an t DHFR gene wh ich is normal ly r e p r e s s e d . - 29 -Of these two a l te rna t i ves the most l i ke l y mechanism is s t r u c t u r a l muta t ions . Ev idence has recen t l y been p resen ted f o r t h i s mechan ism. T h e c D N A fo r an a l te red DHFR p resen t in M T X - r e s i s t a n t mouse 3 T 6 -R400 ce l ls (17) wi th a b i n d i n g a f f i n i t y f o r M T X of 1/270th of tha t of wi ld t y p e c e l l s , has recen t l y been c loned and i ts sequence de termined (21 ) . T h e D N A sequence of the a l te red c D N A d i f f e red f rom the w i l d -t ype c D N A b y a s ing le base s u b s t i t u t i o n : a T - t o - G at pos i t ion 68. T h i s change resu l t s in the inco rpora t ion of an a rg in i ne res idue fo r a leuc ine at pos i t ion 22. Leu-22 has been found to be in h y d r o p h o b i c contac t wi th the t r i az i ne r i ng of 2 , 4 - d i a m i n o - 5 , 6 - d i h y d r o - 6 , 6-d i m e t h y l - 5 - ( 4 ' - m e t h o x y p h e n o l ) - s - t r i a z i n e ( 8 8 ) , a fo late an tagon is t and potent i nh ib i t o r of D H F R . T h u s the replacement of the h y d r o p h o b i c s ide cha in of leuc ine wi th tha t of pos i t i ve l y c h a r g e d a rg i n i ne may d i s r u p t th i s i n t e rac t i on . Some genera l conc lus ions (89) can be d rawn f rom the c u r r e n t l i t e ra tu re on a l te red D H F R s wi th dec reased a f f i n i t y f o r M T X : a) T h e ex ten t of the decrease in a f f i n i t y is v a r i a b l e . b ) Mutant enzymes have d i f f e ren t i soe lec t r i c p rope r t i es and lower spec i f i c a c t i v i t i e s , c ) Molecu lar we igh ts of the mutant and w i l d - t y p e enzymes are not a p p r e c i a b l y d i f f e r e n t . d ) No genera l pa t te rn is seen in o ther s t r u c t u r a l o r ca ta l y t i c parameters such as heat l ab i l i t y , Km v a l u e s , or pH opt imum, e) T h e a l te red enzymes may (90) or may not (17) re ta in a f f in i t ies f o r ant i fo la tes o ther than M T X . It t he re fo re appears that in as much as amino ac id subs t i t u t i ons at va r i ous po in ts in the D H F R molecule can af fect M T X b i n d i n g ( 9 1 ) , a v a r i e t y of a l te red enzymes wh ich d i f f e r in phys i ca l and k ine t i c p roper t i es can be e x p e c t e d . - 30 -B iochemis t r y and Molecu lar Genet ics of D ihyd ro fo la te Reduc tase . B iochemis t r y D ihydro fo la te reduc tase ( D H F R ) has been the sub jec t of in tense research d u r i n g the past ten y e a r s , most ly because it is the i n t r a -ce l lu la r t a rge t fo r the an t i cance r d r u g M T X as well as fo r the a n t i -bac ter ia l d r u g t r ime thop r im , bu t also because of i ts cen t ra l role in the b iosyn thes i s of pu r i nes and p y r i m i d i n e s . D H F R ( t e t r ahyd ro fo l a t e : N A D P H + : o x i d o r e d u c t a s e , E . C . 1 . 5 . 1 . 3 ) ca ta lyzes the N A D P H -dependen t reduc t ion of 7, 8 -d ihyd ro fo la te ( F H 2 ) to 5 , 6 , 7 , 8 - t e t r a -hyd ro fo la te ( F H 4 ) . F H 2 + N A D P H + H + -» F H 4 + N A D P + Fol ic ac id ( F A ) can also be enzymat ica l l y reduced to F H 4 by D H F R at ac id p H , bu t the rate of reduc t ion in the p resence of N A D P H is d r a s t i c a l l y reduced at neut ra l pH fo r most mammalian c e l l s . T h e cent ra l role of D H F R in main ta in ing the i n t race l l u l a r pool of F H 4 o n e - c a r b o n d e r i v a t i v e s fo r t hymidy la te and p u r i n e nuc leo t ide b iosyn thes i s is i l l us t ra ted in F i g . 1-3. T h e consequences of the inh ib i t i on of D H F R have been desc r i bed ea r l i e r in th i s c h a p t e r . D H F R is a ub iqu i t ous enzyme and has been cha rac te r i zed f rom many sources i n c l u d i n g b a c t e r i a , p ro tozoa , bac te r iophages and mammalian c e l l s . D H F R s isolated f rom bacter ia l and mammalian sou rces o c c u r as a s ing le po l ypep t ide cha in wi th molecular we igh ts in the range df 18 ,000-22,000. However the enzymes f rom ce r ta in paras i te pro tozoans have been repor ted to be in the molecular we igh t range of 100,000 to 240,000 based on gel f i l t ra t ion (92 ,93 ) . More recen t l y the D H F R from the pro toza l f lage l la te C i r i t h i d i a f asc i cu la ta has been shown to occu r as a b i func t iona l p ro te in wh ich also conta ins t h y m i d y l -- 31 -ate syn the tase a c t i v i t y . T h e enzyme is a d imer composed of two 57,000 dal ton s u b u n i t s (94 ) . R Plasmid coded D H F R s ex i s t in two d i s t i n c t c l a s s e s , t ype I and t ype II. T h e s e D H F R s con fe r res is tance to t r imethopr im ( T M P ) , a folate an tagon is t wi th a h igh a f f i n i t y f o r bac te r ia l D H F R s bu t a low a f f i n i t y fo r mammalian D H F R s . T h e t ype II enzymes appear to be te t ramers of molecular we ight 34,000 wi th a s u b u n i t molecular we igh t of about 8,500 (95 and re fe rences t h e r e i n ) . T y p e II D H F R s are almost to ta l l y i nsens i t i ve to inh ib i t i on by T M P and M T X . T y p e I enzymes are also h i gh l y i nsens i t i ve to M T X and T M P compared to the host E . Col i enzyme and appear to be a d imer wi th a s u b u n i t molecular we ight of about 18,000. T h e T 4 bac te r i ophage-coded D H F R also f unc t i ons as a s t r u c t u r a l element of the phage ta i l baseplate and is also a d imer of 44,500 wi th a s u b u n i t molecular we ight of about 23,000. T h e use of fo late analogue a f f i n i t y matr ices has g rea t l y f a c i l i -ta ted the isolat ion and pu r i f i ca t i on of D H F R s f rom v a r i o u s s o u r c e s . T h i s method u t i l i zes the i n c r e d i b l y h igh a f f i n i t y of most D H F R s fo r -10 M T X ( K i = 10 M) and is also su i tab le fo r sepa ra t i ng m ix tu res of D H F R s wi th low and h igh a f f in i t ies fo r M T X , s ince low a f f i n i t y D H F R s fa i l to b ind to the column ( 7 9 , 9 6 ) . A major fac to r in the isolat ion of la rge quan t i t i es of D H F R fo r amino acid s e q u e n c i n g , x - r a y c r y s t a l l o g r a p h i c s tud ies and o ther phys i co -chemica l ana lyses f rom bacter ia l and animal sources has been the development of M T X - r e s i s t a n t ce l ls wh ich o v e r p r o d u c e D H F R . T h e amino ac id sequence has been determined fo r D H F R s f rom many sources i n c l u d i n g human cel l l ines (95 and re fe rences t h e r e i n ) and - 32 -the th ree d imensional s t r u c t u r e s of bac ter ia l and av ian enzymes has been e luc ida ted (91 ,88 ) . Tab le 1.1 summarizes some of the molecular p rope r t i es of monomeric D H F R s f rom va r i ous s o u r c e s . T h e molecular weights f o r these enzymes range f rom 18,000 to 22,000 da l tons and conta in f rom 159-184 amino ac id r e s i d u e s . T h e Km va lues fo r d i h y d r o f o l i c ac id and N A D P H lie in the u molar range fo r most reduc tases and the mammalian enzymes have two pH op t ima, 4 .5 and 7 . 5 . Bac te r ia l enzymes have a s ing le ac id ic pH opt imum. Most D H F R s have neut ra l to bas ic i soe lec t r i c po i n t s . Amino acid sequence compar isons of D H F R s f rom v a r i o u s mammalian sources ind ica te a h igh degree of sequence c o n s e r v a t i o n , espec ia l l y in the N- termina l reg ion of the molecu les . A s ment ioned ear l i e r in th i s c h a p t e r , D H F R s wi th a l te red p rope r t i es have been iso lated f rom v a r i o u s M T X - r e s i s t a n t cel l l i nes . T h e a l te ra t ions usua l l y r esu l t in a lower a f f i n i t y fo r M.TX and the p rope r t i es of these have a l ready been d e s c r i b e d . M T X - r e s i s t a n t cel ls may also e x p r e s s D H F R s wi th h i g h e r ca ta l y t i c ac t i v i t i es in the absence of an inc rease in the actual amount of D H F R p ro te in o r an a l te ra t ion in the a f f i n i t y f o r M T X : e g . isoenzyme I f rom M T X -res is tan t f aecu im, and D H F R from M T X - r e s i s t a n t human p romye lo -c y t i c leukemia H L - 6 0 ( R 4 ) ce l ls (123) (see C h a p t e r 5 ) . D H F R s f rom M T X - s e n s i t i v e and some - r e s i s t a n t ce l ls b ind M T X wi th a v e r y h igh a f f i n i t y (97) and u n d e r app rop r i a te c o n d i t i o n s , the b i nd ing is s to ich iomet r ic ( 98 ) . A n o t h e r i n te res t i ng p r o p e r t y of mammalian D H F R s is the ab i l i t y of ce r ta in compounds such as o rgan i c mercu r ia l s to i nc rease t he i r ca ta ly t i c ac t i v i t y in v i t r o (95 ) . T h i s is in con t ras t to bac ter ia l enzymes wh ich are e i the r unaf fec ted by these compounds or are inh ib i ted by them. T h e in te rac t ion of o rgan i c mercu r ia l s wi th D H F R s T A B L E 1.1 Some P rope r t i es of Ve r t eb ra te and Bacter ia l D ihydro fo la te Reductases Sou rce M. Wt. Km (pM) pH S . A c . 3 pi Amino A c i d b ( x 1 0 - 3 ) F A H 2 N A D P H Op t . Res idues A . V e r t e b r a t e 1. Animal L i v e r a . C h i c k e n 21.65 0. 12 1.8 b. Bov ine 21.45 6 15 c . Porc ine 21.45 0. 74 3.2 2. Ce l l L ines a . L1210(R) 21.46 0. .3 1.36 b. Sarcoma 180 ~21 1. 7 5.9 ( A T / 3 0 0 0 ) c . Human WIL2 ~21 0. 04 0.25 d . Human K B •v20 0. 67 5.9 e. Human Hela 21-22 6. .1* 170 B U - 2 5 4 ; 7.4 14 8. .4 189 4 .8 - 5.0 26 6. ,8 186 5 ; 7.6 36 8. .4 186 4 ; 7.5 11 8. ,1 186 4 ; 7.5 27 8. .6 N . D . 4 ; 7.3 - 8.3 15 7. ,7 N . D . <5; 7.2 - 8.2 60 7. .3 N . D . 4 .8 0.4 B . Bac te r ia l ( M T X - r e s i s t a n t ) S . Faecium Isoenzyme I ^20 7.0 20 6 .5 300 Isoenzyme II 19.6 7.5 4.9 5.8 45 167 L casei 18.3 0.36 0.78 6.5 12 6.25 162 E. Co l i (MBI428) 18.25 0.44 6.45 6 .8 36 159 a . E x p r e s s e d in pinoles F A H 2 r e d u c e d / m i n / m g p r o t e i n . b. Based on amino ac id sequence . * Fol ic ac id was used as s u b s t r a t e f o r determinat ion of Km. A d a p t e d f rom Fre ishe im and Matthews (95 ) . - 34 -is t hough t to o c c u r in mammalian enzymes wi th a s ing le cys te i ne res idue at the amino- terminal pos i t ions 6 or 11 . In bac ter ia l D H F R s the re is e i the r a total absence of cys te ine r e s i d u e s , o r if i t is p resen t it is not located near the N- termina l reg ion of the enzyme. T h e p roper t i es of the mercu r i a l - ac t i va ted D H F R have been s tud ied in detai l (99) and the ac t iva ted enzyme has been found to have h i g h e r Km and Vmax va lues fo r the F H 2 / N A D P H combinat ion and g rea t l y i nc reased s e n s i t i v i t y to heat , p ro teo lys i s and ionic env i ronmen t . It has been sugges ted that th i s un ique modi f icat ion of the cys te i ne res idue in the N- termina l reg ion in v i t r o may have an in v i v o c o u n t e r p a r t fo r the cont ro l of D H F R ac t i v i t y in v i v o (99,123 and C h a p t e r 5 ) . A p a r t f rom hav ing a p u r e l y ca ta l y t i c f u n c t i o n , DHFR f rom L.  casei has been shown to have D N A - b i n d i n g p rope r t i es (100) . Two spec i f i c b i n d i n g s i tes have been mapped at the 5' end of the s t r u c t u r a l gene fo r L. casei D H F R approx imate ly 100 base pa i rs ups t ream f rom the s ta r t of the cod ing reg ion (101) . T h e s i gn i f i cance of the DNA b i n d i n g p r o p e r t y in bacter ia l ce l ls is unc lea r and whe ther euca ryo t i c D H F R exh ib i t s s imi lar p rope r t i es has s t i l l to be de te rm ined . In euca ryo t i c c e l l s , D H F R has been found to be pa r t of a mu l t i -enzyme complex ca l led the ' r ep l i t ase " (102) wh ich is located in the nuc leus d u r i n g the S phase of the cel l c y c l e . T h i s ' r ep l i t ase ' molecule has the appa ren t f unc t i on of ca ta l ys i ng the rap id i n c o r p o r -at ion of r i bonuc leos ide d iphospha tes into D N A . T h u s it wou ld appear that d u r i n g the S phase D H F R can also be located in the nuc leus bes ides i ts usual locat ion in the cy top lasm. - 35 -Molecu lar Genet ics of Mammalian D ihyd ro fo la te Reduc tase T h e se lec t i ve ampl i f icat ion of D H F R genes in M T X - r e s i s t a n t ce l ls has fac i l i ta ted the ana l ys i s of the genomic o rgan iza t i on of the mouse, hamste r , as well as human D H F R gene . T h e mouse D H F R gene has been e x t e n s i v e l y cha rac te r i zed by N u n b e r g et a[, (103) and C r o u s e et a[, (104) . T h e cod ing sequence of D H F R mRNA in the mouse is 561 nuc leot ides in length and the longest p reva len t mRNA spec ies is 1600 nuc leo t ides . T h e s ix exons in the mouse gene are d i s t r i b u t e d o v e r 31 Kb of chromosomal DNA and conta in f i ve i n t e r v e n i n g sequences ( i n t r o n s ) . Mul t ip le mouse DHFR m R N A spec ies have been iden t i f i ed (105) , and re f lec t the use of d i f f e ren t po lyadeny la t ion s i tes y i e l d i ng m R N A molecules that d i f f e r in the length of t he i r 3' un t rans la t iona l reg ions (106) . T h e C h i n e s e hamster D H F R gene has a s imi lar genera l o rgan iza t ion (125) . T h e o rgan iza t ion of the human D H F R gene has been recen t l y d e s c r i b e d (107,108) . T h e gene is about 30 k i lobases in length and the cod ing reg ions are separa ted into s ix exons by f i ve i n t r o n s . Sou the rn b lot ana l ys i s of genomic DNA wi th n i ck t r ans la ted 3 2 p -labeled D H F R c D N A resu l t s in v e r y d i f f e ren t r es t r i c t i on f ragment pa t te rns fo r human and mouse D H F R genes (Tab le 1 -2) . T h e human DHFR gene has a t r a n s c r i p t i o n promoter 72 base pa i rs ups t ream f rom the in i t ia t ion c o d o n . T h r e e D H F R mRNA spec ies of 800 - , 1000- , and 3800-nuc leot ides have been iden t i f i ed in human ce l ls and are accounted f o r by the s ing le t r a n s c r i p t i o n s ta r t s i te and th ree p o l y -adeny la t ion s i t e s . A compar ison of the mouse and human D H F R genes has shown tha t sequence homology (approx imate ly 90%) is l imited to the cod ing r e g i o n s , 100 base pa i r s of the 3' un t rans la ted reg ion upto - 36 -T A B L E 1-2 Res t r i c t i on Enzyme A n a l y s i s of Human and Mouse D H F R G e n e . ECOR1 H ind 111 Ps t I Bam HI Bg l 11 H M H M H M H M H M 13 14. 8 22 12. 9 8. 3 3. 9 =32 >20. 5 5 .2 7. 9 6 .0 6. 0 5.8 3. 8 7. 2 2 . 8 17. 6 3.9 4. 4 4 .0 5. 4 4 .3 3. 4 3. 8 2 . 0 4. 8 3.7 4 . 2 1.8 3. 4 1. 6 3 .5 0. 55 1.65 1.0 0.30 T h e res t r i c t i on enzyme f ragments con ta in ing D H F R cod ing sequences are g i v e n in k i l obases . Human ( H ) D H F R res t r i c t i on f ragment s izes have been taken f rom C h e n et al_, (107) excep t fo r B g l II, taken f rom Sr ima tkanada et a[, (108) mouse (M) D H F R res t r i c t i on f ragment s ize have been taken f rom C r o u s e et a l , (104) . - 37 -the f i r s t po lyadeny la t ion s i te and the 5' f l a n k i n g r e g i o n s . A p a r t f rom homologies at the i n t r o n - e x o n b o u n d a r i e s , the i n t rons d i f f e r in leng th as well as sequences and the re is no sequence homology downst ream of the f i r s t po lyadeny la t ion s i te (107) . T h e human genome has been found to h a v e , in add i t ion to func t iona l D H F R g e n e s , severa l i n t ron less genes ca l led pseudogenes d e r i v e d f rom p rocessed mRNA molecules (109 ,110) . These v a r y in s ize and o rgan iza t ion and the ex ten t of homology to the normal cod ing sequences . Desp i te the h igh degree of sequence i den t i t y of most of the pseudogenes to the normal D H F R g e n e , these i n t ron less genes are u n l i k e l y to be func t iona l s ince they do not i nc lude the p romoter . T h e pseudogenes have been found not be ampl i f ied in M T X - r e s i s t a n t ce l ls in wh ich ampl i f icat ion of the func t iona l gene has taken place (107) . A s d i s c u s s e d ear l i e r D H F R genes have been local ized at the expanded reg ions of chromosomes ca l led H S R s in s tab l y M T X - r e s i s t a n t ce l ls wh ich o v e r p r o d u c e D H F R due to gene amp l i f i ca t ion , by us i ng the techn ique of in s i tu h y b r i d i z a t i o n of metaphase chromosome s p r e a d s . T h i s t echn ique has local ized the DHFR gene to chromosome 2 in mouse (111) and C h i n e s e hamster cel ls (112) . Howeve r , H S R ' s may be found at s i tes d i s tan t f rom the s t r u c t u r a l gene and may not be the o r ig ina l s i te of the D H F R gene . B y us i ng en t i r e l y d i f f e ren t t e c h n i q u e s , Maure r et a[, (113) have local ized the human D H F R gene on chromo-some 5 and have shown that the D H F R pseudogenes are located on chromosomes o ther than 5. In a d d i t i o n , these i nves t i ga to r s have shown that, the HSR con ta in ing ampl i f ied D H F R genes are p resen t on chromosomes d i s t i nc t f rom chromosome number 5 (113 and re fe rences t h e r e i n ) . ,These obse rva t i ons sugges t tha t a t rans loca t ion even t f rom - 38 -chromosome 5 to o ther chromosomes has o c c u r r e d in these c e l l s . In fac t F l in to f f et a_[, (122) have p roposed a model whe reby t rans loca t ion and chromosomal rea r rangement may be essent ia l to the in i t ia t ion of the gene ampl i f icat ion even t . T h e D H F R locus appears to be d i p l o i d , at least in C h i n e s e hamster o v a r y ce l ls fo r wh ich the most genet ic data is ava i l ab le . U r l aub et a L (114) have shown that s t a r t i ng wi th a M T X - r e s i s t a n t c lone ( d e s c r i b e d in re fe rences 38 and 59 ) , the ce l ls of wh ich conta in ampl i f ied copies of a mutant D H F R gene tha t codes fo r a M T X -res i s tan t D H F R , mutants l ack ing the en t i re locus f o r D H F R cou ld on ly be iso lated in two s t e p s . T h e f i r s t s tep resu l ted in M T X - s e n s i t i v e hemizygotes wh ich exh ib i t ed a reduced level" of D H F R ac t i v i t y and conta ined a reduced number of D H F R g e n e s . T h e D H F R a c t i v i t y in these ce l ls was M T X - s e n s i t i v e . T h e s e data were i n t e rp re ted as the hemizygotes los ing all copies of the ampl i f ied a l te red D H F R genes and re ta in ing one w i l d - t y p e gene . T h i s w i ld t ype gene cou ld then be deleted in a second mutagenic s t e p . T h e s e obse rva t i ons are on ly cons is ten t wi th the idea tha t the D H F R locus is d ip lo id in these cel ls ( F i g . 1 -4 ) . O t h e r ev idence fo r d i p l o i dy at t h i s locus comes f rom the work of Lewis et a[, (115) who found res t r i c t i on length po lymorph ism in a cel l l ine of Ch inese hamster lung c e l l s . C lones of these ce l ls e x p r e s s e i the r one o r the o ther of two forms of D H F R wh ich d i f f e r in molecular we ight by 1,000 da l tons and in i soe lec t r i c p o i n t s , and are coded by d i s t i n c t mRNA spec ies (116) . M T X - r e s i s t a n t L5178Y ( R 4 ) ce l ls (see C h a p t e r s 2 and 3) code fo r two forms of D H F R , one of wh ich is e x p r e s s e d at low levels and is h i g h l y res i s tan t to M T X ( form 2 ) , and the o ther ( form 1) is e x p r e s s e d at h igh leve ls and resembles - 39 -F I G U R E 1-4 Diagramat ic rep resen ta t ion of delet ion of the d ip lo id locus of Ch inese hamster D H F R . T h e d iagram has been adapted f rom U r l aub et al_, (114) . R r ep resen t s an a l terat ion of th i s D H F R gene such that it codes fo r an enzyme wh ich is less sens i t i ve to inh ib i t i on by metho t rexa te . 40 6 0 C p C p dhfr+ Wild-Type 0 0 amplification d h f r R r j ] CJDdhfr+ Altered Enzyme T dhfr R ED dhfr+ Altered and Amplified 7 Irradiation ^ 7 Irradiation ^ ^ 0 o -< 0 0 O dhfr+ Double Deletion Hemizygote - 41 -the w i l d - t y p e enzyme (18 ) . A t tempts to subc lone th i s ce l l l ine wi th the aim of i so la t ing c lones wh ich conta in e i ther one o r the o ther form of the enzyme have fa i led and the re fo re it would appear that the same cel l codes fo r both forms of D H F R . S im i l a r l y Haber et a | , (17) f ound tha t M T X - r e s i s t a n t 3T6-R400 ce l ls o v e r p r o d u c e d a M T X - i n s e n s i t i v e form of D H F R in the p resence of low leve ls of normal D H F R . A l l of these data are cons i s ten t wi th the p resence of two D H F R a l l e les , e i ther one or both of wh ich can unde rgo gene ampl i f icat ion or s t r u c t u r a l muta t ions . T h e cont ro l of e x p r e s s i o n of D H F R has been s tud ied i n t ens i ve l y s ince the ava i l ab i l i t y of D H F R c D N A , wh ich can be used as a p robe fo r the quan t i f i ca t ion and s t r u c t u r a l ana l ys i s of D H F R m R N A . Serum st imulat ion of qu iescen t (Go ) ce l ls in cu l t u re resu l t s in these ce l ls en te r i ng the cel l c y c l e . T h e level of D H F R inc reases marked l y when these ce l ls en ter S phase (117 and re fe rences t h e r e i n ) . T h e inc rease is due to the rate of s y n t h e s i s of D H F R resu l t i ng f rom an inc rease in the D H F R m R N A con ten t . T h i s i nc rease has been shown to be due to an e levated rate of p roduc t i on of cy top lasmic D H F R mRNA ra the r than an inc rease in the s tab i l i t y of the message. T h e r e appears to be a c lose co r re la t ion between the rate of D H F R mRNA p roduc t i on and the rate of D H F R m R N A s y n t h e s i s , s u g g e s t i n g tha t the inc rease in DHFR gene e x p r e s s i o n is con t ro l led mainly at the level of t r a n s c r i p t i o n (117) . in a separa te r e p o r t , h o w e v e r , the inc rease in D H F R mRNA was shown not to be due to i nc reased mRNA p roduc t i on bu t due to inc reased s tab i l i t y of the mRNA (118) . S u g g e s t i v e as th i s data may b e , o ther forms of con t ro l of D H F R ac t i v i t y cannot be ru led ou t . For i n s t a n c e , an inc rease in D H F R ac t i v i t y cou ld well come about by a sub t le modi f icat ion of the enzyme - 42 -in v i v o , and does not necessa r i l y have to come about by an actual inc rease in D H F R p r o t e i n . In a d d i t i o n , a l though not documen ted , t rans la t iona l con t ro l of D H F R cannot be ru led ou t . What con t ro ls the rate of t r a n s c r i p t i o n of the D H F R gene? T h i s is as yet an unanswered q u e s t i o n , a l though the ana lys i s of the human D H F R promoter , and e n h a n c e r - l i k e sequences p resen t in the in t ron 1 of the human D H F R gene (107) may shed more l i gh t on th i s sub jec t . One mechanism of cont ro l of the e x p r e s s i o n of t i s s u e - s p e c i f i c genes has been D N A methy la t ion (119) ; e g . t i s s u e - s p e c i f i c genes tha t are f ound to be h i gh l y methy la ted in sperm D N A are f ound to be u n d e r -methy la ted in the t i ssue in wh ich they are e x p r e s s e d . H y p o m e t h y l -at ion appa ren t l y leads to i nc reased e x p r e s s i o n of these g e n e s . In con t ras t to t i s sue spec i f i c p r o t e i n s , ' housekeep ing ' p r o t e i n s , of wh ich D H F R is one , are s y n t h e s i z e d in all c e l l s . S te in et a[, (120) recen t l y looked at the pa t te rn of methy la t ion of the mouse D H F R gene and found it to be unmethy la ted in the 5' reg ion in all ce l ls of the mouse, i nc l ud ing s p e r m . T h e gene was heav i l y methy la ted in the res t of gene . T h e s e au tho rs conc luded tha t undermethy la t ion of the 5' reg ion of the ' housekeep ing genes ' may be a n e c e s s a r y cond i t ion f o r i ts e x p r e s s i o n , espec ia l l y s ince it was found tha t the e x p r e s s i o n of the gene cod ing fo r adenine p h o s p h o r i b o s y l t r a n s f e r a s e , ano ther ' housekeep ing ' p r o t e i n , is i nh ib i t ed by D N A methy la t ion in v i v o . T h e r e f o r e , one form of cont ro l of t r a n s c r i p t i o n of the D H F R gene may be hypomethy la t ion of the 5' r e g i o n , pe rhaps the promoter r e g i o n . It is c lear f rom th i s i n t roduc t i on tha t d ihyd ro fo la te reduc tase is one of the most e x t e n s i v e l y s tud ied enzymes . T h i s l i t e ra tu re rev iew has by no means been exhaus t i ve and severa l po in ts not deal t wi th in - 43 -detai l here wil l be d i s c u s s e d in more detai l in the s u b s e q u e n t c h a p t e r s . T h e Scope of th i s T h e s i s T h e s t u d y of a h i g h l y M T X - r e s i s t a n t mouse leukemia cel l l i ne , L5178Y had shown tha t these ce l ls e x p r e s s e d an e levated level of DHFR (18 ) . T h i s i nc reased D H F R ac t i v i t y was composed of two forms of D H F R : form 1, wh ich rep resen ted the major component of the D H F R a c t i v i t y , and form 2 , wh ich was p resen t in smal ler amounts . Whereas form 1 had a h igh a f f i n i t y fo r M T X ( K i = 1 0 ~ 9 M) form 2 had a v e r y low a f f i n i t y fo r M T X ( K i = 5 x 1 0 ~ 4 M ) . T h e two forms were separa ted f rom each o ther by M T X - s e p h a r o s e a f f i n i t y ch romatog raphy (79) and the i r p rope r t i es were cha rac te r i zed (18 ,121 ) . T h e two forms were found to have marked ly d i f f e ren t p r o p e r t i e s ; e g . i soe lec t r i c po i n t s , heat s t a b i l i t y , response to o rgan i c mercu r ia l s and enzyme k ine t ic c o n s t a n t s . Whether the two forms of D H F R were p resen t in d i f f e ren t c e l l s ; i . e . d e r i v e d f rom a heterogeneous cel l popu la t i on , or were e x p r e s s e d in the same c e l l , was not c l ea r . E x t e n s i v e at tempts to subc lone these ce l ls into those e x p r e s s i n g e i ther one o r the o ther of the two forms were not success fu l and the re fo re it would seem tha t both forms are s y n t h e s i z e d in the same c e l l . F i g . 1-5 is a d iagramat ic rep resen ta t ion of the re levance of the exp ress i on of e levated levels of these two forms of D H F R in impar t ing a h igh degree of res is tance to these c e l l s . T h i s p resen t s t u d y was in i t ia ted to cha rac te r i ze the molecular even ts respons ib le fo r the inc reased level of D H F R p resen t in these c e l l s , and more impor tan t l y , to isolate the complementary D N A s ( c D N A ) f o r the two forms of the enzyme from the i r c o r r e s p o n d i n g - 44 -F I G U R E 1-5 Schemat ic rep resen ta t ion of the ef fect of M T X on fo late metabo-lism in 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 c e l l s . A . M T X - s e n s i t i v e c e l l s : M T X is ac t i ve l y taken up by the ce l ls and b inds s t r o n g l y to the s ing le spec ies of ' h i g h - a f f i n i t y ' fo late reduc tase p r e s e n t . Inh ib i t ion of the enzyme resu l t s in the dep le t ion of t e t rahyd ro fo la te pools wi th the consequen t decrease in d T M P l eve l s , lead ing to unba lanced and dec reased D N A s y n t h e s i s . T h e t endency of the inc rease in d i h y d r o f o l i c ac id leve ls (due to the inh ib i t ion of fo late reduc tase ) to d i sp lace M T X from the enzyme can be overcome by sus ta i n i ng f ree i n t race l l u l a r M T X . ' B . M T X - r e s i s t a n t c e l l s : 1) When exposed to ' h i g h ' concen t ra t i ons -4 (10 M) M T X , the d r u g en te rs the cel l a n d , 2) b i nds s t r o n g l y to the e levated levels of ' h i g h - a f f i n i t y ' fo late reduc tase ( H A ) , i nac t i va t i ng it and leav ing v e r y l i t t le f ree i n t race l l u l a r M T X . 3) T h e ' l ow-a f f i n i t y ' fo late reduc tase ( L A ) (wi th a cons ide rab le h ighe r Ki fo r M T X ) p resen t in these ce l ls wi l l con t inue to be func t iona l at the f ree i n t race l l u l a r concen t ra t ions of M T X now p r e s e n t . T h e un inh ib i t ed ac t i v i t y of the ' l ow-a f f i n i t y ' enzyme e n s u r e s the s y n t h e s i s of su f f i c i en t amounts of t e t rahyd ro fo la te co fac to rs and t hus of d T M P . 45 j MTX-SENSITIVE CELL MTX-RESISTANT CELL DNA - 46 -mRNA molecules wi th the ul t imate aim of sequenc ing these c D N A molecu les . T h e resu l t s of th i s pro ject are d e s c r i b e d in chap te rs 2 and 3. In add i t ion to the molecular b io logica l cha rac te r i za t i on of th i s sys tem, a pro jec t was unde r t aken to tes t o ther fo late an tagon is t compounds fo r t he i r i nh i b i t o r y potent ia l of the l o w - M T X - a f f i n i t y ( form 2) D H F R . T h i s s t u d y is d e s c r i b e d in A p p e n d i x I. T h e f i n d i n g tha t M T X - r e s i s t a n t mouse ce l ls can e x p r e s s e levated levels of two forms of D H F R prompted the inves t iga t ion of M T X -res i s tan t human tumour c e l l s . Two cel l l ines were i n v e s t i g a t e d , HeLa (carc inoma of the c e r v i x ) and H L - 6 0 (p romye locy t i c l eukemia ) . In a d d i t i o n , cel l ex t r ac t s of b last ce l ls f rom pat ients w i th acute myelogenous leukemia were assayed fo r the level and s e n s i t i v i t y to M T X of DHFR a c t i v i t y . T h e resu l t s of the s tud ies wi th human tumour ce l ls are d e s c r i b e d in C h a p t e r s 4 and 5. S t r a t e g y fo r the Isolat ion of L5178Y ( R 4 )  Form 1 and Form 2 D H F R c D N A s T h e s t r a tegy taken was one in wh ich the c D N A would be s y n t h e -s ized f rom h i g h l y en r i ched m R N A s cod ing fo r the two forms of D H F R . T h e mRNA would be en r i ched by polysome immunoprec ip i ta t ion us i ng ant ibod ies to form 1 and form 2 D H F R s . In o r d e r fo r th i s s t r a tegy to w o r k , t h ree p r e r e q u i s i t e s had to be f u l f i l l e d . F i r s t l y , the two forms of D H F R had to be p u r i f i e d to a h igh deg ree of homogenei ty . S e c o n d l y , an t ibod ies had to be p r e p a r e d aga ins t these enzymes and assayed fo r po tency as well as degree of - 47 -c r o s s r e a c t i v i t y . T h i r d l y , fo r th i s app roach to be s u c c e s s f u l , the m R N A s cod ing fo r these enzymes would have to be re la t i ve l y abundan t and th i s had not been demonst ra ted as y e t . T h e r e f o r e m R N A f rom the w i l d - t y p e sens i t i ve and M T X - r e s i s t a n t ce l ls had to be iso lated and ana lyzed to determine whether the inc reased D H F R ac t i v i t y in these cel ls resu l ted f rom an inc rease in the m R N A coded by ampl i f ied D H F R g e n e s , as had been shown fo r o ther M T X - r e s i s t a n t cel ls ( 8 ) . T h e overa l l schemat ics of the app roach taken fo r these s tud ies is ou t l ined as a f low d iagram in F i g . 1-6. Sou the rn and N o r t h e r n blot ana lyses d e s c r i b e d in t h i s thes is of genomic D N A and ce l lu la r m R N A , r e s p e c t i v e l y , were c a r r i e d out us i ng a D H F R - c D N A wh ich was p resen t in the p lasmid p D H F R II and was supp l i ed by D r . Robe r t S c h i m k e , S tan fo rd U n i v e r s i t y . F i g . 1-7 shows the map of t h i s c D N A wh ich cons i s ted of the en t i re D H F R cod ing reg ion and approx imate ly 900 bases of 3' n o n - c o d i n g sequences . - 48 -F I G U R E 1-6 A . Ove ra l l schemat ics f o r the determinat ion of the reasons f o r i nc reased D H F R ac t i v i t y in L5178Y ( R 4 ) ce l ls ( d e s c r i b e d in C h a p t e r 2 ) . B . S t r a t e g y fo r enr ichment of m R N A s cod ing f o r Form 1 and Form 2 D H F R s f rom these ce l ls ( d e s c r i b e d in C h a p t e r 3 ) . ; 49 v j Dot Blot ^ Analysis I Gene Amplification? L5178Y Cells (S) (R4) High M Wt. DNA I Digest with Restriction Endonucleases Separate Restriction Fragments by Agarose Gel Electrophoresis I Southern Blot Analysis I Gene Amplification? Total mRNA Translate in vitro -SDS-Page Immunoppt. Dot Blot Analysis Northern Blot Analysis Increased DHFR mRNA? B L5178Y (R 4 ) cells-I Polysomes Purified Form 1 and 2 DHFRs cDNA Translate in vitro I 1D and 2D gels Dissociate Complex Size by Northern Blot Analysis 1gG — Rabbit J Anti Form 1 or Anti Form 2 r J Polysome-Antibody • Complex S. Aureus Protein A 50 j FIGURE 1-7 Map of Double Stranded cDNA clone (p DHFR 11) 630 1600 \ \ AUG H h 5' • T 3' coding region non-coding region - 51 -R E F E R E N C E S 1. Law, L .W. (1956) D i f fe rences between cance rs in terms of evo lu t ion of d r u g res i s t ance . C a n c e r R e s . 16, 698-716. 2. L u r i a , S . E . and D e l b r u c k , M. (1943) Mutat ion of bac te r ia f rom v i r u s sens i t i ve to v i r u s r e s i s t a n c e . G e n e t i c s . 28, 491. 3. Law, L .W. (1952) O r i g i n of the res is tance of leukemic ce l ls to fo l ic ac id an tagon i s t s . N a t u r e . 169, 628-629. 4. Poche , H . , V a r s h a v e r , N . V . and G e i r s l e r , E. (1975) C y c l o -hex imide res is tance in C h i n e s e hamster c e l l s . I. 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(1983) S t r u c t u r e of the d ihyd ro fo la te reduc tase gene in C h i n e s e hamster o v a r y c e l l s . N u c . A c i d s . R e s . V\_, 1997-2012. 126. S t a r k , G . R . and Wah l , G . M . (1984) Gene amp l i f i ca t ion . A n n . R e v . B iochem. 53 , 447-491. - 62 -C H A P T E R 2 O V E R P R O D U C T I O N OF TWO A N T I G E N I C A L L Y D I S T I N C T FORMS OF D I H Y D R O F O L A T E R E D U C T A S E A N D G E N E A M P L I F I C A T I O N IN A H I G H L Y M E T H O T R E X A T E R E S I S T A N T M O U S E L E U K E M I A C E L L L INE I N T R O D U C T I O N T h e an t ineop las t i c agent methot rexate ( M T X ) p roduces cy toc ida l e f fec ts by i nh i b i t i ng the a c t i v i t y of the enzyme d ihyd ro fo la te r e d u c -tase ( 5 , 6 , 7 , 8 - t e t r a h y d r o f o l a t e : N A D P + o x i d o r e d u c t a s e , EC 1 . 5 . 1 . 3 ) , wh ich ca ta lyzes the N A D P H - d e p e n d e n t reduc t ion of d i h y d r o f o l i c ac id (and at a slow ra te , fo l i c ac id ) to t e t r a h y d r o f o l i c a c i d , the metabol ic -a l l y ac t i ve coenzyme form of fo l ic a c i d . Res is tance to M T X has been s tud ied in a v a r i e t y of cel l c u l t u r e sys tems (1 -5 ) and has been asc r i bed to one o r more of a v a r i e t y of mechanisms. T h e s e inc lude decreased up take of M T X by the ce l ls ( 6 - 8 ) , o v e r p r o d u c t i o n of d ihyd ro fo la te reduc tase ( D H F R ) (5 ,9 -12 ) and e x p r e s s i o n of an a l te red form of D H F R resu l t i ng in reduced a f f i n i t y fo r methot rexate ( 4 , 5 , 13 -16 ) . O v e r p r o d u c t i o n of d ihyd ro fo la te reduc tase is accompanied by a para l le l i nc rease in the ce l l u la r concen t ra t ion of D H F R mRNA (10 ,17 ,18) and ampl i f icat ion of the D H F R genes ( 1 1 , 1 7 , 4 1 ) . A l t h o u g h in some cases the o v e r p r o d u c e d enzyme is i n d i s t i n g u i s h a b l e f rom the enzyme s y n t h e s i z e d by the wi ld t y p e sens i t i ve c e l l s , in o the rs the o v e r p r o d u c t i o n is assoc ia ted wi th the e x p r e s s i o n of a l te red D H F R s , some of them wi th a reduced a f f i n i t y fo r M T X ( 5 , 1 4 - 1 6 ) . P r e v i o u s l y , it was found tha t L5178Y mouse leukemia ce l ls se lected fo r ext reme res is tance to M T X (1 mM) e x p r e s s two forms - 63 -( form 1 and 2) of D H F R , wi th one of these forms ( form 2) e x h i b i t i n g a marked reduc t ion in a f f i n i t y fo r M T X ( 5 ) . T h i s res i s tan t form rep resen ted approx imate ly 38% of the total D H F R ac t i v i t y in these cel ls wh ich exh ib i t ed a h i ghe r total spec i f i c a c t i v i t y than the w i l d -t ype sens i t i ve c e l l s . T h e two forms were found to d i f f e r marked l y in the i r K i va lues fo r M T X (5 ,15) bu t not in t he i r Km fo r the s u b s t r a t e s fo l ic ac id and N A D P H . In a d d i t i o n , t he re were o ther d i f f e rences between the two forms of the enzyme i n c l u d i n g i soe lec t r i c po in ts and heat s tab i l i t y (15 ) . In th i s chap te r I demonst ra te that rabb i t serum ant ibod ies p repa red us i ng p u r i f i e d form 1 D H F R d i s p l a y e d a c o n s i d e r a b l y h ighe r a f f i n i t y fo r form 1 D H F R than form 2 D H F R , i nd i ca t i ng a h igh degree of an t igen ic d i v e r s i t y between the two forms of the enzyme. An t i bod ies p repa red aga ins t p u r i f i e d form 2 D H F R d i sp l ayed a h ighe r a f f i n i t y fo r form 2 than fo r form 1 D H F R , a l though not to as g rea t an ex ten t . It was also f ound that the o v e r p r o d u c t i o n of both forms of D H F R is due to the p resence of abundan t D H F R mRNA a c t i v i t y in the M T X - r e s i s t a n t ce l ls as determined by in v i t r o t rans la t i on of total mRNA f rom L5178Y ( S ) and ( R 4 ) c e l l s , and N o r t h e r n blot ana lys i s of th i s RNA. ' A compar ison of S o u t h e r n blot ana lys i s of genomic D N A from ( S ) and ( R 4 ) ce l ls demonst ra ted tha t the D H F R genes have been ampl i f ied in the res i s tan t c e l l s . M A T E R I A L S A N D M E T H O D S Ce l l s Me tho t rexa te -sens i t i ve ( S ) and res i s tan t ( R 4 ) L5178Y cel l l ines were g rown at 37° as suspens ion cu l t u res in p las t i c f l a s k s u n d e r an - 64 -atmosphere of 5% C 0 2 in a i r . T h e g rowth medium was F i s h e r ' s medium supp lemented wi th 10% horse s e r u m , 1% g lu tamine , 100 u n i t s / ml of p e n i c i l l i n , 100 pl /ml of s t rep tomyc in ( G r a n d Is land B io log ica l C o . , G r a n d I s l and , N . Y . ) . T h e R 4 cel l l ine was d e r i v e d f rom S ce l ls by a s tepwise se lect ion p rocess and u l t imate ly g rown in 1 mlVI M T X . Unde r these cond i t ions the doub l i ng time fo r both cel l l ines was approx imate ly 12 h r . Tota l cel l numbers were determined by coun t i ng on a Model ZB1 Cou l t e r C o u n t e r ( C o u l t e r E l e c t r o n i c s , H ia leah , F L . ) . Separa t ion and Pu r i f i ca t i on of D ihyd ro fo la te Reduc tases Ce l l e x t r a c t s were p repa red as d e s c r i b e d p r e v i o u s l y ( 5 ) . Form 1 (h igh a f f i n i t y ) and form 2 ( low a f f i n i t y ) d i hyd ro fo la te reduc tases f rom R 4 ce l ls were separa ted by a f f i n i t y ch roma tog raphy on M T X -sepharose columns as d e s c r i b e d before ( 5 , 1 5 ) . F rac t ions con ta in ing D H F R ac t i v i t y wh ich d id not b ind to the column ( form 2) were pooled and rechromatographed t h r o u g h another M T X - s e p h a r o s e co lumn. Once again f r ac t i ons con ta in ing D H F R ac t i v i t y wh ich d id not b i nd to th i s column were pooled a n d , a f te r c o n c e n t r a t i o n , the p repa ra t i on was f u r t h e r pu r i f i ed by gel f i l t r a t i on t h r o u g h Sephadex G-100 . D H F R ac t i v i t y wh ich bound to the f i r s t M T X - s e p h a r o s e column ( form 1) was also f u r t h e r p u r i f i e d by gel f i l t r a t i o n . F rac t i ons con ta in ing enzyme ac t i v i t y a f ter gel f i l t ra t ion of form 2 d ihyd ro fo la te reduc tase were poo led , c o n c e n t r a t e d , and a f te r d i a l y z i n g aga ins t s t a r t i n g b u f f e r , app l ied to a D E A E af f ige l b lue (B io Rad Labo ra to r i es , R i c h m o n d , C A . ) column (1 .5 x 5.4 cm) wh ich had been washed wi th 50 ml 0.1 M acet ic a c i d , pH 3.0 con ta in ing 1.4 M NaCl and 40% i so -p ropano l and equ i l i b r i a ted wi th 5 volumes of s t a r t i ng bu f f e r (50 mM T r i s - C I , pH - 65 -7 . 5 ) . T h e column was then e lu ted wi th a s tep g rad ien t f rom 1.0 M NaCI to 2 .5 M NaCI in s t a r t i ng b u f f e r . D H F R a c t i v i t y was measured us i ng 3 H - l a b e l e d d i h y d r o f o l i c ac id as s u b s t r a t e . 3 H - d i h y d r o f o l i c ac id was s y n t h e s i z e d in ou r l abo ra to ry by d i th ion i te reduc t ion of [ G - 3 H ] fo l ic ac id (5 C i /mmole) (Amersham C o r p . , O a k v i l l e , O n t . ) acco rd i ng to the method of Hayman et aj_, (54 ) . T h e r a d i o p u r i t y of each p repara t i on of 3 H - d i h y d r o f o l i c ac id was determined by paper ch romatog raphy as d e s c r i b e d in Hayman et a[, (54) and was found rou t i ne l y to be 90% or g r e a t e r . T h e 3 H -d i h y d r o f o l i c ac id was s to red in a l iquots as d r y powder at - 7 0 ° C . When needed , a l iquots were d i sso l ved in 1 ml 20 mM T r i s - H C I , pH 7 .3 and the concen t ra t ion and spec i f i c a c t i v i t y de termined fo r each a l iquot by measur ing the absorbance at 282 nm in 20 mM T r i s - H C I , pH 7 .3 ( t ak i ng the molar ex t inc t ion coef f i c ien t at 282 nm as 28,400) ( 5 5 ) , and by sc in t i l l a t ion c o u n t i n g . T h e d ihyd ro fo la te reduc t ion was assayed in 12.5 mM T r i s - H C I , pH 7 . 5 , 0.2 M K C I , 0 .8 mM N A D P H , and 0.1 mM 3 H - d i h y d r o f o l i c ac id in a total volume of 200 u l . Con t ro l s were per fo rmed in the absence of N A D P H . To measure inh ib i t i on b y methot rexate ( M T X ) the lysa tes were p re incuba ted wi th M T X in the p resence of N A D P H fo r 2 min at room tempera tu re . T h e react ion was s ta r ted by the add i t ion of 3 H -d i h y d r o f o l i c ac id and c a r r i e d out at 37°C fo r 20 m in . T h e react ion was s topped by p lac ing the tubes in ice and 30 ul of un labe l led fo l i c ac id (0.027 M) was added to each t u b e . U n r e d u c e d d i h y d r o f o l i c ac id and fo l ic ac id were p rec ip i ta ted by the add i t ion of 30 ul Z n S 0 4 solu t ion (0 .17 M) and 10 ul g lac ia l acet ic a c i d . A f t e r cen t r i f uga t i on at 1000 g ( r , 20 cm) fo r 1 h r . at 4 ° C , 100 ul of the supe rna tan t - 66 -was added to P C S l iqu id sc in t i l l a t ion cock ta i l (Amersham C o r p . , O a k v i l l e , O n t . ) and c o u n t e d . A l l assays were run in t r i p l i c a t e . One un i t of ac t i v i t y is de f ined as tha t amount of enzyme r e d u c i n g 1.0 nmole of d i h y d r o f o l i c ac id /20 m in . T h e homogenei ty of p u r i f i e d form 1 and 2 d i hyd ro fo l a te reduc tases was determined by Laemmli sodium dodecy l su lpha te po lyac ry lamide gel e lec t rophores i s (20) as d e s c r i b e d be low, excep t tha t the gel was s ta ined fo r p ro te in wi th the h i gh l y sens i t i ve s i l v e r s ta in us i ng a modi f icat ion of the method d e s c r i b e d by Sw i t ze r et a[, ( 21 ) : T h e gel was f i x e d f o r 1 h r . in 25% ( V / V ) methanol/10% ( V / V ) acet ic a c i d . A f t e r t h o r o u g h l y r i n s i n g the gel wi th d i s t i l l ed wa te r , it was f i x e d in 10% ( V / V ) g l u ta ra l dehyde so lu t ion f o r 30 m i n . , r i nsed in d i s t i l l ed water and left o v e r n i g h t in the same. T h e gel was nex t p laced in 200 ml of a so lu t ion con ta in ing 0.07% ( W / V ) sodium h y d r o x i d e , 1.4% ( V / V ) ammonium h y d r o x i d e , 0.81% ( W / V ) s i l v e r n i t ra te and 14.8% ( V / V ) ethanol fo r 15 m i n . , r i nsed wi th d i s t i l l ed wa te r , and then p laced in 500 ml of the deve lop ing so lu t ion con ta in ing 10% ( V / V ) e thano l , 0.005% ( W / V ) c i t r i c ac id and 0.0185% ( V / V ) f o rma ldehyde . T h e gels were pho tog raphed soon a f ter the appearance of the p ro te in b a n d s . P repa ra t i on of An t i bod ies P u r i f i e d form 1 o r form 2 d ihyd ro fo la te reduc tase f rom L5178Y ( R 4 ) ce l ls (1 mg/ml in s te r i le phosphate bu f fe red sa l ine (0 .15 M N a H 2 P 0 4 . H 2 0 ; 0.15 M N a 2 H P 0 4 , pH 7.2 in 0.9% N a C l ) was emuls i f ied wi th an equal volume of complete F r e u n d ' s ad juvan t . Two rabb i t s were each in jected wi th th i s emu ls ion , 2 ml subcu taneous l y at - 67 -two d i f f e ren t si tes, on the b a c k . A t 2 week i n t e r va l s fo r 6 w e e k s , each rabb i t was boosted subcu taneous l y wi th 1 mg of the an t igen as d e s c r i b e d above . T e n days a f ter the f ina l in ject ion the rabb i t s were b led f rom the ear v e i n , the se ra co l l ec ted , d i a l y zed aga ins t s t a r t i n g bu f fe r (0 .02 M T r i s - H C I , pH 8 . 0 , 0.028 M NaCI) and 6 to 10 ml app l ied to a D E A E af f ige l b lue column (2 .5 x 15. cm) . T h e column was e lu ted wi th s t a r t i ng bu f fe r and 6.0 ml f r ac t i ons were co l l ec ted . Pro te in was moni tored by i ts abso rbance at 280 nm. F rac t i ons con ta in ing p ro te ins exc l uded by the column and e lu ted in the vo id vo lume, were poo led , concen t ra ted b y u l t ra f i l t r a t i on (Amicon S y s t e m , L e x i n g t o n , M A . ) t h r o u g h a f i l t e r w i th a molecular we igh t cu t off of 10,000 and d ia l yzed aga ins t phosphate bu f fe red sa l i ne . T h i s p repara t ion (pa r t i a l l y p u r i f i e d IgG) was used in subsequen t expe r imen t s . Immunoassay T h e e n z y m e - l i n k e d immunoabsorbent assay ( E L I S A ) (22,23) was used fo r the detec t ion and determinat ion of the an t igen ic spec i f i c i t y of a n t i - d i h y d r o f o l a t e reduc tase ( form 1 and 2) IgG p r e p a r a t i o n s . Pu r i f i ed an t igen (e i ther form 1 or form 2 D H F R in phosphate bu f f e red sa l i ne , pH 7 . 2 , 10 ug) was adso rbed onto a so l id phase : M ic roe l i sa plates (Dyna tech Labora to r ies L t d . , V i r g i n i a , U S A ) fo r 16 h r . at 4 ° C . Unreac ted p ro te in b i nd ing s i tes were then b locked by wash ing each we l l , twice fo r 15 m in . wi th P B S , pH 7.2 con ta in ing bov ine serum albumin (5 g / l ) and po lyso rba te 20 (1%) ( b l o c k i n g s o l u t i o n ) . An t i bod ies o r an t i body -an t i gen complexes were then al lowed to react wi th the bound an t igen on the plate fo r 1 h r . at room tempera tu re . - 68 -Each well was then washed twice wi th the b l ock i ng so lu t ion p r i o r to the add i t ion of 0.1 ml pe rox idase label led goat a n t i - r a b b i t IgG (3000 fo ld d i lu t ion of s tock ob ta ined f rom C a l b i o c h e m - B e h r i n g , San D iego , C A . ) . T h e react ion was al lowed to take place f o r 1 h r . at room tempera tu re . Each well was then washed f i ve t imes wi th b l ock i ng so lut ion and O.I ml of the s u b s t r a t e f o r pe rox idase a d d e d . T h e subs t ra te so lu t ion cons i s ted of a 1:1 m ix tu re of h y d r o g e n pe rox ide and A B T S (2 .2 ' ^ a z i n o - d i - [ 3 - e t h y l - b e n z t h i a z o l i n e su lphona te (24)] ( K i r k e g a a r d and P e r r y Labo ra to r i es , G a i t h e r s b u r g , M D . ) . T h e react ion was al lowed to p roceed at room tempera tu re fo r 10 m in . and was s topped by the add i t ion of 50 ul 1 M sodium f l uo r i de so lu t i on . A pos i t i ve react ion was ind ica ted by the appearance of a b l u e - g r e e n co lour and the assay was quant i ta ted by d i l u t i n g the con ten ts of each well wi th 0 .5 ml d i s t i l l ed water and measur ing the abso rbance at 414 nm. A p p r o p r i a t e b lanks were determined by b i n d i n g un re la ted p r o t e i n s , e . g . a l bum in , to the p lates ins tead of D H F R . P repara t ion of 3 H - L a b e l l e d D ihyd ro fo la te Reduc tase f rom L5178Y ( R 4 ) Ce l l s g A p p r o x i m a t e l y 5 x 10 ce l ls in logar i thmic g rowth were label led fo r 7 h r . wi th 500 uC'\ of 3 H - l e u c i n e (46 C i /mmol ) in leuc ine f ree medium p lus 10% horse se rum. T h e ce l ls were then pe l l e ted , washed o t h ree t imes wi th P B S , pH 7.2 and mixed wi th 5 x 10 un labe l led c e l l s . T h e combinat ion was washed f u r t h e r in P B S and the pe l le t r e s u s -pended in 10 ml T r i s - H C I , pH 7 . 5 . Me tho t rexa te -Sepha rose a f f i n i t y ch romatog raphy of the cel l e x t r a c t f rom these ce l ls was then c a r r i e d out as d e s c r i b e d e a r l i e r . 3 H incorpora t ion into p ro te in was moni tored - 69 -by t r i ch lo roace t i c ac id p rec ip i ta t ion of an a l iquot of the e x t r a c t and pa r t i a l l y p u r i f i e d D H F R p repara t ions f rom 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 . Isolat ion of Tota l mRNA A l l g l a s s w a r e , p ipet te t ips and eppendor f t ubes were au toc laved and the bu f f e r s au toc laved or f i l t e r s te r i l i zed before u s e . Tota l 9 ce l lu la r R N A was ex t rac ted f rom 2 x 10 ce l ls ( R 4 o r S ) by the g u a n i d i n e - H C I t echn ique i n t roduced by Cox (25) as d e s c r i b e d by Deeley et a[, ( 26 ) , excep t tha t a f te r severa l ethanol p rec ip i t a t i ons of R N A f rom bu f f e r con ta in ing 8 M g u a n i d i n e - H C I and d i t h i o t h r e i t o l , the R N A was f u r t h e r p u r i f i e d by d i s s o l v i n g the ethanol p rec ip i ta te in t h e above bu f fe r con ta in ing 0.4 mg/ml CsCI and l aye r i ng s i x 2 .2 ml a l iquots ove r 1.5 ml cush ions of 5.7 M C s C I . R N A was sedimented by cen t r i f uga t i on at 38,000 r e v s / m i n . fo r 16 h r . at 20° in a Beckman SW 60.1 ro to r , the bottom of the tubes were s l i ced off and the pel le ts were d i sso l ved 0.01 M T r i s - C I pH 7 . 5 , 0 .5 M KCI (5 m l ) . Messenger R N A was p u r i f i e d by ch romatog raphy on Ol igo ( d T ) - c e l l u l o s e acco rd ing to the method of A v i v and Leder (27) excep t tha t the low ionic s t r e n g t h bu f f e r used in the e lu t ion of mRNA (Po l y A +) was prewarmed to 37° . F rac t ions con ta in ing Po ly A + R N A were pooled and made 2% wi th sodium aceta te ; 2 volumes of ethanol were added and the mRNA was al lowed to p rec ip i ta te f o r 16 h r . at - 20 °C . T h e mRNA was pel le ted by cen t r i f uga t i on at 35,000 rpm fo r 30 m in . at 4°C in a Beckman SW 50.1 ro to r . T h e supe rna tan t was decanted and the pel le t s to red at - 2 0 ° . - 70 -Immunoprecip i ta t ion Ind i rec t immunoprec ip i ta t ion of the t rans la t i on p r o d u c t s was c a r r i e d out by the method d e s c r i b e d by Morand i et a[, ( 28 ) . B r i e f l y , samples were d i l u ted wi th an equal volume of N E T b u f f e r (50 mM T r i s - C I , pH 7 . 4 , 150 mM N a C l , 5 mM E D T A , 0.25% ( W / V ) sodium dodecy l s u l p h a t e , 0.75% ( V / V ) t r i t on X - 1 0 0 ) and p rec lea red by i n c u -bat ion fo r 16 h r . at 4°C wi th 10 pi of pre immune rabb i t s e r u m . S . au reus cel l suspens ion ( p a n s o r b i n ce l ls ob ta ined f rom Ca lb iochem-B e h r i n g ) (10% V / V ) was then added and a f te r 20 min . in ice the m ix tu re was c e n t r i f u g e d fo r 1 m in . in an Eppendor f c e n t r i f u g e . T h e supe rna tan ts were mixed wi th 10 pi a n t i - D H F R ( form 1 o r 2) I g G , and incuba ted fo r 3 or 17 h r . at 4°C and then t rea ted wi th S . au reus suspens ion as be fo re . T h e S . au reus t rea ted pel le ts were washed 3 t imes wi th N E T b u f f e r , and the p ro te ins e lu ted wi th 0.063 M T r i s - C I , pH 6 . 8 , 0.002 M meth ion ine , 2% ( W / V ) sodium dodecy l su lpha te at room tempera tu re f o r 1 h r . A f t e r c e n t r i f u g i n g , the supe rna tan ts were removed , and a f ter add ing sample bu f f e r were e lec t rophoresed us ing the Laemmli sodium dodecy l su lpha te po lyac ry lamide gel sys tem ( 8 ) , w i th 13% ( W / V ) po lyac ry lamide in the sepa ra t i ng gel and 4.5% ( W / V ) in the s t a c k i n g gel ( ac r y l am ide : b i s a c r y l a m i d e , 2 9 . 2 : 0.8 W/W .in both g e l s ) . A f t e r e l e c t r o p h o r e s i s , the gels were f i x e d in 10% ( W / V ) T C A , 10% ( V / V ) acet ic ac id and 30% ( V / V ) methano l , p r e p a r e d fo r au to rad iog raphy t reatment wi th the f l uo r E n 3 H a n c e (New Eng land Nuc lear C o r p . , B o s t o n , M A . ) and d r i e d on a s lab gel d r i e r (B io Rad L a b s , R i chmond , C A . ) . Dr ied gels were exposed wi th Kodak X - O m a t A R f i lm at - 7 0 ° . - 71 -In V i t r o T r a n s l a t i o n T r a n s l a t i o n of mRNA from L5178Y ( S ) and ( R 4 ) ce l ls was conduc ted in the wheat germ in v i t r o t rans la t i on system obta ined f rom Be thesda Research Labo ra to r i es , G a i t h e r s b u r g , M D . T r a n s l a t i o n was al lowed to occu r fo r 3 h r . at 25°C us i ng 3 5 S - m e t h i o n i n e . T h e react ions were s topped in ice and then incuba ted f o r 10 m in . at 37°C wi th 30 ug/ml RNase A . Pro te in s y n t h e s i s was moni tored by the inco rpora t ion of 3 5 S - m e t h i o n i n e into co ld T C A inso lub le ma te r ia l , o r by S D S - P A G E and f l u o r o g r a p h y as d e s c r i b e d above . Gel E lec t ropho res i s of R N A and DNA Messenger R N A was e lec t rophore t i ca l l y ana lyzed in 1.5% agarose ge ls con ta in ing 2 .2 M fo rma ldehyde (42 ) . E lec t ropho res i s was c a r r i e d out at 2 .5 v o l t s / c m fo r 18 h r . T h e gel was s ta ined wi th 36 ug/ml ac r i d i ne o range in 10 mM sodium phosphate b u f f e r , pH 6 .5 f o r 10 min . at room tempera tu re and des ta ined in the above bu f f e r (3 x 20 m i n . ) . DNA res t r i c t i on f ragments were e lec t ropho re t i ca l l y separa ted on 0.8% agarose gels in T A E bu f f e r ("0.04 M T r i s - a c e t a t e , 0.001 M E D T A ) . T h e gel was s ta ined wi th e th id ium bromide (10 ug /m l ) and v i s u a l i z e d u n d e r u l t rav io le t l i gh t . T r a n s f e r of R N A and D N A to N i t roce l lu lose  Paper and S u b s e q u e n t H y b r i d i z a t i o n Messenger R N A e lec t rophore t i ca l l y separa ted on f o rma ldehyde -con ta in ing agarose gels was t r a n s f e r r e d to n i t roce l lu lose ( N C ) paper by d i f f us ion f o r 16 h r . in 20 x S S C (1 x S S C is 0.15 M N a C I , 0.015 - 72 -M Na c i t r a t e , pH 7 . 0 ) . T h e NC papers were al lowed to a i r d r y and then baked in vacuo at 80°C fo r 2 h r . A g a r o s e gels con ta in ing e lec t rophore t i ca l l y separa ted DNA res t r i c t i on f ragments were dena tu red in 1.5 M N a C I , 0 .5 N NaOH, neu t ra l i zed in 0.05 M T r i s - H C I , pH 7 . 5 , 1 M NaCI and t r a n s f e r r e d onto NC papers as d e s c r i b e d above (43 ) . T h e papers were al lowed to a i r d r y and then baked in vacuo at 80°C fo r 2 h r . T h e t r a n s f e r papers were p r e h y b r i d i z e d in 3 x S S C , 50% formamide, 0.1% S D S , 5 x Denha rd t ' s so l u t i on , 10 mM T r i s - H C I , pH 7 . 5 , 10% sodium py rophospha te and 100 •ug/ml boi led son icated salmon sperm DNA at 42°C fo r 16 h r . H y b r i d i z a t i o n was c a r r i e d out in the above so lu t ion con ta in ing n i ck t r ans la ted 3 2 P - l a b e l e d c D N A ( 0 . 5 - 1.0 x 1 0 7 c p m ; S . A c . 1 x 1 0 8 c p m / u g ) at 42°C fo r 24 h r . T h e pape rs were then sequen t ia l l y washed in 2 x S S C , 0.1% S D S at room temper -a t u r e ; and 0.1 x S S C , 0.1% S D S at 50°C fo r 30 m in . to 1 h r . , and exposed to x - r a y f i lm ( K o d a k X - O m a t A R ) at -70°c . S y n t h e s i s of 3 2 P - l a b e l e d c D N A A 3 2 P - l a b e l e d DNA compl imentary to a mur ine D H F R c D N A , wh ich was p resen t in a c loned plasmid ( p D H F R II) and k i n d l y supp l i ed by R. S c h i m k e , was s y n t h e s i z e d by n i ck t r a n s l a t i o n . T h e plasmid D N A was d iges ted wi th the res t r i c t i on enzyme Ps t I to remove the i nse r ted D N A and e lec t rophoresed in 1% agarose g e l s . T h e i nse r ted D N A was e x t r a c t e d f rom the gel and n i ck t r ans la ted wi th [ a - 3 2 P ] - d C T P us ing a n i ck t rans la t i on system supp l i ed by Amersham C o r p . - 73 -P repara t i on and Res t r i c t i on A n a l y s i s of C e l l u l a r D N A H igh molecular we igh t DNA f rom L5178Y ( S ) and ( R 4 ) cel l was p r e p a r e d acco rd i ng to B l i n and S ta f f o rd (44 ) . B r i e f l y , approx imate ly g 2 x 10 ce l ls were washed in P B S and resuspended at a concen t ra t ion o of 10 ce l l s /m l in an ice cold so lu t ion of T E bu f f e r (10 mM T r i s - H C I , pH 7 . 4 , 1 mM E D T A ) . Ten volumes of 0 .5 M E D T A , pH 8 . 0 ; 100 | jg/ml p ro te inase K; 0.5% sa rcosy l were added and the m ix tu re was incuba ted at 50°C fo r 3 h r . T h e D N A was then gen t l y ex t rac ted th ree t imes wi th an equal volume of p h e n o l . T h e D N A was d ia l yzed aga ins t severa l changes of 4 l i te rs of a so lu t ion of 50 mM T r i s - H C I , pH 8 . 0 , 10 mM E D T A , and 10 mM N a C l . T h e sample was t rea ted wi th 100 pg/ml of RNase at 37°C fo r 3 h r . , and the D N A was ex t rac ted once again wi th an equal volume of a 1:1 pheno l / ch lo ro fo rm m i x t u r e . A f t e r d i a l y z i n g the sample e x t e n s i v e l y aga ins t T E bu f f e r the concen t ra t ion of DNA was measured by i ts abso rbance at 260 nm. One abso rbance un i t was taken to be equ iva len t to a concen t ra t ion of 50 p g / m l . T h e s ize of the DNA was determined by e lec t ropho res i s on 0.8% agarose gels and was found to migrate more s lowly than in tac t phage X D N A . Res t r i c t i on ana lys i s of the DNA was per fo rmed u n d e r cond i t ions recommended by the enzyme s u p p l i e r s , excep t that the reac t ions were incuba ted at the des igna ted tempera tu res fo r 16 h r . to e n s u r e complete d i g e s t i o n . Chromosome A n a l y s i s Metaphase chromosomes f rom M T X - r e s i s t a n t and sens i t i ve L5178Y ce l ls were p repa red fo r G i e m s a - t r y p s i n - b a n d ana lys i s as fo l lows : Ce l l s - 74 -(3 x 10 c e l l s / 5 ml) were incubated f o r 72 h r . at 37°C in F i s c h e r ' s medium con ta in ing 10% horse se rum. Colcemid was added to a f ina l concen t ra t ion of 16 ng /m l and a f ter 90 m in . at 37° ce l ls were c e n t r i -f uged at 300 g fo r 3 m in . and r e s u s p e n d e d in 5 ml 75 mM KCI (37°C) fo r 8 m in . Ce l l s were c e n t r i f u g e d as above and resuspended gen t l y in 3 ml methano l : acet ic ac id (3:1 V / V ) , c e n t r i f u g e d as before and resuspended in 5 ml of the same so lu t i on . T h e cel l suspens ion was then d r o p p e d on wet s l ides and al lowed to a i r d r y . S l i des were s to red at room tempera tu re fo r 1 week , heated f o r 16 h r . at 56° , i ncuba ted in 25 mM potassium phospha te , pH 6.8 (56° ) f o r 8 m i n . , and then s ta ined wi th G i e m s a - t r y p s i n as per Do ln ick et al_, ( 2 9 ) . S l i des were r i nsed in d i s t i l l ed wa te r , a i r d r i e d , and examined by l igh t m i c roscopy . Dot B lo t A n a l y s i s of Genomic D N A Known amounts (0.1 to 20 ug) of h igh molecular we igh t D N A ( p r e p a r e d as d e s c r i b e d above) were dena tu red in 0 .3 N NaOH at room tempera tu re f o r 18 h r . , c h i l l e d , mixed wi th an equal volume of co ld 2 M ammonium acetate and 80 ul a l iquots were spot ted onto n i t roce l lu lose f i l t e r s us i ng a h y b r i - d o t manifold ( B e t h e s d a Research L a b s . , G a i t h e r s b u r g , M D . ) . T h e f i l t e r s were then washed in 3 M NaCI , 10 mM sodium E D T A , pH 7 . 5 , a i r - d r i e d and then baked u n d e r vacuum at 80°C fo r 2 h r . ( 45 ) . H y b r i d i z a t i o n wi th n i c k - t r a n s l a t e d 3 2 P - l a b e l e d D H F R c D N A was c a r r i e d out as d e s c r i b e d above . A f t e r h y b r i d i z a t i o n the f i l t e r s were washed sequen t ia l l y in 2 x S S C / 0 . 1 % S D S at room tempera tu re , 0.1 x S S C / 0 . 1 % S D S at 50°C, and f i na l l y in 0.1 x S S C at room tempera tu re . S q u a r e s of n i t roce l lu lose c o r r e s p o n d i n g to the spots were counted by l i qu id sc in t i l l a t ion c o u n t i n g . - 75 -R E S U L T S P u r i t y of Form 1 and Form 2 L5178Y ( R 4 ) D H F R In o r d e r to determine the ex ten t of an t igen ic d i v e r s i t y of the two forms of D H F R e x p r e s s e d by h i gh l y M T X - r e s i s t a n t L5178Y c e l l s , ant ibod ies aga ins t these enzymes had . to be p r e p a r e d us i ng h i g h l y pu r i f i ed p repa ra t i ons of the a n t i g e n s . A s d e s c r i b e d p r e v i o u s l y ( 15 ) , form 1 and 2 D H F R s were separa ted f rom each o ther by a f f i n i t y ch romatog raphy on M T X - s e p h a r o s e . To ensu re tha t form 2 D H F R (wh ich fa i led to b ind to co lumn, because of i ts i nab i l i t y to b i nd M T X ) was f ree of any form 1 D H F R wh ich may have fa i led to b ind to the column due to sa tu ra t ion of all the M T X b i n d i n g s i t e s , f r ac t i ons con ta in ing form 2 D H F R ac t i v i t y f rom the M T X - s e p h a r o s e were poo led , concen t ra ted and chromatographed t h r o u g h a s e c o n d , ident ica l M T X -sepharose co lumn. T h i s p rocess resu l ted in the p repa ra t i on of form 2 D H F R f ree of form 1 D H F R as determined by t i t ra t ion aga ins t M T X . Bo th form 1 and form 2 enzymes were f u r t h e r p u r i f i e d by gel f i l t r a t i on t h r o u g h sephadex G-100 as d e s c r i b e d p r e v i o u s l y ( 15 ) . Sodium dodecy l su lpha te po lyac ry lamide gel e l e c t r o p h o r e s i s , fo l lowed by detect ion of the p ro te ins wi th the h i g h l y sens i t i ve s i l v e r s t a i n , showed that form 1 D H F R was homogeneous at th i s s t age , whereas the form 2 D H F R p repara t i on had contaminat ing p ro te ins ( p r e v i o u s l y unde tec ted by s ta in ing wi th coomassie b lue ) ( F i g . 2 - 1 , lane 2 ) . Porc ion dye agarose ma t r i ces , of wh ich C i b a c r o n B lue F3GA is one , are t hough t to b ind enzymes con ta in ing a d inuc leo t ide fo ld (31 ) . D ihyd ro fo la te reduc tase has been iso lated f rom Walker 256 carc inoma ce l ls by ch romatog raphy t h r o u g h a C i b a c r o n B lue F3GA agarose column (32 ) . Form 2 D H F R af ter gel f i l t r a t i o n , was the re fo re - 76 -F I G U R E 2-1 Sodium dodecy l su l pha te -po l yac ry l am ide gel (13%) e lec t rophores i s of form 2 D H F R p repara t i on f rom L5178Y ( R 4 ) ce l ls a f ter pu r i f i ca t i on t h r o u g h Sephadex G100 ( lane B ) and a f ter f u r t h e r pu r i f i ca t i on t h r o u g h D E A E - A f f i g e l B lue ( lane A ) . Lane C : Molecu lar we ight s t anda rds ob ta ined f rom B io rad L a b o r a t o r i e s , R i c h m o n d , C a . (From top to bot tom: phospho ry l ase b, 94 ,000; bov ine serum a lbum in , 68 ,000; o v a l b u m i n , 43 ,000 ; ca rbon i c a n h y d r a s e , 30 ,000; soyabean t r y p s i n i n h i b i t o r , 21 ,000 ; l y sozyme , 14 ,300) . T h e gel was s ta ined by the s i l v e r s ta in ing method d e s c r i b e d in the Mater ia ls and Methods sec t i on . A r r o w ind ica tes the pos i t ion of D H F R in lanes A and B . 77 - 78 -chromatographed t h r o u g h an A f f i ge l b lue ( C i b a c r o n B lue F 3 G A ) (B io Rad L a b s , R i chmond , C A . ) column acco rd ing to Johnson et a[, ( 32 ) . However , r e c o v e r y of enzyme ac t i v i t y was ex t remely low. T h e above au tho rs have repor ted 85% b ind ing and quan t i ta t i ve r e c o v e r y of p u r i f i e d DHFR from Walker 256 carc inoma c e l l s . T h e v e r y low r e c o v e r y of form 2 DHFR in th is case may be a re f lec t ion of the a l te red na tu re of the enzyme. Ch roma tog raphy of the enzyme was the re fo re c a r r i e d out on a D E A E - a f f i g e l b lue column (B io Rad L a b s , R i chmond , C A . ) . D E A E - a f f i g e l b lue is an a f f i n i t y mat r ix made by coup l ing C ibac ron B lue F3GA to D E A E Bioge l A . T h i s p rocedu re resu l ted in the b i nd ing of g rea te r than 70% of form 2 DHFR ac t i v i t y and quan t i ta t i ve e lu t ion wi th 1.5 M KCI in 50 mM T r i s - C I , pH 7 . 5 . Sodium dodecy l su lphate po lyacry lamide gel e lec t rophores is of th i s p repara t ion showed that i t was now f ree of some of the contaminat ing h i ghe r molecular we igh t p ro te ins ( F i g . 2 - 1 ) . However it was not poss ib le to p u r i f y the enzyme to total homogenei ty s ince a minor contaminant , a 14,000 dal ton pro te in b a n d , pe rs i s ted in the p r e p a r a t i o n . Whether the po lypep t ide rep resen ts a b reakdown p roduc t of D H F R , or is a d i f f e ren t pro te in has not been de te rm ined , a l though recent H P L C pu r i f i ca t i on data ind ica tes that it may be a b reakdown p r o d u c t . A n t i g e n i c D i v e r s i t y Pu r i f i ed form 1 D H F R ( f rom sephadex G-100) and form 2 D H F R ( f rom D E A E af f igel b lue) were used to p repa re serum ant ibod ies in r a b b i t s . T h e IgG f rac t i ons were p u r i f i e d f rom the a n t i - s e r a by ch romatography on D E A E A f f i ge l B l u e , and these p repa ra t i ons were - 79 -used fo r all of the s u b s e q u e n t expe r imen t s . F i g . 2-2 ( A ) shows the react ion of form 1 D H F R and form 2 D H F R to i nc reas ing c o n c e n -t ra t ions of a n t i - F o r m 1 I gG . A b s o r b a n c e at 414 nm is a measure of the amount of an t ibody bound to the a n t i g e n . In th i s exper iment e i ther form 1 or form 2 DHFR was bound to the micro E L I S A plate and then reacted aga ins t i nc reas ing concen t ra t ions of an t i - f o rm 1 I gG . T h i s an t ibody reacted s t r o n g l y wi th form 1 D H F R , as would be e x p e c t e d , bu t less s t r o n g l y wi th form 2 D H F R . F i g . 2-2 ( B ) shows the react ion of form 2 D H F R wi th i nc reas ing concen t ra t ions of an t i - f o rm 2 I gG . A s can be seen f rom these two f i g u r e s , the an t i - f o rm 2 IgG was of a much lower t i t e r than an t i - f o rm 1 IgG and in the compet i t ion exper iments to be d e s c r i b e d be low, an t i - fo rm 1 and an t i - fo rm 2 IgG p repa ra t i ons were d i l u ted 500 fo ld and 50 fo ld r e s p e c t i v e l y . A l t h o u g h the re appears to be a d i f f e rence in the a f f i n i t y of an t i - fo rm 1 IgG fo r form 1 and 2 D H F R s ( F i g . 2-2 ( A ) ) , t h i s d i f f e rence cou ld be exp la ined by d i f f e rences in the b i n d i n g e f f i c iences of the two forms of the enzyme to the micro E L I S A p la te . In o r d e r to determine if the two forms were an t i gen ica l l y d i v e r s e , the fo l lowing exper iments were c a r r i e d ou t . In the f i r s t i n s t a n c e , form 1 D H F R (10 ug) was bound to the mic ro t i te r p late and then al lowed to react wi th an t i - f o rm 1 IgG ( f i xed concen t ra t i on : 500 fo ld d i lu t ion of s t ock ) wh ich was mixed wi th an i nc reas ing amount of e i the r form 1 o r form 2 D H F R jus t p r i o r to incuba t ion wi th the plate bound a n t i g e n . T h i s exper iment formed the bas is of a compet i t ion assay and al lowed us to determine the degree of an t igen ic d i v e r s i t y between the two forms of D H F R . F i g . 2-3 ( A ) shows data f rom such an expe r imen t . Whereas - 80 -F I G U R E 2-2 T i t r a t i on of A n t i - D H F R A n t i b o d i e s . A ) T i t r a t i on of an t i - fo rm 1 IgG aga ins t form 1 and form 2 D H F R s us i ng a micro E L I S A assay method d e s c r i b e d in the t e x t . T h e an t igens (e i the r form 1 or form 2 D H F R ) were bound to the mic ro t i te r plate and al lowed to i n te rac t wi th ser ia l d i l u t i ons of an t i - f o rm 1 I gG . A b s o r b a n c e at 414 nm is a measure of the degree in te rac t ion between the an t ibody and ant igen (see Mater ia ls and Methods s e c t i o n ) . ( • ) Form 1; (o) Form 2. B ) T i t r a t i on of an t i - f o rm 2 IgG aga ins t form 2 D H F R . Exper imenta l cond i t ions were as d e s c r i b e d above and in the t e x t . Con t ro l s were c a r r i e d out in each case by b i n d i n g un re la ted p ro te ins ( e . g . bov ine serum a lbum in , or non immune rabb i t serum) to the plate and a l lowing the an t ibod ies to i n te rac t as above . Con t ro l A414 va lues n e v e r exceeded 0 . 0 1 . I ' a i " ) V i T I T R A T I O N O F F O R M 1 A N O F O R M 2 D H F R A G A I N S T A N T I - F C R M 1 IgG A 1.2 -• .1 ' ' ' ' 0.012 0.12 12 12 120 ANTI-FORM 1 DHFR B 0.3 -0.23 2.3 23 230 A N T I - F O R M 2 D H F R (/ig) F I G U R E 2-3 A ) Compet i t ion assay i l l u s t r a t i ng an t igen ic d i v e r s i t y between form 1 and 2 D H F R s us ing the micro E L I S A assay d e s c r i b e d in the t e x t . Form 1 D H F R (10 ug) was bound to the mic ro t i te r plate and al lowed to i n te rac t w i th a f i x e d concen t ra t ion of an t i - f o rm 1 IgG (500 fo ld d i lu t ion of s t ock ) in the p resence of i nc reas ing concen t ra t ions of e i ther form 1 or form 2 D H F R . T h e ex ten t of compet i t ion is rep resen ted by the pe rcen t abso rbance at 414 nm of cont ro l ( i . e . in the absence of compet ing a n t i g e n ) . A b s o r b a n c e at 414 nm is a measure of the degree of i n te rac t ion between the an t ibody and the ant igen (see Mater ia ls and Methods s e c t i o n ) . B ) A s fo r A ) excep t tha t form 2 D H F R (10 ug) was bound to the plate and al lowed to i n te rac t wi th a f i x e d concen t ra t ion of a n t i -form 2 IgG (50 fo ld d i lu t ion of s t o c k ) in the p resence of i nc reas ing concen t ra t ions of e i the r form 2 or form 1 D H F R . FORM 1 OR 2 OHFR (pg) - 84 -f ree form 1 D H F R competed s t r o n g l y wi th the plate bound D H F R ( form 1) fo r the an t i - f o rm 1 I g G , f ree form 2 D H F R on l y pa r t i a l l y competed wi th plate bound D H F R ( form 1) fo r the same a n t i b o d y . The data ind icate tha t f ree form 1 D H F R even tua l l y complete ly sa tu ra tes the a n t i b o d y , leav ing almost none or v e r y l i t t le f ree an t ibody to i n te rac t with the plate bound a n t i g e n . Free form 2 D H F R on l y pa r t i a l l y sa tu ra tes the an t i body because on l y some of the de terminants are s imi lar to form 1 D H F R . T h i s enzyme is unable to compete wi th plate bound form 1 D H F R any f u r t h e r because it has de terminants not recogn ized by th i s a n t i b o d y , leav ing a cons ide rab le amount of f ree an t ibody to in te rac t wi th the plate bound form 1 D H F R , even at h i gh concen t ra t ions of f ree form 2 D H F R . T h e d i f f e rence in the a f f in i t ies of the two forms fo r D H F R fo r an t i - f o rm 1 IgG can be determined to be approx imate ly 65%. A s imi lar exper iment c a r r i e d out by b i n d i n g form 2 D H F R (10 ug) to the mic ro t i te r plate and then a l lowing the two forms of the enzyme to compete fo r an t i - f o rm 2 IgG (50 fo ld d i lu t ion of s tock ) again demonst ra ted a d i f fe ren t ia l a f f i n i t y of the two forms of the enzyme fo r an t ibody to form 2 D H F R , wi th form 2 D H F R hav ing a 25% g rea te r a f f i n i t y than form 1 D H F R . T h e two forms of D H F R , a l though e x h i b i t i n g some c r o s s - r e a c t i v i t y , can c l ea r l y be d i s t i n g u i s h e d as an t igen ica l l y d i s t i n c t molecu les. O v e r p r o d u c t i o n of D H F R We have p r e v i o u s l y shown (5 ,15) tha t the res i s tan t ce l ls had a h i g h e r D H F R spec i f i c ac t i v i t y than the w i l d - t y p e sens i t i ve c e l l s , and tha t th i s ac t i v i t y was con t r i bu ted by both forms of the enzyme. However , we had not determined whe ther th i s i nc rease in spec i f i c - 85 -ac t i v i t y was due to an inc rease in the ca ta ly t i c a c t i v i t y o r an inc rease in the amount of the enzyme. In o r d e r to determine t h i s , the p ro te ins in exponen t ia l l y g row ing res i s tan t ce l ls were label led wi th 3 H - l e u c i n e and S D S - p o l y a c r y l a m i d e gel e lec t rophores i s of the ce l l u la r lysates c a r r i e d ou t . A f t e r a u t o r a d i o g r a p h y , an abundan t p ro te in band c o r r e s p o n d i n g to approx imate ly 19,000 molecular we igh t was c lea r l y v i s i b l e in the separa t ion p ro f i le of the res i s tan t cel l l ysa te ( F i g . 2 - 4 , lane 3 ) . Par t ia l pu r i f i ca t i on of D H F R on a M T X - s e p h a r o s e a f f i n i t y column conf i rmed that th i s band was D H F R ( form 1) because of i ts s t r ong a f f i n i t y to the column ( F i g . 2 - 4 , lane 1 ) . Howeve r , a rad io labe led p ro te in band of approx imat ley 19,000 da l tons was also detected in the pro te in m ix tu re that d id not b i nd to the column and could be form 2 (low M T X - a f f i n i t y ) D H F R ( F i g . 2 - 4 , lane 2 ) . T h e inc reased spec i f i c ac t i v i t y of D H F R in the res i s tan t ce l ls appears to be due to an inc rease in the amount of the enzyme and not due to an inc rease in i ts ca ta ly t i c a c t i v i t y . To determine whether the p resence of h igh levels of D H F R in these res is tan t ce l ls was due to the p resence of abundan t D H F R mRNA ac t i v i t y total mRNA from both the sens i t i ve and res i s tan t ce l ls was t r ans la ted in v i t r o in the p resence of 3 5 S - m e t h i o n i n e , and the p ro te in p roduc t s ana lyzed by S D S - p o l y a c r y l a m i d e gel e lec t rophores i s fo l lowed by a u t o r a d i o g r a p h y . F i g . 2-5 c l ea r l y shows the p resence of an abundan t p ro te in band (~19,000 da l ton ) a f ter t rans la t i on of mRNA f rom the res i s tan t ce l ls ( F i g . 2 - 5 , lane 1 ) , wh ich was not detectab le in the t rans la t i on p r o d u c t s of m R N A f rom the w i l d - t y p e sens i t i ve c e l l s . Immunoprecip i tat ion of the t rans la ted p roduc t s f rom the r e s i s -tant ce l ls us i ng an t i - fo rm 1 D H F R IgG or an t i - fo rm 2 IgG resu l ted in - 86 -F I G U R E 2-4 Sodium dodecy l su l pha te -po l yac r y l am ide gel (13%) e lec t ropho res i s of 3 H - l e u c i n e labeled p ro te ins and pa r t i a l l y p u r i f i e d 3 H - l a b e l e d p repa ra t i ons of form 1 and form 2 D H F R from L5178Y ( R 4 ) c e l l s . A r r o w ind ica tes the pos i t ion of D H F R b a n d . Lane 1: 3 H - f o r m 1 D H F R ( M T X - s e p h a r o s e column bound a c t i v i t y ) ; Lane 2 : 3 H - f o r m 2 D H F R ( M T X - s e p h a r o s e column unbound a c t i v i t y ) ; Lane 3 : 3 H label led p ro te ins f rom R 4 ce l l u la r l y sa te . A f t e r e lec t ro -p h o r e s i s , the gels were p repa red fo r f l u o r o g r a p h y as d e s c r i b e d in the Mater ia ls and Methods sec t i on . 200 92-5K 69K 46K 30K DHFR 14-3K - 88 -F I G U R E 2-5 mRNA d i r ec ted s y n t h e s i s of D H F R in v i t r o . Po ly ( A ) con ta in ing R N A isolated as d e s c r i b e d in the tex t f rom S and R 4 ce l ls was used to p rogram t rans la t i on react ions in v i t r o in a wheat germ s y s t e m . T h e p roduc t s were ana lyzed by e lec t rophores i s t h r o u g h a 13% sodium dodecy l su l pha te -po l yac r y l am ide gel and the gel was sub jec ted to f l u o r o g r a p h y as d e s c r i b e d in the t e x t . Lane 1: T r a n s l a t i o n p roduc t s s y n t h e s i z e d by m R N A (5 ug) f rom ( R 4 ) c e l l s . Lane 2 : T r a n s l a t i o n p roduc t s s y n t h e s i z e d by an equ iva len t c o n c e n -t ra t ion of m R N A (5 ug) f rom ( S ) c e l l s . 89 - 90 -F I G U R E 2-6 Immunoprecip i ta t ion of in v i t r o t r ans la ted D H F R by an t ibod ies to two forms of D H F R . A f t e r t rans la t i on the p roduc t s were immuno-p rec ip i ta ted and e lec t rophoresed t h r o u g h a 13% sodium dodecy l su l pha te -po l yac ry l am ide g e l , fo l lowed by f l u o r o g r a p h y as d e s c r i b e d in the t e x t . Lane 1: 1 4 C - m e t h y l a t e d molecular we igh t p ro te in s t anda rds (Amersham C o r p . , O a k v i l l e , O n t . ) . Lane 2: T r a n s l a t i o n p r o d u c t s s y n t h e s i z e d u n d e r the d i rec t i on of p o l y A - c o n t a i n i n g R N A from R 4 ce l ls (as in F i g . 2 - 5 ) . Lane 3 : T rans l a t i on p roduc t s s y n t h e s i z e d as in lane 2 but immuno-p rec ip i ta ted us i ng an t i - f o rm 2 I gG . Lane 4 : A s in lane 3 bu t immunoprec ip i ta ted us i ng an t i - fo rm 1 I gG . Lane 5: 3 H marker D H F R p repa red f rom L5178Y ( R 4 ) c e l l s . 91 - 92 -the spec i f i c p rec ip i ta t ion of the 19,000 molecular we ight p ro te in ( F i g . 2-6) wh ich co -mig ra ted wi th 3 H - l a b e l l e d D H F R isolated f rom these ce l ls ( F i g . 2 - 6 , lane 5 ) , con f i rm ing tha t the abundan t p ro te in indeed is D H F R (both form 1 and form 2 ) . A l t h o u g h the two forms of D H F R in the res i s tan t ce l ls have been p r e v i o u s l y shown to d i f f e r s l i g h t l y in t he i r molecular we igh t s , wi th form 2 h a v i n g a h i g h e r molecular we igh t than form 1 (15) the p resen t data and subsequen t exper imen ts have shown tha t the two forms have v e r y s imi lar molecular we igh ts (V | 9 ,000) . Cha rac te r i za t i on of Tota l C e l l u l a r D H F R m R N A s Tota l ce l l u la r mRNA was iso lated f rom L5178Y ( S ) and M T X -res i s tan t L5178Y ( R 4 ) c e l l s . T h i s mRNA was e lec t rophoresed on 1.5% agarose gels con ta in ing 2 .2 M fo rma ldehyde and t r a n s f e r r e d onto n i t roce l lu lose p a p e r . T h e t r a n s f e r r e d mRNA was then h y b r i d i z e d to a 3 2 P - l a b e l e d D H F R c D N A ( N o r t h e r n blot a n a l y s i s ) . T h e map of the c D N A is shown in C h a p t e r 1, ( F i g . 1-7) . T h e resu l t s of such an exper imen t are shown in F i gu re 2 -7 . Mu l t ip le mRNA spec ies are p resen t in both 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 c e l l s . T h e s izes of these va r i ous D H F R m R N A s v a r y f rom g rea te r tha t 5 Kb to 0.85 K b . From the in tens i t ies of the h y b r i d i z a t i o n s igna ls and the amount of R N A app l ied to the gels it is c lear tha t the R 4 ce l ls have a h i g h e r content of D H F R mRNA than the ( S ) c e l l s . T h i s is in agreement wi th the h i ghe r m R N A ac t i v i t y i n f e r r e d f rom the in v i t r o t rans la t i on d e s c r i b e d above . T h e two major forms of m R N A s p r e s e n t in both the cel l l ines are the 1.65 Kb and 0.85 Kb fo rms . Two of the e igh t mRNA spec ies ( the 1.05 Kb and 2.2 K b ) p resen t in the res i s tan t ce l ls are absent in the M T X - s e n s i t i v e ce l ls ( F i g . 2-7 ind ica ted b y a r r o w s ) . - 93 -F I G U R E 2-7 No r the rn b lot ana l ys i s of total L5178Y ( S ) and ( R 4 ) Po ly A + R N A . Tota l ce l l u la r R N A was isolated f rom L5178Y ( S ) and ( R 4 ) c e l l s , the Poly ( A ) + R N A was pu r i f i ed by o l i g o - ( d T ) - c e l l u l o s e chromato-g r a p h y and then f rac t iona ted on a 1.5% agarose gel con ta in ing 2 .2 M fo rma ldehyde . T h e f rac t iona ted messenger R N A was t r a n s f e r r e d to n i t roce l lu lose paper and p robed wi th 3 2 P - l a b e l e d D H F R - c D N A as d e s c r i b e d in Exper imenta l P r o c e d u r e s . ( A ) 23 ug total poly ( A ) + R N A from ( S ) c e l l s . ( B ) 2 .5 ug total po ly ( A ) + R N A from ( R 4 ) c e l l s . ( C ) 1.0 ug total po ly ( A ) + R N A from ( R 4 ) c e l l s . A r r o w s ind ica te pos i t ions of m R N A spec ies p resen t in ( R 4 ) ce l ls bu t not v i s i b l e in ( S ) c e l l s . 94 - 95 -D H F R Gene Ampl i f i ca t ion In o r d e r to determine whe the r the inc reased amounts of D H F R m R N A s p resen t in the L5178Y ( R 4 ) ce l ls as compared to the ( S ) ce l ls were due to the p resence of mul t ip le D H F R gene copies ( i . e gene amp l i f i ca t ion ) , h igh molecular we igh t D N A was p r e p a r e d f rom ( S ) and ( R 4 ) ce l ls and d iges ted wi th the res t r i c t i on endonuc leases E c o R 1 , Bam H1 and T a q 1 and Hpa II. T h e res t r i c t i on f ragments t h u s genera ted were separa ted e lec t rophore t i ca l l y on 0.8% agarose ge ls t r a n s f e r r e d onto n i t roce l lu lose paper and h y b r i d i z e d to a 3 2 P - l a b e l e d c loned mur ine D H F R c D N A probe d e r i v e d f rom the p lasmid p D H F R II ( 18 ) . T h e map of the p robe is shown in F i g . 1-7. T h e resu l t s of t h i s exper iment are shown in F i g . 2 -8 . C l e a r l y t he re are many more copies of the D H F R gene in the R 4 ce l ls as compared to the ( S ) c e l l s . T h e number and s izes of the f ragments genera ted b y the res t r i c t i on endonuc leases EcoR1 and Bam H1 ( F i g . 2-8 and Tab le 2 .1 ) agree well w i th pub l i shed v a l u e s . To quant i ta te the degree of ampl i f icat ion of the D H F R genes in ( R 4 ) ce l ls a dot b lot ana lys i s of genomic D N A f rom S and R 4 ce l ls was c a r r i e d ou t . T h e resu l t s of th i s ana l ys i s are shown in F i g . 2 -9 . S q u a r e s of the n i t roce l lu lose paper a round the dots were cu t and counted in a l i qu id sc in t i l l a t ion c o u n t e r . From the s lope of the l ines in F i g . 2-9 it is appa ren t that t he re are 25- fo ld more sequences h y b r i d i z i n g to the D H F R probe in R 4 ce l ls than in S c e l l s . A n a l y s i s of methaphase chromosomes by G i e m s a - t r y p s i n band ing revea led tha t the s tab le inc rease in the amount of D H F R in the res i s tan t ce l ls is accompanied by a homogeneously s ta in ing reg ion ( H S R ) in two of i ts chromosomes ( F i g . 2 - 1 0 ) , co r re l a t i ng well wi th - 96 -F I G U R E 2-8 Sou the rn B lo t A n a l y s i s of L5178Y ( S ) and ( R 4 ) Genomic D N A . H igh molecular we ight DNA was ex t rac ted f rom ( S ) and ( R 4 ) ce l ls as d e s c r i b e d in the t e x t . T h e D N A was d iges ted to complet ion wi th the res t r i c t i on endonuc leases E'coRI, Hap II and Bam H1 and the res t r i c t i on f ragments were separa ted by e lec t rophores i s in a 0.8% agarose gel con ta in ing e th id ium bromide . A f t e r d e n a t u r a t i o n , the gel was neu t ra l i zed and the DNA t r a n s f e r r e d onto n i t roce l lu lose p a p e r . D H F R spec i f i c res t r i c t i on f ragments were v i s u a l i z e d by h y b r i d i z a t i o n wi th n i ck t rans la ted 3 2 P - l a b e l e d D H F R - c D N A probe ( fo r the map of the p r o b e , see C h a p t e r 1 ) . A : L5178Y ( S ) D N A (20 p g ) : A u t o r a d i o g r a p h i c e x p o s u r e was fo r 3 d a y s . Lane 1: Eco R1 d i g e s t . Lane 2 : Hpa II d i g e s t . Lane 3: Bam H1 d i ges t . B : L5178Y ( R 4 ) DNA (5 ug ) : fo r 2 h o u r s . A u t o r a d i o g r a p h i c e x p o s u r e was Lane 4 : Eco R1 d i g e s t . Lane 5: Hpa II d i ges t . Lane 6: Bam H1 d i g e s t . T h e numbers on the r i g h t hand s ide of the f i g u r e rep resen t s izes in k i lobases of DNA molecular we igh t marke rs r u n on the same g e l . 5 4<3 3-4 - 98 -T A B L E 2.1 Res t r i c t i on Endonuc lease Fragment Leng ths H y b r i d i z i n g to 3 2 P - D H F R c D N A . Eco R1 L 5 1 7 8 Y ( R 4 ) T a q 1 Bam H1 >20 a 14 >20 14 12 20 7 9 .5 5 .3 6.1 5.1 3 .9 3.15 2.15 1.9 Eco R1 Pub l i shed V a l u e s * Bam H1 14.8 >20.5 6.0 17.6 5 .4 4 .8 3.4 a . Res t r i c t i on f ragment length va lues are g i ven in k i l obases . From C r o u s e et al (39 ) . - 99 -F I G U R E 2-9 Dot b lot ana lys i s of genomic D N A f rom L5178Y ( S ) and ( R 4 ) c e l l s . Inc reas ing amounts of genomic D N A were app l ied to a n i t r o -ce l lu lose f i l t e r and h y b r i d i z e d to n i ck t r ans la ted 3 2 P - l a b e l e d D H F R -c D N A . S q u a r e s con ta in ing the app l ied DNA were cu t o u t , t r a n s f e r r e d to sc in t i l l a t i on v ia l s and counted in a l i qu id sc in t i l l a t ion c o u n t e r . A L5178Y ( S ) D N A . • L5178Y ( R 4 ) D N A . 100 i J I I I I L_ 2 4 6 8 1 0 12 D N A U g ) - 101 -F I G U R E 2-10 G i e m s a - t r y p s i n s ta ined metaphase chromosome sp read of an L5178Y ( R 4 ) cel l p r e p a r e d as d e s c r i b e d in Mater ia ls and Methods sec t i on . A r r o w s ind ica te prominent homogeneously s ta in ing reg ion ( H S R ) on two of the chromosomes. 102 - 103 -the obse rva t i ons made p r e v i o u s l y by o ther w o r k e r s (36) tha t HSR chromosomes a re p resen t in ce l ls w i th s tab l y ampl i f ied D H F R g e n e s . D I S C U S S I O N The resu l t s d e s c r i b e d in t h i s repo r t ind ica te tha t h i g h l y M T X -res is tan t L5178Y mouse leukemia ce l ls ob ta ined by a mul t is tep se lect ion p rocess o v e r p r o d u c e two an t igen ica l l y d i s t i n c t forms of d i hyd ro fo la te r e d u c t a s e , the chemical and k ine t i c p rope r t i es of wh ich have been d e s c r i b e d in detai l e lsewhere (15 ) . T h e h igh level of res i s tance in these ce l ls is in all l i ke l ihood due to the ex t reme i n s e n s i t i v i t y of one of the forms of D H F R ( form 2) to M T X , a l though the moderate o v e r -p roduc t i on of D H F R may also con t r i bu te to the mechanism of r e s i s t a n c e . I have demonst ra ted tha t serum ant ibod ies p r e p a r e d in the rabb i t aga ins t one form of the enzyme on l y pa r t i a l l y i n te rac t with the o ther f o r m , i nd i ca t i ng cons ide rab le an t igen ic d i v e r s i t y between the two forms of D H F R . From compet i t ion assays ( F i g . 2-3) it can been shown that the an t ibody aga ins t form 1 ( i . e . h igh M T X - a f f i n i t y ) D H F R has a 65% h i g h e r a f f i n i t y fo r th i s form of the enzyme than the a l te red form ( form 2 , i . e . low M T X - a f f i n i t y ) , whereas one would expec t equal a f f in i t ies if the enzymes were an t i gen ica l l y s im i la r . T h e an t ibody aga ins t form 2 D H F R has a 25% h i g h e r a f f i n i t y fo r th i s form than form 1 D H F R . These resu l t s conf i rm ou r p rev i ous obse rva t i ons (15) tha t the two forms d i f f e r in t he i r p r ima ry s t r u c t u r e s , leading to d i f f e ren t 3-d imensional con fo rmat ions . A l t h o u g h i t had p r e v i o u s l y been shown that these M T X - r e s i s t a n t cel ls had a h i ghe r D H F R spec i f i c ac t i v i t y than the w i l d - t y p e sens i t i ve - 104 -c e l l s , it had not been determined whe ther th is was due to gene amp l i f i ca t ion , as has been shown to be the case in o ther M T X -res i s tan t cel l l ines (11 ,14 ,17) w i th the excep t ion of a M T X - r e s i s t a n t HL -60 cel l l ine ( C h a p t e r 5 ) . T h a t the h igh D H F R spec i f i c ac t i v i t y is due to the o v e r p r o d u c t i o n of D H F R pro te in is i l l us t ra ted in F i g . 2 - 4 , wh ich c l ea r l y shows the abundance of D H F R in the ce l l u la r lysate f rom these ce l ls wh ich were label led wi th 3 H - l e u c i n e . A h igh amount of r ad ioac t i v i t y was i nco rpo ra ted into a 19,000 molecular we igh t p ro te in band wh ich was s u b s e q u e n t l y p u r i f i e d by M T X - s e p h a r o s e a f f i n i t y ch romatog raphy ( F i g . 2 - 4 ) . Both forms of D H F R were detected s ince a 19,000 dal ton band is also p resen t in the mater ia l wh ich d id not b ind to the a f f i n i t y co lumn. T h e ques t ion of whe ther two forms of D H F R wi th d i f f e ren t M T X - b i n d i n g a f f in i t ies have s imi lar molecular we igh ts or not is an impor tant one , espec ia l l y in l igh t of the obse rva t i ons of Melera et a_[, (19) who have shown tha t two a n t i -f o l a te - res i s t an t sub l ines of C h i n e s e hamster ce l ls conta in reduc tases of s imi lar molecular w e i g h t s , bu t d i f f e r in t he i r s e n s i t i v i t y to M T X . F l in to f f and Essan i (14) have also shown that a l te ra t ions of enzymolog ica l p rope r t i es of C h i n e s e hamster o v a r y cel l d i hyd ro fo la te reduc tase can o c c u r w i thout obv ious e f fec ts on the molecular we ight of the enzyme. A s p r e v i o u s l y d e s c r i b e d , in all cases of o v e r p r o d u c t i o n of D H F R repor ted so f a r , the e levated levels have been assoc ia ted wi th the p resence of abundan t D H F R mRNA and an inc rease of gene copy number . In these c e l l s , the p resence of a major p ro te in band c o r r e s p o n d i n g to D H F R af ter in v i t r o t rans la t i on of the total m R N A - 105 -f rom the res i s tan t cel ls ( bu t not f rom the w i l d - t y p e sens i t i ve c e l l s , F i g . 2-5) demonst ra tes the abundance of mRNA ac t i v i t y f o r D H F R . Immunoprecip i ta t ion of D H F R f rom the t r ans la ted p roduc t s ind ica tes that the an t ibod ies are p u r e and able to recogn ize an t igen ic s i tes on newly s y n t h e s i z e d po lypep t ide c h a i n s . In a d d i t i o n , an t ibod ies to both forms of - D H F R were able to p rec ip i ta te D H F R , the p rec ip i ta ted p roduc t us i ng an t i - fo rm 2 IgG is p resen t in a lower amount ( F i g . 2 - 5 ) , is of a v e r y s l i gh t l y h i ghe r molecular we igh t than that p rec ip i t a ted by an t i - fo rm 1 IgG and is most p r o b a b l y form 2 D H F R because of the g rea te r a f f i n i t y of an t i - fo rm 2 IgG fo r th i s enzyme (see F i g . 2-3 ( b ) ) , a l though the co -p rec ip i t a t i on of a small amount of form 1 D H F R cannot be ru led ou t . T h e inc rease in the mRNA cod ing fo r D H F R in ( R 4 ) ce l ls was conf i rmed b y N o r t h e r n blot ana lys i s wh ich showed tha t the re are many more copies of mRNA h y b r i d i z i n g to a spec i f i c D H F R c D N A probe in the ( R 4 ) ce l ls than in the ( S ) c e l l s . U t i l i z i ng a dot blot assay (da ta shown in C h a p t e r 5) s imi lar to the one d e s c r i b e d fo r D N A dot ana l ys i s it was found tha t the R 4 ce l ls had a 15- fo ld inc rease in D H F R m R N A . T h e No r the rn blot ana l ys i s ( F i g . 2-7) also showed what has p r e v i o u s l y been demonst ra ted by Se tze r et a[, ( 4 0 ) , i . e . that cons ide rab le s ize he te rogene i ty ex i s t s in the mRNA cod ing f o r DHFR in the mouse. S i x spec ies of D H F R mRNA were found to be p resen t in the ( S ) ce l ls and e igh t in the ( R 4 ) c e l l s . T h e s ize he te rogene i ty has been shown to be due to d i f f e rences in the length of the 3' un t rans la ted (or n o n - c o d i n g ) reg ion (40 ) . Mu l t ip le D H F R m R N A s are not r es t r i c t ed to the mouse bu t are also f ound in Ch inese hamster ce l ls (45) and human cel l l ines ( 4 6 ) . T h e mul t ip le forms ex i s t not on ly in M T X - r e s i s t a n t ce l ls bu t also in the parenta l ce l ls - 106 -and in in tac t mouse l i ve r ( 4 0 ) , a l though in th i s p resen t s t u d y the p resence of two -add i t i ona l , weak ly h y b r i d i z i n g spec ies of D H F R mRNA in the ( R 4 ) ce l ls as opposed to the ( S ) ce l ls ( F i g . 2-7) ra ise the poss ib i l i t y tha t these may code fo r the a l te red enzyme, t hough d i r e c t ev idence is l a c k i n g . On the o ther h a n d , s ince the molecular we igh ts of the normal and a l te red D H F R s are so s im i l a r , one would not expec t any d i f f e rences in the s izes of the m R N A s cod ing fo r t hem. In th i s la t ter case the two add i t iona l m R N A s p resen t in the R 4 ce l ls may resu l t as a consequence of the gene ampl i f icat ion p r o c e s s . T h e m R N A s d i f f e r i ng in the leng ths of the 3' un t rans la ted reg ion are in all l i ke l ihood the resu l t of the p resence of mul t ip le p o l y -a d e n y l a t i o n / t r a n s c r i p t i o n terminat ion s igna ls in the 3' un t rans la ted reg ion (47 ) . A sho r t s e q u e n c e , A A T A A A , was found by Proudfoo t and Brown lee (48) to be p resen t approx imate ly 12 to 15 nuc leo t ides ups t ream of the po lyadeny la t ion s i te in the B g lob in gene . S ince t h e n , t h i s sequence or a va r i an t of it has been found in many o ther genes and is now recogn ized as a bonaf ide po lyadeny la t ion s i g n a l . Whether the m R N A s are genera ted by the p r o c e s s i n g of a mRNA spec ies con ta in ing a large 3' un t rans la ted reg ion o r by random terminat ion of t r a n s c r i p t i o n at the mul t ip le terminat ion s i tes is not ye t c l e a r . Some ev idence ex i s t s fo r the post t r a n s c r i p t i o n a l p r o c e s s i n g of the 3' un t rans la ted r e g i o n . T h e s ign i f i cance of the t r a n s c r i p t i o n of the D H F R gene f a r into the 3' un t rans la ted reg ion is not c lear and whether the p resence or absence of the 3' un t rans la ted reg ions has any b io logical f unc t i on in the s y n t h e s i s of D H F R is not ye t known . Mul t ip le m R N A s d i f f e r i ng in the length of 3' un t rans la ted reg ions are t r a n s c r i b e d f rom o the r ce l lu la r g e n e s : e . g . the gene cod ing f o r - 107 -the mouse n immunoglobul in heavy cha in ( 4 9 ) , a amylase (50 ) , ova lbumin l ike X gene of c h i c k e n s (51) and v iment in gene (52 ) . For the immunoglobul in gene and v iment in gene a b io log ica l f unc t i on can be a t t r i bu ted to the p resence of these mul t ip le m R N A s . In the case of the p immunoglobul in heavy cha in g e n e , the length of the t r a n s c r i p t appears to cont ro l the p roduc t i on of sec re ted o r membrane-bound an t i bod ies . T h e v iment in mul t ip le m R N A s have a t i s s u e - s p e c i f i c d i s t r i b u t i o n and the re fo re in fe r some b io log ica l f u n c t i o n . In add i t ion to the e x p r e s s i o n of two an t i gen i ca l l y d i s t i n c t forms of D H F R , the ( R 4 ) ce l ls have inc reased copies of D H F R genes as demonst ra ted by Sou the rn blot ana l ys i s as well as dot b lot a n a l y s i s . T h e inc rease in the amount of D H F R m R N A can be to ta l ly accoun ted fo r by gene ampl i f i ca t ion . S o u t h e r n blot ana lys i s has shown tha t the res t r i c t i on f ragment leng ths co r re la te well wi th the p u b l i s h e d va lues (Tab le 2 . 1 ) , a l t h o u g h , upon longer au to rad iog raph i c e x p o s u r e an add i t iona l 20 Kb EcoR1 f ragment h y b r i d i z e d wi th D H F R c D N A in the R 4 c e l l s . T h e s ign i f i cance of t h i s f ragment is not c l e a r , a l though it may rep resen t a gene rear rangement of the t y p e repo r ted by Bos tock et a[, (53) who also detected an add i t iona l EcoRI f ragment of s imi lar s ize in a D H F R - g e n e ampl i f ied cel l l i ne . O v e r p r o d u c t i o n of D H F R in M T X - r e s i s t a n t ce l ls has been shown to be assoc ia ted wi th severa l chromosomal anomalies (29 ,33^35) , wi th the p resence of a la rge homogeneously s ta in ing reg ion on one o r severa l chromosomes, o r the p resence of doub le minute chromosomes as the most f r e q u e n t l y o b s e r v e d anomal ies. Sch imke et a[, (36) have shown tha t in s tab l y ampl i f ied cel l l ines the D H F R genes are local ized on the homogeneously s ta in ing reg ions ( H S R ) , whereas in uns tab l y ampl i f ied ce l ls t hey are p resen t on ex t rachromosomal , sel f rep l i ca t i ng - 108 -elements ca l led doub le minute chromosomes (37 ) . In the h i g h l y M T X -res i s tan t L5178Y ce l ls d e s c r i b e d in t h i s repor t the r e s i s t a n c e , as well as the o v e r p r o d u c t i o n of both forms of D H F R have been h i g h l y s tab le . Ce l l s have been g rown con t i nuous l y in the absence of M T X fo r longer than one yea r w i thout the loss of any of these pa ramete rs . G i e m s a - t r y p s i n band ing ana lys i s of chromosomes f rom these ce l ls have indeed revea led HSR reg ions on two of the chromosomes of the res i s tan t ce l ls ( F i g . 2 -10 ) . In summary , t h e r e f o r e , con t inuous g rowth of mouse L5178Y ce l ls in s t ep -w i se i nc reas ing concen t ra t ions of M T X (up to 1 mM) , resu l ted in the se lect ion of cel ls wi th moderate ampl i f icat ion of D H F R g e n e s , some of these hav ing mutat ions wh ich lead to the e x p r e s s i o n of a D H F R wi th a g rea t l y reduced a f f i n i t y fo r M T X ( form 2) ( a p p r o x i -mately 100,000 f o l d ) , a Vmax one - ten th tha t of form 1 enzyme and d i f f e rences in o ther p roper t i es s u c h as i soe lec t r i c po in ts and heat s tab i l i t y ( 15 ) . Form 1 D H F R was shown to be essen t ia l l y s imi lar to that f rom the w i l d - t y p e sens i t i ve ce l ls and rep resen ted the major i ty of the total D H F R e x p r e s s e d by the res i s tan t c e l l s . It is not c lear whether the form 2 enzyme is encoded by normal ampl i f ied genes wh ich s u b s e q u e n t l y underwen t s t r u c t u r a l muta t ions , o r tha t th i s form of the enzyme is coded by a separa te gene wh ich is normal ly r e p r e s s e d in the w i l d - t y p e sens i t i ve c e l l s . T h e ant ibod ies d e s c r i b e d in t h i s chap te r may have some potent ia l l y impor tant u s e s . For i n s t a n c e , they cou ld be used to e n r i c h m R N A s cod ing p redominan t l y fo r e i ther one o r the o ther forms of D H F R fo r subsequen t c D N A s y n t h e s i s (see C h a p t e r 3 ) . In a d d i t i o n , if i t is f ound that on ly ce r ta in t y p e s of s t r u c t u r a l a l t e r -- 109 -at ions r ende r D H F R s more i nsens i t i ve to M T X i n h i b i t i o n , then the an t ibody aga ins t form 2 D H F R may be usefu l in the detec t ion of such enzymes in M T X - r e s i s t a n t c l in i ca l tumour specimens and cel l l ines in c u l t u r e . In o r d e r to determine whether the M T X - i n s e n s i t i v e form 2 D H F R is more sens i t i ve to inh ib i t i on by o ther fo late an tagon i s t s , a v a r i e t y of qu inazo l i ne and subs t i t u t ed t r i az i ne compounds were tes ted aga ins t p u r i f i e d form 2 enzyme. T h e resu l t s of these exper iments are d e s c r i b e d in A p p e n d i x I. - 110 -R E F E R E N C E S 1. H a k a l a , M . T . , Z a k r e s e w s k i , S . F . and N i c h o l , C H . (1961) Relat ion of fo l ic ac id reduc tase to amethopter in res i s tance in c u l t u r e d mammalian c e l l s . J . 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(1979) Co r re l a t i on of d ihyd ro fo la te reduc tase e levat ion wi th gene ampl i f icat ion in a homogeneously s ta in ing chromosomal reg ion in L5178Y c e l l s . J . C e l l . B i o l . 83 , 394-402. 30. D e d h a r , S . , F r e i s h e i m , J . H . , H y n e s , J . B . and G o l d i e , J . H . (1983) Inh ib i t ion of a me tho t rexa te - i nsens i t i ve d ihyd ro fo la te reduc tase f rom L5178Y ce l ls by subs t i t u t ed t r i az i nes and q u i n a z o l i n e s . Biochemical Pharmaco l . 32, 922-924. 31 . T h o m s o n , S . T . , C a s s , K . H . and S t e l l w a g e n , E . (1975) B lue d e x t r a n - s e p h a r o s e : A n a f f i n i t y column fo r the d inuc leo t ide fo ld in p r o t e i n s . P r o c . Nat . A c a d . S c i . U . S . A . 72 , 669-672. 32. J o h n s o n , S . J . , S t e v e n s o n , K . J . and G u p t a , V . S . (1980) Proc ion d y e s as a f f i n i t y l i gands and repo r te r g r o u p s fo r d ihyd ro fo la te reduc tase f rom Walker 256 ca rc inoma. C a n . J . B iochem. 58, 1252-1260. 33. B i e d l e r , J . L . , Me le ra , R .W. and S p e n g l e r , B . A . (1980) Spec i f i ca l l y a l te red metaphase chromosomes in ant i fo la te res i s tan t Ch inese hamster cel ls that o v e r p r o d u c e d ihyd ro fo la te r e d u c t a s e . C a n c e r Gene t . C y t o g e n e t . 2 , 47-60. 34. B o s t o c k , C . J . , C l a r k , E . M . , H a r d i n g , N . G . L . , Moun t s , P . M . , T y l e r - S m i t h , C , van H e y n i n g e r , V . and Wa lke r , P . M . B . (1979) - 113 -T h e development of res i s tance to methot rexate in a mouse melanoma cel l l i ne . I. C h a r a c t e r i z a t i o n of the d ihyd ro fo la te reduc tases and chromosomes in sens i t i ve and res i s tan t c e l l s . Chromosoma. 74, 153-177. 35. T y l e r - S m i t h , C . and B o s t o c k , C . J . (1981) Gene ampl i f icat ion in me tho t rexa te - res i s tan t mouse c e l l s . III. I n te r re la t i onsh ips between chromosome changes and DNA sequence ampl i f ica t ion o r l oss . J . Mo l . B i o l . 153, 237-256. 36. S c h i m k e , R . T . , B r o w n , P . C , K a u f m a n , R . J . , M c G r o g a n , M. and S l a t e , D . L . (1981) Chromosomal and ext rachromosomal local izat ion of ampl i f ied d ihyd ro fo la te reduc tase genes in c l u t u r e d mammalian c e l l s . Co ld S p r i n g H a r b o u r S y m p . Q u a n t . B i o l . X L V , 785-797. 37. K a u f m a n , R . J . , B r o w n , P . C . and S c h i m k e , R . T . (1979) Amp l i f i ed d ihyd ro fo la te reduc tase genes in uns tab l y metho-t r e x a t e - r e s i s t a n t ce l ls are assoc ia ted wi th doub le -m inu te chromosomes. P r o c . Na t . A c a d . S c i . U . S . A . 76, 5669-5676. 38. H a b e r , D . A . and S c h i m k e , R . T . (1981) Uns tab le ampl i f ica t ion of an a l te red d ihyd ro fo la te reduc tase gene assoc ia ted wi th d o u b l e -minute chromosomes. C e l l . 26, 355-362. 39. C r o u s e , G . F . , S imonsen , C C , M c E w a n , R . N . and S c h i m k e , R . T . (1982) S t r u c t u r e of ampl i f ied normal and va r i an t d i hyd ro fo la te reduc tase genes in mouse sarcoma S180 c e l l s . J . B i o l . C h e m . 257, 7887-7897. 40. S e t z e r , D . R . , M c G r o g a n , M . , N u n b e r g , J . H . and S c h i m k e , R . T . (1980) S ize he te rogene i t y in the 3' end of d ihyd ro fo la te reduc tase messenger R N A s in mouse c e l l s . C e l l . 22 , 361-370. 41 . M e l e r a , P . W . , Lew i s , J . A . , B i e d l e r , J . L . and H e s s i o n , C . (1980) A n t i f o l a t e - r e s i s t a n t C h i n e s e hamster c e l l s : Ev idence fo r d ihyd ro fo la te reduc tase gene ampl i f icat ion among i ndependen t l y d e r i v e d sub l i nes o v e r p r o d u c i n g d i f f e ren t d i hyd ro fo la te r e d u c t a s e s . J . B i o l . C h e m . 255, 7024-7028. 42. L e h r a c h , H . , D iamond, D . , Wozney , J . M . and B o e d t k e r , H . (1977) R N A molecular we igh t de terminat ions by gel e l ec t ro -phores is u n d e r d e n a t u r i n g cond i t i ons , a c r i t i ca l reexamina t ion . B i o c h e m i s t r y . 16, 4743-4752. 43. S o u t h e r n , E . M . (1975) Detect ion of spec i f i c sequences among DNA separa ted by gel e l e c t r o p h o r e s i s . J . Mo l . B i o l . 98, 503-517. 44. B l i n , N . and S t a f f o r d , D.W. (1976) Isolat ion of h igh molecular weight D N A . N u c . A c i d R e s . 3 , 2303-2308. 45. Lew i s , J . A . , K u r t z , D . T . and Me le ra , P .W. (1981) Molecu lar c lon ing of the Ch inese hamster d i hyd ro fo la te reduc tase spec i f i c c D N A and the iden t i f i ca t ion of mul t ip le d ihyd ro fo la te reduc tase - 114 -messenger R N A s in an t i f o l a te - res i s tan t Ch inese hamster lung f i b r o b l a s t s . N u c . A c i d . R e s . 9 , 1311-1322. 46. M a s t e r s , J . M . , Y a n g , J . K . , C e l l i n i , A . and A t t a r d i , G . (1983) A human d ihyd ro fo la te reduc tase pseudogene and i ts re la t ionsh ip to the mul t ip le forms of spec i f i c messenger R N A . J . M o l . B i o l . 167, 23-36. 47. S e t z e r , D . R . , M c G r o g a n , M. and S c h i m k e , R . T . (1982) Nuc leot ide sequence s u r r o u n d i n g mul t ip le po lyadeny la t ion s i tes in the mouse d ihyd ro fo la te reduc tase gene . J . B i o l . C h e m . 257, 5143-5147. 48. P roud foo t , N . J . and B r o w n l e e , G . G . (1976) 3' N o n - c o d i n g sequences in euca ryo t i c messenger R N A . N a t u r e . 263, 211-214. 49. E a r l y , P . , R o g e r s , J . , D a v i s , M . , Ca lame, K . , B o n d , M . , Wa l l , R. and Hood , L. (1980) Two m R N A s can be p roduced f rom a s ing le, immunoglobul in (j gene by a l te rna t i ve R N A p r o c e s s i n g p a t h w a y s . C e l l . 20 , 313-319. 50. T o s i , M . , Y o u n g , R . A . , H a g e n b u c h l e , O . and S c h i b l e r , U . (1981) Mul t ip le po lyadeny la t ion s i tes in a mouse a-amylase gene . N u c . A c i d . R e s . 9, 2313-2323. 51 . H e i l i g , R . , P e r r i n , F . , G a n n o n , F . , Mande l . J . L . and C h a m b o n , P . (1980) T h e ova lbumin gene fami l y : s t r u c t u r e of the X gene and evo lu t ion of dup l i ca ted sp l i t g e n e s . C e l l . 20, 625-637. 52. C a p e t a n a k i , Y . G . , N g a i , J . , F l y t z a n i s , C . N . and L a z a r i d e s , F. (1983) T i s s u e spec i f i c e x p r e s s i o n of two m R N A spec ies t r a n s c r i b e d f rom a s ing le v iment in gene . C e l l . 35 , 411-420. 53. B o s t o c k , C . J . and T y l e r - S m i t h , C . (1982) Ampl i f i ca t ion of d ihyd ro fo la te reduc tase genes and o ther D N A sequences in mouse c e l l s . In : Gene Amp l i f i ca t i on , R. Sch imke ( e d . ) , p p . 15 -21 , New Y o r k : Co ld S p r i n g H a r b o u r . 54. Hayman , R . , M c G r e a d y , R . , and V a n d e r Weyden (1978) A rap id rad iometr ic assay fo r d ihyd ro fo la te r educ tase . A n a l . B iochem. 87, 460-465. 55. B l a k e l y , R . L . (1969) T h e . b i o c h e m i s t r y of fo l i c ac id and re la ted p t e r i d i n e s . p. 93 , Wi ley , New Y o r k . - 115 -C H A P T E R 3 P O L Y S O M E I M M U N O P R E C I P I T A T I O N OF L5178Y ( R 4 ) F O R M 1 A N D FORM 2 D I H Y D R O F O L A T E R E D U C T A S E mRNA A N D S Y N T H E S I S OF C O M P L E M E N T A R Y DNA ( c D N A ) I N T R O D U C T I O N In C h a p t e r 2 I have shown tha t a h i g h l y M T X - r e s i s t a n t va r i an t of L5178Y c e l l - l i n e , the L5178Y ( R 4 ) ce l l - l i ne o v e r p r o d u c e s two a n t i -gen ica l l y d i s t i n c t forms of d i hyd ro fo la te reduc tase ( 1 ) , forms 1 and 2 , the la t ter be ing h i g h l y res i s tan t to inh ib i t i on by M T X ( 2 , 3 ) . T h e ( R 4 ) ce l ls have abundan t m R N A spec ies cod ing f o r both forms of D H F R and the re is good co r re la t ion between the m R N A abundance and the ex ten t of DHFR gene ampl i f i ca t ion . Because of the p resence of both forms of the enzyme in the same c e l l , and because form 1 ( M T X - s e n s i t i v e ) DHFR. is p resen t in g rea t excess as compared to form 2 ( M T X - r e s i s t a n t ) D H F R it has been v e r y d i f f i cu l t to p u r i f y the more i n te res t i ng form 2 D H F R fo r amino ac id sequenc ing s t u d i e s . A l t h o u g h the use of p ro te in H P L C has recen t l y resu l ted in a subs tan t i a l l y p u r e r p repa ra t i on and an amino ac id composi t ion ana lys i s has been c a r r i e d out showing t e n t a t i v e l y , tha t the two forms d i f f e r c o n s i d e r a b l y in t he i r amino ac id compos i t ions , a separa te d i rec t i on was taken to ana lyze the form 2 D H F R . T h e ava i l ab i l i t y of c D N A s cod ing fo r the two forms of D H F R , one h i g h l y sens i t i ve and one h i g h l y res i s tan t to M T X would be of use not on l y in the chemotherapeut ic aspec ts of the t reatment of M T X - r e s i s t a n t tumours (by sequenc ing the c D N A one cou ld p red i c t the amino ac id - 116 -sequence and beg in to determine the t y p e s of compounds tha t might be more potent i nh ib i t o r s of t h i s form of D H F R ) , bu t also as dominant se lectab le marke rs in gene t r a n s f e r e x p e r i m e n t s , and in the e l u c i d a -t ion of the D H F R gene o r g a n i z a t i o n . With the ava i l ab i l i t y of spec i f i c an t ibod ies aga ins t the two forms of D H F R , it was dec ided to e n r i c h the m R N A s cod ing fo r the two forms of D H F R by polysome immunoprec ip i ta t ion and s y n t h e s i z i n g c D N A s f rom these en r i ched m R N A s , fo l lowed by c lon ing of the c D N A s into a su i tab le v e c t o r . Polysome immunoprec ip i ta t ion has been used as a t echn ique fo r e n r i c h i n g spec i f i c m R N A s wi th a cons ide rab le amount of success ( 4 - 7 ) . With time the techn ique has been re f ined and the p r o c e d u r e used in th i s p resen t s t u d y has taken elements f rom all of these wi th some mod i f i ca t ions . M A T E R I A L S A N D M E T H O D S Ce l l s L5178Y(S) ce l ls were p ropogated in c u l t u r e as d e s c r i b e d in C h a p t e r 2 . M T X - r e s i s t a n t L5178Y ( R 4 ) cel l l ine was d e r i v e d f rom L5178Y ( S ) cel ls by a s tepwise se lec t ion p rocess and u l t imate ly g r o w n ' in 1 mM M T X . U n d e r these cond i t ions the doub l i ng time fo r both cel l l ines was 12 h r . To ta l cel l numbers were determined by coun t i ng on a Model ZB1 C o u l t e r C o u n t e r ( C o u l t e r E l e c t r o n i c s ) . P repara t i on and A s s a y of An t i bod ies Rabb i t serum ant ibod ies were p repa red aga ins t h i g h l y pu r i f i ed p repa ra t i ons of form 1 ( h i g h - a f f i n i t y ) and form 2 ( l ow -a f f i n i t y ) D H F R and assayed as d e s c r i b e d in C h a p t e r 2 . - 117 -Polysome Isolat ion Polysomes were iso lated by a modi f icat ion of the method of Lee et g a[, ( 8 ) . App rox ima te l y 10 ce l ls were removed f rom suspens ion by cen t r i f uga t i on at 300 g fo r 10 min . T h e ce l ls were then r e s u s p e n d e d and gen t l y homogenized in 1 x HB (25 mM T r i s - H C I , pH 7 . 5 , 25 mM N a C l , 5 mM M g C I 2 , 2% T r i t o n X - 1 0 0 , 1 Mg/ml h e p a r i n ) at 2 ° C . T h e homogenate was immediately c e n t r i f u g e d at 10,000 g fo r 30 s e c . at 4 °C . T h e supe rna tan t was poured into a beake r and the polysomes were p rec ip i ta ted wi th 0.1 M M g C I 2 f o r 1 h r . at 4 ° C . Polysomes were co l lec ted by cen t r i f uga t i on t h r o u g h a 0 .5 M s u c r o s e cush ion in 25 mM T r i s - H C I , pH 7 . 5 , 25 mM N a C l , 5 mM M g C I 2 , 0.5% T r i t o n X - 1 0 0 , 0 .5 Mg/ml hepar in at 10,000 g fo r 15 m in . T h e supe rna tan t was d i s c a r d e d and the pel let was rehomogenized in 10 mM Hepes b u f f e r , pH 7 .5 con ta in ing hepar in (100 u g / m l ) , NaCl (0 .15 M ) , T r i t o n X - 1 0 0 / s o d i u m deoxycho la te (0.5% each) and M g C I 2 (5 mM) . T h e homogenate was c e n t r i f u g e d at 10,000 g fo r 10 m i n . , the supe rna tan t removed and the pel let rehomogenized in the above b u f f e r . A f t e r c e n t r i f u g a t i o n , the supe rna tan t s were combined and s to red in a l iquots at - 70 °C . T h i s method usua l l y y ie lded 100-120 A 2 6 o un i t s /m l g polysomes ( in a total volume of 15 ml) f rom 5 x 10 c e l l s . ' Polysome Immunoprecip i ta t ion L5178Y ( R 4 ) polysome immunoprec ip i ta t ion was c a r r i e d out u s i n g e i ther an t i - fo rm 1 o r an t i - f o rm 2 IgG (see C h a p t e r 2) and S taphy lococcus au reus pro te in A . P r i o r to u s i n g , the S . au reus p repa ra t i ons obta ined f rom C a l b i o c h e m - B e h r i n g were t rea ted acco rd ing to the method of Gough and Adams (4) whe reby the ce l ls were washed - 118 -3 t imes wi th bu f f e r A (0 .15 M N a C I , 5 mM E D T A , 5 mM T r i s - H C I , pH 7 .4 , and 0.05% Nonidet P - 4 0 ) ; 3 t imes wi th bu f f e r B (0 .3 M N a C I , 5 mM M g C I 2 , 50 mM T r i s - H C I , pH 7 . 4 , 0.5% Nonidet P - 4 0 , and 0.5% sodium d e o x y c h o l a t e ) ; and 3 t imes in bu f f e r C (0 .3 M N a C I , 50 mM T r i s - H C I , pH 7 . 4 , 5 mM M g C I 2 and 2 mM E G T A ) . A l l of these bu f f e r s were s te r i l i zed by au toc lav ing before u s i n g . T h e t rea ted S . au reus ce l ls were s to red as a 10% ( W / V ) suspens ion in bu f f e r C at 4 ° C . T h i s t reatment was c a r r i e d out to remove so lub le p ro te in A and r i bonuc lease . T h e deta i led p rocedu re used fo r the immunoprec ip i ta t ion can be best d e s c r i b e d in a f low d iagram shown be low. Deta i led P rocedu re f o r Polysome Immunoprec ip i ta t ion . T h e method desc r i bes immunoprec ip i ta t ion wi th an t i - f o rm 1 IgG. A n ident ica l p rocedu re was c a r r i e d out wi th an t i - fo rm 2 I gG . Polysomes D i lu ted to 25 A 2 6 0 / m l wi th R B u f f e r •l- + Hepar in Polysomes + Hepar in (5 Mg /m l ) + RNase Inh ib i to r T rea ted S . A u r e u s Ce l l s ( 0 .5 packed u l / A 2 6 0 ) 4- 30 m in . 4°C C e n t r i f u g e T h r o u g h 1M S u c r o s e C u s h i o n In R (13 ,000g , 20 m in , 4 °C) Pel let D i s c a r d Supe rna tan t - 119 -60 m in , 4°C wi th gent le agi ta t ion 30 m in , 4°C 4-with gent le ag i ta t ion <- A n t i - f o r m 1 IgG (1 Mg) Polysome - IgG <- T r e a t e d S . A u r e u s ce l ls (1 ml of 10% s u s p e n s i o n ) C e n t r i f u g e T h r o u g h 1M S u c r o s e C u s h i o n In R B u f f e r + 0.25% Nonidet P-40 (13 ,000g , 20 m i n , 4 °C) S u p e r n a t a n t A b o v e S u c r o s e C u s h i o n Polysomes R e - P r e c i p i t a t e d or R N A E x t r a c t e d " N o n - P r e c i p i t a t e d " Pel let ( Immunoprec ip i ta ted Po l ysomes ) . 4-Resuspend in R B u f f e r + 0.25% Nonidet P-40 C e n t r i f u g e T h r o u g h IM S u c r o s e C u s h i o n as A b o v e 4-R e s u s p e n d Dr ied Pel le t In S D S - E D T A -P ro te i nase K B u f f e r at 23°C 4-E x t r a c t R N A R B u f f e r : 10 mM T r i s - H C I , pH 7 . 5 , 50 mM K C I , 1.5 mM M g C I 2 , 0.1 M s u c r o s e , 2 mM d i t h i o t h re i t o l , and 200 un i t s p lacenta l r ibonuc lease i nh ib i t o r (Amersham C o r p . ) . S D S - E D T A - P r o t e i n a s e K B u f f e r : 15 mM E D T A , 10 mM T r i s - H C I (pH 7 . 5 ) , 1% S D S , 0.2 Mg/ml p ro te inase K. - 120 -Isolat ion of Polysomal Messenger R N A Tota l po lysoma l , " n o n - p r e c i p i t a t e d " , o r immunoprec ip i ta ted p o l y -somal R N A was iso lated by r e s u s p e n d i n g the polysomes or po lysome-I g G - S . A u r e u s cel l complex r e s p e c t i v e l y , in S D S / E D T A / p ro te inase K solut ion (10 mM T r i s - H C I , pH 7 . 5 , 15 mM E D T A , 1% S D S , 0.2 Mg/ml Pro te inase K ) . A f t e r incubat ion at 45°C fo r 5 m in . and cen t r i f uga t i on at 10,000 g fo r 10 min . at 25°C, the supe rna tan t was removed , the pel let r e s u s p e n d e d in the above bu f f e r and t rea ted as d e s c r i b e d above . T h e two supe rna tan t s were pooled and the R N A was ex t rac ted wi th an equal volume of pheno l / ch lo ro fo rm ( 1 : 1 ) . T h e aqueous phase was made 2% with sodium acetate and the R N A was co l lec ted by ethanol p rec ip i ta t i on at - 2 0 ° C . T h e R N A was d i sso l ved in 0.01 M T r i s - H C I , pH 7 . 5 , 0 .5 M KCI and the mRNA (po ly A +) was p u r i f i e d by ch romatog raphy on o l i g o - d T ce l lu lose as d e s c r i b e d by A v i v and Leder (11 ) . In V i t r o T r a n s l a t i o n T r a n s l a t i o n of m R N A . was conduc ted in the wheat germ in v i t r o t rans la t i on system obta ined f rom Be thesda Research Labo ra to r i es . T r a n s l a t i o n was al lowed to occu r fo r 3 h r . at 25°C us i ng 3 5 S -methionine in the p resence of 40 un i t s of p lacenta l RNase i nh i b i t o r . T h e reac t ions were s topped in ice and then incuba ted f o r 10 m in . at 37°C wi th 30 pg/ml RNase A . Tota l react ion p roduc t s were ana lyzed on 13% S D S po lyac ry lamide gels as d e s c r i b e d in C h a p t e r 2 . - 121 -Gel E lec t rophores i s Messenger R N A was e lec t rophore t i ca l l y separa ted in 1.5% agarose gels con ta in ing fo rma ldehyde ( 9 ) . E lec t rophores i s was c a r r i e d out at 2 .5 v o l t s / c m fo r 18 h r . T h e gel was s ta ined wi th 36 ug/ml ac r i d i ne o range in 10 mM sodium phosphate b u f f e r , pH 6.5 fo r 10 m in . at room tempera tu re and des ta ined in the above bu f f e r (3 x 20 m i n . ) . T r a n s f e r of R N A and DNA to N i t roce l lu lose  Paper and S u b s e q u e n t H y b r i d i z a t i o n Messenger R N A e lec t rophore t i ca l l y separa ted on f o rma ldehyde -con ta in ing agarose gels was t r a n s f e r r e d to n i t roce l lu lose ( N C ) paper by cap i l l a r y act ion f o r 16 h r . in 20 x S S C (1 x S S C is 0.15 M N a C l , 0.015 M sodium c i t r a t e , pH 7 . 0 ) . T h e NC paper was al lowed to a i r d r y and then baked in vacuo at 80°C fo r 2 h r . T h e t r a n s f e r paper was p r e h y b r i d i z e d in 5 x S S C , 50% formamide, 0.1% S D S , 5 x D e n h a r d t ' s so lu t ion and 100 ug/ml dena tu red yeas t t R N A at 42°C fo r 16 h r . H y b r i d i z a t i o n was c a r r i e d out in the above solut ion con ta in ing 3 2 P n i ck t r ans la ted c D N A (0 .5 -1.0 x 1 0 7 c p m ; S . A c . 6-8 x 1 0 7 c p m / u g ) (see C h a p t e r 2 fo r p repa ra t i on ) at 42°C fo r 24 h r . T h e paper was then sequen t ia l l y washed in 2 x S S C , 0.1% S D S at room tempe ra tu re ; and 0.1 x S S C , 0.1% S D S at 50°C fo r 30 m in . to 1 h r . , and exposed to x - r a y f i lm ( K o d a k X - O m a t A R ) at - 7 0 ° C . S y n t h e s i s of F i r s t - S t r a n d c D N A c D N A f rom h i g h l y en r i ched form 1 and form 2 mRNA f rom L5178Y - 122 -( R 4 ) ce l ls was s y n t h e s i z e d u s i n g a r e v e r s e t r a n s c r i p t a s e ( R N A -dependen t DNA po lymerase) f rom av ian myelob las tos is v i r u s ( p u r c h a s e d f rom Be thesda Research L a b o r a t o r i e s , G a i t h e r s b u r g , M D . ) . T h e reac t ion was c a r r i e d out in a total volume of 50 ul in 100 mM T r i s - H C I , pH 8 . 3 , 10 mM M G C I 2 , 50 mM K C I , 10 mM d i t h i o t h r e i t o l , 0 .5 mM [ 3 H ] o r [ a - 3 2 P ] d C T P , 1 mM each of d G T P , d T T P and d A T P , 3 .5 ug o l igo ( ~ d T ) 1 2 _ 1 8 , 1-5 to 2 .0 ug m R N A , 50 un i t s of RNase i n h i b i t o r , and 250 un i ts r e v e r s e t r a n s c r i p t a s e at 42°C fo r 1 h r . T h e react ion was s topped by the add i t ion of 2 ul 0 .5 M E D T A , pH 8.0 and 25 ul 150 mM N a O H . A f t e r i ncuba t ion fo r 1 h r . at 65°C and neu t ra l i z i ng wi th 25 ul 1 M T r i s - H C I , pH 8.0 and 25 ul INHCI , the total r ad ioac t i v i t y and the rad ioac t i v i t y i nco rpo ra ted into D N A ( T C A p rec ip i t ab le ) was determined by coun t ing an a l iquot of the m i x t u r e . T h e spec i f i c ac t i v i t i es of the c D N A s was 3 x 1 0 5 dpm/ug fo r form 1 D H F R c D N A and 1.5 x 1 0 5 dpm/ug fo r form 2 D H F R c D N A . T h e D N A was ex t rac ted wi th an equal volume of a 1:1 m ix tu re of phenol ch l o ro fo rm . A f t e r c e n t r i f u g i n g , the o r g a n i c phase was r e -ex t rac ted wi th T E N bu f f e r (10 mM T r i s - H C I , pH 8 . 0 , 100 mM NaCI , 1 mM E D T A ) . The aqueous phases were pooled and the labeled c D N A was p u r i f i e d by gel f i l t ra t ion t h r o u g h Sephadex G-100 column (bed volume 2 .0 m l ) . T h e column was e lu ted wi th T E N bu f f e r and the exc l uded peak of rad ioac t i v i t y was made 2% with sodium acetate and the c D N A p rec ip i ta ted wi th 2\ volumes of ethanol at - 2 0 ° C . T h e s ize of the f i r s t s t r a n d c D N A was determined by e lec t rophores i s t h r o u g h a 1.4% a lka l ine agarose gel (10 ) . 3 H - D N A molecular we igh t markers ( p u r c h a s e d f rom New Eng land N u c l e a r , B o s t o n , M a s s . ) were used fo r s ize est imat ions of the c D N A s . - 123 -R E S U L T S A N D D I S C U S S I O N Pur i f i ca t i on of Form 1 and 2 D H F R Polyr ibosomes, and De r i ved m R N A In ea r l i e r exper iments (see C h a p t e r 2) it was shown tha t the m R N A s cod ing f o r form 1 and 2 D H F R s were p resen t in re la t i ve l y h igh a b u n d a n c y in L5178Y ( R 4 ) c e l l s , s u c h that the D H F R s cou ld be read i l y de tec ted immunological ly a f ter in v i t r o t rans la t i on of total m R N A . It t he re fo re seemed poss ib le to e n r i c h fo r m R N A s cod ing fo r these enzymes by immunoprec ip i ta t ing po ly r ibosomes us ing ant ibod ies p repa red aga ins t form 1 and form 2 D H F R s . Polysomes were iso lated as d e s c r i b e d in Mater ia ls and Methods and mRNA isolated f rom such polysomes was able to d i r e c t t rans la t ion in v i t r o of a wide range of p ro te in molecules v a r y i n g in molecular we igh ts f rom g rea te r than 100,000 down to 10,000 da l tons ( F i g u r e 3 - 1 ) . It is c lear f rom F i g . 3-1 that these po ly r ibosomes conta ined h igh levels of mRNA cod ing f o r D H F R s ince an abundan t 19,000 dal ton p ro te in band can be v i s u a l i z e d upon S D S - P A G E of 3 5 S - m e t h i o n i n e labeled in v i t r o t rans la t i on p roduc t s wh ich co -mig ra tes wi th endogenous 3 H - l e u c i n e labeled D H F R p r e p a r e d f rom the same c e l l s . T h e ab i l i t y of the mRNA to d i r e c t p ro te in syn thes i s in v i t r o is a re f lec t ion of i ts func t iona l i n t e g r i t y and because it was iso lated f rom po lysomes, a re f lec t ion on the s t r u c t u r a l i n t e g r i t y of the po lysomes. Init ial at tempts at e n r i c h i n g mRNA by immunoprec ip i ta t ion resu l ted in v e r y poor y i e lds and the p u r i t y of the m R N A s cou ld not be de te rm ined . However , the i nc lus ion of th ree manoeuvres d u r i n g the immunoprec ip i ta t ion p rocess resu l ted in a marked inc rease in y ie ld and p u r i f i c a t i o n . T h e f i r s t two were the inc lus ion of p lacenta l r i b o -nuc lease i nh ib i t o r s in all the bu f f e r s used and the inc lus ion of a h igh - 124 -F I G U R E 3 - 1 . S D S - P A G E of I n -V i t r o T rans la t i on of Tota l Polysomal and Immuno- p rec ip i ta ted Polysomal m R N A . Lane A : 3 H - l a b e l e d DHFR p r e p a r e d f rom L5178Y ( R 4 ) c e l l s . Lane B : 3 5 S - l a b e l e d t rans la t i on p roduc t s of total polysomal m R N A . Lane C : 3 5 S - l a b e l e d t rans la t i on p r o d u c t s of an t i - f o rm 1 D H F R immunoprec ip i ta ted polysomal m R N A . Lane D: A s f o r C excep t an t i - fo rm 2 D H F R u s e d . A r r o w ind ica tes the pos i t ion of D H F R . 125 - 126 -quan t i t y of hepar in (5 Mg /m l ) d u r i n g the po lysome-an t ibody i n t e r -ac t i on . Hepar in not on l y i nh ib i t s RNase ac t i v i t y bu t also decreases n o n - s p e c i f i c b i n d i n g and ent rapment of unwanted po lysomes. T h e use of such h igh amounts of hepar in in i nc reas ing the y ie ld and pu r i f i ca t i on of o ther m R N A s has been documented by B o y e r et a[, ( 7 ) . T h e resu l t of the inc lus ion of these two compounds is shown in F i g . 3-1 wh ich is an au to rad iog raph of S D S - P A G E of total polysomal ( lane B ) , an t i - f o rm 1 ( lane C ) and an t i - fo rm 2 ( lane D) immuno-p rec ip i ta ted n iRNA d i rec ted in v i t r o pro te in s y n t h e s i s p r o d u c t s . C l e a r l y the re is a cons ide rab le amount of en r i chment of the D H F R -m R N A s ; however many o ther m R N A s appear to have co -p rec i p i t a t ed and the fo ld pu r i f i ca t i on of D H F R spec i f i c m R N A s is re la t i ve l y low. T h e t h i r d manoeuvre was the p readso rp t i on of the polysomes wi th S . au reus ce l ls in the p resence of hepar in to remove a subse t of polysomes wh ich may be p red i sposed to n o n - s p e c i f i c b i n d i n g . A l t h o u g h some loss of D H F R - p o l y s o m e s would o c c u r , the degree of enr i chment upon immunoprec ip i ta t ion shou ld i n c r e a s e , and th i s in fac t was the case as shown in F i g . 3 -2 . T h e ex ten t of D H F R mRNA enr ichment is g rea te r than that ach ieved when p readso rp t i on was not c a r r i e d ou t . B y scann ing the pho tog raph i c f i lm wi th a dens i tometer it was determined tha t the mRNA cod ing fo r form 1 D H F R at leas t , was approx imate ly 80% p u r e . It is c lear f rom F i g . 3-2 tha t on ly a small pe rcen tage of the DH F R - m R N A assoc ia ted wi th polysomes was immunoprec ip i ta ted by th i s method. One reason may be tha t because of the h igh abundance of th i s mRNA the amount of an t i body used (3 M9 /A260 po lysome) was below sa tu ra t ion level r esu l t i ng in low r e c o v e r y . However , doub l i ng the amount of an t i body used d id not - 127 -F I G U R E 3-2 S D S - P A G E of I n -V i t r o T r a n s l a t i o n of P r e a d s o r b e d , Immunoprec ip i ta ted  Polysomal m R N A . Lane A : 1 4 C - l a b e l e d molecular Weight p ro te in s t a n d a r d s . Lane B : 3 5 S - l a b e l e d t rans la t i on p roduc t s of total polysomal m R N A . Lane C : 3 5 S - l a b e l e d t rans la t i on p roduc t s of an t i - f o rm 1 D H F R immunoprec ip i ta ted polysomal mRNA wi th p readso rp t i on of polysomes by S . au reus c e l l s . Lane D: A s f o r C excep t an t i - f o rm 2 D H F R was u s e d . A r r o w ind ica tes the pos i t ion of D H F R . 128 - 129 -resu l t in h i ghe r r e c o v e r y . A n a l t e rna t i ve exp lanat ion may be that at any g i ven time not all of the polysomal D H F R - m R N A is engaged in p ro te in s y n t h e s i s resu l t i ng in the p rec ip i ta t ion of on ly tha t m R N A wh ich is assoc ia ted wi th nascen t D H F R po lypep t ide c h a i n s . Nonprec ip i t a ted polysomes conta ined a cons ide rab le amount of D H F R mRNA wh ich cou ld t rans la te into D H F R ( F i g . 3 - 3 ) . T h i s resu l t however does not d i sc r im ina te between the above two a l t e r n a t i v e s . A s can be seen f rom F i g . 3 - 2 , both an t i - f o rm 1 and an t i - f o rm 2 D H F R ant ibod ies were able to immunoprec ip i ta te po lysomes, wi th the mRNA isolated f rom these polysomes be ing able to t rans la te D H F R s in v i t r o . Because of the s im i la r i t y in the molecular we ights of the two forms of D H F R one cannot be su re whe ther the p ro te ins r ep resen t the two forms of D H F R or jus t one . Howeve r , the cons ide rab le d i f fe ren t ia l a f f in i t ies of the an t ibod ies f o r the two an t igens (see C h a p t e r 2) make it l i ke ly tha t an t i - f o rm 1 IgG p rec ip i ta ted polysomes p redominan t l y i nvo l ved in the s y n t h e s i s of form 1 D H F R ( F i g . 3 - 2 , lane C ) , whereas an t i - fo rm 2 IgG p rec ip i ta ted polysomes p r e -dominant ly s y n t h e s i z i n g form 2 D H F R . De f in i t i ve proof however can on ly come by t r ans fo rm ing D H F R - v e ce l ls (12) w i th c D N A s s y n t h e s i z e d f rom these h i g h l y en r i ched m R N A s . M T X - t i t r a t i o n of the DHFR ac t i v i t i es in lysates of these ce l ls would show whe the r the c D N A s coded fo r b o t h , or e i the r one or the o ther form of D H F R . S i ze Determinat ion of E n r i c h e d D H F R mRNA T h e s izes of the h i gh l y en r i ched form 1 and 2 D H F R m R N A s was determined by No r the rn blot ana lys i s us i ng n i ck t r ans la ted 3 2 P -labeled mur ine D H F R - c D N A ( d e s c r i b e d in C h a p t e r 2 ) . A s fo r the - 130 -F I G U R E 3-3 S D S - P A G E of In V i t r o T r a n s l a t i o n of " N o n - P r e c i p i t a t e d " Polysomal  m R N A . Lane A : 3 5 S - l a b e l e d t rans la t i on p roduc t s of total polysomal m R N A . Lane B : 3 5 S - l a b e l e d t rans la t i on p roduc t s of " n o n - p r e c i p i t a t e d " polysomal m R N A . A r r o w ind ica tes pos i t ion of D H F R . 131 - 132 -total ce l l u la r m R N A , polysomal mRNA from the L5178Y ( R 4 ) ce l ls was heterogeneous in s ize ( F i g . 3-4) and the reasons f o r t h i s have been d i s c u s s e d in detai l in C h a p t e r 2. Immunoprecip i ta ted polysomal form 1 D H F R mRNA however was not as heterogeneous and had a s ize of 0.85 Kb ( F i g . 3-4) wh ich is the same s ize as one of the total polysomal D H F R m R N A s . T h e reason fo r the smal ler s ize m R N A to be p re fe ren t i a l l y immunoprec ip i ta ted is not c l ea r . In any case it was func t iona l as determined by in v i t r o t rans la t i on ( F i g . 3 - 2 ) . T h e s ize of the immunoprec ip i ta ted polysomal form 2 mRNA cou ld not be determined by th i s t echn ique s ince it was not poss ib le to obta in a h y b r i d i z a t i o n s igna l wi th the c D N A probe u s e d . E i the r the mRNA was p resen t in v e r y low amounts , o r that the supposed a l tera t ion in i ts p r ima ry sequence resu l ted in a v e r y low degree of homology between i t and the c D N A p robe u s e d , wh ich codes fo r an u n a l t e r e d , h igh M T X a f f f i n i t y D H F R . c D N A S y n t h e s i s T h e h i gh l y e n r i c h e d m R N A s cod ing fo r the two forms of D H F R were used to d i r ec t the s y n t h e s i s of c D N A in an in v i t r o system d e s c r i b e d in Mater ia ls and Methods . U s i n g 3 H - d C T P a time cou rse exper imen t was c a r r i e d out fo r the i nco rpo ra t i on of 3 H - d C T P into cold T C A inso lub le mater ia l . T h e inco rpora t ion was found to be l inear fo r upto 1 h r . at 42°C. 3 2 P - l a b e l e d c D N A was then s y n t h e s i z e d us i ng these mRNA p repara t i ons and the f i r s t s t r a n d c D N A s were s ized by a lka l ine agarose gel e l ec t r opho res i s . T h e average s izes of both the c D N A s ( i . e form 1 and 2) were approx imat ley 600 base pa i rs and agreed well wi th the s ize of the mRNA (at least fo r form 1) used fo r the s y n t h e s i s . - 133 -F I G U R E 3-4 N o r h t e r n Blot A n a l y s i s of Tota l Polysomal and Immunoprec ip i ta ted  Polysomal mRNA f rom L5178Y ( R 4 ) C e l l s . mRNA ex t rac ted f rom polysomes was f rac t iona ted on 1.5% agarose gels con ta in ing 2 .2 (VI f o rma ldehyde , t r a n s f e r r e d to n i t roce l lu lose paper and p robed wi th n i ck t rans la ted 3 2 P - l a b e l e d D H F R c D N A as d e s c r i b e d in the Mater ia ls and Methods sec t i on . Lane A : 2 .0 ug polysomal m R N A . Lane B : A n t i - f o r m 2 D H F R immunoprec ip i ta ted polysomal mRNA from (40 ng ) L5178Y ( R 4 ) c e l l s . Lane C : A n t i - f o r m 1 D H F R immunoprec ip i ta ted polysomal mRNA f rom (40 n g ) L5178Y ( R 4 ) c e l l s . T h e f i g u r e s on the r i gh t hand s ide ind ica te the s izes in k i lobases of marker R N A molecules (23S and 16S p r o c a r y o t i c r ibosomal R N A s ) . 1.54 - 135 -T h e data p resen ted in th i s chap te r shows tha t h i g h l y en r i ched p repa ra t i ons of c D N A s rep resen t i ng p redominan t l y e i t he r one or the o ther form of D H F R have been p repa red by the t echn ique of polysome immunoprec ip i ta t ion . These, can now be c loned into su i tab le v e c t o r s , ampl i f ied and the D N A sequence de te rm ined . - 136 -R E F E R E N C E S 1. D e d h a r , S . and G o l d i e , J . H . (1983) O v e r p r o d u c t i o n of two an t i gen ica l l y d i s t i n c t forms of d ihyd ro fo la te reduc tase in a h i g h l y me tho t rexa te - res i s tan t mouse leukemia cel l l i ne . C a n c e r R e s . 43 , 4863-4871. 2 . G o l d i e , J . H . , K r y s t a l , G . , H a r t l e y , D . , G u d a u s k a s , G . and D e d h a r , S . (1980) A me tho t rexa te - i nsens i t i ve v a r i a n t of fo late reduc tase p resen t in two l ines of me tho t rexa te - res i s tan t L5178Y c e l l s . E u r . J . C a n c e r 16, 1539-1546. 3. G o l d i e , J . H . , D e d h a r , S . and K r y s t a l , G . (1981) P rope r t i es of a me tho t rexa te - i nsens i t i ve v a r i a n t of d ihyd ro fo la te reduc tase d e r i v e d f rom methot rexate res i s tan t L5178Y c e l l s . J . B i o l . C h e m . 256, 11629-11635. 4 . G o u g h , N . M . and A d a m s , J . M . (1978) Immunoprec ip i ta t ion of spec i f i c polysomes us i ng S taphy lococcus a u r e u s : Pu r i f i ca t i on of the immunoglobul in K cha in messenger R N A from the mouse myeloma MPC II. B iochem. 17, 5560-5566. 5 . A l t , F . W . , Ke l lems, R . E . , B e r t i n o , J . R . and S c h i m k e , R . T . (1978) Se lec t i ve mul t ip l i ca t ion of d ihyd ro fo la te reduc tase genes in me tho t rexa te - res i s tan t v a r i a n t s of c u l t u r e d mur ine c e l l s . J . B i o l . C h e m . 253, 1357-1370. 6. R o b s o n , K . J . H . , C h a n d r a , T . , M a c G i l l i v r a y , R . T . A . and Woo, S . L . C . (1982) Polysome immunoprec ip i ta t ion of pheny la lan ine h y d r o x y l a s e mRNA f rom rat l i ve r and c lon ing of i ts c D N A . P r o c . N a t l . A c a d . S c i . U . S . A . 79 , 4701-4705. 7. B o y e r , S . H . , S m i t h , K . D . , N o y e s , A . N . and Y o u n g , K . E . (1983) A d j u v a n t s to immunological methods f o r mRNA p u r i f i -c a t i o n . App l i ca t i on to the isolat ion of mRNA fo r ca rbon i c a n h y d r a s e I f rom rabb i t r e t i c u l o c y t e s . J . B i o l . C h e m . 258, 2068-2071. 8. Lee , D . C . , M c K n i g h t , C . S . and Pa lm i te r , R . D . (1978) T h e act ion of es t rogen and p roges te rone on the e x p r e s s i o n of the t r a n s f e r r i n g e n e . A compar ison of the response in ch i ck l i ve r and o v i d u c t . J . B i o l . C h e m . 253, 3494-3503. 9. L e h r a c h , H . , D iamond, D . , Wozney , J . M . and B o e d t k e r , H . (1977) R N A molecular we igh t determinat ions by gel e lec t ro -pho res i s unde r d e n a t u r i n g c o n d i t i o n s , a c r i t i ca l reexamina t ion . B iochem. 16, 4743-4752. 10. Man ia t i s , T . , F r i t s c h , E . F . and Sambrook , J . (1982) In : Molecu lar C l o n i n g , A Labo ra to ry M a n u a l , New Y o r k : Co ld S p r i n g H a r b o u r . - 137 -11 . A v i v , H . and L e d e r , P . (1972) Pu r i f i ca t i on of b io log ica l l y ac t i ve g lob in messenger R N A by ch roma tog raphy on o l i go -t hym idy l i c a c i d - c e l l u l o s e . P r o c . N a t l . A c a d . S c i . U . S . A . 69, 1408-1412. 12. U r l a u b , G . , and C h a s i n , L . A . (1980) Isolat ion of C h i n e s e hamster cel l mutants de f i c ien t in d i hyd ro fo la te reduc tase a c t i v i t y . P r o c . N a t l . A c a d . S c i . U . S . A . 77, 4216-4220. - 138 -C H A P T E R 4 H E T E R O G E N E I T Y IN T H E S P E C I F I C A C T I V I T Y A N D M E T H O T R E X A T E - S E N S I T I V I T Y OF D I H Y D R O F O L A T E R E D U C T A S E FROM B L A S T C E L L S OF A C U T E M Y E L O G E N O U S L E U K E M I A P A T I E N T S I N T R O D U C T I O N Pheno typ i c res is tance to the an t i - fo la te d r u g methot rexate ( M T X ) is assoc ia ted wi th a number of spec i f i c b iochemical mechanisms tha t have been well cha rac te r i zed in exper imenta l s ys tems . T h e s e have been d i s c u s s e d in detai l in C h a p t e r 1 and inc lude o v e r p r o d u c t i o n of d ihyd ro fo la te reduc tase ( D H F R ) due to gene ampl i f icat ion ( 1 , 2 ) , impaired t r a n s p o r t of the d r u g ( 3 ) , d im in ished capac i t y to s y n t h e s i z e methot rexate po lyg lu tamates ( 4 ) , and the p resence of a l te red forms of d ihyd ro fo la te reduc tase that are c h a r a c t e r i z e d by s i g n i f i c a n t l y less s e n s i t i v i t y to methot rexate inh ib i t i on ( 5 , 6 ) . T h e r e is much less d i r ec t ev idence fo r s imi lar mechanisms of res is tance in c l in i ca l ma l ignanc ies . Ea r l y s tud ies showed ev idence fo r impai red metho-t rexa te t r a n s p o r t in human leukemic ce l ls ( 7 ) , and recen t l y ev idence fo r gene ampl i f icat ion wi th inc reased d ihyd ro fo la te reduc tase p r o d u c -t ion has been d e s c r i b e d in pat ients wi th acute leukemia ( 8 , 9 ) . We have p r e v i o u s l y pub l i shed repo r t s on a h i g h l y methot rexate res i s tan t d ihyd ro fo la te reduc tase p resen t in mur ine L5178Y ce l ls tha t is marked ly res i s tan t to methot rexate inh ib i t i on ( 5 , 1 0 ) . T h e p rope r t i es of th i s enzyme and i ts response to va r i ous c lasses of ant i fo late i nh ib i t o r s have been documented (10-12 and th i s t h e s i s ) . In th i s chap te r I p resen t ev idence that v a r i a n t forms of d ihyd ro fo la te - 139 -reduc tase wi th d imin ished s e n s i t i v i t y to methot rexate inh ib i t i on are p resen t in the b las t ce l ls of some pat ients wi th acute myelogenous leukemia. Ev idence fo r the p resence of th i s enzyme was seen in 3 of 8 pat ients none of whom had been p r e v i o u s l y t rea ted wi th metho-t rexa te as pa r t of t he i r t he rapeu t i c reg imen . In add i t ion the D H F R spec i f i c a c t i v i t y was c o n s i d e r a b l y h i ghe r in the b las t cel ls of two of the 8 pa t ien ts as compared to the res t and also as compared to the a c t i v i t y in the whi te blood ce l ls of norma ls . M A T E R I A L S A N D M E T H O D S Isolat ion of Leukemic Ce l l s B las t ce l ls were obta ined before o r a f te r l eukophores is on a cel l separa to r un i t ( con t inuous f low c e l l t r i f u g e , Amer ican Ins t rument C o . ) , of pe r i phe ra l blood f rom pat ients w i th A M L . B las t ce l ls cons t i t u ted upwards of 60% of the pe r i phe ra l blood l e u k o c y t e s . Hepar in i zed blood (30-50 ml) ( con ta in ing b las t ce l l s ) was d i l u ted two fo ld wi th 0.9% NaCI so lu t ion and 10 ml a l iquots were gen t l y layered onto 10 ml F i c o l l - H y p a q u e ( d e n s i t y = 1.077 g / m l ) . C e n t r i f u -gat ion was c a r r i e d out at 400 g fo r 40 minutes at room tempera tu re . T h e whi te cel l l ayer was gen t l y asp i ra ted and contaminat ing e r y t h r o -cy tes were l ysed by incuba t ion wi th bu f fe red isoton ic N H 4 C I so lu t ion fo r 3 minutes at 37° . A f t e r red cel l l y s i s the b las t ce l ls were pel le ted by cen t r i f uga t i on (5 m inu tes , 300 g ) and washed twice in ice cold P B S (0 .15 M N a H 2 P 0 4 ; 0.15 M N a 2 H P 0 4 , pH 7.2 in 0.9% NaCI) con ta in ing 3.0 mM E D T A . T h e ce l ls were e i ther f r ozen and s to red in l i qu id n i t rogen in 1.5 ml a l iquots in F i s h e r ' s medium con ta in ing 1% ant ib io t i cs ( p e n i c i l l i n - s t r e p t o m y c i n ) , 1% g lu tamine , 10% DMSO and 20% - 140 -fetal cal f serum or were l ysed d i r e c t l y as d e s c r i b e d be low. F rozen ce l ls were r a p i d l y thawed when r e q u i r e d , and l ysed as d e s c r i b e d be low. White blood ce l ls f rom normal hea l thy i nd i v i dua l s were also isolated f rom 45 ml of pe r i phe ra l blood by F i c o l l - H y p a q u e d e n s i t y g rad ien t c e n t r i f u g a t i o n . T h e ce l ls were iso lated as d e s c r i b e d above and l ysed immediate ly . P repara t ion of Ce l l Lysa te T h e p u r i f i e d b las t ce l ls (o r normal whi te blood ce l l s ) were r e s u s p e n d e d in ice co ld 0.05 M T r i s - C I , pH 7 . 5 , 3 mM E D T A and d i s r u p t e d by son icat ion at 20 K H z fo r 30 seconds at 4 ° . ( B r a n s o n S o n i f i e r , cel l d i s r u p t e r 350, B r a n s o n Son ic Power C o . , C o n n . ) . T h e so lub le cy top lasmic f rac t i on was p r e p a r e d by cen t r i f uga t i on of the cel l l ysate at 100,000 g fo r 60 minutes at 4° in a Beckman L5 u l t r a -c e n t r i f u g e . T h e supe rna tan t (so lub le cy top lasmic f r a c t i o n ) was removed and d i v i d e d into f o u r equal volumes of 4 ml each in o r d e r to determine the optimum cond i t ions fo r the p r e s e r v a t i o n of enzyme ac t i v i t y and p reven t i on of endogenous p ro teo ly t i c n i c k i n g . Pheny l methyl su l phony l f l uo r i de ( P M S F ) was added to the supe rna tan t s to d i f f e ren t f ina l concen t ra t ions (see resu l t s s e c t i o n ) , and the lysa tes s to red at - 2 0 ° . Enzyme A s s a y D ihyd ro fo la te reduc tase ac t i v i t y was measured us i ng rad io labe led d i hyd ro fo l i c ac id as the s u b s t r a t e . Us ing d i h y d r o f o l i c ac id resu l ted in an approx imate ly 25% increase in the s e n s i t i v i t y of the assay ove r - 141 -us i ng fo l i c a c i d . T h e p repara t i on of 3 H - d i h y d r o f o l i c ac id and the D H F R assay are d e s c r i b e d in detai l in C h a p t e r 2. Pro te in concen t ra t i ons were determined by the B io Rad pro te in assay method u t i l i z i ng Coomassie b lue (B io Rad L a b s , R i c h m o n d , C A . ) . N o n - D e n a t u r i n g Po lyac ry lamide Gel E lec t rophores i s Samples (75 ul) con ta in ing 50 ug pro te in were e lec t rophoresed on 7.5% acry lamide (ac ry l am ide : methy leneb isac ry lamide 2 9 . 2 : 0 .8) d i sc gels as d e s c r i b e d in Gold ie et a\_, ( 10 ) . T h e gels were s ta ined fo r d ihyd ro fo la te reduc tase .ac t i v i t y in the p resence and absence of M T X as d e s c r i b e d p r e v i o u s l y ( 5 ) . Gels were scanned at 570 nm in a G i l f o rd spec t rophotometer Model 250, equ ipped wi th a g e l - s c a n n i n g un i t Model 2520 ( G i l f o r d Ins t rumen ts , O b e r l i n , O h i o ) . R E S U L T S S tab i l i t y of D ihyd ro fo la te Reduc tase A c t i v i t y D ihyd ro fo la te reduc tase ac t i v i t y in the u n p u r i f i e d cel l l ysa tes of A M L ce l ls was found to be h i g h l y uns tab le . O n l y 44% of the in i t ia l enzyme ac t i v i t i es remained a f ter s to rage of the lysa tes at -20°C fo r one month . T h e reason fo r th is i ns tab i l i t y was most p r o b a b l y p ro teo ly t i c n i c k i n g of the enzyme by endogenous p ro teases . P r e -incubat ion of the cel l l ysates at 37°C in assay bu f f e r f o r v a r i o u s time per iods resu l ted in a r a p i d , almost total inac t i va t ion of the enzyme ( F i g . 4 - 1 ) , p r o b a b l y due to the concomitant tempera tu re dependen t ac t iva t ion of pro tease a c t i v i t y . S to rage of the lysates in the p resence - 142 -F I G U R E 4-1 S tab i l i t y of D H F R A c t i v i t y in B las t Ce l l L y s a t e s . A M L b las t cel l l y sa te , r e p r e s e n t i n g 0.6 mg p r o t e i n , was incuba ted wi th 0.05 M T r i s - H C I , p.H 7.5 fo r v a r i o u s time pe r iods at 37°C. Enzyme react ion was then s ta r ted by the add i t ion of the res t of the components of the react ion m ix tu re and c a r r i e d out as d e s c r i b e d in the Mater ia ls and Methods s e c t i o n . V T / V o : rat io of in i t ia l rate of enzyme a c t i v i t y at t ime T o v e r time z e r o . — • — in the p resence of 0.25 mM PMSF and 1% P E G — 0 — in the absence of PMSF and P E G . 143 - 144 -of 0.25 mM pheny l methyl su l phony l f l uo r i de ( P M S F ) , a pro tease i nh ib i t o r ( 1 3 ) , and po lye thy lene g l yco l ( P E G ) (1%), resu l ted in the re tent ion of 100% of the in i t ia l enzyme ac t i v i t i es a f ter one month at f - 2 0 ° C . In a d d i t i o n , . p re incuba t i on of the lysa tes at 3 7 ° C in the p resence of these compounds resu l ted in 70% of the a c t i v i t y remain ing af ter 10 m in . i n c u b a t i o n , compared wi th 5% in the absence of PMSF and P E G ( F i g . 4 - 1 ) . Po lye thy lene g lyco l t reatment has been found to be use fu l in the pu r i f i ca t i on of o ther enzymes and in min imiz ing the e f fec ts of p ro teo ly t i c contaminat ion (14 ,15 ) . M T X Inh ib i t ion of D ihyd ro fo la te Reduc tase A c t i v i t y D ihydro fo la te reduc tase spec i f i c ac t i v i t y was found to be va r i ab le in the b las t cel ls of the A M L pat ients (Tab le 4-1) and was gene ra l l y h i ghe r by a fac to r of two o r more than the ac t i v i t y in normal te rm ina l l y d i f fe ren t ia ted whi te blood ce l ls (Tab le 4-1 B ) . T h e spec i f i c ac t i v i t i es ranged f rom 0.42 to 8.9 nmoles/20 m i n . / M g pro te in u n d e r the assay cond i t ions u s e d . F i g . 4-2 shows the inh ib i t i on of d i h y d r o -folate reduc tase a c t i v i t y by M T X in the ce l l u la r l ysa tes of the b las t ce l ls f rom th ree pa t i en t s . D ihyd ro fo la te reduc tase ac t i v i t y dec reased s h a r p l y wi th i nc reas ing M T X concen t ra t ion in the b las t cel l l ysa tes of these th ree pat ients ( F i g . 4-2 A - C ) , wi th the I - C . ^ Q va lues ( i . e . the concen t ra t ion of M T X requ i red to decrease the enzyme ac t i v i t y by 50%) r a n g i n g f rom 2-4 x 10~ 9 M M T X (Tab le 4 - 1 ) . Howeve r , the inh ib i t i on of d ihyd ro fo la te reduc tase ac t i v i t y in the b las t cel l l ysa tes of th ree o ther pat ients ( F i g . 4-3 A - C ) fo l lowed a v e r y d i f f e ren t p a t t e r n . T h e inh ib i t i on c u r v e s were f ound to be b iphas i c in each of these c a s e s , wi th the in i t ia l phase be ing sha l lower than those found T A B L E 4-1 A c t i v i t i e s and M T X - i n h i b i t o r y Proper t ies of D ihydro fo la te Reductase A c t i v i t y P resen t in B las t Ce l l Lysa tes f rom A M L Pa t ien ts , and Normal White Blood Cel l L y s a t e s . A M L E N Z Y M E A C T I V I T Y S P E C I F I C A C T I V I T Y L Y S A T E (n moles/20 m i n . / m l ) (n moles/20 m in . /mg p ro te in ) L C . 50 (M) L C . 100 (M) D . R . 72.0 1.7 4 .0 X l O ' 9 1 0 ' 6 S . A . 13.7 0.82 3.0 x -9 10 y 10~ 6 A . P . 23.0 0.73 2.7 x -9 10 3 1 0 "5 Rom 11.4 3 .3 2.7 x -9 10 s 1 0 " 7 K.W. 124.0 8.9 1.8 x 1 0 " 8 5 x 10 D . R o . 7 .0 0.42 5.4 X 1 0 " 8 > 1 0 " 5 J . N . 7 .2 0.91 4 .5 X 1 0 " 8 > 1 0 " 6 J . B . 4 .0 0.98 6.0 X -9 10 3 > 1 0 " 6 N O R M A L * W B C L Y S A T E -10 D . H . 0.92 0.47 4 .0 x 10 10 -10 J . W . 1.12 0.48 6.0 x 10 l u 10 S . D . 1.0 0.43 1.0 x 10~ 9 10 * W B C : White blood c e l l . - 146 -F I G U R E 4-2 Inh ib i t ion of D ihyd ro fo la te Reduc tase A c t i v i t y by M T X in B las t Ce l l  Lysa tes of A M L Pa t i en t s . Lysa tes were incuba ted wi th M T X in the p resence of N A D P H and KCI fo r 2 minutes at room tempera tu re . T h e react ions were then s ta r ted by the add i t ion of 3 H - d i h y d r o f o l i c ac id and the assay c a r r i e d out as d e s c r i b e d in the Mater ia ls and Methods sec t i on . A : Pat ien t D . R . ; B : P a t i e n t s . A . ; C : Pat ient A . P. 147 0 10" 10" 10" 10" 10" [MTX] M - 148 -F I G U R E 4-3 Inh ib i t ion of d ihyd ro fo la te reduc tase a c t i v i t y by M T X in b las t cel l l ysa tes of A M L pa t i en t s . T h e assay was c a r r i e d out as d e s c r i b e d in f i g u r e 4 - 2 . A : Pat ien t D . R o . ; B : Pat ient J . N . ; C : Pat ient J . B . 149 - 1 5 0 -in the o ther th ree pa t i en ts . T h e second phase was sha l lower s t i l l and resu l ted in the re tent ion of o v e r 30% of the in i t ia l d i hyd ro fo la te _g, reduc tase ac t i v i t y even in the p resence of 1 0 M M T X . T h e I . C . ^ Q 8 - 8 va lues in these cases were 5 . 4 x 1 0 M ( F i g . 4 - 3 A ) , 4 . 5 x 1 0 M ( F i g . 4 - 3 B), and 6 . 0 x 1 0 _ 9 M ( F i g . 4 - 3 C ) (about 1 0 fo ld h i ghe r than that in the o ther th ree p a t i e n t s ) . In the b las t cel l l ysa tes f rom pat ients e x h i b i t i n g the monophasic M T X - t i t r a t i o n c u r v e s , total - 6 i nh ib i t i on of d ihyd ro fo la te reduc tase a c t i v i t y was ach ieved by 1 0 M M T X ( F i g . 4 - 2 ) . However , in complete c o n t r a s t to these f i g u r e s , g rea te r than 25% ac t i v i t y remained in the b las t cel l l ysate of the pat ients e x h i b i t i n g the b iphas i c c u r v e s ( F i g . 4 - 3 ) , and I . C . ^ Q Q va lues are c o n s i d e r a b l y h i ghe r than those fo r the f i r s t 3 pat ients (Tab le 4 - 1 ) . Ex t rapo la t ion of the f i r s t i n h i b i t o r y phases showed tha t the - 6 l ine in te rsec ted wi th the X - a x i s at j us t o v e r 1 0 M M T X . T h e r e f o r e it would appear that at least two forms of d ihyd ro fo la te reduc tase are e x p r e s s e d by the b las t ce l ls in these pa t i en t s . F i g . 4 - 4 shows the inh ib i t i on of D H F R a c t i v i t y in the lysa tes of whi te blood ce l ls f rom th ree hea l thy i n d i v i d u a l s . It is c lear that in all these cases the D H F R a c t i v i t y is i nh ib i ted in a monophasic manner - 1 0 - 9 with v e r y h igh a f f i n i t y ( I . C . ^ Q r a n g i n g f rom 4 x 1 0 M to 1 x 1 0 M ) . In add i t ion the D H F R spec i f i c ac t i v i t i es were at least 2 f o l d lower than those found in the A M L b las t cel l l ysa tes (Tab le 4 - 1 ) . T h e D H F R spec i f i c ac t i v i t i es in the b las t cel l l ysa tes of two of the A M L pat ients ( K . W . and Rom) were found to be cons ide rab l y h i ghe r than those found in the res t of the A M L lysa tes or in the normal whi te blood cel l l ysa tes (Tab le 1 ) . - 151 -F I G U R E 4-4 Inh ib i t ion of d ihyd ro fo la te reduc tase ac t i v i t y by M T X in whi te blood cel l l ysa tes of th ree normal hea l thy i n d i v i d u a l s . T h e assay was c a r r i e d out as d e s c r i b e d in f i g u r e 4 - 2 . — O — : D . H . ; A : J . W . ; A: S . D . 152 o cc p- 10CK z O o 80 o 60 > 1— > 40 O < 20 CC LL X Q 0 MTX - 153 -In o r d e r to v e r i f y whether mul t ip le D H F R f o rms , wi th d i f fe ren t ia l sens i t i v i t i es to M T X , ex i s ted in those pat ien ts e x h i b i t i n g b i phas i c M T X - t i t r a t i o n c u r v e s , n o n - d e n a t u r i n g po lyac ry lamide gel e l ec t ro -phoreses of the b las t cel l l ysa tes f rom pat ient D . R o and J . N . , were c a r r i e d ou t . T h e gels were s ta ined fo r d ihyd ro fo la te reduc tase ac t i v i t y as d e s c r i b e d p r e v i o u s l y by us ( 5 ) , in the p resence and absence of M T X . A t least t h ree d i s t i n c t bands of ac t i v i t y were detec ted v i s u a l l y by th i s method in the cel l l ysate of pat ien t D . R o ( F i g . 4-5 A , lane A ) , and these cou ld be f u r t h e r reso lved into f o u r peaks upon scann ing the gel at 570 nm ( F i g . 4-5 B ) . When M T X was ~6 i nc luded at a concent ra t ion of 10 M in the s ta in ing m ix tu re all of the bands were abo l ished excep t f o r one ( F i g . 4-5 A , lane B ) wh ich co r responded to the major band in Lane A . A scan of t h i s gel demonst ra ted that th i s major band had on ly d im in ished in i n tens i t y by 47% upon incubat ion wi th M T X ; 53% of the ac t i v i t y remain ing a f ter 30 min incubat ion at 37°C ( F i g . 4-5 B ) . A dup l i ca te gel s ta ined in the absence of d i h y d r o f o l i c ac id was complete ly c lear i nd i ca t i ng tha t all of the bands o b s e r v e d rep resen ted d ihyd ro fo la te reduc tase a c t i v i t y . S imi la r ana l ys i s of the cel l l ysa te of pat ien t J . N . revea led on l y one major peak of DHFR ac t i v i t y ( F i g . 4-6 B ) , wh ich d id not d im in ish in -6 i n tens i t y upon incubat ion of the gel in the p resence of 10 M M T X ( F i g . 4 - 6 , lane C ) . A cont ro l gel wh ich was s ta ined in the absence of d i h y d r o f o l i c ac id is shown in F i g . 4 - 6 , lane A . A l t h o u g h not v i s i b l e on the g e l s , the gel scan ind ica tes a shou lde r of D H F R ac t i v i t y ad jacent to the main peak ( F i g . 4-6 A ) wh ich does d i sappear upon incubat ion wi th M T X and may rep resen t M T X - s e n s i t i v e D H F R a c t i v i t y . - 154 -F I G U R E 4-5 a . N o n - d e n a t u r i n g po lyac ry lamide gel e lec t ropho res i s of b las t cel l lysate f rom A M L pat ien t D . R o on 7.5% po lyac ry lamide g e l s . Samples (75 pi) con ta in ing 50 pg pro te in were e lec t rophoresed on 7.5% p o l y -acry lamide ge ls in 25 mM T r i s - g l y c i n e , pH 8.3 at 2 .2 m A / t u b e fo r 1.5 h r . at 4 ° C . T h e gels were s ta ined fo r d ihyd ro fo la te reduc tase ac t i v i t y in the p resence or absence of M T X as d e s c r i b e d in Gold ie et a L ( 5 ) . Incubat ion was fo r 30 m in . at 37°C. Lane A : D . R o b las t cel l l ysate - No M T X Lane B : D . R o b las t cel l l ysa te - + 10~ 6 M M T X . b. Scan of 7.5% po lyac ry lamide gels d e s c r i b e d in A at 570 nm us ing a G i l f o rd spect rophotometer model 250 equ ipped wi th a gel scann ing un i t model 2520. A . Gel s ta ined fo r D H F R ac t i v i t y in the absence of M T X . B . Gel s ta ined fo r DHFR ac t i v i t y in the p resence of 10 M M T X . 155 - 156 -F I G U R E 4-6 a . Scan of n o n - d e n a t u r i n g P A G E of b las t cel l l ysate f rom pat ien t J . N . on 7.5% po lyac ry lamide g e l s . T h e e lec t rophore t i c and s ta in ing cond i t ions were as d e s c r i b e d fo r F i g . 1. A : J . N . b las t cel l lysate s ta ined in the absence of d i h y d r o f o l i c ac id ( - ve c o n t r o l ) . B : J . N . b last cel l l ysate s ta ined fo r D H F R ac t i v i t y in the absence of M T X . C : J . N . b las t cel l lysate s ta ined fo r D H F R ac t i v i t y in the p resence of 1 0 " 6 M M T X . 157 - 158 -A n ana lys i s by P A G E of the cel l lysate f rom one of the pat ien ts (pa t ien t A . P . ) showing monophas ic , M T X - s e n s i t i v e D H F R a c t i v i t y is shown in F i g . 4 - 7 . O n l y one D H F R band is v i s i b l e on th i s gel and it is complete ly abo l i shed upon incubat ion wi th M T X ( F i g . 4 - 7 , lane B ) . D I S C U S S I O N In the 8 pat ients s tud ied in t h i s r epo r t t he re was found to be a va r i ab le level of d ihyd ro fo la te reduc tase spec i f i c a c t i v i t y . In 4 of the 8 pa t ien ts the d ihyd ro fo la te reduc tase ac t i v i t y was marked ly sens i t i ve to methot rexate i n h i b i t i o n . Howeve r , in 3 pa t ien ts marked res is tance to methot rexate inh ib i t i on was demons t ra ted . T h e t i t ra t ion c u r v e f o r d r u g inh ib i t i on was essen t ia l l y b iphas i c w i th the inh ib i t i on level p la teau ing at approx imate ly 30% of base l ine a c t i v i t y . In 2 of the 3 pat ients d i s p l a y i n g the s i gn i f i can t methot rexate r e s i s t a n c e , po lyac ry lamide gel e lec t ropho res i s conf i rmed the p resence of at least one band of d ihyd ro fo la te reduc tase ac t i v i t y that p e r s i s t e d ~6 in the p resence of incubat ion wi th 10 molar concen t ra t ion of the d r u g . In one pat ient ( A . P . ) who was sens i t i ve to methot rexate and in wh ich the re was su f f i c i en t D H F R ac t i v i t y to be iden t i f i ed in P A G E , the band of enzyme a c t i v i t y was complete ly abo l ished by incuba t ion wi th d r u g . It would appear reasonable t he re fo re to in fe r tha t the d ihyd ro fo la te reduc tase res i s tan t bands demonst ra ted on e lec t ro -pho res i s were c o n t r i b u t i n g to the res i s tan t por t ion of the d r u g t i t ra t ion c u r v e . A s ment ioned above , the D H F R spec i f i c ac t i v i t i es in b las t ce l ls of the 8 pat ients were va r i ab le and in two of the pat ients (Rom and K . W . ) the ac t i v i t i es were s i g n i f i c a n t l y h i ghe r than the res t (Tab le - 159 -F I G U R E 4-7 N o n - d e n a t u r i n g P A G E of b las t cel l l ysate f rom pat ient A . P. on 7.5% po lyac ry lamide g e l s . T h e e lec t rophore t i c and s ta in ing cond i t i ons were as d e s c r i b e d fo r F i g . 4 - 5 . Lane A : A . P . b las t cel l l ysate s ta ined fo r D H F R a c t i v i t y in the absence of M T X . Lane B : A . P . b las t cel l l ysa te s ta ined fo r D H F R a c t i v i t y in the -fi presence of 10 M M T X . 160 - 161 -4 - 1 ) . These h igh D H F R ac t i v i t i es may be due to the o v e r p r o d u c t i o n of D H F R resu l t i ng f rom DHFR gene amp l i f i ca t ion , as has been repor ted recen t l y ( 8 , 9 ) , o r may be due to the e x p r e s s i o n of a ca ta l y t i ca l l y more ac t i ve D H F R , as has been shown to be the case in a M T X -res is tan t human p romye locy t i c leukemia ( H L - 6 0 ) cel l l ine (16 , and C h a p t e r 5 ) . A s can be seen f rom Tab le 4-1 th i s h igh D H F R spec i f i c a c t i v i t y , espec ia l l y in pat ient K . W . , resu l t s in an appa ren t h i g h e r I . C . J - Q va lue f o r M T X - i n h i b i t i o n . None of the 8 pat ients who were s tud ied had been p r e v i o u s l y exposed to metho t rexa te . T h u s the th ree ins tances of methot rexate res i s tan t D H F R s and the two of i nc reased D H F R spec i f i c ac t i v i t i es appear to be examples of i n t r i n s i c res i s tance not i n t roduced by a p rocess of spec i f i c d r u g se lec t i on . Methot rexa te does not p lay a major role in the p resen t day management of adu l t acute mye lob las t ic leukemia . Howeve r , on the bas is of o lde r data r e g a r d i n g i ts use as s ing le agent t h e r a p y and more recen t pro toco ls in wh ich methot rexate was pa r t of a mu l t i d rug protoco l i t can be i n f e r r e d that methot rexate p r o b a b l y has some c l in i ca l ac t i v i t y at least in a p ropo r t i on of pat ients wi th A M L . If i n t r i n s i c r e s i s t a n c e , due to the p resence of methot rexate i nsens i t i ve d ihyd ro fo la te reduc tase is a common o c c u r r e n c e in pa t ien ts w i th A M L , then th i s cou ld help exp la in the obse rva t i on tha t methot rexate does not appear to be a h i g h l y e f fec t i ve agent in th i s d i sease . In the 3 pat ients ( D . R . , S . A . and A . P . see Tab le 4-1) who demonst ra ted s e n s i t i v i t y to methot rexate i n h i b i t i o n , and low levels of DHFR a c t i v i t y , o ther mechanisms of methot rexate res i s tance cou ld not be e x c l u d e d , i . e . d im in ished d r u g po lyg lu tamat ion or impai red t r a n s p o r t . - 162 -A t the p resen t time the re is i nsu f f i c i en t data to. al low one to make an est imate of the f r e q u e n c y wi th wh ich methot rexate i nsens i t i ve reduc tases occu r in A M L or o ther t y p e s of acute leukemia . Nor can one make any in fe rences about the p rope r t i es of these human low a f f i n i t y reduc tases as compared wi th the p r e v i o u s l y cha rac te r i zed mur ine enzymes . However , the fac t that p re l im ina ry ev idence fo r i nsens i t i ve reduc tase was o b s e r v e d in 3 of 8 pa t ien ts wi th A M L sugges t s tha t it may not be an uncommon mechanism of methot rexate res i s tance in ce r ta in forms of leukemia . If such a l te red enzymes are p resen t in a s i gn i f i can t p ropo r t i on of pa t ien ts wi th methot rexate res i s tan t d isease then it s u p p o r t s the concept of i den t i f y i ng novel t ypes of ant i fo la te compounds tha t may have a h i g h e r degree of spec i f i c i t y f o r the low a f f i n i t y enzyme. A g e n t s wh ich have been iden t i f i ed as hav ing enhanced ac t i v i t y aga ins t methot rexate res i s tan t reduc tase in mur ine sys tems (12) can then also be tes ted f o r a c t i v i t y in c l in ica l specimens tha t show ev idence of a va r i an t i nsens i t i ve r educ tase . - 163 -R E F E R E N C E S 1. A l t , F . W . , Ke l lems, F . R . , B e r t i n o , J . R . and S c h i m k e , R . T . (1978) Se lec t i ve mul t ip l i ca t ions of d i hyd ro fo la te reduc tase genes in me tho t rexa te - res i s tan t va r i an t s of c u l t u r e d mur ine c e l l s . J . B i o l . C h e m . 253, 1357-1370. 2 . F l in to f f , W . F . , Weber , M . K . , N a g a i n i s , C . R . , E s s a n i , K , , R o b e r t s o n , D. and S a l s e r , W. (1982) O v e r p r o d u c t i o n of d i h y d r o -fo late reduc tase and gene ampl i f icat ion in me tho t rexa te - res i s tan t C h i n e s e hamster o v a r y c e l l s . M o l . and C e l l . B i o l . 2 , 275-285. 3. S i r o t n a k , F . M . , K u r i t a , S . and H u t c h i s o n , D . J . (1968) On the na tu re of a t r a n s p o r t a l te ra t ion de te rm in ing res i s tance to amethopter in in L1210 leukemia . C a n c e r R e s . 28, 75-80 . 4 . J o l i v e t , J . , C o w a n , K . H . , C u r t , G . A . , G l e n d e n i n n , N . J . and C h a b n e r , B . J . (1983) T h e pharmaco logy and c l in i ca l use of methot rexa te . New E n g l . J . M e d . 309, 1094-1104. 5. Go ld i e , J . H . , K r y s t a l , G . , H a r t l e y , D . , G u d a u s k a s , G . and D e d h a r , S . (1980) A methot rexate i nsens i t i ve v a r i a n t fo late reduc tase p resen t in two l ines of methot rexate res i s tan t c e l l s . E u r . J . C a n c e r 16, 1539-1546. 6. H a b e r , D . A . , B e v e r l e y , S . M . , K i e l y , M . L . and S c h i m k e , R . T . (1981) P rope r t i es of an a l te red d ihyd ro fo la te reduc tase encoded by ampl i f ied genes in c u l t u r e d mouse f i b r o b l a s t s . J . B i o l . C h e m . 256, 9501-9510. 7. K e s s e l , D . , H a l l , T . C . and R o b e r t s , D. (1968) Modes of up take of methot rexate by normal and leukemia human leukocy tes in v i t r o and t he i r re lat ion to d r u g r e s p o n s e . C a n c e r R e s . 28, 564. 8. H o r n s , R . C , Dower , W . J . and S c h i m k e , R . T . (1984) Gene ampl i f icat ion in a leukemic pat ient t rea ted wi th metho t rexa te . J . C l i n . O n c o l . 2 , 2 -7 . 9. C a r m a n , M . D . , S c h o r n a g e l , J . H . , R i v e s t , R . S . , S r i m a t k a n d a d a , S . , P o r t l o c k , C . S . , D u f f y , T . and B e r t i n o , J . R . (1984) Res is tance to methot rexate due to gene ampl i f icat ion in a pat ient w i th acute leukemia . J . C l i n . O n c o l . 2 , 16-20. 10. G o l d i e , J . H . , D e d h a r , S . , and K r y s t a l , G . (1981) P rope r t i es of a me tho t rexa te - i nsens i t i ve v a r i a n t of d ihyd ro fo la te reduc tase d e r i v e d f rom me tho t rexa te - res i s tan t L5178Y c e l l s . J . B i o l . Chem. 256, 11629-11635. 11 . D e d h a r , S . and Go ld ie , J . H . (1983) O v e r p r o d u c t i o n of two a n t i -gen ica l l y d i s t i n c t forms of d i hyd ro fo la te reduc tase in a h i g h l y me tho t rexa te - res i s tan t mouse leukemia cel l l i ne . C a n c e r R e s . 43, 4863-4871. - 164 -12. D e d h a r , S . , F r e i s h e i m , J . H . , H y n e s , J . B . and G o l d i e , J . H . (1983) Inh ib i t ion of a me tho t rexa te - i nsens i t i ve d ihyd ro fo la te reduc tase f rom L5178Y ce l ls by subs t i t u ted t r i a z i n e and qu inazo l i nes . B iochem. Pharmaco l . 32 , 922-924. 13. M o s s , D . E . and F a h r n e y , D . E . (1978) K ine t i c ana l ys i s of d i f f e rences in b ra in ace ty l cho l i nes te rase f rom f i sh o r mammalian s o u r e c e s . B iochem. Pharmaco l . 27, 997. 14. L i n n , T . C . and S p e r e , P . A . (1979) Ident i f ica t ion of A T P c i t ra te l ysase as a phosphopro te in J . B i o l . C h e m . 254, 1691-1698. 15. L i n n , T . C . (1981) Pu r i f i ca t i on and c r ys ta l l i za t i on of ra t l i ve r f a t t y ac id s y n t h e t a s e . A r c h . B iochem. B i o p h y s . 209, 613-619. 16. D e d h a r , S . , H a r t l e y , D. and Go ld ie , J . H . , (1984) Increased d ihyd ro fo la te reduc tase a c t i v i t y in a me tho t rexa te - res i s tan t human p romye locy t i c leukemia ( H L - 6 0 ) cel l l i ne . Lack of co r re la t ion between inc reased a c t i v i t y and o v e r p r o d u c t i o n . B iochem. J . ( in p r e s s ) . - 165 -C H A P T E R 5 S E C T I O N 1 I N C R E A S E D D I H Y D R O F O L A T E R E D U C T A S E A C T I V I T Y IN M E T H O T R E X A T E - R E S I S T A N T H U M A N P R O M Y E L O C Y T I C L E U K E M I A ( H L - 6 0 ) C E L L S . L A C K OF C O R R E L A T I O N B E T W E E N I N C R E A S E D A C T I V I T Y A N D O V E R P R O D U C T I O N . I N T R O D U C T I O N In chap te rs 2 , 3 and A p p e n d i x 1 I have deal t wi th the ex tens i ve cha rac te r i za t i on of D H F R in a M T X - r e s i s t a n t mouse leukemia cel l l i ne . A s a f u r t h e r model system D H F R e x p r e s s i o n in M T X - r e s i s t a n t human p romye locy t i c leukemia ( H L - 6 0 ) and M T X - r e s i s t a n t human carc inoma of the c e r v i x ( H e L a ) cel l l ines was i n v e s t i g a t e d . T h e res i s tan t ce l ls were se lec ted f rom the parenta l M T X - s e n s i t i v e ce l ls by a s tep -w i se inc rease in M T X concen t ra t ion as fo r the mouse c e l l s . T h e expec ta t ion was that as f ound by o t h e r s , th i s t ype of a se lect ion p rocess would lead to M T X - r e s i s t a n t ce l ls o v e r p r o d u c i n g D H F R due to D H F R gene amp l i f i -c a t i o n , and that at least one res i s tan t cel l l ine would be se lec ted wh ich e x p r e s s e d a more M T X - r e s i s t a n t form of D H F R . T h e M T X - r e s i s t a n t HeLa ce l ls conformed to th i s expec ta t ion i . e . that HeLa ce l ls g row ing in the p resence of 1 uM M T X had an approx ima te ly 30 fo ld h i ghe r D H F R ac t i v i t y than the paren ta l ce l ls and D H F R gene ampl i f icat ion cou ld be demonst ra ted by S o u t h e r n blot a n a l y s i s . T h e o v e r p r o d u c t i o n of D H F R was uns tab le in tha t the D H F R ac t i v i t y in the res i s tan t ce l ls g rown in the absence of M T X fo r 28 cel l doub l i ngs re tu rned to that p resen t in the parenta l c e l l s . - 166 -T h e s u r p r i s e however was the M T X - r e s i s t a n t HL-60^, ce l ls (a lso g row ing in the p resence of 1 uM M T X ) wh ich had an approx imate ly 20 fo ld inc rease in D H F R ac t i v i t y o v e r tha t in the parenta l c e l l s . T h i s inc rease in ac i t i v i t y was complete ly s tab le o v e r 28 d o u b l i n g s in the absence of M T X , and was found not to be due to the o v e r p r o d u c t i o n of D H F R , bu t ra the r to a modi f icat ion of the D H F R such tha t it was more ac t i ve than the D H F R in the paren ta l c e l l s . T h i s t y p e of M T X -res i s tan t pheno type has no t , to my know ledge , been repor ted in the l i t e ra tu re to da te . In th i s chap te r I p resen t data wh ich shows tha t the inc rease in D H F R ac t i v i t y in M T X - r e s i s t a n t H L - 6 0 ce l ls is not due to o v e r -p roduc t i on of D H F R or D H F R gene amp l i f i ca t ion . Sect ion 2 of t h i s chap te r deals wi th some of the d i f f e rences in the p rope r t i es of the DHFR from the res is tan t and sens i t i ve H L - 6 0 c e l l s . M A T E R I A L S A N D M E T H O D S Ce l l C u l t u r i n g and Se lec t ion of M T X - r e s i s t a n t S u b l i n e s H L - 6 0 p romye locy t i c leukemia ce l ls (1) were g rown as suspens ion c u l t u r e s in RPM1 1640 medium supp lemented wi th fetal ca l f serum (10%), g lutamine (2 mM) and 100 un i t s /m l p e n i c i l l i n ; 100 ul /ml s t rep tomyc in (obta ined f rom G r a n d Is land B io log ica l C o . , G r a n d I s l and , N . Y . ) . Ce l l s were in i t i a l l y exposed to 5 nM M T X and subsequen t i nc reases in M T X concen t ra t ions were s tepwise at ha l f - l og i n te r va l s up to 10 uM. H L - 6 0 ( R 4 - ) ce l ls were d e r i v e d f rom R 4 ce l ls and al lowed to grow in the absence of M T X . Hela ce l ls were g rown as monolayer c u l t u r e s in Eag le 's minimum essent ia l medium wi th Ear le ' s sa l ts supp lemented wi th fetal ca l f serum - 167 -(10%), g lutamine (2 mM) and 100 un i t s /m l p e n i c i l l i n ; 100 ul /ml s t r e p t o m y c i n . M T X - r e s i s t a n t ce l ls were se lected as d e s c r i b e d above fo r H L - 6 0 c e l l s . P repara t ion of Ce l l Lysa tes and D H F R A s s a y Ce l l l ysa tes were p r e p a r e d as d e s c r i b e d p r e v i o u s l y ( 2 ) . T h e cel l l ysa tes were a l iquoted out and s to red at -70°C in the p resence of 0.1 mM pheny l methyl su l phony l f l uo r i de ( P M S F ) . D H F R a c t i v i t y was measured u s i n g 3 H - l a b e l e d d i h y d r o f o l i c ac id as d e s c r i b e d in C h a p t e r 2. Pro te in concen t ra t ions were determined by the B io Rad pro te in assay method u t i l i z i ng Coomassie b lue (B io Rad L a b s , R i chmond , C A . ) . Est imat ion of the Ex ten t of D H F R Gene Ampl i f i ca t ion H igh molecular we igh t D N A was ex t rac ted f rom the ce l ls by the method of B l i n and S t a f f o r d , ( 3 ) . Known amounts (0.1 to 20 ug) of DNA were dena tu red in 0 .3 N NaOH at room tempera tu re fo r 18 h r . , c h i l l e d , mixed wi th an equal volume of cold 2 M ammonium acetate and 80 ul a l iquo ts were spot ted onto n i t roce l lu lose f i l t e r s (4) us i ng a h y b r i - d o t manifold (Be thesda Research L a b s . , G a i t h e r b u r g , M D . ) . T h e f i l t e r s were then washed in 3 M NaCI /10 mM s o d i u m - E D T A , pH 7 . 5 , a i r - d r i e d and then baked u n d e r vacuum at 80°C fo r 2 h r . T h e f i l t e r s were p r e h y b r i d i z e d in 5 x S S C (1 x S S C is 0.15 M N a C l / 0.015 M sodium c i t r a t e , pH 7 . 0 ) , 50% formamide, 0.1% S D S , 5 x Denhard ts so lu t ion (1 x Denha rd t s is 0.02% ( w / v ) each of bov ine serum a lbum in , f i c c l l , p o l y v i n y l p y r o l l i d o n e ) and 100 ug/ml son icated - 168 -dena tu red salmon sperm D N A at 42°C fo r 16 h r . H y b r i d i z a t i o n was c a r r i e d out in the above so lu t ion con ta in ing 3 2 P n i ck t r ans la ted 7 8 D H F R - c D N A (0 .5 x 10 c p m / m l , spec i f i c ac t i v i t y 1 x 10 c p m / u g ) at 42°C fo r 24 h r . T h e papers were then sequen t ia l l y washed in 2 x S S C / 0 . 1 % S D S at room tempera tu re , 0.1 x S S C 0.1% S D S at 50°C and f i na l l y in 0.1 x S S C / 0 . 1 % S D S at room tempera tu re and exposed to x - r a y f i lm (Kodak X - O m a t A R ) at - 7 0 ° C . A f t e r a u t o r a d i o g r a p h y , squa res of n i t roce l lu lose c o r r e s p o n d i n g to the spots were counted by l i qu id sc in t i l l a t ion c o u n t i n g . S y n t h e s i s of 3 2 P - l a b e l e d c D N A A 3 2 P - l a b e l e d DNA complementary to a mur ine D H F R c D N A , wh ich was p resen t in a c loned p lasmid (p D H F R II) was s y n t h e s i z e d by n i ck t r a n s l a t i o n . T h e plasmid DNA was d iges ted wi th the res t r i c t i on enzyme Ps t 1 to remove the i nse r ted DNA and e lec t ro -phoresed in 1% agarose g e l . T h e i nse r ted D N A was ex t rac ted f rom the gel and n ick t r ans la ted wi th ( a - 3 2 P ) - d C T P us i ng a n i ck t rans la t i on system supp l i ed by Amersham C o r p . T h e map of the D H F R - c D N A is shown in C h a p t e r 1. Messenger R N A Isolat ion and Determinat ion  of the Re la t i ve D H F R mRNA Con ten t Messenger R N A was isolated f rom M T X - s e n s i t i v e and - r e s i s t a n t HL -60 ce l ls as d e s c r i b e d p r e v i o u s l y ( 5 ) . Re la t i ve D H F R - m R N A content was determined essen t ia l l y as d e s c r i b e d by Co l l i ns et a l , ( 6 ) . B r i e f l y , known amounts of po ly ( A ) + m R N A were b r o u g h t to 20 mM sodium phospha te , pH 6.8 in 50% formamide (de ion ized ) -7% fo rma lde-- 169 -hyde (dena tu ra t ion b u f f e r ) and i ncuba ted at 65°C fo r 15 m in . to dena tu re the R N A . A l i quo t s of 5 to 50 ul were then added to 0 .3 ml of 11 x S S C in 3.5% fo rma ldehyde and the volume was made up to 0 .4 ml wi th dena tu ra t ion b u f f e r . T h e en t i re 0.4 ml was app l ied as d e s c r i b e d above to n i t r o -ce l lu lose f i l t e r s wh ich had been r i nsed wi th 20 x S S C . A f t e r bak i ng the f i l t e r s fo r 2 h r . at 80°C u n d e r v a c u u m , the m R N A was h y b r i d i z e d wi th 3 2 P - l a b e l e d n i c k - t r a n s l a t e d D H F R - c D N A as d e s c r i b e d above . Labe l ing of Ce l l s Exponen t i a l l y g row ing H L - 6 0 ( S ) and ( R 4 ) ce l ls were washed twice in meth ion ine- f ree medium and suspended at approx imate ly 8 x 5 10 ce l l s /m l ( total volume - 50 ml) in the same medium supp lemented wi th 10% fetal cal f s e r u m . A f t e r 90 m in . of incubat ion at 37°C, the cel ls were pel le ted and resuspended in f r e s h meth ion ine- f ree medium con ta in ing 10% fetal ca l f serum and 10 uCi [ 3 5 S ] methionine per ml (1050 C i / m m o l ) . A f t e r 2 h r . of incubat ion the ce l ls were pel le ted and washed th ree times wi th cold phosphate bu f f e red sa l ine ( P B S : 0 . 1 5 M N a H 2 P 0 4 . H 2 O / 0 . 1 5 M N a 2 H P 0 4 , pH 7 .2 /0 .9% ( w / v ) NaCI) con ta in ing 10 mM meth ion ine. Ce l l l ysa tes were then p r e p a r e d as d e s c r i b e d above . Incorpora t ion of rad ioac t i ve label into p ro te in was determined by t r i ch lo roace t i c ac id p r e c i p i t a t i o n . B r i e f l y , a small a l iquot of the labeled lysate was app l ied to a g lass f i b e r ce l lu lose d i sc wh ich was then washed sequen t ia l l y in ice cold 10% ( w / v ) T C A fo r 10 m i n . , 5% ( w / v ) T C A fo r 5 m in . and 95% ( v / v ) e thano l . T h e d i sc was d r i e d , t r a n s f e r r e d to a l i qu id sc in t i l l a t ion v ia l and counted in a l i qu id sc in t i l l a t ion c o u n t e r . - 170 -T h e 3 5 S - l a b e l l e d cel l e x t r a c t s were used fo r immunoprec ip i ta t ion and S D S po lyac ry lamide gel e l ec t r opho res i s . Immunoprec ip i ta t ion T h e immunoprec ip i ta t ion react ions were c a r r i e d out us i ng r a b b i t -an t i -mouse D H F R an t ibody (L5178Y an t i - fo rm 1) as d e s c r i b e d p r e v i o u s l y ( 5 , and C h a p t e r 2 ) . A l i quo t s of the e lu ted labeled p ro te ins f rom the immune complexes were e i ther quant i ta ted by l iqu id sc in t i l l a t ion coun t ing of t r i ch lo roace t i c ac id p rec ip i t ab le mate r ia l , o r ana lyzed by S D S po lyac ry lamide gel e lec t rophores i s and au to -r a d i o g r a p h y ( 5 ) . Immune Dot B lo t A s s a y s T h e amount of DHFR pro te in in H L - 6 0 ( S ) and ( R 4 ) cel l l ysa tes was also quan t i ta ted by a dot blot a s s a y . Known amounts (0.1 to 50 ug) of p ro te in in 0.4 ml T r i s bu f f e red sa l ine ( T B S : 20 mM T r i s / 5 0 0 mM N a C L , pH 7 .5) were app l ied to a n i t roce l lu lose f i l t e r (0 .20 micron d iameter pore s ize ) us i ng a h y b r i - d o t mani fo ld ( B e t h e s d a Research L a b s . , G a i t h e r s b u r g , M D . ) . A f t e r d r y i n g , the f i l t e r paper was immersed in b l ock ing so lu t ion ( T B S con ta in ing 3% ( w / v ) bov ine serum a lbumin) f o r 45 m in . T h e n i t roce l lu lose paper was then t r a n s f e r r e d into a so lu t ion con ta in ing rabb i t a n t i - D H F R ant iserum ra ised aga ins t p u r i f i e d D H F R from mouse leukemia (L5178Y) ce l ls ( 5 ) . A f t e r 1 h r . the paper was washed in T B S fo r 20 m in . (2 - 100 ml washes , 10 min . e a c h ) . T h e paper was nex t t r a n s f e r r e d into a so lut ion con ta in ing h o r s e r a d i s h pe rox idase con jugated goat a n t i - r a b b i t immunoglobul in and al lowed to incuba ted fo r 1 h r . T h e f ree and n o n -spec i f i c bound an t ibody was removed by wash ing the paper in T B S as - 171 -d e s c r i b e d above . T h e n i t roce l lu lose paper was then immersed in a so lu t ion cons i s t i ng of a 1:1 m ix tu re of h y d r o g e n pe rox ide and 2 , 2 ' - a z i n o d i ( 3 -e thy lbenz th iazo l i ne su l fona te ) ( K i r k e g a a r d and P e r r y L a b s . , G a i t h e r s b u r g , M D . ) . Pos i t i ve react ions were ind ica ted by the appearance of p u r p l e d o t s . R E S U L T S Isolation of M T X - r e s i s t a n t H L - 6 0 V a r i a n t s T h e se lect ion of M T X - r e s i s t a n t ce l ls f rom the w i l d - t y p e M T X -sens i t i ve H L - 6 0 ce l ls was in i t ia l l y c a r r i e d out in the p resence of 5 nM M T X , and the rea f te r se lect ion of ce l ls res i s tan t to h i ghe r c o n c e n -t ra t ions of M T X was c a r r i e d out by a s tep -w i se inc rease in M T X c o n c e n t r a t i o n . F i g . 5-1 shows the time taken fo r ce l ls res i s tan t to a p a r t i c u l a r concen t ra t ion of M T X to ou tg row the more sens i t i ve popula t ion of ce l ls at p r o g r e s s i v e l y h i ghe r concen t ra t ions of M T X . T h e time cou rse is compared to tha t taken by the Hela ce l ls at equ iva len t M T X concen t ra t i ons . It would appear tha t the f r e q u e n c y to mutat ion lead ing to M T X res is tance is much lower in H L - 6 0 ce l ls than in HeLa c e l l s , wh ich o v e r p r o d u c e d D H F R by v i r t u e of gene ampl i f icat ion ( F i g . 5 - 2 ) . Quant i f i ca t ion of D H F R A c t i v i t y in H L - 6 0  M T X - S e n s i t i v e and - R e s i s t a n t Ce l l s DHFR a c t i v i t y in ce l lu la r l ysa tes was assayed as d e s c r i b e d in Mater ia ls and Methods at v a r y i n g pro te in concen t ra t i ons . DHFR ac t i v i t y was marked ly e levated in H L - 6 0 ( R 4 ) ce l ls ( r e s i s t a n t to 1 uM M T X ) as compared to that in H L - 6 0 ( S ) ce l ls ( F i g . 5-3 A ) . T h e - 172 -F I G U R E 5-1 Time C o u r s e fo r the Development of M T X Res is tance in HL -60 and  Hela C e l l s . T h e c u l t u r i n g and se lect ion of M T X - r e s i s t a n t ce l ls is d e s c r i b e d in the Mater ia ls and Methods sec t i on . o H L - 6 0 c e l l s ; m Hela c e l l s . 173 Resistance to [MTX] M - 174 -F I G U R E 5-2 Sou the rn B lo t A n a l y s i s of HeLa ( S ) and ( R 4 ) Genomic D N A T h e exper imen t was c a r r i e d out exac t l y as d e s c r i b e d in F i g . 2 - 8 . A . HeLa ( S ) DNA (10 ug) Lane 1: Eco R1 d iges t Lane 2: T a q 1 d iges t Lane 3 : Bam H1 d iges t B . HeLa ( R 4 ) D N A (10 ug) Lane 1: Eco R1 d iges t Lane 2 : T a q 1 d iges t Lane 3: Bam H1 d iges t T h e numbers on the r i gh t hand s ide of the f i g u r e rep resen t s izes in k i lobases of DNA molecular we igh t marke rs r u n on the same g e l . - 176 -F I G U R E 5-3 D H F R A c t i v i t y in Ce l l u l a r L y s a t e s . D H F R ac t i v i t y was measured as d e s c r i b e d in the Mater ia ls and Methods sect ion us i ng i nc reas ing amounts of l ysa te p r o t e i n . A . • H L - 6 0 ( S ) cel l l y sa te ; • H L - 6 0 ( R 4 ) cel l l y sa te . o H L - 6 0 ( R 4 - ) cel l l y sa te . B . o Hela ( R 4 - ) cel l l y sa te ; © Hela ( R 4 ) cel l l y sa te . 177 Protein( tig) Protein( /*g) - 1 7 8 -elevat ion rep resen ts an approx ima te ly 2 0 fo ld inc rease in D H F R spec i f i c a c t i v i t y . F i g . 5 - 3 A also shows the D H F R ac t i v i t y in ( H L - 6 0 ( R 4 - ) c e l l s . These ce l ls were washed f ree of M T X and g rown in the absence of M T X fo r 2 8 cel l d o u b l i n g s . Removal of se lect ion p r e s s u r e d id not resu l t in a d rop in D H F R a c t i v i t y ( F i g . 5 - 3 A ) or M T X -res is tance ( I . D . ^ Q of R 4 ( - ) ce l ls = 5 0 uM as opposed to I . D . ^ Q of S ce l ls = 5 nM) and the re fo re these ce l ls appear to be s tab l y r es i s t an t . Uns tab le r e s i s t a n c e , i . e . a decrease in D H F R ac t i v i t y upon removal of M T X can be detected in 3 0 d o u b l i n g s as shown fo r Hela ce l ls ( F i g . 5 - 3 B ) wh ich are uns tab l y res i s tan t to M T X . It shou ld be noted that the basal D H F R a c t i v i t y in the Hela ce l ls ( i . e . in Hela ( S ) and ( R 4 - ) ce l l s ) is approx imate ly 1 0 fo ld h i ghe r than the basal ac t i v i t y in H L - 6 0 ce l ls ( i . e . H L - 6 0 ( S ) ) ce l ls ( F i g . 5 - 3 ) . T h e mechanism fo r the h i ghe r ac t i v i t y in the Hela ce l ls is not c l e a r . Re la t i ve D H F R Gene Dosage in H L - 6 0 ( S ) and H L - 6 0 ( R 4 ) Ce l l s S i nce an inc rease in D H F R ac t i v i t y is usua l l y assoc ia ted wi th the concomitant ampl i f icat ion of D H F R genes ( 7 ) , we wanted to determine whe ther gene ampl i f icat ion had taken p lace in the H L - 6 0 ( R 4 ) c e l l s . T h e re la t i ve gene dosage was determined by do t -b lo t ana lys i s of genomic DNA f rom H L - 6 0 ( S ) and ( R 4 ) ce l ls us i ng a 3 2 P - l a b e l e d mouse D H F R c D N A probe ( p D H F R II) . T h e ana lys i s was c a r r i e d out by the method d e s c r i b e d by V a r s c h a v s k y ( 4 ) . F i g . 5 - 4 shows tha t the D H F R gene dosage in the H L - 6 0 ( S ) and ( R 4 ) ce l ls is s im i la r . T h e r e is no ind ica t ion of i nc reased gene dosage in the res i s tan t c e l l s . Inc luded in the ana l ys i s was the est imat ion of gene dosage in mouse - 179 -F I G U R E 5-4 Est imat ion of D H F R Gene Dosage . Est imat ion of D H F R gene dosage in H L - 6 0 M T X - s e n s i t i v e and - r e s i s t a n t cel l l ines was determined by dot blot ana l ys i s u s i n g a mouse D H F R - c D N A p r o b e . Inc reas ing amounts of genomic D N A were app l ied to a n i t roce l lu lose membrane and h y b r i d i z e d to n i ck t r ans la ted 3 2 P label led D H F R - c D N A as d e s c r i b e d in Mater ia ls and Me thods . A . A u t o r a d i o g r a p h of D N A do t -b lo t f rom wh ich data fo r B were o b t a i n e d . Lane A : H L - 6 0 ( S ) DNA Lane B : HL -60 ( R 4 ) D N A Lane C : L5178Y ( R 4 ) D N A Bottom to t o p : i nc reas ing amount of DNA app l ied (0.1 -20ug) . B . A f t e r au to rad iog raphy squa res con ta in ing the app l ied DNA were cu t ou t , t r a n s f e r r e d to sc in t i l l a t ion v ia ls and counted in a l iqu id sc in t i l l a t ion c o u n t e r . o H L - 6 0 ( S ) D N A ; e H L - 6 0 ( R 4 ) D N A ; A L5178Y ( S ) D N A ; • L5178Y ( R 4 ) D N A . 100 DNA ( ng)-- 181 -C leukemia L5178Y(S) and L5178Y(R) c e l l s , the la t ter g row ing in 1 mM M T X . T h i s ce l l - l i ne has been shown to o v e r p r o d u c e D H F R (5) and subsequen t l y shown to have inc reased D H F R gene copy numbers by Sou the rn b lot ana l ys i s ( C h a p t e r 2 ) . From F i g . 5-4 B the gene dosage in the L5178Y ( R ) ce l ls can be est imated to be 25 fo ld g rea te r than in the L5178Y ( S ) c e l l s , and ind ica tes tha t th i s method can be used to determine re la t ive gene dosages in d i f f e ren t c e l l s . F i g . 5-4 A shows the actual au to rad iog raph of the dot ana l ys i s f rom wh ich the data p resen ted in F i g . 5-4 B were ob ta i ned . D H F R - m R N A Con ten t in M T X - S e n s i t i v e and - R e s i s t a n t H L - 6 0 Ce l l s Dot b lot ana lys i s of p u r i f i e d m R N A f rom sens i t i ve and res i s tan t ce l ls fa i led to show any d i f f e rences in the D H F R - m R N A conten t in the two cel l l ines ( F i g . 5-5 A and B ) . T h i s f i n d i n g is cons is ten t wi th lack of gene ampl i f icat ion and also wi th the lack of o v e r p r o d u c t i o n of D H F R pro te in (see be low) . Inc luded in the ana lys i s was the D H F R m R N A content of L5178Y ( S ) and L5178Y ( R ) c e l l s , the la t ter has p r e v i o u s l y been shown to have i nc reased D H F R pro te in and m R N A (5 and C h a p t e r 2 ) . A negat ive cont ro l was also c a r r i e d out to show the spec i f i c i t y of the DHFR p r o b e . T h i s enta i led the h y b r i d i z a t i o n of the D H F R p robe to i nc reas ing amounts of g lob in mRNA bound to a n i t r o -ce l lu lose f i l t e r . No au to rad iog raph i c s igna l was v i s i b l e and the rad ioac t i ve coun ts were s imi lar to b a c k g r o u n d c o u n t s . D H F R Con ten t in H L - 6 0 M T X - S e n s i t i v e and - R e s i s t a n t Ce l l s T h e D H F R content in sens i t i ve and res i s tan t ce l ls was determined by two d i f f e ren t p r o c e d u r e s : S D S - P A G E of ce l l u la r l ysa tes and immune dot blot a s s a y s . T h e rate of D H F R s y n t h e s i s was determined - 182 -F I G U R E 5-5 D H F R mRNA Con ten t in M T X - S e n s i t i v e and - R e s i s t a n t H L - 6 0 C e l l s . M e s s e n g e r - R N A from the v a r i o u s cel l l ines was p u r i f i e d by o l i g o - d T ce l lu lose a f f i n i t y ch romatog raphy and i nc reas ing amounts of mRNA were app l ied to a n i t roce l lu lose membrane. T h e membrane-bound mRNA was h y b r i d i z e d to n ick t r ans la ted 3 2 P - l a b e l e d mouse D H F R -c D N A as d e s c r i b e d in Mater ia ls and Me thods . A . A u t o r a d i o g r a p h of mRNA do t -b lo t f rom wh ich data fo r F i g u r e 4A were ob ta i ned . Lane A : L5178Y ( S ) mRNA Lane B : L5178Y ( R 4 ) mRNA Lane C : H L - 6 0 ( S ) mRNA Lane D: H L - 6 0 ( R 4 ) m R N A Lane E: HL -60 ( R 4 - ) mRNA B . A f t e r au to rad iog raphy squa res con ta in ing the app l ied mRNA were cu t ou t , t r a n s f e r r e d to sc in t i l l a t ion v ia l s and counted in a l iqu id sc in t i l l a t ion c o u n t e r . A H L - 6 0 ( R 4 ) m R N A ; • H L - 6 0 ( S ) m R N A ; o L5178Y ( S ) m R N A ; • L5178Y ( R 4 ) m R N A . - 184 -by immunoprec ip i ta t ion of 3 E 1 S - m e t h i o n i n e label led ce l l u la r p r o t e i n s . F i g . 5-6 shows the s i l v e r s ta ined p ro f i l e of S D S - P A G E c a r r i e d out on ce l l u la r l ysa tes f rom H L - 6 0 ( S ) and ( R 4 ) c e l l s . A s can be s e e n , the 20 fo ld inc rease in D H F R a c t i v i t y is not assoc ia ted wi th a concomitant 20 fo ld inc rease in D H F R p r o t e i n . T h e r e does not appear to be a d i f f e rence in the in tens i t ies of the pro te in bands in the S and R 4 lanes c o r r e s p o n d i n g to the mouse D H F R band at approx imate ly 20,000 da l t ons . U s i n g a s imi lar p r o c e d u r e , Domin et a[, (8) were able to detect i nc reased D H F R band in tens i t ies f rom cel ls w i th as l i t t le as 9 fo ld i nc reases in D H F R p r o t e i n . T h e r e f o r e a 20 fo ld inc rease in D H F R pro te in shou ld have been detected by th i s method. A n t i b o d y immunolocal izat ion u s i n g mouse a n t i - D H F R on Western b lots of these gels fa i led to detec t D H F R bands in l ysa tes of all t h ree cel l l i nes , i n d i c a t i n g , e i ther tha t the D H F R content was too low to be d e t e c t e d , or tha t the mouse an t i body fa i led to react wi th the human D H F R . T h e lat ter exp lanat ion is u n l i k e l y s ince g rea te r than 85% homology has been shown between mouse and human D H F R s ( 9 ) . F i g . 5-7 shows the immune dot blot ana lys i s of cel l l ysa tes f rom the sens i t i ve and res i s tan t ce l ls us i ng an t ibody p r e p a r e d aga ins t L5178Y mouse leukemia cel l D H F R ( 5 ) . Inc reas ing amounts of ce l l u la r p ro te in ( f rom 0.1 to 50 ug) were app l ied to n i t roce l lu lose paper and p robed wi th a n t i - D H F R an t ibody as d e s c r i b e d in the Mater ia ls and Methods sec t i on . A s can be seen the re does not appear to be any inc rease in a n t i - D H F R c r o s s - r e a c t i n g mater ial in the res i s tan t cel ls as compared to the sens i t i ve c e l l s . D H F R s y n t h e s i s in M T X - s e n s i t i v e and - r e s i s t a n t ce l ls was d e t e r -mined by immunoprec ip i ta t ion of 3 5 S - p u l s e - l a b e l e d p r o t e i n s . Both the - 185 -F I G U R E 5-6 S D S Po lyac ry lamide Gel E lec t rophores i s of Lysa tes f rom H L - 6 0  ( S ) and ( R 4 ) C e l l s . E lec t ropho res i s was c a r r i e d out in the p resence of S D S in a 13% po lyac ry lamide gel (29 .2 ac ry lam ide : 0.8 b i sac ry lam ide ) in 0.25 M T r i z m a b a s e / 0 . 1 9 2 M g l yc i ne pH 8.1 con ta in ing 0.1% S D S fo r 4 h r . at 30 mA. T h e gel was then s ta ined by the s i l v e r s ta in ing method d e s c r i b e d in C h a p t e r 2 . Lane A : Molecu lar we igh t marke rs ob ta ined f rom B i o - R a d L a b o r a t o r i e s , R i c h m o n d , C A . ( top to bot tom: phospho ry l ase b , 94 ,000 ; bov ine serum a lbum in , 68 ,000 ; o v a l b u m i n , 43 ,000; ca rbon i c a n h y d r a s e , 30 ,000; soybean t r y p s i n i n h i b i t o r , 21 ,000; l ysozyme, 14 ,300) . Lane B : P u r i f i e d mouse D H F R (10 ug) Lane C : HL -60 ( S ) l ysa te (50 ug) Lane D: H L - 6 0 ( R 4 ) l ysate (50 ug) 136 A B C D - 187 -F I G U R E 5-7 Immune Do t -b lo t A n a l y s i s Inc reas ing amounts ( top to bot tom: 0 .1-50 ug) of l ysa te pro te in were app l ied to a n i t roce l lu lose membrane (0 .22 micron pore s i ze ) and the membrane was then p robed wi th rabb i t an t i -mouse D H F R ant iserum (Dedha r and G o l d i e , 1983) as d e s c r i b e d in Mater ia ls and Me thods . A negat i ve cont ro l cons i s t i ng of a p p l y i n g i n c r e a s i n g amounts of bov ine serum albumin was r u n s imu l taneous ly . T h e pos i t i ve cont ro l cons is ted of a p p l y i n g i n c r e a s i n g amounts of p u r i f i e d mouse D H F R aga ins t wh ich the an t ibody was p r e p a r e d ( lane E ) . Lane 1: H L - 6 0 ( S ) l ysa te Lane 2 : H L - 6 0 ( R 4 ) l ysate Lane 3 : H L - 6 0 ( R 4 ) l ysa te Lane 4: H L - 6 0 ( R 4 - ) l ysate Lane 5 : p u r i f i e d mouse D H F R - 189 -sens i t i ve and res i s tan t cel l l ines i nco rpo ra ted a s imi lar amount of label into total so lub le p ro te in (Tab le 5 - 1 ) , and in a d d i t i o n , the res i s tan t ce l ls wh ich have inc reased D H F R ac t i v i t y d i d not s y n t h e s i z e t h i s p ro te in at an inc reased rate compared to the sens i t i ve ce l ls (Tab le 5 - 1 ) . S D S P A G E fo l lowed by au to rad iog raphy con f i rm these resu l t s ( F i g . 5 - 8 ) . T h e r e is no inc rease in the i n tens i t y of bands c o r r e s p o n d i n g to the mouse D H F R ( lane B ^ 20,000 da l tons ) in the R 4 lane ( lane D) a n d , in a d d i t i o n , immunoprec ip i ta t ion of the p ro te ins wi th an t i -mouse D H F R an t ibody fa i led to p rec ip i ta te a more in tense pro te in band c o r r e s p o n d i n g to DHFR f rom the R 4 l ysa te ( lane E) as compared to S lysate ( lane F ) . D I S C U S S I O N I have desc r i bed here some of the p rope r t i es of a M T X - r e s i s t a n t cel l l ine d e r i v e d f rom a M T X - s e n s i t i v e H L - 6 0 p romye locy t i c leukemia cel l l i ne . T h e M T X - r e s i s t a n t cel l l ine cha rac te r i zed ( H L - 6 0 R 4 ) g rows in the p resence of 1 uM M T X wi th a doub l i ng time approx imate ly that of the sens i t i ve cel l l ine f rom wh ich it was d e r i v e d . A 20 fo ld inc rease in D H F R ac t i v i t y was o b s e r v e d in the res i s tan t cel l l i ne . S tepwise se lect ion of M T X - r e s i s t a n t va r i an t s usua l l y resu l t s in the select ion of ce l ls wi th i nc reased D H F R a c t i v i t y , and in e v e r y case repor ted to date the inc rease is assoc ia ted wi th o v e r p r o d u c t i o n of D H F R pro te in r esu l t i ng f rom an inc rease in the D H F R gene copy number ( fo r a recent r ev i ew , see 7 ) . Howeve r , in the HL -60 M T X - r e s i s t a n t va r i an t d e s c r i b e d here the i nc rease in D H F R ac t i v i t y appears not to be assoc ia ted wi th an o v e r -p roduc t i on of D H F R p r o t e i n . I have shown th i s to be the case by - 190 -T A B L E 5-1 T-ABLE 1. SYNTHESIS OF DHFR Cell line cpm incorporated into total soluble protein/mg protein cpm incorporated into DHFR/mg protein immunopptd. Ratio. DHFR incorporation/ total protein incorporation DHFR Synthesized in resistant cells/ OHFR synthesized in sensitive cells HL-60(S) 2.2 x 107 7.47 x 104 0.0034 1.0 HL-60(R4) 1.5 * 107 6.8 x 10* 0.0045 1.33 Ce l l s were incuba ted in the p resence of 10 uC i /m l 3 5 S - m e t h i o n i n e in meth ion ine- f ree medium fo r 2 h r . a f ter wh ich the cel ls were pe l le ted and l y s e d . Pro te in concen t ra t ion was determined and incorpora t ion of the rad io label into p ro te ins was determined by cold t r i ch lo roace t i c ac id p rec ip i ta t ion of an a l iquot of the ce l l u la r lysates fol lowed by sc in t i l l a t ion c o u n t i n g . Ce l l l ysates r e p r e s e n t i n g equ iva len t amounts of pro te in were immunoprec ip i ta ted wi th rabb i t an t i -mouse DHFR a n t i s e r u m , and cold t r i ch lo roace t i c ac id p rec ip i t ab le counts in the immunoprec ip i ta tes were de te rm ined . - 191 -F I G U R E 5-8 A u t o r a d i o g r a p h of S D S Po lyac ry lamide Gel E lec t ropho res i s of  3 5 S Meth ion ine- labe led Ce l l u l a r P r o t e i n s . C e l l u l a r p ro te ins were label led in v i v o by i ncuba t i ng ce l ls in the p resence of 3 5 S - m e t h i o n i n e (10 u C i / m l ) in methionine f ree medium as d e s c r i b e d in Mater ia ls and Me thods . Equ iva len t amounts of label led l y s a t e s , or immunoprec ip i ta ted lysa tes were app l ied to a 13% p o l y -acry lamide gel and e lec t rophoresed as d e s c r i b e d fo r F i g u r e 5 -6 . A f t e r e lec t rophores i s the gel was p rocessed fo r f l u o r o g r a p h y as d e s c r i b e d in C h a p t e r 2 . Lane A : 1 4 C - m e t h y l a t e d molecular we ight p ro te in marke rs (Amersham C o r p . , O a k v i l l e , O n t . ) . From top to bot tom: 92 ,500 , 69 ,000, 46 ,000, 30,000 and 14,300 da l t ons . Lane B : 3 H - l e u c i n e labeled mouse D H F R (band at ~ 20,000 da l t ons , ind ica ted by a r r o w ) . Lane C : 3 S S - m e t h i o n i n e labeled H L - 6 0 ( S ) cel l l ysa te (800,000 cpm a p p l i e d ) . Lane D: 3 5 S - m e t h i o n i n e labeled H L - 6 0 ( R 4 ) cel l l ysa te (800,000 cpm a p p l i e d ) . Lane E: 3 5 S - m e t h i o n i n e labeled H L - 6 0 ( S ) lysate immuno-p rec ip i ta ted wi th a n t i - D H F R an t i se rum. Lane F: 3 5 S - m e t h i o n i n e labeled H L - 6 0 ( R 4 ) lysate immuno-p rec ip i t a ted wi th a n t i - D H F R a n t i s e r u m . A B C D E F - 193 -two d i f f e ren t approaches (see Resu l t s sec t ion) and in each case the amount of D H F R pro te in in the sens i t i ve and res i s tan t ce l ls is s imi lar and rep resen t s a v e r y small pe rcen tage of the total ce l l u l a r p r o t e i n . T h e rate of D H F R s y n t h e s i s in H L - 6 0 ( S ) and ( R 4 ) ce l ls is s imi lar (Tab le 5-1) nega t ing the poss ib i l i t y that the inc reased D H F R ac t i v i t y is due to an inc rease in the rate of D H F R s y n t h e s i s . S ince the s teady -s ta te level of D H F R pro te in is also s imi lar in both cel l l ines it is u n l i k e l y tha t the inc rease in ac t i v i t y cou ld be due to a dec reased degrada t ion rate of DHFR in the ( R 4 ) c e l l s . In a d d i t i o n , the D H F R m R N A content and D H F R gene dosage in the sens i t i ve and res i s tan t ce l ls were est imated u s i n g a mouse mur ine D H F R c D N A . Bo th of these are s imi lar in the two cel l l i nes . T h e est imat ions were determined by do t -b lo t assays and it has been shown tha t any inc reases in D H F R mRNA or D N A in the res i s tan t ce l ls would have been detected by th i s method. S imul taneous assay of L5178Y M T X - s e n s i t v e and - r e s i s t a n t c e l l s , wh ich have p r e v i o u s l y been shown to o v e r p r o d u c e D H F R pro te in ( 5 ) , c l ea r l y show an approx imate ly 25 fo ld inc rease in D H F R gene-dosage in the res i s tan t L5178Y ce l ls ( F i g . 5-4 and C h a p t e r 2 ) . It is u n l i k e l y tha t the gene dosage has been underes t imated because of the use of mouse D H F R c D N A to p robe human sequences s ince almost 90% homology ex i s t s between mouse and human D H F R cod ing sequences ( 9 ) . T h e f i n d i n g tha t D H F R ac t i v i t y can be enhanced up to 20 fo ld in the absence of an inc rease in D H F R pro te in has not been repo r ted p r e v i o u s l y . T h e inc rease in D H F R ac t i v i t y in th i s case is not modulated by M T X as has been shown by Domin et a[, ( 1 0 ) , s ince g rowth of ce l ls in the absence of M T X for up to 30 cel l doub l i ngs d id - 194 -not d imin ish the ac t i v i t y ( F i g . 5-3 A ) . It would t he re fo re appear that the inc rease in D H F R ac t i v i t y is a s tab le one . T h i s pheno type d i f f e r s f rom tha t of a Hela res i s tan t cel l l ine tha t has a comparable inc rease in D H F R ac t i v i t y ove r tha t in the Hela sens i t i ve ce l ls wh ich decreases upon removal of M T X ( F i g . 5-3 B ) . T h e inc rease in ac t i v i t y in t h i s la t ter case is due to D H F R gene-ampl i f i ca t ion ( F i g . 5-2) and may rep resen t uns tab le D H F R gene-ampl i f i ca t ion (11 ) . T h e s e data ind ica te tha t the H L - 6 0 res i s tan t ce l ls e x p r e s s an enzyme wi th a 20 fo ld h i ghe r i n t r i n s i c spec i f i c ac t i v i t y than tha t p resen t in the sens i t i ve c e l l s . T h i s cou ld come about by one of two a l t e r n a t i v e s : the res i s tan t ce l ls e x p r e s s an a l tered form of D H F R lead ing to a more ac t i ve enzyme, o r , tha t the DHFR in the res i s tan t ce l ls is ac t iva ted by an as ye t u n c h a r a c t e r i z e d in v i v o modulator of D H F R . T h e lat ter mechan ism, i . e . one of in v i v o ac t i va t ion has an in v i t r o p r e c e d e n t . It has been known fo r qu i te some time that mammalian D H F R s can be ac t i va ted up to 10 fo ld in v i t r o by compounds such as o rgan i c mercur ia l s (12,13) and te t ra th iona te (14) wh ich react wi th a s ing le s u l p h y d r y l g roup located near the amino termina l end of the molecule. T h e in v i t r o ac t i va ted conformer has been recen t l y cha rac te r i zed in detai l (13) and found to exh ib i t a marked ly i nc reased s e n s i t i v i t y to hea t , p r o t e o l y s i s , and the ionic env i ronment as compared to the nat ive form of D H F R . A s wil l be d e s c r i b e d in the nex t sect ion of th i s chap te r the D H F R from HL -60 ( R 4 ) ce l ls is also h i gh l y sens i t i ve to heat as compared to tha t in the H L - 6 0 ( S ) c e l l s . A n i nc reas ing number of enzymes have been shown recen t l y to be modif ied by s u l p h y d r y l react ion in v i v o , e . g . l i ve r phospho ry l ase phosphatase (15 ) , ra t b ra in adeny la te cy lase ( 1 6 ) , euca ryo t i c in i t ia t ion fac to r 2 ( 17 ) . It is poss ib le that the pu ta t i ve - 195 -phys io log ica l modulator normal ly r egu la t i ng the ac t i v i t y of D H F R in v i v o has been permanent l y " t u r n e d o n " in the H L - 6 0 M T X - r e s i s t a n t ce l ls lead ing to the ac t i va t ion of normal D H F R . It is not c lear as to what ex ten t the 20 fo ld inc rease in D H F R ac t i v i t y would have on the res i s tance of these ce l ls to M T X . O t h e r mechan isms, such as impai red i n f l ux of M T X (18) and dec reased p o l y -g lutamat ion of M T X (19) cannot be ru led ou t . In any case the ava i l ab i l i t y of a . cel l l ine wh ich e x p r e s s e s a more ac t i ve D H F R may lead to a be t te r u n d e r s t a n d i n g of the regu la t ion of D H F R ac t i v i t y in v i v o and may also be of re levance in chemotherapy wi th fo late an tagon i s t s . - 196 -R E F E R E N C E S 1. C o l l i n s , S . J . / Ga l l o , R . C . and G a l l a g h e r , R . E . (1977) Con t i nuous g rowth and d i f fe ren t ia t ion of human myelo id leukemic ce l ls in suspens ion c u l t u r e . Na tu re 270, 347-349. 2. G o l d i e , J . H . , D e d h a r , S . and K r y s t a l , G . (1981) P rope r t i es of a me tho t rexa te - i nsens i t i ve v a r i a n t of d i hyd ro fo la te rduc tase d e r i v e d f rom methot rexate res i s tan t L5178Y c e l l s . J . B i o l . C h e m . 256, 11629-11635. 3. B l i n , N . and S t a f f o r d , D.W. (1976) Isolat ion of h igh molecular we igh t D N A . Nuc le ic A c i d R e s . 3 , 2303-2308. 4. V a r s c h a v s k y , A . (1981) Pho rbo les te r d ramat ica l l y inc reases inc idence of me tho t rexa te - res i s tan t mouse c e l l s : poss ib le mechanisms and re levance to tumor p romot ion . Ce l l 25, 561-572. 5. D e d h a r , S . and G o l d i e , J . H . (1983) O v e r p r o d u c t i o n of two an t igen ica l l y d i s t i nc t forms of d i hyd ro fo la te reduc tase in a h i g h l y me tho t rexa te - res i s tan t mouse leukemia cel l l i ne . C a n c e r R e s . 43, 4863-4871. 6. C o l l i n s , M . L . , W u , R . J . , San t i ago , C , H e n d r i c k s o n , S . L . and J o h n s o n , L . F . (1983) Delayed p rocess ing of d ihyd ro fo la te reduc tase heterogeneous nuc lea r R N A in amino a c i d - s t a r v e d mouse f i b r o b l a s t s . Mo l . and C e l l . B i o l . 3 , 1792-1802. 7. S c h i m k e , R . T . (1984) Gene amp l i f i ca t ion , d r u g r e s i s t a n c e , and c a n c e r . C a n c e r R e s . 44, 1735-1742. 8. Domin , B . A . , G r i l l , S . P . and C h e n g , Y . (1983) Es tab l i shment of d i hyd ro fo l a te r e d u c t a s e - i n c r e a s e d human cel l l ines and re la t ionsh ip between d ihyd ro fo la te reduc tase levels and gene c o p y . C a n c e r R e s . 43 , 2155-2158. 9. C h e n , M . , Sh imada , T . , Mou l ton , A . D . , C l i n e , A . , H u m p h r i e s , R . K . , Ma i ze l , J . and N i e n h u i s , A . W . (1984) T h e func t iona l human d ihyd ro fo la te reduc tase gene . J . B i o l . C h e m . 259, 3933-3943. 10. Domin , B . A . , G r i l l , S . P . , Bas tow , K . F . and C h e n g , Y . C . (1982) Ef fec t of methot rexate on d ihyd ro fo la te reduc tase ac t i v i t y in me tho t rexa te - res i s tan t human K B c e l l s . Mo l . Pharmaco l . 2 1 , 478-482. 11 . Kau fman , R . J . , B r o w n , P . C . and S c h i m k e , R . T . (1981) Amp l i f i ed d ihyd ro fo la te reduc tase genes in uns tab l y metho-t r e x a t e - r e s i s t a n t ce l ls are assoc ia ted wi th doub le -m inu te chromosomes. P r o c . N a t l . A c a d . S c i . U S A 76, 5669-5673. - 197 -11. Me le ra , P . W . , Lew i s , J . A . , B i e d l e r , J . L . and H e s s i o n , C . (1980) An t i fo la te res i s tan t C h i n e s e hamster o v a r y c e l l s : ev idence fo r d ihyd ro fo la te reduc tase gene ampl i f icat ion among i n d e p e n -den t l y d e r i v e d sub l ines o v e r p r o d u c i n g d i f f e ren t d i hyd ro fo la te r e d u c t a s e s . J . B i o l . C h e m . 255, 7024-7028. 12. Kau fman , B . T . , K u m a r , A . A . , B l a n k e n s h i p , D . T . and F r e i s h e i m , J . H . (1980) Ac t i va t i on of bov ine and c h i c k e n l i ve r d i hyd ro fo la te reduc tases and i ts re la t ionsh ip to a spec i f i c cys te ine res idue in t he i r N H 2 - t e r m i n a l amino ac id s e q u e n c e s . J . B i o l . C h e m . 255, 6542-6545. 13. B a r b e h e n n , E . K . and Kau fman , B . T . (1982) C h i c k e n l i ve r d ihyd ro fo la te r educ tase : ac t iva t ion and a l tera t ion of enzymat i c -p rope r t i es as a resu l t of react ion wi th m e t h y l m e r c u r y . A r c h . B iochem. B i o p h y s . 219, 236-247. 14. B a r b e h e n n , E . K . and Kau fman , B . T . (1980) A l te ra t i on of the p roper t i es of c h i c k e n l i ve r d i hyd ro fo la te reduc tase as a - r e s u l t of modi f icat ion b y te t ra th iona te . J . B i o l . C h e m . 255, 1978-1984. 15. Usami , M . , M a t s u s h i t a , H . and S c h i m a z u , T . (1980) Regu la t ion of l i ve r phospho ry l ase phosphatase by g lu ta th ione d i s u l p h i d e . J . B i o l . C h e m . 255, 1928-1931. 16. B a b a , A . , Lee , E . , M a t s u d a , T . , K i h a r a , T . and Iwata, H . (1978) Reve rs i b l e inh ib i t i on of adeny la te cyc lase ac t i v i t y of ra t b ra in caudate nuc leus by ox i d i zed g lu ta th ione . B iochem. B i o p h y s . R e s . Commun. 85, 1204-1210. 17. J a g u s , R. and S a f e r , R. (1981) A c t i v i t y of euca ryo t i c in i t ia t ion fac to r 2 is modi f ied by a p rocess d i s t i nc t f rom p h o s p h o r y l a t i o n . II A c t i v i t y of euca ryo t i c in i t ia t ion fac to r 2 in lysate is modi f ied by ox ida t ion reduc t ion state of i ts s u l p h y d r y l g r o u p s . J . B i o l . C h e m . 256, 1324-1329. 18. S i r o t n a k , F . M . , K u r i t a , S . and H u t c h i s o n , D . J . (1968) On the na tu re of a t r a n s p o r t a l tera t ion de te rmin ing res is tance to amethopter in in L1210 leukemia . C a n c e r R e s . 28, 75-80 . 19. J o l i v e t , G . , S c h i l s k y , R . L . , B a i l e y , B . D . , D r a k e , J . C . and C h a b n e r , B . A . (1982) S y n t h e s i s , re tent ion and b io logica l ac t i v i t y of methot rexate po lyg lu tamates in c u l t u r e d human b reas t cancer c e l l s . J . C l i n . Invest'. 70, 351-360. - 198 -C H A P T E R 5 S E C T I O N 2 P R O P E R T I E S OF A N A C T I V A T E D F O R M OF D I H Y D R O F O L A T E R E D U C T A S E FROM A M E T H O T R E X A T E - R E S I S T A N T H U M A N P R O M Y E L O C Y T I C L E U K E M I A ( H L - 6 0 ) C E L L L INE I N T R O D U C T I O N One i n te res t i ng p r o p e r t y of mammalian D H F R s is that t hey can be ac t iva ted in v i t r o by ce r ta in compounds such as o rgan i c mercur ia l s ( 3 ) , te t ra th ion i te ( 4 ) , iodine ( 5 ) , and urea and th iou rea ( 6 ) . T h e ac t iva t ion resu l t s f rom cer ta in modi f icat ions of a s ing le s u l p h y d r y l g roup located in the N- termina l reg ion of D H F R ( 7 ) . T h e o rgan i c me rcu r i a l - ac t i va ted form of D H F R has been cha rac te r i zed (3) and was found to be marked ly sens i t i ve to heat , p ro teo lys i s and the ionic env i ronment as compared to the nat ive enzyme. T h e Vmax and Km va lues of the modif ied enzyme were also found to be 10 to 15 fo ld h i g h e r . It has been sugges ted that the la rge changes in the p rope r t i es of the in v i t r o modi f ied enzyme may re f lec t a potent ia l form of regu la t ion of D H F R ac t i v i t y in v i v o . In th i s sect ion I desc r i be some of the p rope r t i es of D H F R f rom a M T X - r e s i s t a n t human p romye locy t i c leukemia cel l l ine wh ich exh ib i t s a 20- fo ld h i ghe r ac t i v i t y than the enzyme p resen t in the M T X - s e n s i t i v e c e l l s . In sect ion 1 of th is chap te r I have demonst ra ted tha t t h i s 20- fo ld inc rease in ac t i v i t y is not due to the o v e r p r o d u c t i o n of the enzyme pro te in ( 8 ) , and th i s unusua l f i n d i n g prompted the i n v e s t i -- 199 -gat ion of the p roper t i es of the more ac t i ve enzyme. It has been found tha t as fo r the in v i t r o ac t i va ted enzyme, the D H F R in the M T X - r e s i s t a n t ce l ls has h i ghe r Vmax and Km va lues (when d i h y d r o -fo l ic ac id is used as s u b s t r a t e ) than tha t f rom the M T X - s e n s i t i v e ce l ls and that i t is marked l y more sens i t i ve to heat . T h e s e resu l t s ind ica te that the enzyme may be p resen t in a more ac t i ve conformat ion in the M T X - r e s i s t a n t c e l l s . M A T E R I A L S A N D M E T H O D S Ce l l s H L - 6 0 human p romye locy t i c leukemia ce l ls (9) were p ropogated in cu l t u re and M T X - r e s i s t a n t ce l ls se lected f rom the paren t sens i t i ve ce l ls as d e s c r i b e d in sect ion 1 of th i s c h a p t e r . P repa ra t i on of Ce l l Lysa tes and D ihdy ro fo la te Reduc tase A s s a y Ce l l l ysa tes were p r e p a r e d as d e s c r i b e d before (10 , and C h a p t e r 2) T h e assay method used fo r the determinat ion of D H F R ac t i v i t y has also been d e s c r i b e d in chap te r 2. One un i t of D H F R ac t i v i t y is de f ined as tha t amount of enzyme reduc ing 1 nmole of d i h y d r o f o l i c ac id per 20 min . Heat S tab i l i t y of D ihyd ro fo la te Reduc tase A c t i v i t y Equ i va len t amounts of cel l l ysa te p ro te in o r M T X - s e p h a r o s e a f f i n i t y column pu r i f i ed D H F R pro te in f rom H L - 6 0 ( S ) and M T X -res i s tan t H L - 6 0 ( R 4 ) ce l ls were incuba ted at 50°C f o r v a r i o u s time p e r i o d s , ch i l l ed in ice and assayed f o r D H F R a c t i v i t y . - 200 -M T X - S e p h a r o s e A f f i n i t y Ch roma tog raphy D H F R f rom H L - 6 0 ( S ) and ( R 4 ) ce l ls was pa r t i a l l y p u r i f i e d by M T X - s e p h a r o s e a f f i n i t y ch roma tog raphy . M T X - s e p h a r o s e was s y n t h e -s ized as d e s c r i b e d p r e v i o u s l y (11) u s i n g 50 mg of M T X . Ce l l l ysa tes were app l ied to M T X - s e p h a r o s e columns (1 x 12 cm) in 50 mM T r i s -H C I , pH 7 . 5 . T h e columns were e lu ted at e i ther 4°C or at room tempera tu re (see r e s u l t s ) sequen t ia l l y wi th 30 ml 50 mM T r i s - H C I , pH 7 .5 /10 urn N A D P H ( b u f f e r 1 ) , fo l lowed by 30 ml 0.2 M T r i s - g l y c i n e , pH 9 . 5 / 2 M KC I /10 uM N A D P H ( b u f f e r 2 ) . T h e column bound D H F R was e lu ted wi th 30 ml 0.2 M T r i s - g l y c i n e , pH 9 . 5 / 2 M K C I / 5 mM d i h y d r o f o l i c ac id ( b u f f e r 3 ) . T h e bu f f e r 3 f r ac t i ons were d ia l yzed e x t e n s i v e l y aga ins t 50 mM T r i s - H C I , pH 7 .5 at 4°C to remove the d i h y d r o f o l i c ac id before assay i ng fo r D H F R a c t i v i t y . T h e f r ac t i ons con ta in ing D H F R ac t i v i t y were poo led , d i a l y zed aga ins t 5 mM T r i s -H C I , pH 7.5 at 4 ° C , l yoph i l i zed and s to red at -20°C unt i l r e q u i r e d . T h e l yoph i l i zed powder was d i sso l ved in water and s to red in a l iquots at - 70 °C . R E S U L T S A N D D I S C U S S I O N Par t ia l Pu r i f i ca t i on of D H F R from H L - 6 0 ( S ) and ( R 4 ) Ce l l s D H F R s can usua l l y be p u r i f i e d in two o r t h ree s teps because of the use of M T X - s e p h a r o s e a f f i n i t y ch romatog raphy wh ich makes use of the v e r y h igh a f f i n i t y of M T X fo r D H F R , and the r e v e r s i b i l i t y of the b i n d i n g of D H F R to M T X by an excess of the s u b s t r a t e , d i h y d r o f o l i c a c i d . D H F R f rom M T X - s e n s i t i v e and - r e s i s t a n t H L - 6 0 ce l ls was p a r t i a l -ly p u r i f i e d by 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 , the deta i ls of - 201 -which are shown in Tab le 5 -2 . T h e ch romatog raphy was c a r r i e d out at e i the r 23°C (Tab le 5-2 A ) or at 4°C (Tab le 5-2 B ) . When c a r r i e d out at 23°C the r e c o v e r y was poor in both cases i . e . H L - 6 0 ( S ) and ( R 4 ) . Howeve r , whereas a 6- fo ld overa l l pu r i f i ca t i on was ach ieved fo r the H L - 6 0 ( S ) D H F R , a loss of ac t i v i t y o c c u r r e d upon pu r i f i ca t i on of the H L - 6 0 ( R 4 ) D H F R even though more a c t i v i t y was app l ied to the co lumn. D H F R ac t i v i t y was not de tec ted in e i ther the bu f f e r 1 or bu f f e r 2 f r ac t i on of S or R 4 co lumns . When ch romatog raphy was c a r r i e d out at 4°C the r e c o v e r y of D H F R ac t i v i t y was about the same (•v 40%) f o r both H L - 6 0 ( S ) and ( R 4 ) D H F R . In add i t ion an overa l l pu r i f i ca t i on of both D H F R s was a c h i e v e d . T h e poor r e c o v e r y and actual loss of D H F R ac t i v i t y f rom R 4 ce l ls when chromotographed at 23°C ind ica ted that t h i s D H F R ac t i v i t y may be more uns tab le than that f rom the ( S ) c e l l s , o r tha t the ch romatog raphy resu l ted in the inac t i va t ion of some o ther component p resen t in R 4 ce l ls wh ich kept the D H F R in a more ac t i ve s ta te . T h e h i g h e r r e c o v e r y of ac t i v i t y when chromotographed at 4°C does not d i s t i n g u i s h between the above two a l t e r n a t i v e s , s ince at 4°C e i the r the enzyme i tse l f o r the 'ac t i va to r ' cou ld be more s tab le . T h e s e data also f u r t h e r s u p p o r t the ev idence a l ready p resen ted (Sec t ion 1) (8) tha t the inc rease in D H F R ac t i v i t y in R 4 ce l ls is not due to the o v e r p r o d u c t i o n of the enzyme s ince even at 4°C the actual amount of p ro te in recove red f rom the a f f i n i t y columns was the same fo r H L - 6 0 ( S ) and ( R 4 ) ce l l s . T A B L E 5-2 M T X - S e p h a r o s e A f f i n i t y Ch roma tog raphy of DHFR from HL -60 ( S ) and ( R 4 ) c e l l s . S . A c , in lysate A c t i v i t y A c t i v i t y % Recove ry S . A c , in par t ia l l y Fold ( u n i t s / M g p ro te in ) app l ied recovered pu r i f i ed prepara t ion pu r i f i ca t i on ( u n i t s ) ( u n i t s ) ( u n i t s / M g pro te in) A HL -60 ( S ) 2 .0 6.25 1.08 17.3% 12.0 6 HL-60 ( R 4 ) 40 .0 143 3.82 2.6% 28.3 0.7 B HL -60 ( S ) 2.1 57 23 40% 33.3 16 HL-60 ( R 4 ) 55 .4 1795 746 42% 622 11 Equ iva len t amounts of H L - 6 0 ( S ) and ( R 4 ) lysate pro te in were app l ied onto M T X - s e p h a r o s e a f f i n i t y columns and ch roma tog raphy was c a r r i e d out as d e s c r i b e d in the Mater ia ls and Methods sec t ion . A : C h r o m a t o g r a p h y c a r r i e d out at 23°C. B : Ch roma tog raphy c a r r i e d out at 4 °C . - 203 -Heat S tab i l i t y of D H F R A c t i v i t y T h e d i s c o v e r y that in v i t r o ac t i va ted form's of D H F R (3 ,4 ) are v e r y much more heat labi le than the pa ren t forms prompted us to determine whe ther the more ac t i ve enzyme p resen t in the H L - 6 0 ( R 4 ) ce l ls is also more heat labi le as compared to tha t f rom H L - 6 0 ( S ) c e l l s . T h e heat s tab i l i t y exper iment was in i t ia l l y c a r r i e d out us i ng the cel l l y s a t e s . F i g . 5-9 A shows that when the cel l l ysa tes were incuba ted at 50°C fo r va r i ous t ime per iods the D H F R ac t i v i t y in the ( S ) cel l l ysate was s tab le fo r up to 30 m i n . , whereas the D H F R in the R 4 cel l lysate was rap id l y inac t i va ted w i th in 10 m in . i n c u b a t i o n . T h e ac t i v i t y d id not d imin ish f u r t h e r and was s tab le at th i s level f o r up to 30 m in . T h e R 4 D H F R a c t i v i t y dec l ined f rom its 20- fo ld h i ghe r level to tha t p resen t in the S ce l ls ( F i g . 5-9 B ) . T h i s data is cons is ten t wi th th ree poss ib i l i t i es i . e . , ( i ) a component not p resen t in the S ce l ls is respons ib le f o r the ac t i va t ion of the D H F R in R 4 ce l ls and it is th i s component that is r a p i d l y heat i n a c t i v a t e d , r e t u r n i n g the level of D H F R a c t i v i t y to that in S c e l l s ; ( i i ) t he re are two forms of D H F R p resen t in the R 4 c e l l , one of wh ich is more a c t i v e ; ( i i i ) the R 4 D H F R i tse l f is p resen t in an ac t iva ted conformat ion and is more uns tab le in t h i s conformat ion (as is the case fo r the in v i t r o ac t i va ted D H F R ) . In o r d e r to test these poss ib le exp lana t ions an attempt was made to p u r i f y the D H F R s in S and R 4 ce l ls by 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 , the deta i ls of wh ich are g i ven in Tab le 5 -2 . T h e pa r t i a l l y p u r i f i e d D H F R f rom S ce l ls was s t i l l heat s tab le ( r e ta i n i ng 80% of i ts ac t i v i t y a f ter 10 m in . at 50°C) ( F i g . 5-10 A ) . However the - 204 -F I G U R E 5-9 Heat S tab i l i t y of HL -60 ( S ) and ( R 4 ) D H F R A c t i v i t y in Ce l l L y s a t e s . C e l l u l a r l ysa tes were incuba ted at 50°C and a l iquots r e p r e s e n t i n g 60 ug p ro te in were removed at va r i ous time p e r i o d s , ch i l l ed r a p i d l y in ice and then assayed fo r D H F R ac t i v i t y as d e s c r i b e d p r e v i o u s l y ( C h a p t e r 2 ) . A . Plot of V ( ve loc i t y of react ion at t ime t ) ove r Vo ( in i t ia l ve loc i t y ) aga ins t t ime. A H L - 6 0 ( R 4 ) l y s a t e ; o H L - 6 0 ( S ) l y sa te . B . Plot of D H F R ac t i v i t y aga ins t t ime. A H L - 6 0 ( R 4 ) l y sa te ; o H L - 6 0 ( S ) l ysa te . 205 - 206 -pa r t i a l l y p u r i f i e d D H F R f rom R 4 ce l ls was once again i n a c t i v a t e d , a l though c o n s i d e r a b l y more s lowly than in the cel l l ysate ( F i g . 5-10 B ) . T h e r e f o r e , a l though the R 4 D H F R is more heat uns tab le than the S D H F R , the pa r t i a l l y p u r i f i e d p repara t i on exh ib i t s s lower inac t i va t ion k ine t i cs than the u n p u r i f i e d p repara t i on (35% of the a c t i v i t y remain ing af ter 5 m in . as opposed to 5% in the cel l l y s a t e , F i g . 5-10 B ) . T h e s e data do not g i ve unequ ivoca l ev idence fo r any of the above ment ioned a l t e r n a t i v e s , a l though the s lower inac t i va t ion k ine t i cs of the pa r t i a l l y p u r i f i e d p repara t i on s u g g e s t the par t ia l removal of a component respons ib le f o r the ext reme i ns tab i l i t y of the D H F R in the R 4 cel l l y sa te . Enzyme k ine t ic s tud ies were c a r r i e d out on the pa r t i a l l y p u r i f i e d D H F R p repa ra t i ons of H L - 6 0 ( S ) and ( R 4 ) c e l l s . A s fo r the in v i t r o ac t i va ted D H F R (3) the D H F R from R 4 ce l ls had marked ly h i ghe r Vmax and Km va lues than tha t f rom the S cel ls when d i h y d r o f o l i c a c i d / N A D P H were used as s u b s t r a t e s (Tab le 5 - 3 ) . T h e I.C.^Q f o r M T X however was the same fo r D H F R s f rom both cel l l ines (Tab le 5 - 3 ) . T h e enzymes f rom both cel l l ines were ac t i va ted by KCI (3 - fo ld ac t iva t ion by 0 .2 M K C I , data not shown) and p a r a c h l o r o m e r c u r i -pheny l su lphonate ( p C M S ) . KCI i nh ib i ted the ac t i va t ion of both enzymes by p C M S ( F i g . 5 -11 ) . T h i s e f fect has p r e v i o u s l y been demonst ra ted fo r o ther D H F R s ( 4 ) . T h e fac t that the R 4 D H F R cou ld be f u r t h e r ac t i va ted by p C M S ( in the absence of KCI ) sugges t s that the modi f icat ion resu l t i ng in the h i ghe r basal a c t i v i t y of R 4 D H F R in v i v o p r o b a b l y does not i nvo l ve the s u l p h y d r y l g roup modi f icat ion of a cys te i ne res idue as is the case f o r in v i t r o ac t i va ted D H F R s . - 207 -F I G U R E 5-10 Compar ison of the Heat S tab i l i t y of D H F R A c t i v i t y in Ce l l Lysa tes  and Pa 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 . T h e exper imen t was c a r r i e d out as d e s c r i b e d fo r F i g . 5 -9 . Pa r t i a l l y p u r i f i e d p repa ra t i ons of D H F R were obta ined by M T X -sepharose a f f i n i t y ch romatography c a r r i e d out at 4 ° C . A . H L - 6 0 ( S ) : A , cel l l y s a t e ; o , pa 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 . B . HL -60 ( R 4 ) : o , cel l l y s a t e ; A , pa r t i a l l y pu r i f i ed p r e p a r a t i o n . 203 A Time (min.) Time (min.) - 209 -T A B L E 5-3 K ine t i c P rope r t i es Vmax Km ( D i h y d r o f o l i c ac id ) L C - 5 0 M T X (nmoles/20min/60ug p ro te in ) (uM) (nM) H L - 6 0 ( S ) Q 2 1 ± Q Q 5 D H F R 69 ± 5 4 .5 ± 1.0 H L - 6 0 ( R 4 ) 20 ± 1 380 ± 1 0 5.0 + 1.0 D H F R K ine t i c p roper t i es of pa r t i a l l y p u r i f i e d D H F R from H L - 6 0 ( S ) and H L - 6 0 ( R 4 ) c e l l s . DHFR ac t i v i t y was assayed as d e s c r i b e d p r e v i o u s l y ( C h a p t e r 2 ) . - 210 -F I G U R E 5-11 Ef fec t of p C M S on the D H F R A c t i v i t y f rom H L - 6 0 ( S ) and ( R 4 ) Ce l l s  in the P resence and A b s e n c e of K C I . ^ Equ iva len t amounts of ( S ) and ( R 4 ) l ysa te p ro te in (60 ug) were incubated wi th i nc reas ing amounts of p C M S at 23°C fo r 2 m in . in the p resence or absence of 0.2 M K C I . T h i s m ix tu re was then added to the res t of the components of the enzyme assay and the react ion was s ta r ted by the add i t ion of 3 H - d i h y d r o f o l i c a c i d . o© R 4 + K C I ; © R 4 - K C I ; A S + K C I ; A S - K C I . 211 - 212 -C l e a r l y , the D H F R in H L - 6 0 ( R 4 ) ce l ls e x h i b i t s d i f f e ren t p rope r t i es to tha t p resen t in the paren t HL -60 ( S ) c e l l s . T h e data are s u g g e s t i v e of an in v i v o ac t i va t ion of D H F R in the R 4 ce l ls by a component not p resen t in the S c e l l s , a l though the e x p r e s s i o n of an a l t e r e d , more ac t i ve form of D H F R cannot be ru led ou t . We are c u r r e n t l y a t tempt ing to d i s t i n g u i s h between these two a l t e r n a t i v e s . - 213 -R E F E R E N C E S 1. S c h i m k e , R . T . (1982) in_ Gene Ampl i f i ca t ion ( R . T . S c h i m k e , e d . ) , p p . 317-333, New Y o r k : Co ld S p r i n g H a r b o u r L a b o r a t o r y . 2 . S c h i m k e , R . T . (1984) Gene amp l i f i ca t ion , d r u g r e s i s t a n c e , and c a n c e r . C a n c e r R e s . 44, 1735-1742. 3 . B a r b e h e n n , E . K . and Kau fman , B . T . (1982) C h i c k e n l i ve r d ihyd ro fo la te r e d u c t a s e : ac t iva t ion and a l te ra t ion of enzymat ic p rope r t i es as a resu l t of react ion wi th m e t h y l m e r c u r y . A r c h . B iochem. B i o p h y s . 219, 236-247. 4 . B a r b e h e n n , E . K . and Kau fman , B . T . (1980) A l te ra t i on of the p rope r t i es of c h i c k e n l i ve r d i hyd ro fo la te reduc tase as a resu l t of modi f icat ion by te t ra th iona te . J . B i o l . C h e m . 255, 1978-1984. 5. Kau fman , B . T . (1966) S tud ies on d ihyd ro fo la te r e d u c t a s e , II. T h e ac t iva t ion of d ihyd ro fo la te reduc tase f rom c h i c k e n l i ve r by iod ine . P r o c . N a t l . A c a d . S c i . U . S . A . 56, 695-700. 6. Kau fman , B . T . (1968) S tud ies on d ihyd ro fo la te r educ tase . J . B i o l . C h e m . 243, 6001-6008. 7. F re i she im , J . H . , K u m a r , A . A . , B l a n k e n s h i p , D . T . and Kau fman , B . T . (1979) in C h e m i s t r y and B io logy of P te r i d i nes ( R . L . K i s l i u k and G . M . B r o w n , e d s . ) , p p . 419-424, E l sev ie r Sc ien t i f i c P u b l i s h i n g C o . , Ams te rdam. 8. D e d h a r , S . , H a r t l e y , D. and Go ld ie , J . H . (1984) Increased d ihyd ro fo la te reduc tase ac t i v i t y in me tho t rexa te - res i s tan t human p romye locy t i c leukemia ( H L - 6 0 ) c e l l s . Lack of co r re la t ion between inc reased ac t i v i t y and o v e r p r o d u c t i o n . B iochem. J . ( in p r e s s ) . 9. C o l l i n s , S . J . , Ga l l o , R . C . and G a l l a g h e r , R . E . (1977) Con t i nuous g rowth and d i f fe ren t ia t ion of human myelo id leukemic cel ls in suspens ion c u l t u r e . Na tu re 270, 347-349. 10. G o l d i e , J . H . , D e d h a r , S . and K r y s t a l , G . (1981) P rope r t i es of a methot rexate i nsens i t i ve va r i an t of d ihyd ro fo la te reduc tase d e r i v e d f rom me tho t rexa te - res i s tan t L5178Y c e l l s . J . B i o l . C h e m . 256, 11629-11635. 11 . G o l d i e , J . H . , K r y s t a l , G . , H a r t l e y , D . , G u d a u s k a s , G . and D e d h a r , S . (1980) A methot rexate i nsens i t i ve v a r i a n t of fo late reduc tase p resen t in two l ines of methot rexate res i s tan t c e l l s . E u r . J . C a n c e r 16, 1539-1546. - 214 -A P P E N D I X 1 Inh ib i t ion of the M e t h o t r e x a t e - i n s e n s i t i v e (Form 2) D ihyd ro fo la te Reduc tase f rom L5178Y ( R 4 ) Ce l l s by S u b s t i t u t e d T r i a z i n e s and Qu inazo l ines T h e demonst ra t ion tha t M T X - r e s i s t a n t mouse leukemia L5178Y cel ls e x p r e s s two forms of D H F R one of wh ich ( form 2) is h i g h l y i nsens i t i ve to M T X ( 1 ) , prompted the inves t iga t ion of the i n h i b i t o r y potent ia l of o ther c lass ica l and non -c l ass i ca l fo late a n t a g o n i s t s . T h e rat ionale fo r c a r r y i n g out these s tud ies was the f o l l ow ing : the p resence of s i gn i f i can t amounts of M T X - i n s e n s i t i v e D H F R in these ce l ls is p r o b a b l y an impor tant fac to r in p r o d u c i n g pheno typ i c d r u g r e s i s t a n c e . A t ex t r ace l l u l a r d r u g concen t ra t ions su f f i c i en t to i nh ib i t -9 the M T X - s e n s i t i v e reduc tase ( form 1, K i fo r M T X , 10 M ) , the -4 i nsens i t i ve enzyme ( K i fo r M T X , 7.5 x 10 M) would be almost to ta l ly ac t i ve . Because i t has been shown that on ly 5% of the normal D H F R ac t i v i t y is r equ i r ed to genera te su f f i c i en t t e t rahyd ro fo la te co fac tors to maintain cel l v i ab i l i t y ( 2 , 3 ) , the M T X - i n s e n s i t i v e enzyme a c t i v i t y would be su f f i c i en t to allow cel l g rowth in the p resence of h igh concen t ra t ions of M T X . To k i l l such res i s tan t cel ls would r equ i r e the s imul taneous inac t i va t ion of both forms of D H F R . T h u s , i f a potent i nh ib i t o r of the M T X - i n s e n s i t i v e D H F R cou ld be f o u n d , it w o u l d , in combinat ion wi th M T X , p rove to be an e f fec t i ve cy to tox i c agent fo r these c e l l s . T h i s i nves t iga t ion became even more pe r t i nen t in l igh t of the f i n d i n g tha t b las t ce l ls f rom some pat ients w i th A M L also e x p r e s s mul t ip le forms of D H F R d i f f e r i n g in t he i r s e n s i t i v i t y to M T X (see C h a p t e r 4 ) . - 215 -M A T E R I A L S A N D M E T H O D S T h e t r i az ine and qu inazo l i ne compounds were obta ined f rom D r . James H . F r e i s h e i m , Depar tment of B io log ica l C h e m i s t r y , U n i v e r s i t y of C i n c i n n a t i , Co l lege of Medic ine and D r . John H y n e s , Depar tment of Pharmaceut ica l S c i e n c e s , Medical U n i v e r s i t y of Sou th C a r o l i n a . M T X - i n s e n s i t i v e ( form 2) D H F R was pu r i f i ed as d e s c r i b e d in C h a p t e r 2 . D ihyd ro fo la te reduc tase ac t i v i t y was assayed by the rad io labe led d i h y d r o f o l i c ac id assay d e s c r i b e d in C h a p t e r 2 , or by the s p e c t r o -photometr ic assay d e s c r i b e d in Dedhar et aj_, ( 4 ) . S tock so lu t ions of M T X were made up in 0.05 M T r i s - H C I , pH 7 .5 a l i quo ted , and s to red at - 7 0 ° C . S tock so lu t ions of qu inazo l ines were made up in 1% DMSO in 0.05 M T r i s - H C I , pH 7.5 and s to red as fo r M T X . S u b s t i t u t e d t r i az i nes were d i sso l ved in 1% N , N - d i m e t h y l -formamide in water and also s to red at - 7 0 ° C . R E S U L T S A N D D I S C U S S I O N T h e common cores of the compounds tes ted are shown in F i g . 1. Two genera l t ypes of qu inazo l ine compounds were t e s t e d : those wi th para -aminobenzoy l g lutamate moieties ( t ype 1 ) , and abb rev ia ted qu inazo l ines wi th on l y the qu inazo l ine r i n g s ( t ype 2 ) . 35 compounds were tes ted in all and the resu l t s are shown in Tab les 1 to 3. A n in dep th ana lys i s of the reasons f o r the g rea te r e f fec t i veness of ce r ta in compounds o v e r o thers cannot be made at t h i s s tage because we do not as yet know the na tu re of the amino ac id sequence changes tha t have o c c u r r e d in t h i s enzyme resu l t i ng in a marked decrease in the a f f i n i t y fo r M T X . - 216 -L E G E N D S FOR F I G U R E S F I G U R E 1 S t r u c t u r a l fo rmula of M T X and core s t r u c t u r e s of qu inazo l ine and t r i az ine compounds . 217 Common Core of Quinazoline Compounds - Type 1 Common Core of Quinazoline Compounds Type 2 N H 2 H 2 N -V N — R C H 3 C H 3 Common Core of Substituted Diaminodihydrotriazines T A B L E 1 Q U I N A Z O L I N E S ( T Y P E 1) Compound [Enzyme] Ri R2 R3 R4 I .C.so I .C . 1 0o % Inh. I .C . • so M T X uM (uM) (uM) B y 1 uM I .C . .50 Q U I N A Z O L I N E M T X 0.00006 0.45 5.0 68% 1.0 1 0.00006 N H 2 H C H 2 N H G L U 0.026 1.0 98% 17.3 2 0.00006 N H 2 H C H 2 N ( C H 3 ) G L U 0.032 1.0 90% 14.0 3 0.00006 N H 2 H C H 2 N ( C H O ) G L U 0.24 2 .0 83% 1.9 4 0.00006 N H 2 H N H C H 2 G L U 1.1 >10.0 48% 0.41 5 0.00006 N H 2 H N ( C H 3 ) C H 2 G L U 0.06 0.7 100% 7 .5 6 0.00006 N H 2 H N ( C H O ) C H 2 G L U 0.54 8.0 66% 0.83 7 0.00006 OH H N H C H 2 G L U >1.0 >10.0 23% <0.5 8 0.00006 OH H N ( C H 3 ) C H 2 G L U >1.0 >10.0 23% <0.5 9 0.00006 OH H N ( C H O ) C H 2 G L U >1.0 >10.0 32% <0.5 10 0.00006 OH H C H 2 N H G L U >1.0 >10.0 32% <0.5 11 0.00006 OH H C H 2 N ( C H 3 ) G L U 1.7 >10.0 45% 0.26 12 0.00006 OH H C H 2 N ( C H O ) G L U 0.2 4 .0 75% 2.25 13 0.00006 OH C H 3 N H C H 2 G L U >1.0 >10.0 20% <0.5 ...12 T A B L E 1 c o n t i n u e d . . . 14 0.00006 OH C H 3 N ( C H 3 ) C H 2 G L U >1.0 >10.0 10% <0.5 15 0.00006 N H 2 C H 3 N H C H 2 OH 0.28 9.0 68% 1.6 M T X 0.0001 10.0 >10.0 43% 1.0 16 0.0001 N H 2 C L C H 2 N H OH 0.12 5.0 85% 83.0 17 0.0001 N H 2 C L C H 2 N H G L U 0.125 >10.0 79% 80.0 18 0.0001 N H 2 C L C H 2 N H O C 4 H 9 N . D . N . D . 50% N . D 19 0.0001 N H 2 C H 3 C H 2 N H G L U 0.13 N . D . 65% 76.0 20 0.0001 N H 2 C L N H C H 2 G L U 0.3 N . D . 72% 33.0 21 0.0001 OH C L N H C H 2 G L U >100.0 N . D . 0% T A B L E 2 Q U I N A Z O L I N E S ( T Y P E 2) Compound [Enzyme] R x R 2 R 3 l - C . 5 0 l - C . 1 0 0 % Inh . I . C . s n M T X  uM uM |JM B y 1 uM I . C 5 0 Q U I N A Z O L I N E M T X 0.000056 0.7 1.2 90% 1.0 1 0.000056 C H 3 CI H 0.8 10.0 56% 0.88 2 0.000056 C H 3 B r H 0.42 4 .0 69% 1.67 3 0.000056 C H 3 H B r >10.0 >10.0 0% 4 0.000056 CI B r H 0.85 3.4 56% 0.82 5 0.000056 C H 3 H CI >10.0 >10.0 0% 6 0.000056 CI CI H >1.0 >10.0 33% <0.5 T A B L E 3 S U B S T I T U T E D DI AMI N O T R I A Z I N ES Compound [ENZ]uM R I . C s o uM I .C . 1 0 o uM % Inh . B y 1 uM I . C . ™ M T X I . C . 5 o T R I A Z I N E M T X 0.000057 1.0 3.0 50% 1 0 C H 3 -(T>( C H 2 ) 2 - C - N H - (o^-S0 2 F 1 0.000057 0.018 1.0 100% 55 2 0.000057 0 ^ - ( C H 2 ) 2 - C - N H - Q - S 0 2 F CI 0.018 1.0 100% 55 3 0.000057 CI S 0 2 F { ^ ( C H 2 ) 4 - ( ^ ) - 0.12 1.0 100% 8 4 0.000057 - ( 7 ^ - ( C H 2 ) 3 - o - ^ o ) - S 0 2 F CI 0 0.35 2.0 80% 2.8 5 0.000057 - ^ o ) - ( C H 2 ) 2 - C - N H ^ o y s 0 2 F 0.8 8.0 55% 1.25 6 0.000057 133071 0.1 1.0 100% . 10 T A B L E 3 c o n t i n u e d . 7 M T X 0.0001 10.0 >10.0 43% 1.0 8 0.0001 £ ^ ( C H 2 ) 4 - < £ o ] > - S C > 2 F 0.32 10.0 73% 31 C l C l C l 0.0001 S 0 2 F - ( ^ ( C H ^ - ^ C I 0.35 N . D . 67% 29 - 223 -Some genera l conc lus ions can be made however and are the f o l l ow ing : 1. Many of the compounds , both qu inazo l i nes and t r i a z i n e s , are more potent than M T X at i nh ib i t i ng M T X - i n s e n s i t i v e D H F R ( form 2 ) . The inc rease in po tency ranges f rom jus t o v e r 1 to almost a 100 fo ld g rea te r than M T X . T h i s f i n d i n g i tse l f is in con t ras t to the fac t tha t normal h i g h - M T X a f f i n i t y forms of D H F R are not i nh ib i t ed more po ten t ly than M T X by these compounds . T h e s e resu l t s show tha t the form 2 D H F R has unde rgone ce r ta in changes at the M T X - b i n d i n g s i te wh ich resu l t in decreased b i n d i n g of M T X bu t s t i l l re ta in h igh a f f i n i t y fo r b i nd ing ce r ta in of those compounds . 2. With the q u i n a z o l i n e s , the ones wi th a ch lo r i ne at the number 5 pos i t ion and the b r i d g e connec t ing the p te r i d i ne r i ng to the benzo ic ac id moiety in the normal o r ien ta t ion ( i . e . C H 2 N H ) appear to have the h ighes t i n h i b i t o r y po ten t ia l . T h e p resence or absence of the glutamate moiety in these compounds d id not appear to make any d i f f e rence (compare compounds 16 and 17, Tab le 1 ) . Subs t i t u t i on of the amino g r o u p at number 4 pos i t ion wi th a h y d r o x y l g r o u p in ch lo r i ne con ta in ing qu inazo l ines resu l ted in a d r a s t i c decrease in i n h i b i t o r y potent ia l (compare compounds 17 and 21 , Tab le 1 ) . 3 . In those qu inazo l ines l ack ing the ch lo r i ne at the number 5 pos i t i on , the subs t i t u t i on o n , and the or ien ta t ion o f , the b r i d g e connec t ing the p te r i d i ne r i n g to the benzo ic a c i d , appears to be impor tan t . T h e i n h i b i t o r y potent ia l decreases wi th i n c r e a s i n g b u l k i n e s s of the b r i d g e (compare compounds 1, 2 and 3 , Tab le 1 ) . A l l of the compounds lack ing the amino g roup at the number 4 pos i t ion were less e f fec t i ve than M T X . - 224 -4. A b b r e v i a t e d qu inazo l ines ( t ype 2) were most ly less e f fec t i ve than M T X rega rd less of the t ypes of subs t i t u t i ons on the p te r i d i ne r i n g (Tab le 2 ) . It would appear t he re fo re tha t the n o n - p t e r i d i n e pa r t of qu inazo l ines is impor tant in the inh ib i t i on of t h i s form of D H F R . 5. T h e subs t i t u t ed t r i a z i n e s , one of wh ich (compound 8, T a b l e 3) has been shown to b ind cova len t l y v ia T y r o s i n e - 3 1 to the c h i c k e n l i ve r D H F R (5) (a h igh M T X - a f f i n i t y D H F R ) , appeared to be bet te r overa l l than the qu inazo l ines as i nh ib i t o r s of the M T X - i n s e n s i t i v e ( form 2) D H F R (Tab le 3 ) . Of those t e s t e d , the most potent were compounds 1 and 2 (Tab le 3) w h i c h , a l though on ly 55 fo ld be t te r than M T X when I . C . ^ Q va lues were c o n s i d e r e d , were the on ly two compounds resu l t i ng in 100% inh ib i t i on of enzyme ac t i v i t y by 1 uM d r u g c o n c e n t r a t i o n . T h i s is even bet te r than qu inazo l i ne compounds 16 and 17 (Tab le 1) wh ich are bet te r when the I . C . ^ Q va lues are cons ide red bu t fai l to i nh ib i t the enzyme ac t i v i t y complete ly at 1 uM d r u g c o n c e n t r a t i o n . T h e t r i az ines 1 and 2 are the most h y d r o p h o b i c of all the t r i az i nes tes ted and s u g g e s t that the enzyme ac t i ve s i te has become more h y d r o p h o b i c . A l t h o u g h the in v i v o e f fec t i veness of these compounds has not been de te rm ined , these data show that the M T X - i n s e n s i t i v e D H F R can be inh ib i ted qu i te s t r o n g l y wi th pharmaco log ica l l y ach ievab le levels of some of these compounds , and en fo rce the idea tha t a systemat ica l s y n t h e s i s and ana l ys i s of o ther compounds may lead to even more potent i nh ib i t o r s of t h i s a l te red form of D H F R , and may be of va lue in the t reatment of M T X - r e s i s t a n t mal ignanc ies such as A M L . - 225 -R E F E R E N C E S 1. G o l d i e , J . H . , K r y s t a l , G . , H a r t l e y , D . , G u d a u s k a s , G . and D e d h a r , S . (1980) A methot rexate i nsens i t i ve v a r i a n t of fo late reduc tase p resen t in two l ines of methot rexate res i s tan t c e l l s . E u r . J . C a n c e r 16, 1539-1546. 2. J a c k s o n , R . C . and H a r r a p , K . R . (1973) S tud ies wi th a mathematical model of fo late metabol ism. A r c h . B iochem. B i o p h y s . 158, 827-841. 3. Whi te , J . C . and Go ldman , I .D. (1976) Mechanism of act ion of methot rexate IV. Free i n t r ace l l u l a r methot rexate r e q u i r e d to s u p p r e s s d ihyd ro fo la te reduc t ion to t e t rahyd ro fo la te by E h r l i c h asc i tes tumour ce l ls in v i t r o . Mo l . Pharmaco l . 12!, 711-719. 4 . D e d h a r , S . , F re i she im , J . H . , H y n e s , J . B . and G o l d i e , J . H . (1983) Inh ib i t ion of a me tho t rexa te - i nsens i t i ve d ihyd ro fo la te reduc tase f rom L5178Y ce l ls by subs t i t u t ed t r i a z i n e s and qu inazo l i nes . B iochem. Pharmaco l . 32 , 922-924. 5 . K u m a r , A . A . , Mangum, J . H . , B l a n k e n s h i p , D . T . and F re i she im , J . H . (1981) A f f i n i t y labe l ing of c h i c k e n l i ve r d ihyd ro fo la te reduc tase by a subs t i t u ted 4 , 6 -d iam inod ihyd ro t r i az jne bear ing a terminal su l phony l f l u o r i d e . J . B i o l . C h e m . 256, 8970. - 226 -S U M M A R Y A N D C O N C L U S I O N T h i s thes i s has d e s c r i b e d the biochemical and molecular cha rac te r i za t i on of the e x p r e s s i o n of e levated levels of s t r u c t u r a l l y normal and a l te red d ihyd ro fo la te reduc tases in M T X - r e s i s t a n t c e l l s . T h e s t u d y was c a r r i e d out us i ng th ree cel l c u l t u r e model s y s t e m s , one mouse and two human. In add i t ion a less deta i led ana l ys i s of D H F R was c a r r i e d out in the b las t ce l ls f rom pat ients w i th acute myelogenous leukemia ( A M L ) , a ma l ignancy wh ich does not usua l l y respond to M T X . T h e s t u d y of the mouse model s y s t e m , the M T X - r e s i s t a n t L5178Y ( R 4 ) c e l l - l i n e , was prompted b y ear l i e r f i n d i n g s that these cel ls conta in i nc reased D H F R ac t i v i t y a por t ion of wh ich is h i g h l y i nsens i t i ve to M T X . In the p resen t i nves t iga t ion the two forms of D H F R were p u r i f i e d to near homogenei ty and rabb i t serum ant ibod ies were p repa red aga ins t them. T h e two forms were found to be a n t i -gen ica l l y d i s t i n c t . T h e inc reased D H F R ac t i v i t y in these ce l ls was found to be due to the o v e r p r o d u c t i o n of D H F R r e s u l t i n g f rom D H F R gene amp l i f i ca t ion , and the abundan t D H F R mRNA p resen t in these ce l ls could, be t rans la ted into D H F R recogn ized by an t ibod ies to both forms of D H F R . A h igh degree of D H F R mRNA enr ichment was ach ieved by polysome immunoprec ip i ta t ion us i ng an t ibod ies to both forms of the enzyme. T h e total polysomal and immur ioprec ip i ta ted m R N A was cha rac te r i zed and f i r s t s t r and complementary DNA ( c D N A ) was p r e p a r e d f rom the en r i ched m R N A s . 4n add i t ion to these molecular b io logica l s t u d i e s , a v a r i e t y of qu inazo l ine and t r i az i ne fo late an tagon is t compounds were tes ted f o r t he i r i n h i b i t o r y potent ia l of the h igh ly - B ^TX- insens i t i ve form of D H F R - 227 -p resen t in these c e l l s . Some of these compounds were found to be much bet te r i nh ib i t o r s of t h i s a c t i v i t y than M T X and may p rove to be usefu l in the t reatment of mal ignanc ies wh ich fa i l to respond to M T X by v i r t u e of the e x p r e s s i o n of an a l te red l e s s - s e n s i t i v e form of D H F R . T h e s e exper iments have also shed l ight on the t y p e s of compounds needed fo r even g rea te r inh ib i t i on of t h i s form of D H F R . In o r d e r to determine whe ther M T X - i n s e n s i t i v e forms of D H F R are p resen t in mal ignant neoplasms known fo r t he i r poor response to M T X , DHFR ac t i v i t y in the b las t ce l ls of pat ients wi th A M L was assayed fo r the level of ac t i v i t y and M T X - s e n s i t i v i t y . T h e resu l t s were s u r p r i s i n g in tha t t h ree out of e igh t pat ients showed ev idence fo r the p resence of low M T X - a f f i n i t y forms of D H F R . T h i s is a re la t i ve l y h igh f r e q u e n c y espec ia l l y s ince these pat ien ts had neve r been t rea ted wi th M T X . Two of the e igh t pat ients had subs tan t i a l l y h i ghe r leve ls of D H F R a c t i v i t y than the r e s t , a l though the mechanism fo r the e x p r e s s i o n of the h igh levels was not de te rm ined . T h e e x p r e s s i o n of both h igh D H F R ac t i v i t y and M T X - i n s e n s i t i v e forms of DHFR re f lec t an i nhe ren t genet ic i ns tab i l i t y of A M L c e l l s . T h e second model sys tem used was M T X - r e s i s t a n t human carc inoma of the c e r v i x (He la) and human p romye locy t i c leukemia ( H L - 6 0 ) cel l l i nes . T h e M T X - r e s i s t a n t Hela ce l ls were shown to have e levated levels of normal D H F R due to gene amp l i f i ca t ion . T h e inc reased ac t i v i t y was uns tab le s ince the D H F R ac t i v i t y dec reased to tha t in the p a r e n t a l , M T X - s e n s i t i v e ce l ls when g rown in the absence of M T X . M T X - r e s i s t a n t H L - 6 0 ce l ls also exh ib i t ed i nc reased D H F R a c t i v i t y wh ich in th is case was stable in tha t i t was mainta ined at a h igh level - 228 -when the ce l ls were g rown fo r ex tens i ve per iods in the absence of M T X . S u r p r i s i n g l y the inc reased D H F R a c t i v i t y in these ce l ls was not due to the o v e r p r o d u c t i o n of D H F R and the re was no ev idence fo r gene ampl i f i ca t ion . T h e inc reased ac t i v i t y appeared in t h i s case to be due to the modi f icat ion of the enzyme i tse l f such tha t i t was more a c t i v e . T h e enzyme from the res i s tan t ce l ls d i f f e red in some of i ts phys i ca l and k ine t i c p rope r t i es f rom tha t in the parenta l sens i t i ve ce l ls and i ts p rope r t i es resembled some of those of in v i t r o ac t i va ted forms of D H F R . To my knowledge th i s is the f i r s t repo r t of the e x p r e s s i o n of i nc reased ac t i v i t y of mammalian D H F R due to modi f icat ion of D H F R ra the r than o v e r p r o d u c t i o n of the enzyme. Whether th i s D H F R is e x p r e s s e d by an a l te red gene or is modi f ied pos t - t r ans la t i ona l l y is not c l e a r , a l though the pu r i f i ca t i on arid the heat s tab i l i t y data s u g g e s t that a heat labi le ce l lu la r component may be respons ib le fo r the h ighe r i n t r i n s i c spec i f i c a c t i v i t y of the DHFR in the res i s tan t c e l l s . In any case the ava i l ab i l i t y of a cel l l ine e x p r e s s i n g a D H F R wi th a h i ghe r i n t r i n s i c spec i f i c ac t i v i t y shou ld fac i l i ta te the s t u d y of the mechanisms of modulat ion of D H F R a c t i v i t y in v i v o . A s is of ten the c a s e , answers to sc ien t i f i c ques t ions lead one to ask many more q u e s t i o n s . T h e work d e s c r i b e d in t h i s thes is demonst ra tes the cons ide rab le amount of ' p l as t i c i t y ' at the genet ic locus of d ihyd ro fo la te reduc tase in tumour c e l l s . Whether t h i s p r o p e r t y is un ique to mal ignant ly t r ans fo rmed ce l ls as opposed to un t rans fo rmed ce l ls is not c lear and would be d i f f i cu l t to de te rm ine . T h e na tu re of the a l terat ion in the p r ima ry sequence of the small amount of M T X - i n s e n s i t i v e D H F R p resen t in mouse L5178Y ( R 4 ) ce l ls - 229 -st i l l remains a m y s t e r y . Howeve r , the ava i l ab i l i t y of the en r i ched c D N A cod ing fo r th i s form of the enzyme , shou ld resu l t in a so lu t ion to th is m y s t e r y . How common is the e x p r e s s i o n of l o w - M T X a f f i n i t y D H F R s in acute myelogenous leukemia , and fo r tha t matter o ther mal ignanc ies wh ich do not respond to M T X ? Would the fo late an tagon is ts wh ich appear to be good i nh ib i t o r s of t h i s form of mouse D H F R be use fu l in v i vo? A r e the h igh leve ls of D H F R ac t i v i t y p resen t in the b las t ce l ls of some of the A M L pat ients due to gene amp l i f i ca t ion , or the e x p r e s s i o n of a more ac t i ve enzyme, as has been demonst ra ted in th i s t hes i s fo r an A M L ce l l - l i ne ( H L - 6 0 ) . What is the na tu re of the modi f icat ion of the D H F R lead ing to a h i g h e r ca ta l y t i c ac t i v i t y? Is the modi f icat ion genet ic or c a r r i e d out by another ce l lu la r p roduc t? T h e answers to these and o ther ques t ions shou ld add f u r t h e r to our knowledge of th i s v e r y i n te res t i ng enzyme and hope fu l l y help in overcoming res is tance to M T X and o ther fo late an tagon i s t s . 

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