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Biochemical and cytological effects of the anticancer alkaloid, acronycine, on tumor cells in culture Dunn, Bruce Partridge 1974

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BIOCHEMICAL AND CYTOLOGICAL EFFECTS OF THE ANTICANCER ALKALOID, ACRONYCINE, ON TUMOR CELLS IN CULTURE by BRUCE PARTRIDGE DUNN B . S c , U n i v e r s i t y of B r i t i s h Columbia, 1969 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Department of Biochemi s t r y We accept t h i s t h e s i s as conforming t o the requ i red standard THE UNIVERSITY OF BRITISH COLtJMBIK January, 1974 In presenting th i s thesis in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the L ibrary shal l make it f ree ly ava i lab le for reference and study. I further agree that permission for extensive copying of th is thesis for scholar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i cat ion of this thesis for f inanc ia l gain sha l l not be allowed without my written permission. Department of Biochemi s t r y The Univers i ty of B r i t i s h Columbia Vancouver 8, Canada Date January 23, 1974 i ABSTRACT Acronycine is an acr idone a l k a l o i d o c cu r r i n g in the bark of the A u s t r a l i a n scrub ash. It has been reported t o have cons ide rab le a n t i -tumor a c t i v i t y aga ins t a v a r i e t y of exper imental neoplasms, and i s c u r r e n t l y undergoing c l i n i c a l t r i a l s . L i t t l e i s known of the mechanisms re spons ib le f o r i t s ant i tumor a c t i v i t y . The cu r ren t study descr ibes some of the c y t o l o g i c a l and biochemical e f f e c t s of the a l k a l o i d on tumor c e l l s in c u l t u r e . The growth i n v i t r o of L5I78Y mouse and IRC ra t leukemia c e l l s was i n h i b i t e d by acronyc ine at concent ra t i ons of 3 to 12 yg/ml. This i n h i b i t i o n was p a r t l y due to the f a i l u r e of acronyc ine t r e a t ed c e l l s t o d i v i d e a t c y t o k i n e s i s , r e s u l t i n g in the accumulat ion of b i nuc lea ted c e l l s in the c u l t u r e s . This i n t e r f e r ence with c e l l d i v i s i o n was not immediate, and s tud ie s w i th synchronized c u l t u r e s i nd i ca ted t ha t at low a l k a l o i d concent ra t ions i t might be delayed f o r up to one and one-ha l f c e l l generat ion t imes . Acronycine s l i g h t l y extended the G| phase, but o t h e r -wise had l i t t l e e f f e c t on the progress of c e l l s through the G|, S, and phases of the c e l l c y c l e . Although acronyc ine caused mi tochondr ia l s w e l l i n g , i t d id not g r e a t l y a f f e c t the oxygen consumption of c e l l c u l t u r e s or appear to i n t e r f e r e with the immediate energy s upp l i e s of the c e l l . The i n h i b i t i o n of c e l l c u l t u r e growth by acronyc ine was c o r r e l a t e d with a marked i n h i b i t i o n of the i nco rpo ra t i on of e x t r a c e l l u l a r nuc leos ides i n to n u c l e i c a c i d s . This i n h i b i t i o n d id not appear t o r e s u l t from an i n h i b i t i o n of n u c l e i c ac id synthes i s ; no evidence could be obta ined in c e l l - f r e e systems t ha t acronyc ine i n t e r ac ted with DNA or a l t e r e d i t s template a c t i v i t y , and the drug d id not i n h i b i t the i nco rpo ra t i on of i n t r a c e l l u l a r precursors i n to n u c l e i c a c i d s . Acronycine s t r ong l y i n h i b i t e d i i the i nco rpo ra t i on of e x t r a c e l l u l a r nuc leos ides i n to i n t r a c e l l u l a r n u c l e i c ac id precursor pools - t h i s e f f e c t appeared to account e n t i r e l y f o r the i n h i b i t i o n of the i n co rpo ra t i on of these precursor s i nto n u c l e i c a c i d s . The mechanism by which L5I78Y c e l l s u t i l i z e e x t r a c e l l u l a r u r i d i n e and by which acronycine i n t e r f e r e s with t h i s process were s t ud i ed . The r e s u l t s of i n v e s t i g a t i o n s i n to the k i n e t i c s and temperature dependence of u r i d i n e u p t a k e by whole c e l l s a n d of u r i d i n e p h o s p h o r y l a t i o n b y c e l l e x t r a c t s suggested t h a t : ( i ) the t r an spo r t of u r i d i n e across the plasma membrane i s a step independent from i t s subsequent phosphory l a t i on , and (:i.t) t h i s t r an spo r t i s normally ra te -1 imi t i ng in the uptake of t h i s nuc leos ide . Acronyc ine, a t concent ra t i ons which markedly i n h i b i t e d u r i d i n e uptake in whole c e l l s , had l i t t l e or no e f f e c t on the phos-pho r y l a t i on of u r i d i n e in c e l l e x t r a c t s . This and other evidence ( i n c l ud i ng the e f f e c t of acronyc ine on the temperature dependence and k i n e t i c s of u r i d i n e uptake ) suggested t h a t the i n h i b i t i o n by acronyc ine of the u t i l i z a t i o n by L5I78Y c e l l s of e x t r a c e l l u l a r u r i d i n e r e s u l t s from an i n t e r f e rence with the t r an spo r t of the nuc leos ide across the plasma membrane. A s i m i l a r mechanism may a l s o account f o r the i n h i b i t i o n of the uptake of o ther nuc leos ides , as we I I as f o r a s l i g h t i n h i b i t i o n of cho l i ne and i n o s i t o l uptake which was a l s o observed in t h i s study. I 4 C-acronycine was prepared and shown t o be bound r a p i d l y and r e v e r s i b l y to L5I78Y c e l l s . It was a l s o bound to non-dia IysabIe serum components, in which form i t appeared t o no longer be a v a i l a b l e f o r i n t e r a c t i o n with c e l l s . This l a t t e r e f f e c t may have i m p l i c a t i o n s f o r the use of acronycine in chemotherapy. Most or a l l of the observed e f f e c t s of acronyc ine can t e n t a t i v e l y be exp la ined on the bas i s of a Ika Io id- induced a l t e r a t i o n s in membrane f u n c t i o n . Acronycine i s a r e l a t i v e l y w a t e r - i n s o l u b l e , l i p o p h i l i c compound, and as such may reasonably be expected to i n t e r a c t w i th l i p i d s and/or hydrophobic regions of p r o t e i n s . It i s specu lated t ha t i n t e r a c t i o n s of t h i s type with membrane components may be re spons ib le f o r the b i o l o g i c a l e f f e c t s of t h i s compound. i V TABLE OF CONTENTS Page ABSTRACT i TABLE OF CONTENTS . iv LIST OF TABLES . v i i i LIST OF FIGURES . • i x. ACKNOWLEDGEMENTS x i i i ABBREVIATIONS USED x i v INTRODUCTION I Chemotherapy . . . . . . . . . I The ceI I eye Ie . • . . . . . . 4 I n ve s t i ga t i on s i n t o the mechanism of a c t i on of a n t i n e o p l a s t i c agents . . . . . . 7 Acronyci ne . . . . . . . . . . II The cu r ren t problem . . . . . . . . 16 Incorporat ion of e x t r a c e l l u l a r nuc leos ides i n to nucIei c ac i ds . . . . . . . . . 18 Mechanism of a c t i on of drugs which i n t e r f e r e with n u c l e i c ac id s yn thes i s . . . . . . . . . 19 METHODS AND MATERIALS 26 MATERIALS 26 II.) Chemicals • 26 2) Rad ioac t i ve I y l a b e l l e d chemicals . . . . . 26 3) T i s sue c u l t u r e s upp l i e s . . . . . . 27 4) M a t e r i a l s 27 Page GENERAL METHODS 28 1) Routine maintainance of c e l l s in t i s s u e c u l t u r e . . 28 2) P repa ra t i on of acronyc ine s o l u t i o n s . . . • 29 3) Determinat ion of r a d i o a c t i v i t y . . . . . 30 METHODS USED IN SECTION I OF RESULTS 3 2 1) Determinat ion of the frequency of normal, m i t o t i c , and '. b i nucleated~ee I Is i.nssuspens i.on c u l t u r e s . . . 32 2) Synchron i za t i on of c e l l c u l t u r e s . . . . 33 3) Mon i to r ing of DNA syn thes i s in synchronized c u l t u r e s . 34 4) E l e c t r on microscopy . . . . . . . 35 5) Re sp i r a t i on of c e l l c u l t u r e s . . . . • . 35 14 6) P repa ra t i on of C-acronyc ine . . . . . 35 7) P r o t e i n determinat ion . . . . . . . 38 I 4 8) Determinat ion of the b ind ing of C-acronycine t o L5I78Y eel Is 3 8 METHODS USED IN SECTIONS 2 AND 3 OF RESULTS . . . 39 1) Incubation of c e l l s w i th r a d i o a c t i v e precursors . . 39 2) Determinat ion of the i n co rpo ra t i on of "^H-uridine i n to RNA • . 40 3) Determinat ion of the i n co rpo ra t i on of r a d i o a c t i v e precur sor s i n t o t o t a l , a c i d - s o l u b l e , and a c i d -i n s o l u b l e mate r i a l using c e n t r i f u g a t i o n procedures . 40 4) S o l u b i l i z a t i o n of c e l l s l a b e l l e d with r a d i o a c t i v e precursor s . . . . . . . . . 41 5) RNA synthes i s in v i t r o . . . . . . . 42 6) E x t r a c t i o n and a n a l y s i s of acj d-so I ub I e mater ia l from L5I78Y c e l l s incubated wi th H -u r id ine . . . 43 7) Assay of u r i d i n e phosphory la t ion in v i t r o . . . 45 RESULTS AND DISCUSSION 50 Organ i za t i on of the r e s u l t s and d i s cu s s i on s e c t i o n . 50 V i Page RESULTS - SECTION I . 50 E f f e c t of acronyc ine on popu la t ion growth in L5I78Y and IRC suspension c u l t u r e s . . . . . . . 50 Induct ion of b i nuc lea ted c e l l s by acronyc ine . . . 51 S tud ies of the e f f e c t of acronyc ine on p a r t i a l l y synchronized popu lat ions of tumor c e l l s . . . . 57 Cytology of acronyc ine t r e a t ed L5I78Y c e l l s . . . 65 Re sp i r a t i on of acronyc ine t r ea ted L5I78Y c u l t u r e s . . 69 Inf luence of horse serum concent ra t i on on the g r o w t h - i n h i b i t o r y p r ope r t i e s of acronyc ine . . . 69 14 B inding of C l a b e l l e d acronyc ine to L5I78Y c e l l s and to serum p ro te i n s . . . . . . . 73 D i scus s ion - Sec t ion I . . . . . . 81 RESULTS - SECTION 2 88 E f f e c t of acronyc ine on the i n co rpo ra t i on of nuc leos ides i n t o n u c l e i c ac id s . . . . . . 88 I n t e r ac t i on of acronyc ine with DNA in v i t r o . . . 90 E f f e c t of acronyc ine on RNA syn thes i s in v i t r o . . 93 E f f e c t of acronyc ine on the i n co rpo ra t i on of e x t r a c e l l u l a r nuc leos ides i n to the a c i d - s o l u b l e mate r i a l of L5I78Y c e l l s 98 E f f e c t of acronyc ine on the t r a n s f e r of r a d i o a c t i v i t y from p r e l a b e l l e d i n t r a c e l l u l a r a c i d - s o l u b l e pools i n to n u c l e i c ac id s . . . . . . . . . 99 E f f e c t of acronyc ine on the d i s t r i b u t i o n of r a d i o a c t i v i t y w i t h i n the a c i d - s o l u b l e pool of c e l l s l a b e l l e d with H -u r id ine . . . . . . . . . . 108 K i n e t i c s of the i n h i b i t i o n by acronyc ine of the i n co rpo ra t i on of u r i d i n e and thymidine i n to t o t a l e e l l u l a r mate r i a l . . . . . . . . 109 Re l a t i on sh i p between the i n h i b i t i o n of u r i d i n e uptake by acronyc ine and the concent ra t i on ' of serum in c u l t u r e media . . . . . . . . . 113 R e v e r s i b i l i t y of the i n h i b i t i o n of u r i d i n e uptake . . 116 V i i Page E f f e c t of acronyc ine on the uptake of compounds other . than nuc leos ides . . . . . . . . 119 D i scuss ion - Sec t ion 2 . . . . . . 122 RESULTS - SECTION 3 - INTRODUCTION 129 Mechanism of nuc leos ide uptake by c e l l s . . . 130 Ra t i ona l e of i n v e s t i g a t i o n s i n t o the mechanism of nuc leos ide uptake . . . . . . . 136 RESULTS - SECTION 3 I 39 K i n e t i c s of the accumulat ion of u r i d i n e as n o n - d i f f u s i n g metabo l i te s by L5I78Y c e l l s and of u r i d i n e phosphory la t ion by ceI I homogenates . . . . . . . . 139 E f f e c t of temperature on u r i d i n e uptake and u r i d i n e phosphory la t ion . . . . . . . . 140 E f f e c t of acronyc ine on the phosphory la t ion of u r i d i n e in v i t r o . . . . . . . . 143 Inf luence of membrane fragments on the i n h i b i t i o n of u r i d i n e phosphory la t ion by acronyc ine . . . . 145 E f f e c t of temperature on u r i d i n e uptake in the presence of acronyc ine . . . . . . . 1 4 6 D i scus s ion - Sec t ion 3 . . . . . . 149 Nature of u r i d i n e uptake in L5I78Y c e l l s . . . . 149 . Nature of the i n h i b i t i o n of u r i d i n e uptake by ac ronyc ine . 152 Mechanism by which acronyc ine i n h i b i t s the t r an spo r t of u r i d i n e through the plasma membrane . . . . . 156 GENERAL DISCUSSION AND CONCLUSIONS 161 Mechanism by which acronyc ine i n h i b i t s the growth of tumor ceI Is . . . . . . . . . 165 Imp l i ca t i on s of the i n v e s t i g a t i o n s f o r the use of acronyc ine in the^treatment:ovf*human neoplasms . .. 167' P o t e n t i a l f o r the employment of acronyc ine in i n v e s t i g a t i o n s of c e l l b io logy . . . . . . 168 BIBLIOGRAPHY ' . . 1 7 1 v i i i LIST OF TABLES Tab Ie Page I D i s t r i b u t i o n of r a d i o a c t i v i t y w i t h i n the a c i d - s o l u b l e mate r i a l of L5I78Y c e l l s incubated wi th "^H-uridine a t 7° 105 II E f f e c t of acronyc ine on the d i s t r i b u t i o n of r a d i o a c t i v i t y w i t h i n the a c i d - s o l u b l e mate r i a l of L5 I 78Y c e l l s incubated wi th "^hhuridine . . . I l l II E f f e c t of acronyc ine on the i nco rpo ra t i on of c ho l i n e and i n o s i t o l i n t o t o t a l c e l l u l a r mate r i a l of L5I78Y ceI Is. . . . . . . . . . . 120 IV E f f e c t of acronyc ine on the i nco rpo ra t i on of deoxygIucose i n to the t o t a l c e l l u l a r mate r i a l of L5 I 78Y ce I I s 121 V E f f e c t of acronyc ine on the phosphory la t ion of u r i d i n e by e x t r a c t s of L5I78Y c e l l s . . . 144 VI E f f e c t of acronyc ine on the phosphory la t ion of u r i d i n e by e x t r a c t s of L5I78Y c e l l s : e f f e c t of membrane fragments . . . . . . . 147 ix LIST OF FIGURES Fi gure Page 1 The ceI I eye Ie . . . . . . . . 6 2 S t r u c t u r a l formulae of a l k a l o i d s of Acronychia baueri and of r e l a t e d compounds . . . . . . 12 3 Incorporat ion of an e x t r a c e l l u l a r nuc leos ide i n to a c i d -s o l ub l e and a c i d - i n s o l u b l e mate r i a l . . . . 20 4 S t r u c t u r a l formulae of acronyc ine and of compound " 3e " of Hoffman-LaRoche compared with those of compounds which i n t e r a c t w i th DNA 24 5 Two dimensional chromatography of a c i d - s o l u b l e mate r i a l i s o l a t e d from L5I78Y c e l l s : l o ca t i on of re ference compounds . . . . . . . . . 46 6 E f f e c t of magnesium' c o n c e n t r a t i o n , ATP c oncen t r a t i o n , and pH on u r i d i n e phosphory la t ion by e x t r a c t s of L5I78Y eel Is 48 7 Time course of u r i d i n e phosphory lat ion by e x t r a c t s of L5I78Y c e l l s a t two d i f f e r e n t e x t r a c t d i l u t i o n s . . 49 8 E f f e c t of acronyc ine on c e l l popu la t ion growth in L5I78Y and IRC suspension c u l t u r e s . . . . 52 9 Appearance of normal and b inuc lea ted L5I78Y c e l l s . 53 10 E f f e c t of ac ronyc ine , c o l c h i c i n e , and acronyc ine p lus c o l c h i c i n e on c e l l popu la t ion growth and percent m i t o t i c and b i nuc lea ted c e l l s in an asynchronous L5I78Y c u l t u r e 56 11 E f f e c t of adding acronyc ine to a p a r t i a l l y synchronized IRC c u l t u r e when v i a b l e c e l l s were mainly in the G| and e a r l y S phases . . . . . . . 59 12 E f f e c t of adding acronyc ine t o a p a r t i a l l y synchronized IRC c u l t u r e when c e l l s were mainly in the S phase . 62 X Figure Page 13 E f f e c t of adding acronyc ine t o a p a r t i a l l y synchronized L5I78Y c u l t u r e when c e l l s were mainly in the e a r l y S phase . . . . . . . . . . 64 14 U l t r a s t r u c t u r e of normal L5I78Y c e l l s . . . . 67 15 U l t r a s t r u c t u r e of L5I78Y c e l l s t r e a t ed wi th acronyc ine ( 6 yg/ml ) f o r 6 hours . . . . . . 6 8 16 E f f e c t of acronyc ine on the oxygen consumption and c e l l popu la t ion growth of L5I78Y c u l t u r e s . . . 70 17 Inf luence of serum concen t ra t i on on the growth-i n h i b i t o r y p r ope r t i e s of acronyc ine in L5I78Y c u l t u r e s 72 14 18 B i o l o g i c a l a c t i v i t y of C-acronyc ine . . . . 7 4 I 4 19 Scatchard p l o t of the b ind ing of C-acronyc ine to non-dia IysabIe components of horse serum . . . 77 I 4 20 B ind ing of C-acronyc ine t o L5I78Y c e l l s in the presence and absence of 10 % horse serum . . . 79 21 E f f e c t of acronyc ine on the i n co rpo ra t i on of u r i d i n e and thymidine i n to the RNA and DNA of L5I78Y and I RC ce I I s 89 22 E f f e c t of DNA on the absorpt ion spec t ra of a c r i d i n e orange and acronyc ine . . . . . . . 92 23 E f f e c t of acronyc ine and a c r i d i n e orange on the me l t i ng of c a l f thymus DNA . . . . . . 94 24 Re l a t i on sh i p between DNA template concent ra t i on and the s yn thes i s of RNA in v i t r o . . . . . 96 25 E f f e c t of acronyc ine and eth id ium bromide on RNA synthes i s in v i t r o . . . . . . . 97 26 E f f e c t of acronycine~6n the i n c o r p o n a t t o n ! o f ~ u r i d i n e i n to a c i d - s o l u b l e and a c i d - i n s o l u b l e mate r i a l of L5I78Y eel Is 100 x i Fi gure Page 27 Incorporat ion of "^H-uridine in to t o t a l and a c i d -i n s o l ub l e mate r i a l of L5I78Y c e l l s a t var ious temperatures . . . . . . . . . 103 28 Incorporat ion of ^H-thymidine in to t o t a l and a c i d -i n s o l ub l e mater ia l of L5I78Y c e l l s at va r ious temperatures . . . . . . . . . 104 29 E f f e c t of acronyc ine and act inomycin D on the l a b e l l i n g of a c i d - i n so I ub I e mate r i a l a t 37° from an a c i d -s o l ub l e pool pre labe l . led with ^H-u r id ine . . . 106 30 E f f e c t of acronyc ine on the l a b e l l i n g of a c i d - i n s o l u b l e mate r i a l at 37° from an a c i d - s o l u b l e pool p r e l a b e l l e d I 4 w i th C-thymidine . . . . . . . 107 31 Re l a t i on sh ip between the i nco rpo ra t i on of u r i d i n e in to the tota I ceI IuIar materi a I of L5178Y ceI Is and t ime . 112 32 K i n e t i c s of the i n h i b i t i o n of u r i d i n e uptake by acronyc i ne . . . . . . . . 114 33 K i n e t i c s of the i n h i b i t i o n of thymidine uptake by acronyc i ne . . . . . . . . 115 34 Inf luence of horse serum concent ra t i on on the i n h i b i t i o n by acronyc ine of urid'hne uptake . . . . . 1.17 35 Recovery of u r i d i n e uptake on removal of acronyc ine . 118 36 Mechanism of the accumulat ion of u r i d i n e as non-d i f f u s i n g metabo l i te s . . . . . . . 132 37 K i n e t i c s of the phosphory lat ion of u r i d i n e ' by an e x t r a c t of L5I78Y c e l l s 141 38 Inf luence of temperature on u r i d i n e uptake by L5I78Y c e l l s and on u r i d i n e phosphory lat ion by c e l l e x t r a c t s 142 39 Inf luence of temperature on u r i d i n e uptake by L5I78Y c e l l s in the presence of acronyc ine . . . . 148 Figure x I I Page 40 S t r u c t u r a l formulae of compounds which are compet i t i ve i n h i b i t o r s of nuc leos ide t r an spo r t . . . . 158 ACKNOWLEDGEMENTS X I I I I would l i k e to express my s i n ce re g r a t i t u d e to Dr. C T . Beer f o r guidance, suggest ions , d i s c u s s i on s , and u n f a i l i n g encouragement dur ing the course of the i n v e s t i g a t i o n s reported in t h i s t h e s i s . I would l i k e to thank Dr. R.L. Noble, D i r e c t o r of the Cancer Research Center , U . B . C , f o r the use of f a c i l i t i e s in the Center. I would e s p e c i a l l y l i k e t o thank Mr. Peter Gout of the Cancer Research Center f o r e x c e l l e n t t e c h n i c a l and s c i e n t i f i c i n s t r u c t i o n . Thanks are a l s o g iven to Drs. J . F . R ichards , M. Smith, G.M. Tener, J . P . Kutney, and N. Auersperg f o r va luab le advi 'ce in the course of the work and in the p repara t ion of the t h e s i s , I 4 and t o Mr. Harald Hansen f o r help in the p repara t ion of C-acronyci I would l i k e to ded icate t h i s t h e s i s to my w i f e Do r i s , who has o f f e r e d u n f a i l i n g moral support and encouragement throughout the course of my s t u d i e s . The support of the Nat iona l Cancer I n s t i t u t e dur ing the course of the research i s g r a t e f u l l y acknowledged. x i V ABBREVIATIONS USED aero. - acronyc ine BSS - balanced s a l t s o l u t i o n cone. - concent ra t i ons G - g ene ra l l y l a b e l l e d ( r e f e r r i n g t o r a d i o a c t i v e chemicals ) HU - hydroxyurea PCMB - para-ch loromercur ibenzoate PCS - brand name of premixed s c i n t i l l a t i o n f l u i d manufactured by Amersham/SearIe - " Phase Combining System " POPOP - I ,4 -d i ( -2 (5 -pheny loxazoy I ) ) -benzene ( secondary s c i n t i l l a t o r f o r p repara t ion of s c i n t i l l a t i o n f l u i d ) PPO - 2,5-diphenyIoxazoIe ( primary s c i n t i l l a t o r f o r p repa ra t i on of s c i n t i l l a t i o n f l u i d ) UDP-NAG - UDP-N-acetyl glucosamine VLB - v i n b l a s t i n e ( former ly v i nca Ieucob Ia s t i ne ) Other abb rev i a t i on s which are wide ly usedpw<Mih®wthdefi nat ion in the biochemical l i t e r a t u r e ( e .g . ATP, DNA e t c . ) are not s p e c i f i c a l l y def ined here. I INTRODUCTION Chemotherapy: Today there are th ree w e l l - e s t a b l i s h e d methods of t r e a t i n g n e o p l a s t i c d i sease in humans : ( i ) surgery, ( i i ) r a d i a t ion. t reatment , and ( i i i ) chemotherapy. The removal of a tumor mass by surgery was the f i r s t e f f e c t i v e treatment f o r n e o p l a s t i c d i sease , and remains the most widely used method of cancer therapy. Surgery however s u f f e r s from severa l l i m i t a t i o n s : ( i ) i t cannot be used aga in s t d isseminated tumors such as leukemias, ( i i ) i t cannot be used f o r tumors in s u r g i c a l l y i n a c c e s s i b l e areas , or in cases where the e f f e c t i v e removal of the tumor mass would nece s s i t a t e the removal of some v i t a l organ, and ( i i i ) i t u sua l l y does not prov ide a cure in cases in which the n e o p l a s t i c t i s s u e has undergone metastas i s t o o ther areas of the body. Rad ia t i on treatment ( X-rays or gamma-rays ) and chemotherapy l a r ge l y avoid these I i m i t a t i o n s . Chemotherapy of mal ignant d i sease with the use of c y t o t o x i c drugs which destroy tumor c e l l s in the body dates from the l a te 1940s, when f o l i c a c i d antagon i s t s and n i t rogen mustard were f i r s t used t o t r e a t leukemias ( 1,2 ). Ea r l y successes w i th these treatments s t imu la ted the search f o r new drugs, and over the f o l l o w i n g years a v a r i e t y of drugs whose ant i tumor p r ope r t i e s were d i scovered in va r ious ways entered the ranks of c l i n i c a l l y usefu l agents. Today chemotherapy represents the treatment of cho ice f o r c e r t a i n types of neoplasms. Thus treatment with a n t i n e o p l a s t i c drugs i s the primary form of treatment f o r c e r t a i n leukemias and lymphomas ( 3,4 ). In a d d i t i o n to i t s use as a primary mode of t reatment, chemotherapy i s o f ten employed a f t e r surgery t o attempt to 2 • e l i m i n a t e c e l l s missed by the primary removal of the tumor ( 5 ). The development dur ing the 1940s of h i gh l y e f f e c t i v e and r e l a t i v e l y non - tox i c a n t i b i o t i c s and s u l f a drugs f o r use aga ins t b a c t e r i a l i n f e c t i o n s led to hopes t h a t s i m i l a r " magic b u l l e t s " could be found f o r the complete e l i m i n a t i o n of n e o p l a s t i c c e l l s from the body. To a large ex ten t , these hopes of e a r l y i n v e s t i g a t o r s have not been r e a l i z e d . A n t i n e o p l a s t i c drugs in cu r ren t use have a d i s a p p o i n t i n g l y narrow spectrum of a c t i v i t y . Chemotherapy in general i s usefu l in the treatment of on ly a r e s t r i c t e d range of neoplasms ( mainly those with a high growth ra te ) and i n d i v i d u a l drugs are even more r e s t r i c t e d in the tumors whose growth they can i n h i b i t . Although in c e r t a i n r e l a t i v e l y ra re tumors such as chor iocarc inoma complete cures are po s s i b l e ( 6 ), such s i t u a t i o n s do not represent the usual r e s u l t of t reatment of a p a t i e n t wi th a n t i n e o p l a s t i c drugs. In most cases , the best t ha t chemotherapy can do i s to prolong l i f e , or make i t more comfor tab le . Furthermore, an t i c ance r drugs are as a r u l e h i gh l y t o x i c , and in many cases are on ly marg i na l l y more damaging to n e o p l a s t i c c e l l s than to c e r t a i n normal body c e l l s . Drug treatment can have severe e f f e c t s on i n t e s t i n a l and o ra l mucosa, can lead t o nausea and n e u r o t o x i c i t y , and can damage the blood forming elements of the body. Depression of the immune system can lead to secondary compl icat ion ' s such as severe b a c t e r i a l and v i r a l i n f e c t i o n s . Although drug t o x i c i t y i s one of the major problems in chemotherapy, i t i s u s u a l l y managable by i n t en s i v e p a t i e n t c a re , . ca re tu I cho i ce of treatment schedules and doses, and constant moni tor ing of the t o x i c e f f e c t s of the drug. More c r i t i c a l in the f a i l u r e of a n t i n e o p l a s t i c agents to ach ieve cures i s the development of popu la t ions of r e s i s t a n t tumor c e l l s . 3 Prolonged treatment with an ant i tumor agent ac t s as a s e l e c t i o n procedure f o r c e l l s r e s i s t a n t to the l e tha l e f f e c t s of the drug ( 7 ). When such r e s i s t a n t c e l l s appear, they r a p i d l y become the predominant form of, tumor c e l l because they are be t te r ab le t o p r o l i f e r a t e dur ing the course of drug t reatment. When t h i s happens, the drug in use must be abandoned in favor of o the r , po s s i b l y less e f f e c t i v e t reatments . In recent year s , the most notable advances in chemotherapy, have been made when agents are used in combination drug therapy. The use of more than .one drug a t a t ime has p o t e n t i a l advantages in overcoming the problems of boitih drug t o x i c i t y and drug r e s i s t ance ( 7,8 ). The development of dtiug r e s i s t a n c e i s reduced, as s imultaneous mutation in a s i n g l e c e l l t o r e s i s t a n c e to two or more agents i s much less l i k e l y than mutation to r e s i s t a n c e to a s i n g l e agent. T o x i c i t y can be reduced by using together drugs which have d i f f e r e n t forms of host t o x i c i t y . By using these a t i n d i v i d u a l drug l e ve l s which do not cause se r i ous t o x i c i t y of each form, the t o x i c i t y to the host can be d i s t r i b u t e d in t o l e r a b l e amounts to va r i ou s bod i l y systems, wh i l e the tumor c e l l i s s ub jec t to a m u l t i p l e a t t a c k . The use of cu r r en t a n t i n e o p l a s t i c agents i s l im i t ed by host t o x i c i t y , the development of r e s i s t a n t tumor c e l l s , and the f a c t t h a t ' in most cases ant i tumor agents are q u i t e l im i t ed in t h e i r spectrum of a c t i o n . Because of these I i m i t a t i o n s , over the past decade there has been ex ten s i ve e f f o r t expended on searches f o r new ant i tumor agents. Research in t h i s area has been p a r t i c u l a r l y i n t en s i v e s i nce the other two w e l l -e s t ab l i s hed methods of cancer therapy — surgery and r a d i a t i o n treatment -are r e l a t i v e l y wel l explored f i e l d s , and there seems l i t t l e l i k e l i h o o d of a r a d i c a l breakthrough in them. In the f i e l d of chemotherapy however, 4 the p o s s i b i l i t y s t i l l e x i s t s t ha t the next drug d i scovered w i l l have s i g n i f i c a n t advantages over e x i s t i n g agents. Some of the research has involved the r a t i o n a l m o d i f i c a t i o n of e x i s t i n g drugs and s yn thes i s of l i k e l y a n t i m e t a b o l i t e s f o r t e s t i n g . A large p ropo r t i on of the research however has concentrated on the p o s s i b i l i t y of d i s cove r i ng fundamental ly new types of ant i tumor agents ( 9 ). The most f r u i t f u l I source has been t h e ' f i e l d of natura l products , mainly a n t i b i o t i c s • or enzymes from m i c r ob i a l sources, but a l s o i nc l ud ing agents from higher p l a n t s . The best known example of the l a t t e r are the V inca a l k a l o i d s , ' v i n b l a s t i n e and v i n c r i s t i n e , which are obta ined from the t r o p i c a l p l an t V;itnca rosea ( " p e r i w i n k l e " )( 10 ). The eel I e ye l e : In o rder f o r a chemical substance t o be an e f f e c t i v e ant i tumor agent, i t must have some s e l e c t i v e a c t i onaaga i n s t tumor c e l l s . Th i s may be achieved i f the substance can e x p l o i t any q u a l i t a t i v e or q u a n t i t a t i v e d i f f e r e n c e between normal and tumor.eel Is. One such d i f f e r e n c e which may account f o r some of the s e l e c t i v e t o x i c i t y of a n t i n e o p l a s t i c drugs i s the f a c t t h a t tumor c e l l s g ene r a l l y have a higher r e p l i c a t i o n r a te than most ( a lthough not a l l .) normal c e l l s . Th i s bas i s of s e l e c t i v e t o x i c i t y i s r e f l e c t e d in the spectrum of tumors which respond to chemotherapy - in most instances s low-growing tumors are r e l a t i v e l y r e s i s t a n t to drug therapy,. whiIe those most s u s c e p t i b l e to t h i s form of treatment are r e l a t i v e l y r a p i d l y growing. D i v i d i n g c e l l s undergo a v a r i e t y of biochemical and morphological processes which do not occur in non -d i v i d i ng 1 ceI Is. These processes, which are those involved in the d u p l i c a t i o n of the contents of the c e l l in p repa ra t i on f o r c e l l d i v i s i o n , and the d i v i s i o n process i t s e l f , may 5 prov ide po in t s of a t t a ck f o r chemotherapeutic agents. In a repeated ly d i v i d i n g c e l l , the per iod between succes s i ve c e l l . d i v i s i on s i s " comprised of an ordered s e r i e s of processes and events, which c o l l e c t i v e l y are known as the c e l l c y c l e ( II, 12 ). There are two major processes in the c e l l c y c l e which a c t as " markers " and de f i ne a t what stage of the c y c l e the c e l l i s i n . These are i ) the s yn thes i s of DNA ( which can be fo l l owed by moni tor ing the i n co rpo ra t i on of r a d i o a c t i v e precursor s i n to DNA ), and i i ) the morphological process of c e l l d i v i s i o n i t s e l f ( which can be recognized by the presence in the c e l l of condensed chromosomes ). The c e l l c y c l e i s o u t l i n e d in f i g u r e I. The process of c e l l d i v i s i o n i s termed the " M " o r ' m i t o t i c phase. At the complet ion of the m i t o t i c phase, the newly formed daughter c e l l s enter the G| phase in which p o s t - m i t o t i c r eo r gan i z a t i o n takes p l a ce , and in which c e l l s make p repara t i ons f o r the rep I i c a t ion- of t h e i r DNA ( II ). There i s no s p e c i f i c marker f o r the G| phase - the phase i s def ined as the per iod between the complet ion of m i t o s i s , and the beginning of DNA synthes i s in the next phase of the c e l l c y c l e , the " S " phase. During the S phase, the c e l l compIeteIy 'dupI i c a te s i t s genome in a per iod of severa l hours ( I I ). Upon complet ion of DNA s yn the s i s , c e l l s enter the " " phase of the c y c l e , in which p reparat ions f o r the morphological processes of c e l l d i v i s i o n take p lace ( 12 ). Once the c e l l i s ready to d i v i d e , the nuc lear membrane breaks down, and the DNA becomes condensed in to chromosomes. The chromosomes are then separated into two i n d i v i d u a l genomes by a co -o rd ina ted system of mic rotubu les known as the m i t o t i c s p i n d l e . These processes are v i s i b l e in the l i g h t microscope, and form the one stage in the c e i l c y c l e which i s morpho log i ca l l y d i s t i n g u i s h a b l e from the o t he r s . Upon complet ion of chromosome movement, the c e l l c o n s t r i c t s 6 F i gu re I The c e l l c y c l e . •M - condensed chromosomes v i s i b l e in the l i g h t microscope - c y t o k i n e s i s ( a t end of M per iod ) G| - p o s t - m i t o t i c r e o r g a n i z a t i o n - p repa ra t i on s f o r DNA s yn the s i s - en t ry p o i n t i n t o c e l l c y c l e of GQ c e l l s S - s yn the s i s of DNA - p repa ra t i on s f o r m i t o s i s G N - n o n - p r o l i f e r a t i n g c e l l s ( e . g . d i f f e r e n t i a t e d c e l l s ) 7 to form two daughter c e l l s - t h i s process bears the d i s t i n c t i v e name of " c y t o k i n e s i s " . The ma jo r i t y of normal c e l l s in the body as wel l as a c e r t a i n p ropor t i on of c e l l s in a tumor are not a c t i v e l y p r o l i f e r a t i n g . These are sa id to be in the " G Q " s ta te , - . C e l l s may remain in t h i s s t a t e ( e . g . -normal d i f f e r e n t i a t e d body c e l l s ), or may resume p r o l i f e r a t i o n by en te r i ng t h e G j stage of the eel I c y c l e ( 13 ). N o n - p r o l i f e r a t i n g c e l l s , i n c l ud i ng those which are n e o p l a s t i c , are r e s i s t a n t , to the c y t o t o x i c e f f e c t s of those drugs which ac t on ly on c e l l s which are in s p e c i f i c phases of the c e l l c y c l e . I n ve s t i ga t i on s in to the mechanism of a c t i o n of a n t i n e o p l a s t i c agents: Many f a c t o r s which a f f e c t the t he r apeu t i c usefu lness of an a n t i -n e o p l a s t i c drug are concerned wi th i t s behaviour in the body. The drug must f i r s t be absorbed ( i f i t i s g iven o r a l l y ) and then must be t ranspor ted to the s i t e of the tumor. The importance of t r an spo r t w i t h i n the body i s demonstrated by.the f a c t t h a t most a n t i n e o p l a s t i c drugs are i n e f f e c t i v e aga in s t b ra in tumors because of t h e i r f a i l u r e to be t ranspor ted across the b l ood -b ra i n b a r r i e r . The metabolism of the drug to i n a c t i v e compounds and i t s e x c r e t i o n from the body s i m i l a r l y i i a f f e c t the a n t i n e o p l a s t i c e f f e c t s of a compound. Studies on the r e l a t i o n -sh ip between f a c t o r s of t h i s nature and the ant i tumor p r ope r t i e s of an agent must of neces s i t y be done in v i v o . Once an ant i tumor agent has reached the t a r g e t c e l l , a second set of f a c t o r s a f f e c t s the ant i tumor p r ope r t i e s of the agent, namely those concerned with the mechanism by which i t k i l l s c e l l s . Although i t i s p o s s i b l e f o r a drug to be usefu l in the treatment of cancer and not know the mechanism by which i t i n h i b i t s the growth of tumor c e l l s , the 8 most e f f e c t i v e use of a drug o f t en on ly can be made once t h i s i s understood ( 14 ). Knowledge of the bas i s of the c y t o t o x i c i t y of a drug can be of advantage in p lanning dosage schedules ( e i t h e r a lone or in combination wi th other agents ), in dec id ing what other drugs an agent may po s s i b l y be used w i t h , in avociid i ng host t o x i c i t y , and in understanding and po s s i b l y c i rcumvent ing the mechanisms of r e s i s t a n c e of tumor eel Is t o the agent ( 15, 19 ). In c o n t r a s t t o s tud ie s on the t ranspor t , , metabol ism, or e x c r e t i o n of an agent in the body, s t ud i e s on the i n t e r a c t i o n of a drug wi th the tumor ce I I i t se I f usua I I y are most eas i I y done J_n_ v i t r o . Tumor ce I I s may be removed from exper imental animals f o r i n v e s t i g a t i o n s in v i t r o . Although t h i s technique a l l ows the use of large numbers of c e l l s , c e r t a i n types of experiments cannot be performed s i nce once the c e l l s are removed from the animal they normally do not r e p l i c a t e and have a l im i t ed l i f e span. For stud ies in v i t r o on r e p l i c a t i n g c e l l s , use may be made of one of a number of c e l l l i n e s which grow in c u l t u r e . Most of these c e l l l i n e s have o r i g i n a t e d from neoplasms in l aboratory an imals , wh i l e a few are of human o r i g i n . These.eel I l i ne s have been adapted to growing in an a r t i f i c i a l medium con ta i n i n g amino a c i d s , carbohydrates , v i t am in s , and other e s s e n t i a l f a c t o r s ( 16 ). In order f o r these c e l l l i n e s to grow in c u l t u r e , i t i s in most cases necessary to supplement the medium wi th 10 to 20 % animal serum ( u s ua l l y horse or f e t a l c a l f serum ) which prov ides c e r t a i n g l y c o p r o t e i n f a c t o r s : needed f o r eel I r e p l i c a t i o n ( 16 ). C e l l s grown in c u l t u r e may e i t h e r r e p l i c a t e attached t o the su r face of the c u l t u r e c on t a i ne r , or in the case of c e r t a i n c e l l l i n e s ( mainly i those der i ved from leukemias ) f r e e in suspens ion. ' The use of eel Is growing in c u l t u r e has a number of advantages f o r 9 i n v e s t i g a t i o n s of the mechanism of the c y t o t o x i c i t y of ant i tumor agents ( 17 ). Compl i ca t ing f a c t o r s occur ing in the whole animal such as the ab so rp t i on , metabol ism, and e x c r e t i o n of a drug are e l im ina ted and thus drug concent ra t i on s can be p r e c i s e l y c o n t r o l l e d . If d e s i r e d , i t i s p o s s i b l e to i n v e s t i g a t e the e f f e c t on tumor c e l l s of high concent ra t i on s of drug which could not-be used in an experimental an'iimal because of whole body t o x i c i t y . The ex te rna l environment of the c e l l s under study can be p r e c i s e l y c o n t r o l l e d with re spect to pH, t empe ra tu r e , , i o n i c s t r eng th , a v a i l a b i l i t y of n u t r i e n t s e t c . Furthermore, the p o s s i b i l i t y t h a t the behaviour of tumor c e l l s removed from i n d i v i d u a l tumor-bear ing animals may vary g r e a t l y from experiment t o experiment i s e l i m i n a t e d . C e l l s grown in c u l t u r e form a homogeneous popu lat ion whose c h a r a c t e r i s t i c s do not change much with t ime. The use of c e l l s growing in c u l t u r e a l l ows the use of c e r t a i n procedures and techniques which are not po s s i b l e wi th tumors in v i v o , o r w i th c e l l s removed from an animal and maintained i n v i t r o . One of the most important techniques i s the use of synchronized c u l t u r e s to i n v e s t i g a t e the e f f e c t of chemotherapeutic agents on the progress ion of c e l l s through the c e l l c y c l e . Many agents a c t at-„specif ic stages of the c e l l c y c l e , and the extent to which exposure to a drug i s l e tha l to a c e l l may be h i gh l y dependent on the phase 'of the c y c l e i t i s in ( 18 ). Thus i n h i b i t o r s of DNA syn thes i s are most t o x i c to c e l l s in the S phase, wh i l e i n h i b i t o r s of RNA synthes i s are most t o x i c to c e l l s a t the Gj/S boundary ( 18 ). Information on the c e l l c y c l e s p e c i f i c i t y of a n t i n e o p l a s t i c agents can lead to important advances in t h e i r use both wi th respect t o dosage schedules , and wi th respect to t h e i r use wi th other agents ( 19 ). Cel I s growi ng ji_n v i t r o or J_n_ v i vo are norma I ly " asynchronous " , 10 i . e . the eel Is. in a c u l t u r e at any one time are found in a l l phases of the c e l l c y c l e , and the growth of a popu la t ion i s cont inuous as new c e l l s c on s t an t l y reach m i t o s i s . S ince asynchronous c u l t u r e s con ta i n c e l l s in a l l phases of the c e l l c y c l e , i t i s u s ua l l y d i f f i c u l t and o f t en not po s s i b l e to study the e f f e c t of a drug on c e l l s in-any p a r t i c u l a r -" phase. To overcome t h i s l i m i t a t i o n , use i s made of s pec i a l techniques to a r t i f i c i a l l y produce popu la t ions of c e l l s in which a l l the c e l l s are w i t h i n a r e s t r i c t e d po r t i on of the c e l l c y c l e . Once such a popu la t ion i s formed, the c e l l s in i t tend to progress through the c e l l c y c l e together and d i v i d e a t approximately the same t ime. The progress of the c e l l s through the c y c l e can be fo l lowed by moni tor ing DNA syn thes i s ( the marker f o r the S phase ) and by f o l l o w i n g the appearance of m i t o t i c f i g u r e s and the r i s e in c e l l popu la t ion dens i t y t h a t mark the M pe r i od . P a r t i a l l y synchronized popu lat ions of p r o l i f e r a t i n g c e l l s can be produced by a va r ie ty , of methods. A l l techniques work on one of two bas ic p r i n c i p l e s ( 20 ) : i ) to s e l e c t by some means from an asynchronous c u l t u r e on l y those c e l l s which are in a c e r t a i n phase of the c e l l c y c l e ( " synchrony by s e l e c t i o n " ), or i i ) t o t r e a t an asynchronous c u l t u r e with chemical or phys i ca l methods which ac t s e l e c t i v e l y to slow or h a l t the progress of c e l l s through c e r t a i n phases of the c e l l c y c l e so as t o . induce a c e l l popu la t ion to become synchronous ( " synchrony by induct ion " ). With monolayer c u l t u r e s , a common method of ob t a i n i n g a p a r t i a l l y synchronized c e l l popu la t ion i s based on the f a c t t ha t c e l l s in the M per iod adhere less t i g h t l y to the su r face on which they are growing than a t other phases of the c e l l c y c l e ( 2 1 ). A popu la t ion of eel Is which are near ly a l l in the M phase of the c y c l e can be s imply 11 produced by gen t l y a g i t a t i n g an asynchronous c u l t u r e and c o l l e c t i n g the detached m i t o t i c c e l l s . With suspension c u l t u r e s t h i s method i s not a p p l i c a b l e , and chemical t reatments designed t o bunch c e l l s a t a p a r t i c u l a r phase of the c e l l c y c l e or t o s e l e c t f o r c e l l s in p a r t i c u l a r phases of the c y c l e are necessary. Acronyci ne: Acronyc ine ( f i g u r e 2, s t r u c t u r e a_ ) i s an a n t i n e o p l a s t i c a l k a l o i d o r i g i n a l l y i s o l a t e d as a natura l product ( 22 ), but which can now be made s y n t h e t i c a l l y ( 26-28 ). Along with a number of other a l k a l o i d s , i t was ex t rac ted in the l a te 1940s by A u s t r a l i a n chemists from the bark of the na t i ve A u s t r a l i a n t r e e Acronychia baueri ( commonly known as the scrub ash or scrub ye l lowwood") . These workers e s t ab l i s hed the phys i ca l p r o p e r t i e s and in most cases *he s t r u c t u r e s of the i s o l a t e d a l k a l o i d s , but d id not r epo r t on any b i o l o g i c a l a c t i v i t i e s ( 22-25 ). The s t r u c t u r e s of the p r i n c i p l e a l k a l o i d s from Acronychia baue r i , and of r e l a t e d compounds are shown in f i g u r e 2, s t r u c t u r e s a to h . Although acronyc ine has been known f o r ! o v e r ; t w o decades, the p r e c i s e s t r u c t u r e of the compound was not e s t ab l i s hed with c e r t a i n t y u n t i l r e c e n t l y . In 1949 Drummond and Lahey ( 25 ) demonstrated t h a t ac ronyc ine was an N-methyl acr idone ( the s t r u c t u r e of acronyc ine may be compared wi th those of- a c r i d i n e and ac r i done , which are s t r u c t u r e s e_ and f_ r e s p e c t i v e l y in f i g u r e 2 ). T h e i r data i nd i ca ted t h a t i t must have one of two po s s i b l e s t r u c t u r e s , d i f f e r i n g on ly in the o r i e n t a t i o n of the benzpyran r i n g ; they were unable however t o ' d e c i d e between the two s t r u c t u r e s , which are shown in f i g u r e 2 as s t r u c t u r e s a_ and g_. Th i s ambiguity was not reso lved u n t i l 1966, when MacDonald and Robertson ( 29 ) showed t ha t o x i d a t i v e degradat ion of acronyc ine y i e l ded a product O OCH2CH2N-(CH3)2 cj_ i soacronyc i ne Compounds r e l a t e d to ac ronyc ine 3 3 h_ compound " 3e " of Hoffman-LaRoche F i gu re 2 S t r u c t u r a l formulae of a l k a l o i d s of Acronych ia baueri and of r e l a t e d compounds. 13 i n con s i s t en t with s t r u c t u r e g_, i n d i c a t i n g t h a t s t r u c t u r e a_ was c o r r e c t . I soacronycine ( s t r u c t u r e g_ ) has s i nce been synthes ized and has been shown' to be without b i o l o g i c a l a c t i v i t y ( 34 ). In 1966, the E l i L i l l y drug company, which mainta ins an ex tens i ve program of research o r i en ted towards f i n d i n g compounds of medical i n t e r e s t in p l a n t s , reported t h a t ac ronyc ine had con s i de rab l e ant i tumor a c t i v i t y aga in s t a v a r i e t y of exper imental mouse neoplasms ( 30 ). Crude e x t r a c t s of the bark of Acronychia baueri had been screened f o r b i o l o g i c a l a c t i v i t y because of the Aust ra Man repor t s of the high a l k a l o i d a l content of t h i s m a t e r i a l . Although de fa t ted e t h a n o l i c e x t r a c t s of Acronychia bark d id not e x h i b i t any ant i tumor a c t i v i t y in r ou t i ne t e s t s us ing the P-I 534 leukemia, the Mecca-Iymphosarcoma, and adenocarcinoma 755, the un f rac t i ona ted mater i a l d id show an i n t e r e s t i n g c e n t r a l nervous system depressant a c t i v i t y in a r ou t i ne mouse behaviour t e s t ( 30 ). Treated mice e x h i b i t e d symptoms s i m i l a r to those produced by known hypnot i c s . A program was i n i t i a t e d to f r a c t i o n a t e the e x t r a c t s in an attempt to determine the nature of the mate r i a l possess ing t h i s b i o l o g i c a l a c t i v i t y . Rout ine screening o f ' m a t e r i a l from va r ious stages of t h i s f r a c t i o n a t i o n revealed s i g n i f i c a n t a n t i n e o p l a s t i c a c t i v i t y in some samples. Fur ther i n v e s t i g a t i o n s showed t h a t the ant i tumor a c t i v i t y r e su l t ed from the presence of acronyc ine - no other mate r i a l in the e x t r a c t s i n h i b i t e d the growth of the tumors te s ted ( 30, '31 ). A subsequent paper reported t h a t the c e n t r a l nervous system depressant a c t i v i t y which had prompted the f r a c t i o n a t i o n of the e x t r a c t s came from acronyc ine and a l s o from a second a l k a l o i d , ac ronyc id i ne ( f i g u r e 2, s t r u c t u r e d_ ), ( 32 ). Acronyc ine was shown ( 30 ) to be a c t i v e aga in s t a very wide range of exper imental neoplasms in mice, i n h i b i t i n g the growth of 12 of 17 14 tumors t e s t e d , i n c l ud i ng both s o l i d and leukemia t ypes . Svoboda and co-workers have noted ( 30, 37 ) t ha t severa l aspects of the a c t i v i t y of acronyc ine are of p a r t i c u l a r i n t e r e s t : i ) Acronyc ine has an unusua l ly broad spectrum o f , a c t i v i t y , and i n h i b i t s the growth of the C-1498 leukemia, a tumor which i s h i gh l y r e s i s t a n t to chemotherapy. In c on t r a s t to most chemotherapeutic agents however, i t i s l a r ge l y i n a c t i v e aga in s t a s c i t e s tumors. i i ) Acronyc ine i s a c t i v e when admin i s tered by a v a r i e t y of routes ( o r a l l y , i n t ravenous l y , subcutaneous Iy, or i n t r a p e r i t o n e a I l y ). i i i i ) In experiments in which acronyc ine therapy i s delayed and the tumor i s wel l e s tab I i shed -in the host, acronyc ine st<M I ihas the a b i l i t y to i n h i b i t the growth, of the neoplasm. Th i s i s in c o n t r a s t to many' chemotherapeutic agents, which are on Iy| e f f e c t i v e in the e a r l y stages of tumor development. iv ) Acronyc ine has chemical f ea tu re s unre lated to those of o ther known a n t i n e o p l a s t i c agents. These p r o p e r t i e s suggest t h a t acronyc ine i s in severa l ways u n l i k e c u r r e n t l y used ant i tumor agents, and thus may represent a new lead in the development of chemotherapeutic agents. One of the major problems encountered in the determinat ion of the b i o l o g i c a l p r ope r t i e s of acronyc ine and other a l k a l o i d s from Acronychia  baueri was the f a c t t h a t they are very s pa r i n g l y s o l ub l e in aqueous • media* The a l k a l o i d s are on ly very weakly ba s i c , and do not form water * It i s of i n t e r e s t to note tha t the water i n s o l u b i l i t y of the a l k a l o i d s of Acronychia baueri probably con s i de rab l y delayed t h e i r i n v e s t i g a t i o n f o r b i o l o g i c a l a c t i v i t y . One of the e a r l y A u s t r a l i a n workers w i th acronyc ine s tated in 1952 t h a t because of the water 15 s o l ub l e s a l t s a t p h y s i o l o g i c a l pH. Tests f o r ant i tumor a c t i v i t y in mice were c a r r i e d out w i th uniform suspensions of the s o l i d a l k a l o i d s prepared by g r i nd i n g the compounds wi th small amounts of a non - tox i c non - i on i c d i spe r san t ( " Emulphor " ) and d i l u t i n g the r e s u l t i n g s l u r r y w i th water or s a l i n e s o l u t i o n ( 30 ). Because of the d i f f i c u l t i e s of admin i s t e r i ng the compound in t h i s form, there were hopes t ha t water -s o l ub l e but a c t i v e d e r i v a t i v e s or analogs of ac ronyc ine could be found. In 1966 Svoboda reported on t e s t s of the a n t i n e o p l a s t i c a c t i v i t y of a number of acronyc ine d e r i v a t i v e s and s t r u c t u r a l analogs, some of them water s o l ub l e ( 3 1 ). The s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p f o r acronyc ine was found to be extremely s t r i c t ( 3 1 ). None of the compounds te s ted -other than acronyc ine . showed any a n t i n e o p l a s t i c a c t i v i t y . Th i s strong dependence on the s t r u c t u r e of the a l k a l o i d was r e c e n t l y confirmed in a second group of acronyc ine analogs synthes ized by the Hoffman-LaRoche drug company ( 34 ). Only a s i n g l e compound in the s e r i e s of analogs synthes i zed by these workers was a c t i v e ( compound " 3e " , f i g u r e 2, s t r u c t u r e h_ ), and i t showed no more ant i tumor a c t i v i t y than the parent compound,.-Recent l y , workers from E l i L i l l y and Company reported on a more s u i t a b l e method of prepar ing acronyc ine f o r a d m i n i s t r a t i o n to animals ( 35 ). Acronyc ine was co -p rec i pa ted from ethanol with p o l y v i n y l -p y r r o l i d o n e , a polymer which i s known t o increase the s o l u b i l i t y of i n s o l u b i l i t y of the a l k a l o i d s these workers had not i n ve s t i g a ted t h e i r pharmacological p r o p e r t i e s , and indeed thought t h a t " The i r main b i o l o g i c a l i n t e r e s t w i l l probably l i e in the mode of format ion in the p l an t " ( 33 ). 16 aromatic compounds. The r e s u l t i n g complex was more s o l ub l e than acronyc ine a lone, and had a g rea te r ant i tumor a c t i v i t y than the equ i v a l en t amount of pure a I k a l o i d . S u l l i v a n e t aj_. have r e c e n t l y reported on the metabolism of acronyc ine in a number of mammalian spec i e s , i n c l ud i ng the r a t , p i g , dog, mouse, and man ( 36 ). Rad i oac t i ve acronyc ine i a b e l l e d in the N-methyl or 14 O-methyl groups wi th C was s yn the s i zed , and admin i s tered to the animals by va r i ou s rou te s . The e x c r e t i o n of r a d i o a c t i v e products in the feces and u r i ne were then f o l l o w e d . The metabo l i t e s were separated by chromatographic procedures, and i d e n t i f i e d by mass spectrometry and nuclear magnetic resonance techn iques . Most of the admin i s tered acronyc ine was hydroxylated in one or more p o s i t i o n s , and the metabo l i t e s were excreted in conjugated form i n the b i l e . The cu r r en t problem: Although ex tens i ve in format ion i s a v a i l a b l e on the chemistry o f acronyc ine and r e l a t e d compounds ( 24-29, 34, 36 ), and con s i de rab le research has been done in i n v e s t i g a t i n g i t s ant i tumor p r ope r t i e s in exper imental animals ( 30, 31, 34, 35, 37 ), there i s very l i t t l e in format ion ava i Iab I e as to the mechanism by which i t i n h i b i t s the growth of tumor ce I I s./SvoBoda et aj_. have noted t ha t the a l k a l o i d was c y t o t o x i c to HeLa and Chinese hamster c e l l s growing in c u l t u r e ( 30).( The mechanism by which the a l k a l o i d k i l l e d c e l l s was not determined, although i t was shown t h a t in c o n t r a s t t o the a l k a l o i d s v i n b l a s t i n e and v i n c r i s t i n e , ac ronyc ine was not a m i t o t i c i n h i b i t o r ( 30 ). In rev iewing the p r ope r t i e s of ac ronyc ine , Johnson has s tated t h a t acronyc ine does not seem to i n h i b i t tumor growth by any obvious c y t o l o g i c a l e f f e c t ( 37 ). He has furthermore i nd i ca ted tha t workers a t E l i L i l l y and Company speculated 17 t h a t the mechanism of a c t i o n of acronyc ine might po s s i b l y be r e l a t ed t o t ha t of act inomycin D ( an a n t i b i o t i c which i n t e r a c t s with DNA and thereby i n h i b i t s RNA synthes i s ), but t ha t no work had been done on the po s s i b l e b ind ing of acronyc ine to DNA ( 37 ). In view of the i n d i c a t i o n s t ha t ac ronyc ine was not on ly a potent ant i tumor agent but a l s o might represent a new lead in the development of a n t i n e o p l a s t i c agents ( 30, 37 ), i t was decided to i n v e s t i g a t e the e f f e c t s of acronyc ine on tumor c e l l s in c u l t u r e , and i f po s s i b l e determine the bas i s of i t s c y t o t o x i c \ a c t i o n . The t i m e l i n e s s of t h i s d e c i s i o n was i nd i ca ted dur ing the course of the s t u d i e s , when i t was reported t h a t acronyc ine was " unde r ' c I i n i ca I i n v e s t i g a t i o n f o r po s s i b l e use in the t reatment of human mal ignanc ies ( 38 ). Two c e l l l i ne s were used f o r the i n v e s t i g a t i o n . The f i r s t was the L5I78Y mouse lymphocytic leukemia ( 39 ), a tumor whose growth i s i n h i b i t e d i n v ivo by acronyc ine ( 30 ). Th i s tumor was obta ined from Dr. G.A. F i s c h e r , and ha's been maintained in c u l t u r e s i nce t h a t t ime. The second was a r a t monocytic leukemia. Th i s tumor, r e f e r r ed to in t h i s t h e s i s as the " IRC " . . c e l l l i n e , i s a v a r i a n t of the Dunning leukemia ( 40 ), and has been induced to grow in c u l t u r e by Dr. R.L. • Noble of the Cancer Research Cent re , U.B.C. Both these tumor l i n e s grow r e a d i l y f r e e in suspension in F i s c h e r ' s medium supplemented wi th 10 % horse serum. A b r i e f p r e l im i na r y i n v e s t i g a t i o n by the author before the beginning- .. of the present study had suggested t h a t acronyc ine might d i s t u r b the i n co rpo ra t i on of extraceI ' I u Iar nuc leos ides i n to n u c l e i c a c i d s of tumor c e l l s ( 4 1 ). A large number of c y t o t o x i c drugs ( i n c l ud i ng many a n t i -n e o p l a s t i c agents ) i n h i b i t n u c l e i c ac id s ynthes i s and thus the i n co rpo ra t i on 18 i n to a c i d - i n s o I ub I e mate r i a l of e x t r a c e l l u l a r r a d i o a c t i v e precursor s ( 42-45 ). It seemed po s s i b l e t h e r e f o r e t ha t an i n h i b i t i o n of t h i s type might be re spons i b l e f o r the c y t o t o x i c i t y of ac ronyc ine . Sect ions 2 and 3 of t h i s t h e s i s de s c r i be i n v e s t i g a t i o n s i n to t h i s p o s s i b i l i t y . In view of t h i s , • t h e pathways by which nuc leos ides are incorporated i n to n u c l e i c a c i d s , and the mechanism of a c t i o n of a number of rep resenta t i ve , i n h i b i t o r s of n u c l e i c a c i d s yn thes i s w i l l be b r i e f l y reviewed here. Incorporat ion of e x t r a c e l l u l a r nuc leos ides i n to n u c l e i c a c i d s : C e l l s of most types ( mammalian, a v i an , b a c t e r i a l e t c . ) are not normal ly dependent on a supply of e x t r a c e l l u l a r nuc leos ides , but are capable of s yn the s i z i n g a l l nuc leo t i de s needed f o r growth by de novo pathways ( 46 ). In s p i t e of t h i s c a p a b i l i t y , most c e l l s r e a d i l y take up e x t r a c e l l u l a r nuc leos ides i f they are a v a i l a b l e . These are phosphory-l a t e d , and then • incorporated in to nuc leos ide t r i pho spha te pools by normal i n t r a c e l l u l a r pathways of nuc l eo t i de metabolism ( 46 ). In c on t r a s t to nuc leo s ide s , e x t r a c e l l u l a r nuc leo t i de s are not taken up by c e l l s . Extens ive use has been made of the f a c t t h a t c e l l s w i l l take up and u t i l i z e nuc leos ides f o r the i n v e s t i g a t i o n of n u c l e i c a c i d s yn the s i s . When c e l l s are incubated with r a d i o a c t i v e e x t r a c e l l u l a r nuc leo s ide s , the i n t r a c e l l u l a r nuc l eo t i de poo l ( s ) are Iabel Ied. ' When l a b e l l e d nuc leo t i de s from t h i s a c i d - s o l u b l e pool are incorporated in to n u c l e i c a c i d s , t h e i r r a d i o a c t i v i t y i s t r a n s f e r r e d i n to a c i d - i n s o l u b l e m a t e r i a l . The process of n u c l e i c ac id s yn thes i s can thus be monitored by i n v e s t i g a t i n g the i n co rpo ra t i on of r a d i o a c t i v i t y from l abe l l ed nuc leos ides i n to a c i d - i n s o I ub I e m a t e r i a l , or i f de s i red in to s p e c i f i c n u c l e i c ac id s or n u c l e i c ac id f r a c t i o n s . Changes in the r a t e of n u c l e i c ac id s yn thes i s w i l l be r e f l e c t e d in changes in the r a te of i n co rpo ra t i on of r a d i o a c t i v i t y 19 in to the n u c l e i c a c i d s . The bas ic pathway by which a r ep re sen ta t i v e e x t r a c e l l u l a r nuc leos ide , u r i d i n e , i s incorporated into a c i d - s o l ub I e and a c i d - i n s o l u b l e mate r i a l i s shown in f i g u r e 3. U r i d i n e or o ther nuc leos ides can c ros s the plasma membrane in both d i r e c t i o n s by s imple d i f f u s i o n through the plasma membrane ( 100-104, 108 ), or with the a id of s p e c i f i c membrane t r a n s p o r t systems ( 98, 100-116 ). The c h a r a c t e r i s t i c s of these t r an spo r t modes w i l l be f u r t h e r d i scussed in the i n t r oduc t i o n t o s e c t i o n 3 of t h i s t h e s i s . Ins ide the c e l l , u r i d i n e or o ther nuc leos ides are phosphoryIated to the nucleoside. monophosphates, which are intermediates in the normal pathways supply ing precursor s f o r n u c l e i c ac id s yn the s i s . Once a nuc leos ide has been phosphoryIated, i t cannot . leave the c e l l un less i t i s f i r s t degraded back to the nuc leo s ide , s i nce the c e l l membrane i s not permeable to nuc leos ide phosphates. The phosphory la t ion r e a c t i o n thus " t r ap s " the u r i d i n e w i t h i n the c e l l . UMP i s success i ve Iy .phosphoryIated to the nuc leos ide diphosphate and then to the nuc leos ide t r i pho spha te , which i s the immediate precursor of n u c l e i c a c i d s . UTP, along with other nuc leos ide t r i pho spha te s , i s a sub s t ra te f o r DNA dependent n u c l e i c ac id polymerases. R a d i o a c t i v i t y from UMP can a l s o enter n u c l e i c a c i d s a f t e r convers ion to CTP, dCTP, and TTP ( 46 ) ( not shown in f i g u r e 3 ). An i n h i b i t i o n of the i n co rpo ra t i on of an e x t r a c e l l u l a r nuc leos ide into n u c l e i c a c i d s could r e s u l t from an i n h i b i t i o n of any one of the steps between the e x t r a c e l l u l a r nuc leos ide and a c i d - i n s o l u b l e m a t e r i a l . Mechanism of a c t i o n of drugs which i n t e r f e r e with n u c l e i c ac id s yn the s i s : One of the main t a r ge t s of c y t o t o x i c drugs i s the s yn thes i s of n u c l e i c a c i d s from low molecu lar weight 'p recursors . R e p l i c a t i o n of tumor c e l l s r equ i r e s repeated d u p l i c a t i o n of the c e l l u l a r genome, as extrace I 1uIar f l u i d u r i d i ne^: p I asma membrane low mo I ecu l a r weight precursors cytopI asm de novo synthes i s ^ u r i d i n e • UMP • UDP- ->UTP-CTP" GTP ATP DNA template 1 a c i d - s o l u b l e mate r i a l nuc Ie i c a c i d poIymerases -•RNA L J ac i d - i nsoIubI< mater ia I F i gure 3 Incorporat ion of an e x t r a c e l l u l a r nuc leo s ide i n t o a c i d - s o l u b l e and a c i d - i n s o l u b l e mate r i a l 21 wel l as ex tens i ve RNA s yn thes i s to supply messenger, r ibosomal , and t r a n s f e r RNA f o r p r o t e i n s yn thes i s involved in the d u p l i c a t i o n of the c e l l u l a r content s . I n te r rup t i on of DNA synthes i s prevents the format ion of v i a b l e progeny and can lead to damaging " unbalanced growth " ( 47 ), wh i l e i n t e r r u p t i o n of RNA s yn thes i s can r e s u l t in the rap id death of the c e l l due to consequent d i s tu rbances in p r o t e i n s yn the s i s . . There are b a s i c a l l y th ree mechanisms by which a drug can i n h i b i t n u c l e i c ac id s yn thes i s : i ) i n t e r f e r ence wi th the s upp l i e s of nuc leos ide t r i phosphate s necessary f o r n u c l e i c a c i d s yn the s i s , i i ) i n t e r f e r ence with the f u n c t i o n of n u c l e i c ac id polymerases, and i i i ) i n t e r f e r ence wi th the template a c t i v i t y of DNA. The mapner in which a number of drugs ( i n c l ud i ng some used in the treatment of cancer ) ac t in c e l l s to i n h i b i t the s yn thes i s of n u c l e i c ac id s by these th ree bas ic mechanisms are b r i e f l y reviewed here: i ) Nuc leos ide t r i phosphate s upp l i e s : Although c e l l s can r e c y c l e nuc l eo t i de u n i t s produced as the r e s u l t of n u c l e i c ac id degradat i ve processes and can u t i l i z e e x t r a c e l l u l a r nuc leos ides i f they are a v a i l a b l e , the main s upp l i e s of n u c l e i c a c i d precursor s in r a p i d l y d i v i d i n g c e l l s are synthes ized " de novo " . To the extent t h a t de novo pathways are e s s e n t i a l f o r the product ion of nuc l eo t i de u n i t s , an i n h i b i t i o n of these pathways w i l l r e s u l t in an i n h i b i t i o n of n u c l e i c ac id s yn the s i s . S ince the po l ymer i z a t i on process r equ i re s a l l four nuc leos ide t r i pho spha te s , an i n t e r f e r e n c e with the s yn thes i s of on ly one of the precursor s i s necessary to i n h i b i t the i n co rpo ra t i on of a l l . Most i n h i b i t o r s of the de novo pathways of nuc l eo t i de s yn thes i s can be c l a s s i f i e d as " a n t i m e t a b o l i t e s " as they are s t r u c t u r a l analogs of normal i n t r a c e l l u l a r compounds. They mimic these compounds and can 22 i n h i b i t the a c t i v i t y of enzymes involved in nuc leo t i de s yn the s i s . The c l a s s i c a l example of such an a n t i m e t a b o l i t e , and one of the o l d e s t and most useful ant i tumor drugs i s the compound methotrexate ( otherwise known as amethopterin ) ( 4 8 ' ) . Th i s drug i s a s t r u c t u r a l .• analog of f o l i c a c i d and binds f a r more s t r ong l y to the enzyme d ihydrofo I ate reductase than does the natura l compound. Th i s enzyme i s r e spons ib le f o r conver t ing d i h y d r o f o l i c ac id t o t e t r a h y d r o f o I i c a c i d , and i t s i n h i b i t i o n consequently r e s u l t s in a rap id dep l e t i on of c e l l u l a r s upp l i e s of the l a t t e r . The shortage of te t rahydro fo I ate r e s u l t s in the i n h i b i t i o n of a number of b i o s y n t h e t i c r e a c t i o n s . The most c r i t i c a l of these appears to be the convers ion of deoxy-UMP to TMP ( 48 ) ; an i n h i b i t i o n of t h i s r e a c t i on r e s u l t s in a dep l e t i on of TTP s u p p l i e s , which i n h i b i t s DNA but not RNA s yn the s i s . Nuc leos ide and nuc leos ide base analogs form an important group of a n t i m e t a b o l i t e s which i n t e r f e r e with n u c l e i c ac id s yn the s i s . Besides i n h i b i t i n g the synthes i s of n u c l eo t i de s , some of these analogs may g i ve r i s e to " f r audu len t " nuc leo t i de s which' are incorporated in to n u c l e i c ac id s with t o x i c r e s u l t s . Perhaps the most important compound of t h i s c l a s s i s the hypoxanthine ana log, 6-mercapto pu r i ne . Th i s analog i s converted in t race I Iu Iar Iy to the r i b o n u c l e o s i d e , which i n h i b i t s severa l of the enzymes involved in the s yn thes i s of pur ines ( 49 ). A second ana log, 5 - f I uo rourac i I, i s a f t e r convers ion to 2 ' - d e o x y - 5 - f I u o r o u r i d y I i c a c i d capable of i n h i b i t i n g thymidy la te synthetase ( 50 ). i i ) Nuc l e i c ac id polymerases : There are a l im i t ed number of drugs which i n h i b i t the f unc t i on of these enzymes. In most cases they are not ant i tumor agents. Among these are the compounds a Ipha-amanit in ( 51 ) which i n h i b i t s s p e c i f i c euka r yo t i c RNA polymerases, the r i f amyc i n and • 23 s t r e p t o v a r i c i n compounds which i n h i b i t b a c t e r i a l and mi tochondr ia l RNA polymerases ( 43 ), and the t umor i c i da l compound kanchanomyc i n' •' ( 43 ) which i n a c t i v a t e s RNA polymerases. The on ly c l i n i c a l l y important ant i tumor agent which a f f e c t s the f u n c t i o n i n g of n u c l e i c ac id polymerases i s the compound c y to s i ne a r ab i no s i de . Th i s nuc leos ide i s aneanalog of c y t o s i n e , and i s phosphoryIated to araCTP which i s a compet i t i ve i n h i b i t o r of DNA polymerase ( 52 ). i i i ) DNA template a c t i v i t y : Several ant i tumor drugs, as wel l as a wide v a r i e t y of c y t o t o x i c compounds which are useful as a n t i b a c t e r i a l , a n t i m a l a r i a l and ant i t rypanosomal agents, i n t e r f e r e with nucleqc ac id s ynthes i s by i n t e r a c t i n g with doubIe-stranded-^DNA and thus i n t e r f e r i n g wi th i t s a b i l i t y to ac t as a template f o r n u c l e i c ac id polymerases ( f o r reviews see 42-44 ). These agents i n t e r a c t with the n u c l e i c ac id by i n t e r c a l a t i n g between adjacent base pa i r s of the double h e l i c a l s t r u c t u r e , or in some cases by a t t a ch i n g to the e x t e r i o r of the polymer. Most of the s imple compounds which i n t e r c a l a t e between base pa i r s of DNA have a 2 or 3 membered r i n g system of which a t l ea s t one r i n g i s h e t e r o c y c l i c . These compounds inc lude a c r i d i n e orange, eth id ium brom<i;de, p r o f l a v i n e , and the ant i tumor agent M i r a c i l D ( fi.gure 4, s t r u c t u r e s c_ to f_, shown wi th the s t r u c t u r e s of acronyc ine ( s t r u c t u r e a_ ) and compound " 3e " of Hoffman-LaRoche ( s t r u c t u r e b_ ) .for comparison ). A number of more complex compounds a l s o i n t e r c a l a t e , but have in a d d i t i o n to a p o l y c y c l i c chromophore other groupings which are thought to help s t a b i l i z e the DNA-drug complex. Among these compounds are the ant i tumor agents act inomyc in D and daunomycin ( f i g u r e 4, s t r u c t u r e s 3_ and h_ ). The i n t e r a c t i o n of drugs wi th DNA causes c h a r a c t e r i s t i c changes in 24 H 2 N e eth id ium bromide f_ a c r i d i n e orange pept ide pept ide i i l H2 3_ a c t i nomyci n D ( chromophore ) O OH H3CO O OH amino sugar h_ daunomycin ( chromophore ) F i gu re 4 S t r u c t u r a l formulae of ac ronyc ine and of compound " 3e " of Hoffman-LaRoche compared w i th those of compounds which i n t e r a c t w i th DNA. 25. the p r ope r t i e s of both the drug and the n u c l e i c a c i d . The a l t e r e d environment of the bound drug ( r e l a t i v e to drug molecules f r ee in s o l u t i o n ) can change i t s u l t r a v i o l e t and v i s i b l e absorpt ion spectrum ( 53-59 ), i t s f l u o r e s c e n t p r ope r t i e s ( 60 ), and i t s chemical r e a c t i v i t y ( 61 ). Changes are a l s o produced in the phys i ca l p r ope r t i e s of the DNA molecule as a r e s u l t of drug b i nd ing . The i n t e r c a l a t i o n of drug molecules f o rce s the double h e l i x to unwind s l i g h t l y ( 62 ). Th i s lengthens the DNA, a process which can be detected by an increase in v i s c o s i t y ( 58, 59, 63 ), by u I t r a c e n t r i f u g a t i o n ( 62, 63 ), or d i r e c t l y by autoradioagraphy of r a d i o a c t i v e I y l a b e l l e d DNA ( 64 ). The i n t e r c a l a t e d drug molecules help s t a b i l i z e the double h e l i x and thus r a i s e the me l t ing temperature of double-stranded DNA ( 52, 54, 59 ), and bound drugs may lessen the s e n s i t i v i t y of DNA to nucleases ( 65 ). The b ind ing of drugs to DNA lessens the a b i l i t y of the DNA to a c t as a template f o r DNA or RNA synthes i s - t h i s may be detected in v i t r o using app rop r i a te systems ( 43 ). The r e l a t i v e s e n s i t i v i t i e s of DNA or RNA synthes i s to a DNA-binding agent may vary , depending on the molecu lar s t r u c t u r e of the drug and the exact mode of i t s b ind ing to DNA . 26 METHODS AND MATERIALS MATERIALS 1) Chemi ca I s : Non - rad ioac t i ve nuc leo s ide s , n u c l eo t i de s , and nuc l eo t i de sugars were purchased from Calbiochem, Los Angeles, C a l i f , or the Sigma Chemical Company, St . Lou i s , Mo. Act inomycin D, a c r i d i n e orange, eth id ium bromide, d i t h i o t h r e i t o I, and c a l f thymus DNA were from Calbiochem. RNase ( bovine p a n c r e a t i c , 5 x r e c r y s t a l l i z e d ) and bovine serum albumin ( f r a c t i o n V ) were from Sigma. Hydroxyurea was from N u t r i t i o n a l B iochemica I s , CI eve I and,0hio, wh i l e v i n b l a s t i n e and acnonye i theewere g i f t s from E l i L i l l y and Co., I nd ianapol i s , Ind. C o l c h i c i n e was from B r i t i s h Drug Houses L t d . , Poo le , England. PPO and POPOP f o r the p repara t ion of s c i n t i l l a t i o n f l u i d were from Kent Labo ra to r i e s , Vancouver, B.C.. " Aquasol " premixed s c i n t i l l a t i o n f l u i d was from the New England Nuclear Corp. , DorvaI Que. and " PCS " premixed s c i n t i l l a t i o n f l u i d was from Amecsham/SearIe, Don M i l l s , C O n t . Unless otherwise noted, a l l o ther reagents were obta ined from e i t h e r the F i she r Chemical Company or Canlab in Vancouver,f.B.C. 2) Rad ioac t i ve I y l a b e l l e d chemica l s : The f o l l o w i n g r a d i o a c t i v e I y l a b e l l e d chemicals with the s p e c i f i c a c t i v i t i e s noted were purched from the New England Nuclear Corp. A l l r a d i o a c t i v e I y l a b e l l e d chemicals were e i t h e r used on r e c e i p t , or i f s tored were p u r i f i e d before use by paper chromatography. rad i oac t i ve I y l a b e l l e d chemical s p e c i f i c a c t i v i t y  u r i d i n e - 6 - 3 H 10.4 Ci/mmole u r i d i n e - 5 - 3 H 25-28 Ci/mmole I 4 t hymid ine -2 - C 52.2 mCi/mmole 27 r a d i o a c t i v e I y l a b e l l e d chemical s p e c i f i c a c t i v i t y thym i d i ne-methyI- J H 14 adenosine t r i p h o s p h a t e - 8 - C 2-deoxy-D-gIucose-^H (G) I 4 methyl i o d i d e - C i nuI i n- H cho I i ne-methyI - H myo-i nos i t o I-2-^H 20.0 49.8 7.2 3.0 1.0 2.84 mC i/mmoIe mC i/mmoIe C i/mmoIe mC i/mmoIe 15.8 mCi/gram C i/mmoIe Ci/mmoIe 3) T i s sue c u l t u r e s u p p l i e s : F i s c h e r ' s medium f o r the leukemic c e l l s of mice, and p e n i c i l l i n / s t reptomyc in s o l u t i o n ( 100 x concentrated ) were obta ined from the Grand Istband B i o l o g i c a l Co., Oakl and,Ca I i f . The powdered medium was d i s s o l ved in d i s t i l l e d water, and the s o l u t i o n then s t e r i l i z e d by f i l t r a t i o n through a M i I Ii pore f i l t e r ( pore s i z e 0.45 nm ). Horse serum was purchased from North American B i o l o g i c a l s Inc.,North Miami, F l o r i d a . 4) Mater ia Is: G lass f i b e r f i l t e r s ( 2.4 cm, type 934 AH ) were from the Reeve Angel Co., Cl i fton,N.c) .-. M i l l i p o r e f i l t e r s ( 2.5 cm, pore s i z e 0.65 nm ) and M i l l i p o r e f i l t e r ho lders ( model XX1002500 ) were from the M i l l i p o r e F i l t e r Corp. , Mont rea l , Que.. C e l l u l o s e t h i n layer chromatography • p l a t e s '(•' 20 cm by 20 cm, c e l l u l o s e MN 300 on p l a s t i c backings ) were obta ined from Macherey-NageI and Co., Duren, Germany,through Canlab, Vancouver, B.C. 28 GENERAL METHODS I) Rout ine maintainance of c e l l s in t i s s u e c u l t u r e : Both the L5I78Y and IRC c e l l l i ne s were maintained at 37° in suspension c u l t u r e in F i s c h e r ' s medium supplemented wi th horse serummC 10 % ), p e n i c i l l i n ( 50 units/ml ), and s t reptomyc in ( 50 yg/ml ). S t e r i l e p recaut ions were observed throughout ; t e s t s f o r mycoplasma at i n t e r v a l s throughout the study us ing commerc ia l ly -prepared media ( North American B i o l o g i c a l s , Inc. ) were negat i ve . C e l l s were quan t i t a ted by count ing samples of c e l l suspensions in a hemacytometer ; on ly i n t a c t , r e f r a c t i le c e l l s were accepted. A l t e r n a t i v e l y , a l i q u o t s of c e l l suspensions were d i l u t e d wi th 0.9 % NaCl and counted in an e l e c t r o n i c p a r t i c l e counter ( Model B, Cou l t e r E l e c t r o n i c s , S t . H i a l e ah , F l o r i d a ). Stock c u l t u r e s of c e l l s were maintained in 10 ml po r t i on s of medium in 50 ml erlenmeyer f l a s k s stoppered with s i l i c o n e rubber s toppers . C e l l s were iirno'cuj|!a?t'edJ i n to f re sh medium ( pH 7.8 ) to g ive i n i t i a l c e l l concent ra t i on s of 10-50 x 10^ c e l l s / m l and the c u l t u r e s were a l lowed t o grow f o r 2 or 3 days to no more than I.0 x 10 c e l l s /m l ( L5I78Y ) or 0.5 x IO 6 c e l l s /m l ( IRC ). By t h i s t ime the pH of the medium had, dropped to cs 6.8, and there was l i t t l e f u r t h e r increase in the c e l l p opu l a t i on . When large numbers of c e l l s were needed f o r exper iments, c e l l s were innoculated at appropr i a te concent ra t i ons i n to 30-50 ml po r t i on s of supplemented F i s c h e r ' s medium conta ined in 100 ml stoppered b o t t l e s . The b o t t l e s were ro ta ted at 4 rpm i n c l i n e d 30° from the h o r i z o n t a l , 5 and the c e l l s were grown to a concent ra t i on of 3-5 x 10 c e l l s / m l ( L5I78Y ) 5 or 2-3 x 10 c e l l s /m l ( IRC ). Unless otherwise noted, a l i q u o t s of the c e l l suspensions were then d i s t r i b u t e d in f l a t -bo t tomed g la s s v i a l s of 12 ml c a p a c i t y . C e l l c u l t u r e s prepared in t h i s manner were in the 29 exponent ia l phase of growth at the time of t h e i r use in exper iments. When, in c e r t a i n exper iments, i t was necessary to t r a n s f e r c e l l s to new medium, they were c o l l e c t e d by c e n t r i f u g a t i o n f o r 3 minutes a t £. 800 g. A f t e r removal of the supernatant f l u i d , the c e l l s were resuspended in the des i red volume of f re sh medium. When i t was necessary t o remove drugs or r a d i o a c t i v e p recu r so r s , t h i s procedure was repeated a second t ime. 2) P repa ra t i on of acronyc ine s o l u t i o n s : Acronyc ine was rece ived from Etli;i: L i l l y and Co. as a dry ye l low powder, and was s tored at 4 ° . I n i t i a l attempts to prepare aqueous s o l u t i o n s of the drug by d i s s o l v i n g the s o l i d compound in the des i red medium were f r u s t r a t e d by i t s water i n s o l u b i l i t y . It was found however t h a t the drug was r e a d i l y s o l ub l e in e t h a n o l , and t h a t with ca re , aqueous s o l u t i o n s of acronyc ine could be prepared by f i r s t d i s s o l v i n g the a l k a l o i d in a r e l a t i v e l y small volume of e t h a n o l , and then c a r e f u l l y adding the ethanol s o l u t i o n to aqueous media. In i n i t i a l exper iments, the ethanol s o l u t i o n was prepared f re sh each day. Acronycine ( 2-3 mg ) was d i s s o l ved in s u f f i c i e n t ethanol t o g ive an approximate concent ra t i on of 10 mg/ml. The p r e c i s e concent ra t i on of t h i s s tock s o l u t i o n was determined s pec t r opho tomet r i c a l l y a f t e r d i l u t i n g a l i q u o t s with ethanol ( E^sO^ = : 39,800 ) ( 30 ). It was subsequently found t h a t acronyc ine was s t a b l e over a per iod of months when s tored as an e t h a n o l i c s o l u t i o n a t a reduced temperature and was p ro tected from l i g h t ( as shown by t h i n layer chromatography and c o n s i s t e n t b i o l o g i c a l p r ope r t i e s ). In l a t e r work t h e r e f o r e , aqueous s o l u t i o n s of acronyc ine were prepared from a s tock s o l u t i o n - o f acronyc ine in ethanol ( 12 mg/ml ) which was s tored 30 at 4 ° . To prepare aqueous s o l u t i o n s of ac ronyc ine , a predetermined volume of s tock s o l u t i o n of the a l k a l o i d in ethanol was s lowly discharged from a micrometer sy r inge o r m ic rop ipe t i n to a po r t i on of v i go rou s l y s t i r r e d bu f f e r or medium. Inadequate s t i r r i n g or an exces s i ve ra te of d i scharge of the a l co l i i o l i c s o l u t i o n r e su l t ed in the immediate p r e c i p i t a t i o n of the drug as a c l e a r l y v i s i b l e c loud of f i n e c r y s t a l s . So l u t i on s of acronyc ine in aqueous media could c o n s i s t e n t l y be prepared at concent ra t i on s of up t o approximately 30 ug/ml . Attempts to prepare h igher concent ra t i on s r e su l t ed in the immediate p r e c i p i t a t i o n of the drug. Because of the l im i t ed s o l u b i l i t y of acronyc ine in aqueous media and the care needed in adding an e t h a n o l i c s o l u t i o n of the drug, i t was necessary t o add the drug t o c e l l c u l t u r e s or assays in a r e l a t i v e l y large volume of t i s s u e c u l t u r e medium or b u f f e r . For experiments w i th c e l l s , normal ly 2 volumes of F i s c h e r ' s medium con ta i n i n g acronyc ine was added t o 2 or 3 volumes of c e l l suspens ion. S i m i l a r r a t i o s were used t o introduce the drug i n to the r eac t i on mixtures used in enzyme assays. With in a g iven exper iment, the concent ra t i on of ethanol in a l l c u l t u r e s or assay tubes was made up t o t h a t in the tube con ta i n i ng the h ighest amount of a l k a l o i d . Contro l expe r iment s ' e s tab I i s hed t h a t , with the except ion of the RNA polymerase r eac t i on ( which was i n h i b i t e d c_. 10 % by the concen t ra t i on of a l coho l used ), the amount of ethanol used ( u sua l l y £. 0.I % ) had no de tec tab le e f f e c t on the parameters measured. 3) Determinat ion of r a d i o a c t i v i t y : R a d i o a c t i v i t y was determined by s c i n t i l l a t i o n count ing us ing 31 e i t h e r a Packard T r iCa rb Model 3003, o r a Nuclear Chicago Mark IIA l i q u i d s c i n t i l l a t i o n spectrophotometer. Counts were co r rec ted f o r background, and where appropr i a te were converted t o dpm using the channels r a t i o method with a graph of e f f i c i e n c y of count ing vs. 3 14 channel r a t i o der ived from count ing a se t of quenched H or C standards purchased from Amersham/Searle. Rad ioac t i ve samples were counted in s c i n t i l l a t i o n f l u i d of three bas i c types : I 4 i ) to luene based : Ac idp inso Iub Ie mate r i a l l a b e l l e d with C which had been trapped on M i l l i p o r e f i l t e r s ( RNA polymerase assay ) was counted by p l a c i n g the f i l t e r s in 10 ml of a to luene based s c i n t i l l a t i o n f l u i d ( composit ion : 1000 ml t o l uene , 4 g PPO, 100 mg POPOP ). i i ) t o Iuene/ethanoI based: a) Glass f i b e r f i l t e r s r e t a i n i n g mate r i a l I 4 l a b e l l e d with C and b) 0.4 ml a l i q u o t s of aqueous samples con ta i n i ng 14 3 e i t h e r C or H were counted in 10 ml of a water -compat ib le s c i n t i l l a t i o n f l u i d ( composit ion : 600 ml t o l uene , 400 ml e t h a n o l , 4 g PPO, 100 mg POPOP ) he rea f t e r r e f e r r ed to as " t o Iuene/ethanoI " s c i n t i l l a t i o n f l u i d . Some d i f f i c u l t y was o c c a s i o n a l l y exper ienced with to Iuene/ethanoI s c i n t i l l a t i o n f l u i d when i t was used to count aqueous s o l u t i o n s of c e r t a i n r a d i o a c t i v e l y l a b e l l e d po l a r compounds. When samples were d i s s o l ved in t h i s s c i n t i l l a t i o n f l u i d and counted repeatedly over a per iod of days the i nd i ca ted amount of r a d i o a c t i v i t y ( cpm ) in the v i a l s dec l i ned in some cases by up to 50 %. It was found t ha t the " miss ing " r a d i o -a c t i v i t y had become adsorbed onto the w a l l s of the g la s s s c i n t i l l a t i o n v i a l s , from which i t could be q u a n t i t a t i v e l y recovered by removing the s c i n t i l l a t i o n f l u i d from the v i a l s and washing the v i a l w a l l s w i th an 32 aqueous s o l u t i o n . S i m i l a r d i f f i c u l t i e s were encountered when dioxane based s c i n t i l l a t i o n f l u i d s were t r i e d . Because of t h i s po s s i b l e loss of counts with t ime , samples counted in to Iuene/ethanoI s c i n t i l l a t i o n f l u i d were not mixed w i th the s c i n t i l l a t i o n f l u i d u n t i l s h o r t l y before count ing , and only the f i r s t count ( u sua l l y 10 minutes ) was used in the c a l c u l a t i o n of the amount of r a d i o a c t i v i t y in the samples. i i i ) xy lene based : In l a t e r exper iments, aqueous samples were r o u t i n e l y counted in one of two r e cen t l y a v a i l a b l e commercia l ly prepared xy lene based s c i n t i l l a t i o n f l u i d s ( " Aquasol " , from the New England Nuclear Corp. , or " PCS " , from Amersham/SearIe ). Both s c i n t i l l a t i o n f l u i d s conta in high concent ra t i on s of detergents which a l l ow the s o l u b i l i z a t i o n of r e l a t i v e l y large q u a n t i t i e s of water. Counting e f f i c i e n c i e s in these two s c i n t i l l a t i o n f l u i d s were found t o be c o n s i s t e n t l y h igher than in t o Iuene/ethanoI o r dioxane based s c i n t i l l a t i o n f l u i d s , and more important ly aqueous samples of l a b e l l e d compounds prepared in them showed no loss of counts of extended per iods of s to rage. The a b i l i t y of these s c i n t i l l a t i o n f l u i d s t o r e t a i n po l a r compounds in s o l u t i o n i s probably due t o t h e i r r e l a t i v e l y high detergent content . Both Aquasol and PCS proved e n t i r e Iy s a t i s f a c t o r y f o r count ing both aqueous and non-aqueous samples, w i th PCS being p re fe r r ed as i t gave h igher count ing e f f i c i e n c i e s , could s o l u b i l i z e cons ide rab l y more water, and was s l i g h t l y less expens ive. METHODS USED IN SECTION I OF RESULTS I) Determinat ion of the frequency of normal, m i t o t i c , and b inuc lea ted  c e l l s in suspension c u l t u r e s : C e l l s from suspension c u l t u r e s were concentrated by c e n t r i f u g a t i o n , and a small drop of the concentrated suspension ( c_. 100 x 10 c e l l s /m l ) smeared on a g la s s s l i d e . The smears were a i r - d r i e d , f i x e d wi th methanol, and s ta ined with Wr i gh t ' s s t a i n using standard procedures ( 66 ). 1000 t o 2000 c e l l s were then examined t o determine the frequency of normal, m i t o t i c , and b inuc lea ted c e l l s . 2) Synchron i za t ion of c e l l c u l t u r e s : C e l l s growing in t i s s u e c u l t u r e are normally " asynchronous " -i ^e . thence I I s i,n=a cu I tu re .at any^one t.ime are found i n a l l phases of the c e l l c y c l e , and the growth of a popu la t ion i s continuous as new c e l l s c on s t an t l y reach m i t o s i s . In c e r t a i n s t ud i e s reported in t h i s t h e s i s , s p e c i f i c techniques were used t o produce p a r t i a l l y synchronized c u l t u r e s of L5I78Y and IRC c e l t s in which most of the c e l l s in the c u l t u r e s are a t any one time mainly w i t h i n a r e s t r i c t e d segment of the c e l l c y c l e . The c e l l s in such popu la t ions tend t o progress through the c e l l c y c l e together and d i v i d e a t approximately the same t ime. S ince i t i s not po s s i b l e t o synchron ize c e l l s growing in suspension c u l t u r e by the common procedure of m i t o t i c detachment ( 21 ), L5I78Y and IRC c u l t u r e s were p a r t i a l l y synchronized by us ing agents which have a s e l e c t i v e t o x i c i t y f o r c e l l s in c e r t a i n phases of the c e l l c y c l e . a) P a r t i a l s ynch ron i za t i on of IRC c u l t u r e s : The method used t o p a r t i a l l y synchronize IRC c u l t u r e s was developed in t h i s l abo ra to ry , and i s based on the a b i l i t y of hydroxyurea to s e l e c t i v e l y damage c e l l s in the S phase of the c e l l c y c l e ( '67 ). Couch showed t ha t i f IRC ceI Is were incubated wi th hydroxyurea at a concent ra t i on of 10 mM, a shor t t reatment of three hours was s u f f i c i e n t t o damage c e l l s in the S phase t o such an extent t h a t they lysed w i t h i n a few hours of removal of the drug ( 68 ). C e l l s which were in the G~, M, and G phases of the c e l l 34 c y c l e a t the time of i n t r oduc t i on of the drug remained undamaged a f t e r a 3 hour t reatment, and on removal of the drug entered the S phase together as a p a r t i a l l y synchronized c e l l p opu l a t i on . On the bas i s of these f i n d i n g s , IRC c e l l s were synchronized by incubat ing them with hydroxyurea f o r 3 hours, then removing the drug by resuspending the c e l l s in f re sh medium. b) P a r t i a l s ynch ron i za t i on of L5I78Y c u l t u r e s : The method used t o ob ta i n p a r t i a l l y synchronized L5I78Y c u l t u r e s - i s based on the a b i l i t y of the a l k a l o i d v i n b l a s t i n e ( VLB ) to a r r e s t c e l l s a t m i t o s i s . Several workers ( 69, 70 ) have shown in o ther systems t h a t VLB at appropr i a te concent ra t i on s a r r e s t s c e l l s a t or near the metaphase stage of c e l l d i v i s i o n without i n t e r f e r i n g wi th t h e i r passage through other phases of the c e l l c y c l e . It has been found in t h i s laboratory t ha t t h i s a l s o app l i e s when L5I78Y c e l l s are t r ea ted with VLB at a. concent ra t i on of 0.02 ug/ml. It was a l s o observed wi th t h i s c e l l l i n e t ha t a f t e r the drug was removed a high p ropor t i on of the a r re s ted c e l l s r a p i d l y l y sed, whereas those c e l l s which had not reached the po in t of a r r e s t cont inued as a p a r t i a l l y synchronized popu l a t i on . On the bas i s of these f i n d i n g s L5I78Y c u l t u r e s were p a r t i a l l y synchronized by incubat ing them with VLB ( 0.02 pg/ml ) f o r 6 hours ( £. two - t h i r d s of a c e l l generat ion t i m e ' ) . The drug was then removed by resuspending the c e l l s in d rug - f ree medium. The c e l l s not lysed by t h i s procedure proceeded through the c e l l c y c l e as a p a r t i a l l y synchronized popu l a t i on . 3) Mon i to r ing of DNA syn thes i s in synchronized c u l t u r e s : I r vo rdeor to monitor DNA syn thes i s in synchronized c u l t u r e s , c e l l s I 4 were incubated f o r 45 minutes with C- thymid ine. Nucleos ide concent ra t i on s and o ther exper imental d e t a i l s are g iven in the legends t o i n d i v i d u a l 35 f i g u r e s . The c u l t u r e s were then c h i l l e d t o 0° ; c on t r o l experiments e s t a b l i s h e d t h a t t h i s reduct ion in temperature e f f e c t i v e l y stopped the i n co rpo ra t i on of r a d i o a c t i v i t y i n to ac i d - i n so I ub I e m a t e r i a l . C e l l s were c o l l e c t e d f o r the a n a l y s i s of a c i d - i n s o l u b l e r a d i o a c t i v i t y using g la s s f i b e r f i l t e r s . The f i l t e r s were mounted in Mi I I I pore m i c roana l y s i s f i l t e r ho lde r s , which were in tu rn mounted in a mani fo ld capable of app ly ing s u c t i on to e i g h t f i l t e r ho lders s imu l taneous l y . The holders were f i l l e d with 10 ml s a l i n e ( 0° ), and 1.0 ml of c e l l suspension was added. A gent le s uc t i on was a p p l i e d , and the c e l l s c o l l e c t e d on the f i l t e r . The trapped c e l l s were washed at 0° with i s o t o n i c s a l i n e ( 2 x 15 ml ), 5%% TCA ( 2 x 10 ml ), s a l i n e ( 2 x 1 5 ml ), and water ( 2 x 15 ml ). The f i l t e r s were d r i ed .in a vacuum, and the trapped ac i d- i nspl ub le r a d i o a c t i v i t y counted by p l a c i n g the f i l t e r s d i r e c t l y in to Iuene/ethanoI s c i n t i I I at ion f l u i d . 4) E l e c t r on microscopy : C e l l s were c o l l e c t e d by c e n t r i f u g a t i o n , and then f i x e d f o r 1.5 hours in 5 % gIutaraIdehyde in 0.07 M phosphate b u f f e r , pH 7.2. The gIutaraIdehyde f i x e d c e l l s were embedded in 5 % agar in b u f f e r , and small p ieces of sample then f i x e d in I % osmium t e t r o x i d e in bu f f e r f o r 1.5 hours. The samples were dehydrated in a s e r i e s of s o l u t i o n s of i nc rea s ing ethanol c oncen t r a t i o n , and then i n f i l t r a t e d with propylene ox ide and embedded in Epon 812. Sect ions were cut using a g la s s k n i f e on a SorvaI I MT2 mictrotpme, and mounted on carbon f i l m s on copper g r i d s . The sec t i on s were- s ta ined wi th uranyl acetate and lead c i t r a t e , and examined in a low-r e s o l u t i o n P h i l l i p s 75C e l e c t r o n microscope. 5) R e s p i r a t i o n of c e l l c u l t u r e s : The consumption of oxygen by c e l l s was measured using standard 36 techniques ( 71 ). Samples of L5I78Y c u l t u r e s at c e l l d e n s i t i e s of 0.5 to I.0 x 10^ c e l l s /m l were placed in the sample chamber of a C l a r k oxygen e l e c t r o d e assembly ( type YSI 4004, Yel low Spr ings Instrument Corp. , Yel low Spr ings , Ohio ) thermostatted at 37 ± 0 . 1 ° . The oxygen e l e c t r ode assembly was attached t o a G i l s o n oxygraph ( model KM,, G>i I son Medical E l e c t r o n i c s , M idd le ton , Wis. ). The sample chamber was sealed ( t a k i n g care to remove any trapped a i r bubbles ) and the c e l l suspension s t i r r e d magnet i ca l l y ; the consumption of oxygen by the c e l l s was monitored over per iods of c. 10 minutes, dur ing which time about 10 % of the oxygen in the sample was consumed. The response of the oxygraph was s tandard ized by record ing t r ace s with a i r - s a t u r a t e d d i s t i l l e d water in the sample chamber, and.with oxygen-depleted c e l l c u l t u r e medium ( produced by d r i v i n g the oxygen tens ion t o zero by adding a small amount of g lucose ox idase to the g l uco se - con ta i n i ng medium ). It was found t ha t when the apparatus was used in t h i s manner to monitor small changes in oxygen tens ion over extended per iods of t ime, the r e s u l t s could g r e a t l y be improved by p l a c i n g two p l a s t i c membranes ( t e f l o n , each 0.001 inch t h i c k ) over the C l a r k e l e c t r o d e , ra ther than the usual s i n g l e membrane. This gave markedly smoother t r a c e s , a t the expense of a g rea te r lag in response to rap id changes in oxygen t e n s i o n . Th is lag was not an o b j e c t i o n a b l e fea tu re in the type of s tud ie s being performed. I 4 6) P repa ra t i on of C -acronyc ine: Acronycine i s h i gh l y f l u o r e s c e n t , and attempts were made to use t h i s property to q u a n t i t a t e i t in experiments i n vo l v i n g the b ind ing of the a l k a l o i d to c e l l s and macromoI ecu Ies. However, cons ide rab le d i f f i c u l t i e s were exper ienced in e x t r a c t i n g the a l k a l o i d in a form s u i t a b l e f o r s pec t r opho to f I uo r ime t r i c a n a l y s i s . To f a c i l i t a t e t h i s type of study 37 t h e r e f o r e , r a d i o a c t i v e l y l a b e l l e d acronyc ine was prepared using the procedure of Su I I i van et. aj_. ( 36 ) with mi nor mod i f i c a t i ons . Noracronycine ( O-desmethyIacronycine ; see f i g u r e 2 f o r the s t r u c t u r e of acronyc ine ) was prepared by the method of Brown et a I. ( 24 ). The hydroch lo r ide of acronyc ine ( 500 mg ) was formed by d i s s o l v i n g the a l k a l o i d in hot 10 %, iv/ivi 'ajjiuepus HCL. and a l l ow ing the s o l u t i o n t o c o o l . The orange-red c r y s t a l l i n e s a l t which separated on c oo l i n g was c o l l e c t e d by f i l t r a t i o n . The acronycine 'HCI was a i r - d r i e d , and then heated at 135 t o 145° f o r 30 minutes ; CH^CI was l i b e r a t e d leav ing noracronycine as a b r i g h t yedxbow powder. The crude product was r e c r y s t a l l i z e d by d i s s o l v i n g i t in a minimum volume of hot e thano l / CHCI^ ( 5:1 v/v ) and s lowly removing the CHCI^ with a stream of n i t rogen ( o v e r a l l y i e l d 361 mg ; 69 % ). I 4 C-acronyc ine was prepared by remethyIat ing a po r t i on of the I 4 noracronyc ine wi th C-methyI iod ide accord ing to the procedure of Su I I i van .et _aj_. ( 36 ) with minor m o d i f i c a t i o n s . Noracronyc i ne ( 102 mg, 0.333 mmoles ) and NaH ( 20 mg, 50 % in mineral o i l , 0.416 mmole ) were added to dimethyIformamide ( 4 ml ) in a narrow-necked g l a s s r eac t i on I 4 tube. C-methyI iod ide ( 47.3 mg, 0.333 mmole, I mCi a t 3.0 mCi/mmole ) d i s s o l ved in an a d d i t i o n a l 2 ml of dimethyIformamide was added, and the r eac t i on tube sealed with a f lame. The sealed r eac t i on vessel was p laced at 55° f o r 22 hours f o r the remethyIat ion of the noracronyc ine. The r eac t i on tube was then coo led , opened, and the contents t r a n s f e r r e d to a small f l a s k . V o l a t i l e - r a d i o a c t i v i t y was removed under reduced pres sure , and the remaining mater ia l p a r t i t i o n e d between CHCI^ and water to separate the product from wate r - s o l ub l e m a t e r i a l . The CHCI^ layer was concentrated to a small volume and app l i ed as a s t reak to the 38 o r i g i n of two p repa ra t i ve s i l i c a gel t h i n layer chromatography p l a t e s ( absorbant th i ckne s s I mm ) ; the p l a t e s were developed with e thy l I 4 acetate:benzene ( 1:1 ). C-acronycine ran as a pale ye l low band at 0.4 ; the s i l i c a gel in t h i s area was scraped from the p l a t e s and e l u ted with methanol. The methanol was concent ra ted, y i e l d i n g the crude product ( 83.5 mg, 77% y i e l d ). Th is mate r i a l was r e c r y s t a I I i z e d to a constant s p e c i f i c a c t i v i t y of 22.2 x 10^ dpm/mg. The product co-chromatographed with au then t i c ac ronyc ine , and autoradioagraphy of t h i n layer chromatograms i nd i ca ted aoradiochemical p u r i t y of at l ea s t 99 %. 7) P r o t e i n de te rmina t i on : The concent ra t i on of serum p ro te i n s in e q u i l i b r i u m d i a l y s i s experiments was determined by the Fo I i n -C ioca I teau method, us ing standard procedures ( 72 ). The response of the assay was s tandard ized each t ime, using f r e sh l y - p repa red standards of bovine serum albumin. I 4 8) Determinat ion of the b ind ing of C-acronycine t o L5I78Y c e l l s : 5 C e l l s were grown to a concent ra t i on of c. 5 x 10 c e l l s / m l , c o l l e c t e d by c e n t r i f u g a t i o n , and resuspended at a c e l l dens i t y of 10 x 10^ c e l l s / m l in F i s c h e r ' s medium supplemented with horse serum to a concen t ra t i on of 0 o r 10 %. 1.0 ml a l i q u o t s of t h i s suspension were I 4 mixed with 1.0 ml a l i q u o t s of C-acronyc ine s o l u t i o n of va r i ous concent ra t i on s in s i m i l a r media. The suspensions were incubated in 2 ml c e n t r i f u g e tubes a t 37° , then cen t r i f u ged at 1500 g f o r 2 minutes. The supernatant medium was removed by p i p e t , the tubes b r i e f l y r e cen t r i f uged to remove medium wett ing the wail,'Is of the tube, and the l a s t t r a ce s of supernatant removed with a c o n s t r i c t e d pasteur p i p e t . The p e l l e t s were resuspended in medium, and together with a l i q u o t s of the supernatant 39 medium were radioassayed to determine the amount of C-acronyc ine present . The amount of r a d i o a c t i v i t y in the p e l l e t s was co r rec ted f o r the r a d i o a c t i v i t y in adhering supernatant medium, the volume of which was determined in con t ro l experiments using ^ H - i n u l i n as a measure of e x t r a c e l l u l a r space. This c o r r e c t i o n amounted t o only 0.5 to 2.0 % of the t o t a l r a d i o a c t i v i t y in the p e l l e t s . METHODS USED IN SECTIONS 2 AND 3 OF RESULTS I) Incubation of c e l l s w i th r a d i o a c t i v e p recur so r s : Incubations of c e l l s w i th r a d i o a c t i v e precursors were c a r r i e d out in f l a t -bo t tomed g la s s v i a l s of 12,ml c a p a c i t y . The cond i t i o n s of the incubat ions v a r i ed somewhat between var ious exper iments, as noted in the legends to i n d i v i d u a l f i g u r e s o r t a b l e s . In g ene r a l , the precur sor was added in 0.2 or 0.5 ml medium t o 4.0 ml c e l l suspens ion. The incubat ions were c a r r i e d out e i t h e r f o r 45 minutes in s i l i c o n e rubber stoppered v i a l s ro ta ted at 4 rpm in an incubator , or f o r 30 minutes in open v i a I s .wi th f requent a g i t a t i o n in a water bath. Unless otherwise noted, incubat ions were at 37° . At the end of the i n cuba t i on , the v i a l s were c h i l l e d t o 0 ° , and the c e l l s analyzed f o r the uptake of r a d i o a c t i v i t y i n to va r ious c e l l u l a r f r a c t i o n s as descr ibed below. In experiments i n v e s t i g a t i n g the i nco rpo ra t i on of u r i d i n e and thymidine at va r ious temperatures, and in k i n e t i c s tud ie s us ing these two p recu r so r s , an a d d i t i o n a l technique besides c h i l l i n g was used t o stop i nco rpo ra t i on at the end of the incubat ion pe r i od . C e l l suspensions were c h i l l e d to 0° by adding them t o p rev i ou s l y prepared c e n t r i f u g e tubes in ice which conta ined s u f f i c i e n t non - r ad i oac t i ve nuc leos ide in 0.5 ml medium t o g i ve a f i n a l concent ra t i on of I mM. The r e s u l t i n g reduct ion in 40 s p e c i f i c a c t i v i t y of the l a b e l l e d precursor s was h i gh ly e f f e c t i v e in s topping the i nco rpo ra t i on of r a d i o a c t i v i t y . 2) Determinat ion of the i nco rpo ra t i on of "^H-uridine in to RNA: C e l l s were incubated wi th "^H-uridine f o r 45 minutes as descr ibed above, and then c o l l e c t e d on g la s s f i b e r f i l t e r s as descr ibed p rev i ou s l y f o r the moni tor ing of DNA synthes i s in synchronized c u l t u r e s . The c e l l s on the f i l t e r were washed in a s i m i l a r manner with s a l i n e , TCA, s a l i n e , and water t o remove a c i d - s o l u b l e r a d i o a c t i v i t y . The r a d i o a c t i v i t y in the RNA was then s e l e c t i v e l y re leased in a c i d - s o l u b l e form by incubat ing the c e l l s on the f i l t e r f o r 16 hours with RNase ( 700 ug/ml in 0.05 M phosphate b u f f e r , pH 6.5, 37° ). Cold TCA was then added t o glhve a f i n a l concent ra t i on of 5 % w/v, and non-hydro I yzed ac i d-i-nso I ub le mate r i a l ( i n c l ud i ng DNA ) removed by c e n t r i f u g a t i o n . The a c i d - s o l u b l e r a d i o a c t i v i t y in the supernatant was determined by l i q u i d s c i n t i l l a t i o n count ing in to Iuene/ethanoI s c i n t i l l a t i o n f l u i d . It has been p rev i ou s l y shown t h a t t h i s procedure does not re lease in a c i d - s o l u b l e form r a d i o a c t i v i t y from DNA, and t h a t i t re leases over 90 % of the r a d i o a c t i v i t y on the f i l t e r s which i s not w i t h i n DNA.( 41 ). 3) Determinat ion of the i n co rpo ra t i on of r a d i o a c t i v e precursors i n to  t o t a l , a c i d - s o l u b l e , and ac i d - i n so I ub I e mate r i a l using c e n t r i f u g a t i o n  procedures: » . C e l l s were incubated with r a d i o a c t i v e precursor s as descr ibed in the legends to i n d i v i d u a l f i g u r e s or t a b l e s . A f t e r i n cuba t i on , the c e l l suspensions were q u a n t i t a t i v e l y t r a n s f e r r e d to c h i l l e d 15 ml c e n t r i f u g e tubes, and the c e l l s c o l l e c t e d by c e n t r i f u g a t i o n at 1500 g f o r 2.5 minutes. The supernatant medium was removed by a s p i r a t i o n , and the c e l l s washed two times by resuspension in balanced s a l t s o l u t i o n 4! ( BSS, r e f . 73 ) at 0 ° . Radioassay of the c e l l p e l l e t a f t e r process ing in t h i s manner gave the t o t a l r a d i o a c t i v i t y taken up by the c e l l s and re ta ined in n o n - d i f f u s i n g form. In cases in which i t was des i red to determine only the r a d i o a c t i v i t y incorporated in to a c i d - i n s o l u b l e m a t e r i a l , c e l l s were c o l l e c t e d and washed two times with BSS as descr ibed above and then resuspended in 5 ml of 0.5 M TCA at 0° f o r 30 minutes. A c i d -i n s o l ub l e mate r i a l was then p e l l e t e d by c e n t r i f u g a t i o n , washed tw ice with co ld 0.5 M TCA, and rad ioassayed. When i t was des i red t o determine the amount of r a d i o a c t i v i t y incorporated in to a c i d - s o l u b l e m a t e r i a l , r e p l i c a t e samples of c e l l s were processed as descr ibed to determine both the t o t a l amount of r a d i o a c t i v i t y ( a c i d - s o l u b l e p lus a c i d - i n s o l u b l e ) and the amount of a c i d - i n so I ub I e r a d i o a c t i v i t y . The amount of a c i d - s o l u b l e r a d i o a c t i v i t y was then c a l c u l a t e d by s u b t r a c t i n g the amount of a c i d -i n s o l ub l e r a d i o a c t i v i t y from the t o t a l r a d i o a c t i v i t y . 4) S o l u b i l i z a t i o n of c e l l s l a b e l l e d with r a d i o a c t i v e p recur so r s : C e l l s which had been washed as descr ibed in the preceding s ec t i on wi th BSS, or BSS p lus TCA were s o l u b i l i z e d f o r radioassay by one of two procedures: i ) P e l l e t s of c e l l s which had been l a b e l l e d with n u c l e i c a c i d precursor s were resuspended in 3.0 ml of 0.5 M TCA, and heated at 70° f o r I hour. A l i q u o t s ( 0.4 ml ) of the hydrolyzed mater i a l were counted in 10 ml of to Iuene/ethanoI s c i n t i l l a t i o n f l u i d . i i ) C e l l s were resuspended <bn 0.5 ml of 1.0 M K0H, and heated at 90° f o r I hour. Thisuprocedure r e s u l t e d in the complete s o l u b i l i z a t i o n of the c e l l u l a r m a t e r i a l . The e n t i r e d i ge s t was d i s s o l ved in 5 ml Aquasol o r PCS s c i n t i l l a t i o n f l u i d s , each con ta i n i ng 4 % g l a c i a l a c e t i c a c i d . The mixture was t r a n s f e r r e d to a s c i n t i l l a t i o n v i a l , and a second 5 ml 42 po r t i on of a c i d i f i e d s c i n t i l l a t i o n f l u i d was used as a wash to t r a n s f e r the remains of the sample to the v i a l . N e u t r a l i z a t i o n of the a l k a l i n e d i ge s t with a c e t i c a c i d was found t o be necessary both to a l low i t t o be d i s s o l ved r e a d i l y in the s c i n t i l l a t i o n f l u i d , and t o prevent a l k a l i -induced chemi Iuminescence from occu r r i n g in subsequent s c i n t i l l a t i o n count i ng. 5) RNA synthes i s in v i t r o : The e f f e c t of acronyc ine and o ther drugs on RNA synthes i s in v i t r o was examined inaan RNA s yn the s i z i n g system u t i l i z i n g E_. co I i RNA polymerase with c a l f thymus DNA as a template. P u r i f i e d E_. col i RNA polymerase ( prepared accord ing t o Burgess ( 74 ) and r e t a i n i n g the a f a c t o r ) was obta ined from M i l e s Labora to r ie s ( Kankakee,11 I. ) as a s o l u t i o n in 50 % g l y c e r o l . The enzyme was s tored in 60 % g l y c e r o l at -20° ; when s tored in t h i s manner the enzyme e x h i b i t e d no apparent loss in a c t i v i t y or change in enzymatic c h a r a c t e r i s t i c s over a per iod of 3 months. Enzymatic a c t i v i t y was assayed as descr ibed by Burgess ( 74 ), w i th minor m o d i f i c a t i o n s . Assay tubes conta ined 0.25 ml of s o l u t i o n of compos i t i on . : 40 mM Tr i s -C I ( pH 7.9 ), 10 mM MgCI 2 , 0.1 mM EDTA, 0.1 mM d i t h i o t h r e i t o l , 0.15 M KCI, 0.4 mM•K2HP0 , 0.5 mg/ml bovine serum albumin, 0.!l>5 mM UTP, GTP, andCTP, 0.15 mM ATP -8 - ' 4 C ( 5 mCi/mmole )•, enzyme ( 0p5 u n i t s , where I u n i t of enzyme ca t a l y ze s the i nco rpo ra t i on of I nmo.le of r a d i o a c t i v e ATP i n to ac i d - i nso I ub I e mater i a l in 10 minutes ), and c a l f thymus DNA in amounts i nd i ca ted in the r e s u l t s s e c t i o n . The r eac t i on was i n i t i a t e d by adding the enzyme in a volume of 0.05 ml. Tubes were incubated f o r 10 minutes a t 37° ; the r eac t i on was then terminated by heat ing the tubes a t 90° f o r 1.5 minutes. A f t e r c oo l i n g the tubes to 0 ° , 0.5 mg c a r r i e r RNA was added in 0.5 ml b u f f e r , and the ac id - i n so I ub Ie 43 mater i a l then p r e c i p i t a t e d by adding co ld TCA to a f i n a l concent ra t i on of 5 •%. The p r e c i p i t a t e was c o l l e c t e d on Mi I I I pore f i l t e r s * ( pore s i z e 0.65 nm ), and the f i l t e r s were washed three times wi th 5 ml of co ld 5 % TCA con ta i n i ng 0.01 M sodium pyrophosphate. The f i l t e r s were then duied and counted in to luene based s c i n t i l l a t i o n f l u i d . When assayed I 4 as descr ibed above, the i n co rpo ra t i on of r a d i o a c t i v i t y from ATP- C i n to a c i d - i n s o l u b l e product was a near ly l i n e a r f unc t i on of t ime over a 20 minute pe r i od . The i n co rpo ra t i on was dependant on the presence of a l l four nuc leos ide t r i pho spha te s , upon added enzyme, and upon template. 6) E x t r a c t i o n and a n a l y s i s of a c i d - s o l u b l e mate r i a l from L5I78Y c e l l s  incubated wi th ^H -u r i d i ne : Cu l tu re s con ta i n i ng 4 t o 5 m i l l i o n c e l l s which had been incubated with u r i d i ne -5 - ^H were cen t r i f u ged and the c e l l s washed tw ice by resuspension in BSS ( 0° ). A c i d - s o l u b l e mate r i a l was ex t r a c ted from the c e l l s by resuspending them in 0.2 ml of 5 % TCA f o r 30 minutes a t 0 ° . The samples were then c e n t r i f u g e d , the supernatant f l u i d removed, and the p e l l e t s washed tw ice with an a d d i t i o n a l po r t i on of TCA ( 0.1 ml each time The TCA e x t r a c t s were combined, and e x t r a c t ed with 5 two ml po r t i on s of e the r t o remove the TCA. S c i n t i l l a t i o n count ing of samples of the e ther i nd i ca ted t ha t no r a d i o a c t i v i t y was l o s t dur ing t h i s e x t r a c t i o n procedure I ne .'.•.TI..-:C"? ^ " T - T T '• ,• * »• • ; -,-.• ' .••ed. * M i l l i p o r e f i l t e r s quench the s c i n t i l l a t i o n process app rec i ab l y and I i • . i: e are r e l a t i v e l y expens ive. Ma l t and M i l l e r ( 75 ) have reported t ha t g la s s f i b e r f i l t e r s gave good r e s u l t s in t r app ing p r e c i p i t a t e d RNA, wh i le c o s t i l e s s , quenching l e s s , and having a f a s t e r f i l t e r i n g ra te than M i l l i p o r e f i l t e r s . However, in s p i t e of these reported p o t e n t i a l advantages, g la s s f i b e r f i l t e r s were found to be u n s a t i s f a c t o r y in the cu r ren t study f o r t r app ing the products of the RNA polymerase r eac t i on performed accord ing to the method of Burgess ( 74 ); they f a i l e d to t rap a l l the precipa-ted r a d i o a c t i v i t y , and gave e r r a t i c r e s u l t s in r e p l i c a t e assays. 44 The aqueous po r t i on s were then Iyophi I i z ed . In p r e l im i na r y experiments the d i s t r i b u t i o n of r a d i o a c t i v i t y among the var ious components of the a c i d - s o l u b l e mate r i a l e x t r a c ted from the c e l l s was i n ve s t i g a ted using paper chromatography and var ious so l ven t systems. However the r e s o l u t i o n of one-dimensional paper chromatography proved i n s u f f i c i e n t to separate the var ious r a d i o a c t i v e components encountered ; as an a l t e r n a t i v e , a two-dimensional chromatography system employing c e l l u l o s e t h i n - l a y e r chromatography p l a te s was developed in the cu r ren t study f o r t h i s a n a l y s i s . Based on the r e s u l t s of p r e l im ina r y exper iments, the procedure e ven tua l l y adopted was as f o l l ows : l y o p h i l i z e d samples were d i s s o l ved in 0.2 ml water, and a l i q u o t s ( 0.01 ml ) were mixed w i th non - r ad i oac t i ve chromatography reference compounds d i s s o l ved in water ( approximately 5 yg of each standard ). The samples were app l i ed as a spot 3 cm from each edge in one corner of a p l a s t i c backed c e l l u l o s e t h i n layer chromatography sheet ( 20 cm by 20 cm, c e l l u l o s e type MN 300 ) which was developed in the f i r s t dimension with a so l vent system c o n s i s t i n g of i s o b u t y r i c a c i d : |IM NH^OH : 0.2 M EDTA ( 75: 45 : 0.6 ). A f t e r they had d r i ed f o r twelve hours at room temperature, the •_• chromatograms were developed at r i g h t angles with a s o l ven t c o n s i s t i n g of ethanol : It.'M ammonium acetate ( pH 6.5 ) con ta i n i ng I g/1 EDTA ( 60 : 40 ). The reference compounds were located under u l t r a v i o l e t l i g h t and the c e l l u l o s e in these areas then scraped o f f f o r rad ioassay. The r e s t of the c e l l u l o s e was a l s o removed in s ec t i on s f o r rad ioassay. The q u a n t i t a t i v e removal of s e l e c ted areas of the c e l l u l o s e layer was f a c i l i t a t e d by f i r s t spray ing the p l a s t i c backs of the chromatograms wi th a commercial a n t i - s t a t i c spray ( P r i c e -D r i s c o I I Corp. , Farmingdale, N.Y. ). The r a d i o a c t i v i t y in the c e l l u l o s e samples was determined by 45 e l u t i n g them with 0.5 ml of 0.1 M HCI ( 3 7° , 24 hrs ), and then count ing the c e l l u l o s e and e l ua te together in 5 ml Aquaso.l or PCS. The recovery of r a d i o a c t i v i t y from the e n t i r e chromatogram was 95 t o 100 %, of which 80 to 85 % was a s soc ia ted with the reference compounds used. The r e s o l u t i o n of a t y p i c a l chromatogram i s shown in f i g u r e 5 . 7) Assay of u r i d i n e phosphory lat ion in v i t r o : A procedure f o r assay ing u r i d i n e k inase from L5I78Y c e l l s was developed, using as a bas i s the reported methods f o r the assay of t h i s enzyme from other c e l l types ( 76-78 ). Based on the r e s u l t s of p r e l im i na r y exper iments, the f o l l o w i n g procedures were e ven tua l l y adopted: F ive m i l l i o n L5I78Y c e l l s were c o l l e c t e d by e e n t r i f u g a t i o n from a l o g a r i t h m i c a l l y growing c u l t u r e , and washed once in BSS ( 0° ). The c e l l p e l l e t was suspended in 1.0 o r 2.0 ml of bu f f e r of compos i t ion: 20 mM Tr i s -C I ( pH 7.5 or 9.0 ), 10 mM MgCI 2 > I mM d i t h i o t h r e i t o I. The ceI Is were a I lowed t o swelI f o r 10 minutes a t 0° and then d i s rupted with 50 s t rokes of a g la s s p e s t l e in a g la s s t i s s u e g r i nde r ( pe s t l e c learance 0.004 to 0.006 inches ; Kontes Glass Co., Vine I and,N. J . ). The homogenate was cen t r i f u ged 5 minutes at 1506 g t o remove p a r t i c u l a t e mate r i a l ( as judged by phase con t r a s t microscopy ), and 0.05 ml of supernatant was added t o 0.20 ml of an assay mixture of composit ion 20 mM Tr i s -C I ( pH 7.5 or 9.0 ), 10 mM MgCI 2 , I mM d i t h i o t h r e i t o I, 2.5 mM ATP, and u r i d i n e - 5 - 3 H in amounts which are i nd i ca ted in the r e s u l t s s e c t i o n . The tubes were incubated 15 minutes at 37°"and the r eac t i on then terminated by adding 0.1 ml of 17.5 % TCA. Ac id - i n so Iub Ie mate r i a l was removed by c e n t r i f u g a t i o n and the supernatants were ex t r a c ted wi th e the r ( 5 x 2 ml ) to remove the TCA. A l i q u o t s of the aqueous samples 46 ^ ^ c y t i d i ne CMP and/ " \ / " "~ )ur id i ne CDP-cho l ine V J V — +-C D P O C T P O UDP 0 UMP r-N C3 /~ ) l lDP -NAG I O o c <D > UDPG O 01 X ». orig in solvent 2 F i gu re 5 Two dimensional chromatography of a c i d - s o l u b l e ma te r i a l i s o l a t e d from L5I78Y c e l l s : l o c a t i o n of r e fe rence compounds So lvent I : i s o b u t y r i c a c i d : I M NH OH : 0.2 M EDTA ( 75 : 45 : 0.6 ) So lvent 2/: ethanol : I M ammonium ace ta te con ta i n i n g I g/l EDTA ( 60 : 40 ) were mixed wi th reference compounds and chromatographed on c e l l u l o s e t h i n layer chromatography sheets with the so l ven t system n-butanol : water ( 86 : 14 ). U r i d i ne migrated with an R^ . of c_. 0.3, wh i le phosphoryIated d e r i v a t i v e s of u r i d i n e remained at the o r i g i n . The c e l l u l o s e was removed from the p l a t e s in s ec t i on s and radioassayed by e l u t i n g i t w i th 0.5 ml of 0.I M HCl ( 24 hours at 37° ) and count ing the e l ua te and c e l l u l o s e together in 5 ml of Aquasol o r PCS s c i n t i l l a t i o n f l u i d s . The convers ion of u r i d i n e to t o t a l phosphoryIated d e r i v a t i v e s was taken as a measure of the u r i d i n e k inase r e a c t i o n . F igure 6 shows the c h a r a c t e r i s t i c s of the u r i d i n e kinase r eac t i on assayed in t h i s manner. The r eac t i on e x h i b i t e d a near ly abso lute r e q u i r e -ment f o r added magnesium and ATP ( t r ace s of a c t i v i t y in the absence of added ATP or M g + + may be due to the presence of smaI I amounts of these ma te r i a l s in the c e l l homogenate ). The f i n a l concent ra t i ons of ATP and M g + + used in the experiments reported in the r e s u l t s s e c t i on of t h i s t h e s i s ( 2 mM and 10 mM r e s p e c t i v e l y ) represent the optimum values der ived from these exper iments. The r a te . o f r eac t i on was maximal a t or near pH 9.0 . Most assays were acco rd ing l y performed at t h i s pH, although in view of the f a c t t ha t t h i s pH might be cons idered unphysio-l o g i c a l some experiments were c a r r i e d out a t pH 7.5 as w e l l . F igure 7 shows the r e l a t i o n s h i p between the phosphory la t ion of u r i d i n e and time at two d i l u t i o n s of the c e l l homogenate. The rate of format ion of phosphoryIated d e r i v a t i v e s of u r i d i n e was near ly constant over a 20 minuter p e r i o d , a l l ow ing the extent of the r eac t i on at 15 minutes t o be used as a measure of the ra te of the r e a c t i o n . CJ_+ 5 10 IS 20 6 0.125 0^5 2 5 10 20 6 7 8 9 10 Mg concent ra t i on (mM) ATP concen t ra t i on (mM) pH F i gu re 6 E f f e c t of magnesium c o n c e n t r a t i o n , ATP c o n c e n t r a t i o n , and pH on u r i d i n e phosphory lat ion by e x t r a c t s of L5I78Y c e l l s . Assays were performed as descr ibed in the t e x t . F i n a l u r i d i n e concen t r a t i on was 0.4 yM ; a l l o ther c ond i t i on s were as f o l l ows : p a r t M q + + cone. ATP cone. pH bu f f e r  A va ry ing 2 mM 9.0 20 mM T r i s -C I , I mM d i t h i o t h r e i t o I B 10 mM vary ing 9.0 20 mM T r i s - C I , I mM d i t h i o t h r e i t o I C 10 mM 2 mM vary ing e i t h e r 20 mM Na phosphate, 20 mM T r i s - C I , o r 20 mM g l y c i n e , a l I p lu s I mM d i t h i o t h r e i t o I. 49 CD <D C > T3 +-— (D 1- > 3 — I l_ X © -o H- X> O 03 +-C (0 o — — >. I- o > ex. o o O -C CL V*. o 0 5 10 15 20 T i m e ( m i n ) F igure 7 Time course of u r i d i n e phosphory la t ion by e x t r a c t s of L5 I78Y c e l l s a t two d i f f e r e n t e x t r a c t d i l u t i o n s . U r i d i n e phosphory la t ion was assayed as desc r ibed in the t e x t , us ing two d i f f e r e n t d i l u t i o n s of c e l l e x t r a c t . U r i d i n e c oncen t r a t i on was 0.4 uM. 1 6 • B 5 x 10 c e l l s homogenized in 1.0 ml b u f f e r , pH 9 .0 • • 5 x 10^ c e l l s homogenized in 2 .0 ml b u f f e r , pH 9 .0 50 RESULTS AND DISCUSSION  Organ i za t ion of the r e s u l t s and d i s cu s s i on s e c t i o n : The r e s u l t s of t h i s t h e s i s w i l l be presented in th ree s e c t i o n s : 1) Experiments on a) the c y t o l o g i c a l and b i o l o g i c a l e f f e c t s of ac ronyc ine , i n c l ud i ng i t s e f f e c t s on : c e l l popu la t ion growth, the cyto logy of tumor c e l l s , the progress ion of c e l l s through the c e l l c y c l e , and the r e s p i r a t i o n of tumor c e l l s , and b) the b ind ing of r a d i o a c t i v e I y l a b e l l e d acronyc ine t o c e l l s and to serum components. 2) Experiments on the i n h i b i t i o n by acronyc ine of the i nco rpo ra t i on of nuc leos ides and other compounds i n to c e l l u l a r f r a c t i o n s , and the po s s i b l e mechanisms of t h i s i n h i b i t i o n . 3) Experiments i n v e s t i g a t i n g the mechanism by which u r i d i n e i s accumulated by c e l l s as n on -d i f f u s i n g me tabo l i t e s , and the mechanism by which acronyc ine i n h i b i t s t h i s process. In a d d i t i o n , t h i s s e c t i on has a separate i n t r oduc t i on p e r t a i n i n g to cu r ren t concepts of the mechanism of nuc leos ide uptake, and to the r a t i o n a l e of experiments i n v e s t i g a t i n g t h i s uptake. At the end of each s e c t i o n , there i s a b r i e f d i s cu s s i on p e r t a i n i n g to the experimental r e s u l t s of t h a t s e c t i o n , and at the end of the t h e s i s there i s an o v e r a l l d i s cu s s i on on the r e s u l t s of the i n v e s t i g a t i o n s as a whole. RESULTS 2 SECTION I E f f e c t of acronyc ine on popu lat ion growth in L5I78Y and IRC suspension  cu I t u re s : The e f f e c t of acronyc ine at va r ious concent ra t i ons on the growth of L5I78Y cu I tu res over a 24 hour per iod i s shown in f i g u r e 8. While 51 a concent ra t i on of I yg/ml had no e f f e c t on c e l l popu la t ion growth, concent ra t i ons of 3 ug/ml or h igher slowed or a r re s ted the growth of the c u l t u r e s . A f t e r some hours of exposure t o acronyc ine at h igher drug concen t r a t i on s , c e l l s began to l y se , thus reducing the number of c e l l s in the c u l t u r e s to below the o r i g i n a l inoculum.? The e f f e c t of acronyc ine on the growth of IRC ra t leukemia c e l l s was a l s o examined ( f i g u r e 8 ). TheggDowth ..of th i s : tumor was a l s o i n h i b i t e d by the drug, but not to such a great extent as t ha t of the L5I78Y c e l l l i n e . Whereas a concent ra t i on of 6 yg/ml prevented more than a s l i g h t increase in popu la t ion in t r ea ted L5I78Y c u l t u r e s and caused the l y s i s of most of the ceI Is over a per iod of 24 hours, the same concent ra t i on of acronyc ine d id not i n h i b i t popu la t ion growth in IRC c u l t u r e s u n t i l 15 hours had e lapsed. While h igher concent ra t i ons of the drug ( 12 yg/ml ) i n h i b i t e d popu la t ion growth w i t h i n 4 to 8 hours, there was s t i l l no mass l y s i s of c e l l s as was observed in L5I78Y c u l t u r e s t r ea ted with the drug. Induction of b i nuc lea ted c e l l s by ac ronyc ine : When L5I78Y c e l l s which had been incubated with acronyc ine f o r 3 to 4 hours at a concent ra t i on of 6 yg/ml were s ta ined and examined under the l i g h t microscope, i t was found tha t many c e l l s conta ined two wel l - fo rmed nuc le i of about normal s i z e ( f i g u r e 9 ). The number of these b inuc lea ted c e l l s in the c u l t u r e s t e a d i l y increased on f u r t h e r incubat ion with the drug, u n t i l a f t e r 8 hours of incubat ion they comprised c_. 30 % of the c e l l p opu l a t i on . S i m i l a r b i nuc lea ted c e l l s a l s o accumulated in IRC c u l t u r e s when these were incubated with the a l k a l o i d a t the same concen t r a t i o n , although in t h i s case they appeared much l a t e r ( c_. 15 hours a f t e r the a d d i t i o n of the drug ). 52 F i gu re 8 E f f e c t of ac ronyc ine on c e l l popu la t i on growth in L5I78Y and IRC suspension c u l t u r e s . L5I78Y and IRC c e l l s were incubated a t c e l l popu l a t i on dens i t y determined a t i n t e r v a l s , ac ronyc ine concen t r a t i on s are in yg/ml. 37° The , and the i nd i cated 53 F igure 9 Appearance of normal and b i nuc l ea ted L5I78Y c e l l s C e l l smears were prepared and s t a i ned w i th W r i g h t ' s s t a i n as; desc r ibed in the methods s e c t i o n a) b i nuc l ea ted c e l l s in an L5I78Y c u l t u r e incubated wi th acronyc ine ( 6 yg/ml ) f o r 8 hours ( 1000 x ) b) norma I L5I78Y ceI Is ( I 000 x ) 54 In both c e l l l i ne s the appearance of b inuc lea ted c e l l s co i nc ided approximately in t ime with the ce s sa t i on of popu la t ion growth in the cu I t u re s . In view of the po s s i b l e r e l a t i o n between the formation of these b inuc lea ted c e l l s and the ant i tumor p r ope r t i e s of ac ronyc ine , i t was of i n t e r e s t t o i n v e s t i g a t e the nature of the mechanism by which they were formed. There are b a s i c a l l y three mechanisms which might po s s i b l y account f o r the format ion of b inuc lea ted c e l l s : i ) f ragmentat ion of the normal interphase nucleus i n to two nuc le i ( although t h i s would appear u n l i k e l y in view of the normal s i z e and morphology of both of the nuc le i of the b i nuc l ea ted c e l l s ), i i ) f u s i on of two c e l l s t o form a s i n g l e c e l l w i th two n u c l e i , and i i i ) i n t e r f e rence wi th the process of c y t o k i n e s i s ( the stage in c e l l d i v i s i o n i n vo l v i n g the sepa ra t i on of daughter ceI Is ). To ob ta in evidence bear ing on these p o s s i b i l i t i e s , andexperimeht was performed using c o l c h i c i n e to make a d i s t i n c t i o n between events occur ing before and a f t e r the metaphase stage of c e l l d i v i s i o n . ; C o l c h i c i n e i s a p l an t product which i s wel l known t o i n h i b i t the f unc t i on of the m i t o t i c s p i nd l e and thus prevent the segregat ion of chromosomes and the format ion of daughter nuc le i ( 79 ). C e l l s poisoned with c o l c h i c i n e are unaf fected in t h e i r progress through the c e l l c y c l e ( 80 ), but do not proceed past the metaphase stage of c e l l d i v i s i o n and do not attempt c y t o k i n e s i s . It was hypothesized t h a t i f b i nuc lea ted c e l l s were being formed e i t h e r by mechanism i ) or i i ) above ( nuc lear f ragmentat ion or c e l l f u s i on ) t ha t c o l c h i c i n e should have no e f f e c t on the a b i l i t y of acronyc ine t o induce the format ion of b i nuc lea ted c e l l s . If on the other hand the b inuc lea ted c e l l s were being formed by 55 the f a i l u r e of c y t o k i n e s i s ( i e . nuc lear d i v i s i o n without c e l l d i v i s i o n ) then c o l c h i c i n e should s p e c i f i c a l l y prevent the formation of b i nuc lea ted c e l l s by prevent ing the segregat ion of c e l l u l a r DNA i n to two n u c l e i . F igure 10 shows the e f f e c t of ac ronyc ine , c o l c h i c i n e , and acronyc ine plus c o l c h i c i n e on the popu la t ion growth, m i t o t i c index, and percentage of b i nuc lea ted c e l l s in an L5I78Y c u l t u r e . R e l a t i v e to the c o n t r o l , the popu lat ion in the acronyc ine t r ea ted c u l t u r e increased on ly s l i g h t l y in the f i r s t four hours and t h e r e a f t e r s lowly d e c l i n e d . The m i t o t i c index of the acronyc ine t r ea ted cu I t u r e , i n i t i a l l y i d e n t i c a l wiith t ha t of the c o n t r o l , dec l i ned s lowly throughout the e i gh t hours of the experiment. In c u l t u r e s t r ea ted e i t h e r with c o l c h i c i n e alone or c o l c h i c i n e p lus ac ronyc ine , the c e l l popu la t ion remained e s s e n t i a l l y unchanged throughout the exper iment, and c e l l s accumulated at m i t o s i s . The s l i g h t l y s lower rate of accumulat ion of m i t o t i c f i g u r e s in the c u l t u r e s t r ea ted with acronyc ine p lus c o l c h i c i n e i s an i n d i c a t i o n t ha t in the presence of acronyc ine the c e l l s may have taken rather, longer t o reach m i t o s i s ; t h i s could po s s i b l y e x p l a i n the s l i g h t d e c l i n e in m i t o t i c index in the c u l t u r e s t r e a t ed with the a n t i n e o p l a s t i c agent a lone. A f t e r four hours, b inuc lea ted c e l l s appeared in the acronyc ine t r ea ted c u l t u r e s , r i s i n g in number t o c, 30 % of the popu la t ion at ^eight hours. The presence of c o l c h i c i n e in a d d i t i o n to acronyc ine completely blocked the induct ion of b i nuc lea ted c e l l s by the l a t t e r . The - fac t t ha t c o l c h i c i n e i s able to prevent the format ion of b i nuc lea ted c e l l s in an acronyc ine t r ea ted c u l t u r e suggests t ha t the two nuc le i of acronyc ine- induced b inuc lea ted c e l l s a r i s e through the normal process of nuc lear d i v i s i o n a t m i t o s i s , and t ha t consequently c e l l s must become b inuc lea ted through a f a i l u r e of the c e l l s t o subsequently undergo normal c y t o k i n e s i s . 2 F igure 10 E f f e c t of a c ronyc i ne , c o l c h i c i n e , and ac ronyc ine pi c o l c h i c i n e on c e l l popu l a t i on growth and percent m i t o t i c and b i nuc l ea ted c e l l s in an asynchronous L5I78Y c u l t u r e . The compounds were added at hour 0, and the i nd i c a t ed parameters determined at two hour i n t e r v a l s . O O c o n t r o l e • ac ronyc ine ( 6 ug/ml ) A A c o l c h i c i n e ( 10 yg/ml ) A A ac ronyc ine ( 6 yg/ml ) p lus c o l c h i c i n e ( 10 yg/ml ) 57 Stud ies of the e f f e c t of acronyc ine on p a r t i a l l y synchronized popu lat ions  of tumor ceI Is : It was of i n t e r e s t t h a t a t c e r t a i n lower acronyc ine concen t r a t i on s , there could be a delay of a number of hours before b inuc lea ted c e l l s appeared and popu la t ion growth in L5I78Y and IRC c u l t u r e s was a f f e c t e d . Thus IRC c u l t u r e s incubated with acronyc ine at a concent ra t i on of 6 yg/ml cont inued to grow f o r up to 15 hours ( f i g u r e 8 ), and b inuc lea ted c e l l s d id not appear u n t i l approximately t h i s t ime. S i m i l a r l y , L5I78Y c u l t u r e s incubated with acronycine at a concent ra t i on of 3 yg/ml cont inued to grow at near ly con t ro l rates f o r severa l hours before c e l l popu la t ion growth was a f f e c t e d ( f i g u r e 8 ). These delayed b i o l o g i c a l e f f e c t s of acronyc ine occurred mainly a t lower acronyc ine concent ra t i ons ; at h igher acronyc ine concent ra t i ons ( i e . L5I78Y, 6 yg/ml ; IRC, 12 yg/ml ) c e l l popu la t ion growth was r a p i d l y i n h i b i t e d and b inuc lea ted c e l l s appeared w i t h i n 2 to 4 hours of the a d d i t i o n of the drug ( e .g . f i g u r e 10 ). The e f f e c t s of these low concent ra t i ons of acronyc ine are of p a r t i c u l a r i n t e r e s t in r e l a t i o n to the e f f e c t s of the drug in v i v o , s i nce acronyc ine i s e x t e n s i v e l y metabol ized in the body ( 36 ) and the l e ve l s of acronyc ine present in v i vo may be r e l a t i v e l y low in comparison t o those which can e a s i l y be obta ined in experiments in v i t r o . The e f f e c t of low concent ra t i ons of acronyc ine on the passage of IRC and L5I78Y c e l l s through t h e i r r e p l i c a t i o n c y c l e was t he r e f o r e s tud ied using p a r t i a l l y synchronized popu la t ions of tumor c e l l s . F igure II shows the e f f e c t of adding acronyc ine ( 6 yg/ml ).to a p a r t i a l l y synchronized IRC popu la t ion when the v i a b l e c e l l s in the c u l t u r e were mainly in the Gj and e a r l y S phases. A bulk c u l t u r e of IRC c e l l s was p a r t i a l l y synchronized by a 3 hour treatment with hydroxyurea ( see 58 the methods s e c t i on f o r d e t a i l s of the method of the induct ion of synchrony ). At the end of the th ree hour t reatment, the hydroxyurea was removed by c e n t r i f u g i n g the c e l l suspensions and resuspending the c e l l s in f re sh d rug - f ree med i um.": The resuspended c e l l s - w e r e then-;e ither d i s t r i b u t e d as such in r e p l i c a t e a l i q u o t s i n to v i a l s , or f i r s t d i l u t e d with acronyc ine s o l u t i o n ( f i n a l concent ra t i on 6 yg/ml ) or an equ i va l en t volume of medium before d i s t r i b u t i o n i n to v i a l s . At i n t e r v a l s t h e r e a f t e r , the I 4 i n co rpo ra t i on of C-thymidine i n to a c i d - i n s o l u b l e m a t e r i a l , the c e l l c oncen t r a t i on s , and the percentages of m i t o t i c and b inuc lea ted c e l l s were determined on a l i q u o t s of the und i l u ted c u l t u r e , the acronyc ine t r ea ted c u l t u r e , and the d i l u t e d con t ro l c u l t u r e . On the upper panel of f i g u r e II are i nd i ca ted : i ) the i n t e r v a l dur ing which hydroxyurea ( " HU " ) was present , and i i ) the rate of DNA s yn the s i s , and the changes in c e l l popu la t ion dens i ty and m i t o t i c index in the und i l u ted con t ro l c u l t u r e dur ing the per iod f o l l o w i n g the removal of the hydroxyurea. Fo l lowing the removal of the drug, a large p ropor t ion of the c e l l s in the c u l t u r e l y sed, as i nd i ca ted by the drop in c e l l popu la t ion dens i ty from hours 4 t o 8. These I etha hj-yc'damaged c e l l s were those which had been in the S phase at the time of the a d d i t i o n of the i n h i b i t o r ( 68 ). The remaining c e l l s ( which had accumulated at the G|/S boundary dur ing the per iod in which DNA synthes i s was i n h i b i t e d by hydroxyurea ) entered the S phase on the removal of the drug. The thymidine i n co rpo ra t i on curve in the upper panel df f i g u r e II i n d i c a te s the amount of DNA syn thes i s by these c e l l s , and thus i s an approximate measure of the p ropor t i on of the c e l l popu la t ion in the S phase. At approximately hour 12 of the experiment ( measuring from the time at which the hydroxyurea had been added ) the v i a b l e c e l l s in the c u l t u r e d i v i d e d . F igure II IRC c e l l s were grown in bu lk , and synchron ized by a 3 hour t reatment w i th hydroxyurea as i n d i c a t e d in the top panel ( see methods s e c t i o n f o r a d e s c r i p t i o n of the method of i nduc t i on of synchrony ). P o r t i o n s of the synchron ized c u l t u r e were d i l u t e d w i th ac ronyc ine s o l u t i o n ( f i n a l c oncen t r a t i on 6 pg/ml ) o r an e q u i v a l e n t volume of medium, and a l i q u o t s were d i s t r i b u t e d i n to i n d i v i d u a l v i a l s . The v i a l s were stoppered and r o t a t ed a t 3 7 ° . 14 At i n t e r v a l s , C-thymid ine ( f i n a l c oncen t r a t i on I.6 uM ) was added and the c e l l s incubated w i th the p recur so r f o r 45 minutes. At the i n d i c a t e d t imes v i a l s were removed from the i ncubato r , and po r t i on s of the c e l l suspension analyzed f o r the i n co rpo r a t i on of r a d i o a c t i v i t y i n t o a c i d - i n s o l u b l e m a t e r i a l , f o r c e l l popu la t i on d e n s i t y , and f o r percent m i t o t i c and b i nuc l ea ted c e l l s . c on t r o l acronyc i ne ( 6 ug/ml ) c e i I popu la t i on dens i t y A-A --A - A 14 C-thymi d i ne i n co rpo ra t i on -O percent m i t o t i c or b i nuc l ea ted ceI Is B -F igure II E f f e c t of adding acronyc ine t o a p a r t i a l l y synchron ized IRC c u l t u r e when v i a b l e c e l l s were mainly in the Gj and e a r l y S phases. For exper imenta l d e t a i l s , see f a c i n g page 60 This i s i nd i ca ted by the increase in c e l l popu la t ion and the r i s e in m i t o t i c index at t h i s t ime. During t h i s p e r i o d , DNA synthes i s was at a minimum. It may be noted t ha t DNA synthes i s does not drop t o ze ro , as would be expected of a p e r f e c t l y synchronized c u l t u r e in which a l l t h e . c e l l s completed the S phase together . Th is i s because the IRC c u l t u r e in t h i s experiment i s not p e r f e c t l y synchronized and the popu la t ion at hours I 2 to 14 conta i ns not on Iy eel Is in G^, M, and G|, but a I so ceI Is which are s t i l l in the S phase of the f i r s t c y c l e , as wel l as those which have a l ready entered the S phase of the second c y c l e . At hour 16, the amount of DNA synthes i s increases again as the ma jo r i t y of c e l l s enter the second S phase. The lower panels of f i g u r e II demonstrate thymidine i n c o r p o r a t i o n , c e l l popu la t ion den s i t y , and the percentage of m i t o t i c and b inuc lea ted c e l l s in both the d i l u t e d con t ro l c u l t u r e and the acronyc ine t r e a t ed c u l t u r e . Acronycine was added one hour a f t e r the removal of the hydroxyurea, at a t ime when the ma jo r i t y of the v i a b l e c e l l s in the c u l t u r e were in the G| or e a r l y S phases of the c e l l c y c l e . The second panel from the top of f i g u r e II shows t ha t the maximum rate at which thymidine was incorporated i n to the ac i d - i n so I ub I e mate r i a l of c e l l s in the acronyc ine t r e a t ed c u l t u r e was somewhat less in the f i r s t S phase than in con t ro l c e l l s . The areas under the thymidine i nco rpo ra t i on curves however were approximately equa l , i n d i c a t i n g t h a t approximately the same amount of r a d i o a c t i v i t y was incorporated in the S phase of the t r e a t ed c u l t u r e . a s in the con t ro l c u l t u r e . Acronycine delayed the pa t te rn of DNA synthes i s in the S phase by approximately 1.5 hours, and the t ime of the subsequent m i t o s i s of the acronyc ine t r ea ted cu l tures ( as determined by the increase in popu la t ion and m i t o t i c index Ouwasocorrespdhdmg I y l a t e r than t ha t of 61 the c o n t r o l . The m i t o t i c per iod otherwise appeared normal, w i th the increase in c e l l popu la t ion being near ly the same in t r e a t ed as in con t ro l c u l t u r e s , and no b inuc lea ted c e l l s appearing a t t h i s t ime. A con s i de ra t i on however of the po in t s a t .hour 28 ( a f t e r the second m i t o t i c per iod of the synchronized c u l t u r e ) shows t h a t a t t h i s t ime wh i l e the con t ro l c u l t u r e had undergone a second increase in c e l l p opu l a t i on , the t r e a t e d c u l t u r e had undergone l i t t l e f u r t h e r i nc rease , and s ub s t an t i a l numbers of b i nuc lea ted c e l l s had appeared. Th is suggests t ha t the drug had i n t e r f e r e d with normal c e l l d i v i s i o n a t the end of the second c y c l e . F igure 12 shows the e f f e c t of adding the same concent ra t i on of acronyc ine ( 6 yg/ml ) to a synchronized IRC c u l t u r e a t a time when most of the c e l l s were in the S phase. The experimental d e t a i l s were the same as those in f i g u r e I I, except t ha t the hydroxyurea-f ree c e l l suspension was not d i l u t e d wi th acronyc ine s o l u t i o n or medium u n t i l hour 8 of the exper iment. Acronycine had no e f f e c t on the rate a t which the c e l l s completed DNA synthes i s in the f i r s t c y c l e , nor d id i t a f f e c t the progress of the c e l l s through the f i r s t d i v i s i o n . The m i t o t i c index curve and the time and extent of the popu la t ion increase were e s s e n t i a l l y i d e n t i c a l w i th those of the c o n t r o l , and no b inuc lea ted c e l l s appeared. The i n h i b i t o r y e f f e c t s of t h e . a l k a l o i d began to appear e a r l y in the second c y c l e as shown by a delay of £. 1.5 hours in the ent ry of c e l l s i n to the S phase. S ince the t r ea ted c e l l s underwent m i t o s i s a t the same time as the c o n t r o l s , t h i s i s an i n d i c a t i o n t ha t the G| phase was extended. However the t r e a t ed c u l t u r e then proceeded through the S phase at about the normal ra te with no s ign of any i n t e r f e rence in DNA s yn the s i s . When the c e l l s reached the second m i t o s i s the growth a r r e s t i n g p r ope r t i e s of the a l k a l o i d were c l e a r l y demonstrated by an i n h i b i t i o n of c. 60 % in the I N C U B A T I O N T I M E : HOURS F igure 12 E f f e c t of adding acronyc ine to a p a r t i a l l y synchron ized IRC c u l t u r e when c e l l s were mainly in the S phase. I Procedures were as noted in the legend t o f i g u r e I I, except t h a t ac ronyc ine was added a t hour 8. eel I popuI a t ion dens i ty I 4 C-thymi d i ne i n co rpo ra t i on percent m i t o t i c o r b i nucIeated ceI Is contro I acronyc i ne ( 6 yg/ml ) A-ir- -A O B-63 popu la t ion inc rease , and the appearance of b inuc lea ted c e l l s in the cu I t u re . In an experiment s i m i l a r to those shown in f i g u r e s II and 12, acronyc ine ( 6 yg/ml ) was added to a synchronized IRC c u l t u r e a t a time when the c e l l s were in the G,-,-M-G| region of the c e l l c y c l e ( i e . 9.5 hours a f t e r the removal of the hydroxyurea ). The t r ea ted c e l l s completed the ongoing m i t o s i s in an e n t i r e l y normal f a s h i on , i nc reas ing in number to the same extent as in the con t ro l c u l t u r e . No b inuc lea ted c e l l s appeared. However, the entry of these c e l l s i n to the S phase of the f o l l o w i n g c e l l c y c l e was delayed by one or two hours, although DNA syn thes i s i t s e l f d id not appear to be i n h i b i t e d . These r e s u l t s with synchronized IRC c u l t u r e s show tha t wh i l e acronyc ine at a concent ra t i on of 6 yg/ml might slow popu la t ion growth somewhat by lengthening the G. phase, the a r r e s t of growth i s due mainly t o an i n t e r f e r ence at o r near c e l l d i v i s i o n . The formation of b i nuc lea ted c e l l s suggests an i n h i b i t i o n of c y t o k i n e s i s . Th is e f f e c t was not immediate, and might not appear f o r up to one and one-ha l f c e l l c y c l e s a f t e r the a d d i t i o n of the a l k a l o i d . The length of t h i s time lapse in synchronized c u l t u r e s i s the same as t ha t of the time lapse seen in asynchronous IRC c u l t u r e s between the a d d i t i o n of acronyc ine at a s i m i l a r concent ra t i on and the appearance of b inuc lea ted c e l l s and the ce s sa t i on of popu la t ion growth. S i m i l a r but less ex tens i ve s tud ie s were made in L5I78Y c u l t u r e s which had been p a r t i a l l y synchronized by a treatment with v i n b l a s t i n e ( as descr ibed in the methods s e c t i on ). F igure 13 shows the e f f e c t of adding acronyc ine ( 3 yg/ml ) t o a p a r t i a l l y synchronized c u l t u r e in which the eel Is were in the e a r l y S phase ( note : the e f f e c t of 3 yg/ml of 64 F i gu re 13 E f f e c t of adding ac ronyc ine t o a p a r t i a l l y s ynchron i zed L5I78Y c u l t u r e when c e l l s were main ly in the e a r l y S phase. L5I78Y c e l l s were grown in bu l k , and synchron ized by a 6 hour t reatment w i t h V L B ending a t hour 0 ( see methods s e c t i o n f o r a d e s c r i p t i o n of the method of i nduc t i on of synchrony ). P o r t i o n s of the synchron i zed c u l t u r e were d i l u t e d w i th ac ronyc ine s o l u t i o n ( f i n a l c o n c e n t r a t i o n 3 ug/ml ) o r an e q u i v a l e n t volume of medium. A l i q u o t s of the d i l u t e d c u l t u r e were incubated a t 3 7 ° , 14 and the i n c o r p o r a t i o n of C - thymid ine ( f i n a l c o n c e n t r a t i o n 1.6 uM ) i n t o a c i d i n s o l u b l e m a t e r i a l and c e l l p opu l a t i on den s i t y determined a t the i n d i c a t e d t i m e s . eel 1 popu1 a t ion d e n s i t y 1 4 C-thymi d i ne i n co rpo ra t i on c o n t r o l ac ronyc ine ( 3 ug/ml ) A A A A 65 acronyc ine on popu la t ion growth in L5I78Y c u l t u r e s i s s i m i l a r to t ha t of 6 yg/ml in IRC c u l t u r e s ). The a l k a l o i d d id not delay the progress of the c e l l s through the f i r s t c y c l e , as judged by the synthes i s of DNA and the subsequent increase in popu l a t i on . However, the i n i t i a t i o n of DNA syn thes i s in the second c y c l e was de layed, and the f o l l o w i n g increase in popu la t ion was i n h i b i t e d . Th is a c t i on of acronyc ine on L5I78Y c u l t u r e s resembles t ha t found in IRC c u l t u r e s at a h igher drug c oncen t r a t i on . Cytology of acronyc ine t r ea ted L5I78Y c e l l s : Examination of s ta ined smears of L5I78Y c e l l s t r ea ted with c y t o t o x i c concent ra t i ons of acronyc ine not on ly revealed large numbers of b inuc -leated c e l l s , but a l s o suggested t h a t the drug induced some degree of cy top la smic v a c u o l a t i o n . The l i m i t e d r e s o l u t i o n of the l i g h t microscope and the nature of the mater ia l being examined ( smears ra ther than t h i n s ec t i on s ) prevented any d e t a i l e d examination of the nature or o r i g i n of these vacuo les . In con junct ion wi th a course in ba s i c e l e c t r o n microscopy being taken by the author, a p re l im ina r y examination of the u I t r a s t r u c t u r e of t r e a t ed and con t ro l c e l l s was t he re f o re undertaken. L5I78Y c e l l s were incubated with acronyc ine f o r 6 hours at a concent ra t i on of 6 yg/ml ( c ond i t i on s which lead to the formation of large numbers of b inuc lea ted c e l l s and which a l s o produce vacuoles in the c e l l s ). They were then recovered by c e n t r i f u g a t i o n , f i x e d , dehydrated, embedded, s e c t i oned , and s ta ined by standard e l e c t r o n m ic ro -scope techniques ( see methods s e c t i o n ) . The sec t i on s were examined in a l ow- re so l u t i on e l e c t r o n microscope ( P h i l l i p s 75 C ). F igures 14 and 15 show the u I t r a s t r u c t u r e of t r ea ted L5I78Y c e l l s , and of con t ro l c e l l s prepared in the same manner. The con t ro l c e l l s 66 ( f i g u r e 14, micrograph a_ ) had wel l def ined nuc le i and n u c l e o l i , with numerous mitochondr ia and f a t d rop le t s in the cytoplasm. The mitochondr ia showed a mat r i x of a s l i g h t l y g rea te r dens i ty than t h a t of the cytoplasm, and f r equen t l y showed c r i s t a e ( f i g u r e 14, micrograph b_ ). The nuc le i of acronyc ine t r ea ted c e l l s were b a s i c a l l y s i m i l a r in form t o those of con t ro l c e l l s , w i th the except ion t ha t b inuc lea ted c e l l s occurred ( f i g u r e 15, micrograph a_ ). However, in many although not a l l cases, acronyc ine t r e a t ed c e l l s showed ex tens i ve mi tochondr ia l s w e l l i n g , leading t o breakage of the i n t e rna l c r i s t a e and a reduct ion in the dens i ty of the mat r i x ( f i g u r e 15, micrographs a_ and b_ ). These swol len and damaged mitochondr ia corresponded in s i z e , number, and approximate l o ca t i on to the vacuoles seen in s ta ined smears of t r ea ted c e l l s , and are probably i d e n t i c a l w i th the l a t t e r . As ide from the occurrence of b inuc lea ted c e l l s and the i n d i c a t i o n s of mi tochondr ia l s w e l l i n g , no other d i f f e r e n c e s were seen between t r ea ted and con t ro l c e l l s in t h i s l i m i t e d , l ow - re so l u t i on study. Subsequent to t h i s p r e l im ina r y examination of the u l t r a s t r u c t u r e of acronyc ine t r e a t ed c e l l s , a more d e t a i l e d examination of the nature of the changes o ccu r r i n g in L5I78Y c e l l s when t r e a t ed with acronyc ine was performed by P. Tan in the Cancer Research Cent re , U.B.C. These r e s u l t s have now been the t o p i c of an undergraduate t h e s i s ( 81 ), and in con junc t i on with i n v e s t i g a t i o n s by Dr. N. Auersperg of the Cancer Research Centre i n to the e f f e c t s of acronyc ine on t i s s u e c u l t u r e c e l l s growing as monolayers form a paper on the c y t o l o g i c a l e f f e c t s of acronyc ine ( 82 ). The r e s u l t s of these i n v e s t i g a t i o n s w i l l be d e a l t with in more d e t a i l in the d i s cu s s i on at the end of s e c t i on I of the r e s u l t s . F igure 14 U I t r a s t r u c t u r e of norma! L5I78Y c e l l s . 68 a_ B inuc lea ted L5I78Y c e l l w i th swo l len mi tochondr ia ( 7,000 x ) b L5I78Y c e l l w i th swo l len mi tochondr ia bes ide one e x h i b i t i n g normal m i tochondr i a l morphology F igure 15 U I t r a s t r u c t u r e of L5I78Y c e l l s t r e a t e d w i th acronyc ine ( 6 yg/ml ) f o r 6 hours. 69 Re sp i r a t i on of acronyc ine t r ea ted L5I78Y c u l t u r e s : In view of the u I t r a s t r u c t u r a I evidence which i nd i c a te s t ha t acronyc ine i s capable of causing mi tochondr ia l s w e l l i n g , i t was of i n t e r e s t t o determine i f t h i s d i s tu rbance of m i tochondr ia l s t r u c t u r e was a s soc ia ted with any evidence of a l t e r e d mi tochondr ia l f u n c t i o n . Because mhe changes in mi tochondr ia l s t r u c t u r e do not occur immediately a f t e r the a d d i t i o n of acronyc ine but only a f t e r a minimum of I t o 2 hours incubat ion with the drug ( 81 ), i t was f e l t adv i s ab le to study mi tochondr ia l f unc t i on in the i n t a c t c e l l . The e f f e c t of acronyc ine on the r e s p i r a t i o n of L5I78Y c u l t u r e s was t he re f o re i n v e s t i g a t e d . The consumption of oxygen by acronyc ine t r ea ted and con t ro l c u l t u r e s was measured using oxygen e l e c t r ode techn iques , as descr ibed in the methods s e c t i o n . F igure 16 shows t ha t ac ronyc ine , at concent ra t i ons of up to 12 yg/ml, has l i t t l e e f f e c t on the oxygen consumption of L5I78Y c u l t u r e s f o r up t o 4 hours. The small d i f f e r e n c e s which e x i s t between t r ea ted and con t ro l c u l t u r e s may be more a t t r i b u t a b l e to the i n h i b i t i o n of c e l l popu la t ion growth by acronyc ine than to any a l t e r a t i o n in the r e s p i r a t i o n of ceI Is. Inf luence of serum concent ra t i on on the g r o w t h - i n h i b i t o r y p r ope r t i e s  of acronyc ine : In the cu r ren t s tudy, L5I78Y and IRC c e l l s were normally grown and experiments were normally performed in F i s c h e r ' s medium supplemented with 10 % horse serum. In view of the f a c t t ha t t h i s e x t r a c e l l u l a r environment d i f f e r s cons ide rab ly from tha t in which the tumor c e l l s e x i s t in the host animal ( which invo lves a much h igher leve l of e x t r a c e l l u l a r serum components ) the i n f l uence of serum concent ra t i on on the i n h i b i t i o n of the growth of L5I78Y c u l t u r e s by acronyc ine was i n v e s t i g a t e d . 70 ^ 11 "j Ac ronyc ine OXYGEN CONSUMPTION 8-1 1 1— 900-CELL POPULATION GROWTH E 500-4 Time ( hours ) F i gu re 16 E f f e c t of ac ronyc ine on the oxygen consumption and c e l l popu l a t i on growth of L5I78Y c u l t u r e s . Bulk c u l t u r e s of L5I78Y c e l l s were incubated a t 37° w i th ac ronyc ine a t the concen t ra t i on s i n d i c a t e d . At I hour i n t e r v a l s , samples of the c e l l suspensions were removed f o r the de te rminat i on of c e l l popu la t i on dens i t y and oxygen consumption as desc r ibed in the methods s e c t i o n . Note - Readings on the th ree c u l t u r e s are o f f s e t from one another by 10 minutes each as on ly a s i n g l e oxygraph was a v a i l a b l e f o r the dete rminat ion of oxygen consumption. 71 C e l l suspensions at a concent ra t i on of 180 x 10 c e l l s /m l were prepared in F i s c h e r ' s medium supplemented with 5, 10, 15, or 20 % horse serum. These c u l t u r e s were then d i s t r i b u t e d in a l i q u o t s i n to i n d i v i d u a l v i a l s , and an equal volume of acronyc ine s o l u t i o n ( 0 to 10 yg/ml, prepared in medium supplemented wi th the same concent ra t i ons of horse serum ) was added. The r e s u l t i n g c u l t u r e s thus comprised four s e r i e s ( one at each horse serum concent ra t i on ), with each s e r i e s con ta i n i ng v i a l s with acronyc ine at a concent ra t i on of 0, 1 , 2 , 3, 4, or 5 yg/ml. The c u l t u r e s were then incubated f o r 24 hours, by which time the c e l l popu la t ion dens i ty in the four v i a l s con ta i n i ng no acronyc ine had increased to approximately 700 x 10^ c e l l s / m l . F igure 17 shows the e f f e c t of serum concent ra t i on on the f i n a l c e l l popu lat ion dens i ty of c u l t u r e s incubated with va r ious acronyc ine concen t r a t i on s . S ince the con t ro l values in the absence of acronyc ine were somewhat a f f e c t e d by the horse serum concen t ra t i on i t s e l f ( as t abu la ted at the bottom of f i g u r e 17 ), the f i n a l c e l l popu la t ion d e n s i t i e s in the acronyc ine t r ea ted c u l t u r e s are expressed as a percentage of the con t ro l va lues f o r each horse serum concen t r a t i on . The growth i n h i b i t o r y p r ope r t i e s of acronyc ine were g rea te s t at the lowest serum concent ra t i on ( 5 % ), and became p rog re s s i v e l y less as the serum concent ra t i on in the medium was inc reased. Thus in the presence of 5 % serum, acronyc ine a t ' a concent ra t i on of I yg/ml slowed popu la t ion growth to the extent t ha t the f i n a l c e l l popu la t ion dens i ty was only 65 % of t h a t of the con t ro l va lue ( i e . in the absence of ac ronyc ine , the c u l t u r e grew in 24 hours from a c e l l dens i t y of 90 x 10^ c e l l s / m l to 667 x 10^ c e l l s / m l , wh i l e in the presence of I yg/ml of acronyc ine i t grew to only 434 x 10^ ce l l s /m l ). The same reduct ion in f i n a l c e l l popu la t ion dens i ty ( 65 % ) requ i red an acronyc ine concent ra t i on 72 1 1 1 1 r 0 1 2 3 4 5 Acronyc ine c o n c e n t r a t i o n ( yg/ml ) F i gu re 17 In f luence of serum concen t r a t i on on the g r o w t h - i n h i b i t o r y p r o p e r t i e s of ac ronyc ine in L5I78Y c u l t u r e s . C e l l suspensions c o n t a i n i n g 90 x 10^ c e l l s / m l and va r y i n g concen t ra t i on s of ac ronyc ine and horse serum ( as i nd i ca ted ) were incubated a t 37° f o r 24 hours. At the end of t h i s p e r i o d , the c e l l popu la t i on dens i t y in the c u l t u r e s was determined. Data are expressed as a percentage of c on t r o l va lues a t each serum concen t r a t i on in the absence of ac ronyc ine - these are t abu l a t ed below. i concen t r a t i on of horse serum c e l l popu l a t i on den s i t y in c o n t r o l s a f t e r 24 hours i ncubat ion 5 % 667 x 10 eel Is/ml 10 ji 707 " 15 % 728 11 20 % 692 " 73 of 2.4 yg/ml in the presence of 10 % horse serum, 3.3 yg/ml in the presence of 15 % serum, and 4.2 yg/ml in the presence of 20 % serum. This suggests t h a t serum e i t h e r p ro tec t s the c e l l s from the growth-i n h i b i t o r y p r ope r t i e s of the a l k a l o i d , or t h a t i t a f f e c t s the a v a i l a b i l i t y of the drug to the eel Is. I 4 B ind ing of C l a b e l l e d acronyc ine to L5I78Y c e l l s and t o serum  components : 14 14 C-acronyc ine was synthes i zed from noracronycine and C-methyI iod ide as descr ibed in the methods s e c t i o n . The product was p u r i f i e d by t h i n layer chromatography and r e c r y s t a l l i z a t i o n . The f i n a l product co-chromatographed with au then t i c ac ronyc ine , and autoradioagraphy of t h i n layer chromatograms i nd i ca ted a radiochemical p u r i t y in excess of 99 %. S ince the s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p of acronyc ine has been found to be very st rong ( 31, 34 ) a d d i t i o n a l evidence t ha t the s y n t h e t i c product was indeed genuine acronyc ine was ob ta inab le from a comparison of the b i o l o g i c a l a c t i v i t y of the r a d i o a c t i v e I y l a b e l l e d mate r i a l with t ha t of au then t i c ac ronyc ine . F igure 18 demonstrates t ha t when added to L5I78Y c u l t u r e s a t concent ra t i ons of from I to 4 yg/ml, the r a d i o a c t i v e I y l a b e l l e d drug had e s s e n t i a l l y the same g r o w t h - i n h i b i t o r y p r ope r t i e s as non - r ad i oac t i ve ac ronyc ine . The evidence t ha t i nc rea s ing the amount of horse serum present in t i s s u e c u l t u r e medium s i g n i f i c a n t l y d imin i shed the c y t o t o x i c i t y of acronyc ine suggested t ha t the a l k a l o i d might po s s i b l y be bound by I 4 serum components. The b ind ing of C-acronycine to non-dia IysabIe serum components was t he r e f o r e i n ve s t i ga ted using e q u i l i b r i u m d i a l y s i s techn iques . To s imu la te as c l o s e l y as po s s i b l e the cond i t i on s of experiments i n vo l v i n g the e f f e c t of acronyc ine on c e l l s , a l l d i a l y s i s experiments were 74 60O E 500 Q) O I O 400H t 300 0) c C O 200H ro CL O CX. © 100' o a acronycine -O C-acronycine - Original population level ~0~ T T T Acronycine concentration ( yg/ml ) 14 F i gu re 18 B i o l o g i c a l a c t i v i t y of C -ac ronyc ine L5I78Y c u l t u r e s c o n t a i n i n g 80 x 10^ c e l l s / m l and va r y i ng 14 concen t r a t i on s of C -ac ronyc ine and n o n - r a d i o a c t i v e ac ronyc ine were prepared in stoppered v i a l s , and incubated a t 37° f o r 24 hours. At the end of t h i s p e r i o d , the c e l l popu l a t i on d e n s i t i e s in the c u l t u r e s were determined. 75 performed using F i s c h e r ' s medium with a n t i b i o t i c s , r a ther than j u s t b u f f e r . A l i q u o t s of horse serum ( 1 0 % in F i s c h e r ' s medium ) were placed in lengths of d i a l y s i s tub ing which had been p rev i ou s l y b o i l e d and r in sed in d i s t i l l e d water. The lengths of tub ing were sea led and I 4 then placed in s o l u t i o n s of C-acronyc ine ( 0 to 15 yg/mI,-prepared in F i s c h e r ' s medium without serum ). The conta ine r s were stoppered and ro ta ted at 4 rpm at 37° f o r 48 hours. At the end of t h i s p e r i o d , a l i q u o t s of the i n t e rna l and ex te rna l s o l u t i o n s were taken f o r the determinat ion of r a d i o a c t i v i t y and p r o t e i n concent ra t i on ( see methods s e c t i o n ' ) . During the incubat ion at 37° , the concent ra t i on of p r o t e i n i n s i de the d i a l y s i s tub ing dec l i ned from 9.4 mg/ml ( the concent ra t i on of 10 % horse serum ) t o 7.5 t o 8.5 mg/ml. As no p r o t e i n was found ou t s i de the d i a l y s i s t u b i n g , the d e c l i n e in p r o t e i n concent ra t i on in the i n t e r na l s o l u t i o n s most probably represents a net f low of f l u i d i n to the tub ing as a r e s u l t of osmosis. At the end of the 48 hour incubat ion p e r i o d , acronyc ine was found to be present i n s i de the d i a l y s i s bags a t concent ra t i ons which were 3 to 4 t imes the ex te rna l c oncen t r a t i on . Th is suggests t ha t the drug was b ind ing to non-dia IysabIe serum components w i t h i n the d i a l y s i s bags. Incubations of longer than 48 hours d id not f u r t h e r increase the concent ra t i on of r a d i o a c t i v e I y l a b e l l e d mate r i a l w i t h i n the d i a l y s i s bags, i n d i c a t i n g t ha t t h i s per iod was s u f f i c i e n t to a l low the system to come to e q u i l i b r i u m . Using accepted procedures ( 83 ) the concent ra t i on of acronyc ine in the ex te rna l s o l u t i o n was taken to be the concent ra t i on of f r ee acronyc ine at e q u i l i b r i u m , wh i le the concent ra t i on of drug in the i n t e rna l p r o t e i n - c o n t a i n i n g s o l u t i o n was taken as the sum of the f r ee acronyc ine and the bound ac ronyc ine . The amount of bound acronyc ine was 76 expressed as micrograms of drug bound per m i l l i g r a m of p r o t e i n , and the data were p l o t t e d accord ing to the method of Scatchard ( 83 ). This method of g raph i ca l r ep re sen ta t i on expresses the amount of bound drug per u n i t concent ra t i on of f r ee drug as a f unc t i on of the amount of bound drug. When the data were p l o t t e d in t h i s manner ( f i g u r e 19 ), as the amount of bound drug increased, the amount of b ind ing per un i t concen t ra t i on of f r ee drug decreased. Th is behaviour i s t ha t which would be expected i f the acronyc ine b ind ing s i t e s became saturated with drug as the acronyc ine concent ra t i on was inc reased. C a l c u l a t i o n s by the method of Scatchard ( 83 ) based on : i ) the s lope of the l i n e , and i i ) i t s i n t e r cep t with the ho r i z on ta l a x i s a t " bound aero. / unbound a e r o . " = 0 gave r e s p e c t i v e l y the b ind ing constant of the process , and the maximum amount of drug bound ( a t i n f i n i t e drug concent ra t i on ). The b ind ing constant ( numer i ca l l y equ i va l en t to the concent ra t i on of f r ee acronyc ine necessary t o ha I f - s a t u r a t e the a v a i l a b l e b ind ing s i t e s ) was found to be 18.6 yg/ml, and the maximum amount of b ind ing was found to be 6.42 yg acronyc ine per m i l l i g r a m serum p r o t e i n . Using these parameters, i t may be c a l c u l a t e d t h a t , in the range of acronyc ine concent ra t i on s normal ly used in the experiments in t h i s t h e s i s ( i'.,e. 3 to 12 yg/ml ) in the presence of 10 % horse serum, about 75 % of the acronyc ine present i s a c t u a l l y not f ree in s o l u t i o n , but i s a s soc i a ted with serum components. Other experiments showed t ha t p u r i f i e d bovine serum albumin 14 could a l s o bind app rec i ab le amounts of C-acronyc ine ; t h i s b ind ing was not however i n ve s t i g a ted q u a n t i t a t i v e l y . I 4 The b ind ing of C-acronyc ine to L5I78Y c e l l s was a l s o i n v e s t i g a t e d . I 4 In p r e l im i na r y exper iments, c e l l s were incubated with C-acronyc ine and then c o l l e c t e d by f i l t r a t i o n . These experiments suggested t ha t L5I78Y 77 14 F i gu re 19 Scatchard p l o t of the b ind ing of C -acronyc ine to non-d i a l y s a b l e components of horse serum. B ind ing was determined as desc r ibed in the t e x t . P r o t e i n concen t r a t i on was measured as desc r ibed in the methods s e c t i o n . 78 c e l l s very r a p i d l y bound s u b s t a n t i a l amounts of the drug. More d e t a i l e d i n v e s t i g a t i o n s were not however po s s i b l e using f i l t r a t i o n procedures, as both M i l l i p o r e f i l t e r s and g la s s f i b e r f i l t e r s re ta ined app rec i ab le I 4 amounts of C-acronyc ine which was not a s soc ia ted with c e l l s , and which could not be removed without washing procedures which a l s o removed c e l l -a s soc ia ted r a d i o a c t i v i t y . For t h i s reason, the b ind ing of r a d i o a c t i v e I y l a b e l l e d acronyc ine to c e l l s was i n ve s t i g a ted using c e n t r i f u g a t i o n procedures ( as descr ibed in the methods s e c t i on ). Since e q u i l i b r i u m d i a l y s i s experiments had i nd i ca ted t ha t acronyc ine could bind to non-d i a l y s a b l e components of horse serum, b ind ing s tud ie s were done in both the presence and absence of the normal amount of horse serum ( 10 % ). I 4 The b ind ing of C-acronyc ine to L5I78Y c e l l s was found to be extremely r a p i d , being complete w i t h i n the minimum per iod wlithin which c e l l s could be analyzed by c e n t r i f ugat ion procedures ( c_. 2 min ). I 4 F igure 20 demonstrates the b ind ing of C-acronyc ine to L5I78Y c e l l s incubated f o r 15 minutes in the presence and absence of 10 % horse serum. P e l l e t s of c e l l s which had been incubated with the drug in the presence of serum were found to conta in approximately 10 t imes the amount of acronyc ine as was present in an equ i va l en t volume of supernatant f l u i d ( i e . a p e l l e t of 10 x 10 c e l l s had a volume of approximately 0.015 ml , and conta ined the same amount of acronyc ine as 0.15 ml of supernatant ). The corresponding f i g u r e f o r p e l l e t s of c e l l s which had been incubated wi th the drug in medium wi thout serum was approximately 40 t imes . Both in the presence and absence of serum, the amount of b ind ing increased in a near ly l i n e a r fash ion with i nc reas ing drug I 4 c oncen t r a t i on . A Scatchard p l o t of the b ind ing of C-acronycine to c e l l s incubated with the drug in medium without serum showed some s l i g h t 79 F i gu re 20 B ind ing of C -acronyc ine t o L5I78Y c e l l s in the presence and absence of 10 % horse serum. 14 6 B ind ing of C -acronyc ine t o i n d i v i d u a l samples of 10 x 10 c e l l s was determined as desc r ibed in the methods s e c t i o n . 80 i n d i c a t i o n s of s a t u r a t i o n of b ind ing ; t h i s e f f e c t was i n con s i s t en t and numer i ca l l y small and so was not f u r t h e r ana lyzed. At a l l drug concent ra t i on s the amount of b ind ing in the presence of 10 % horse serum was on ly 23 t o 24 % of the amount of b ind ing t a k i n g p lace at the same drug concent ra t i on in medium con ta in i ng no horse serum. This f i g u r e i s in very good agreement with the r e s u l t s of c a l c u l a t i o n s based 14 on the Scatchard p l o t of the b ind ing of C-acronycine t o serum components, which suggest t ha t a t drug concent ra t i ons of 0 to 12 yg/ml, only c_. 25 % of the t o t a l acronyc ine present in F i s c h e r ' s medium supplemented with 10 % horse serum i s a c t u a l l y f ree in s o l u t i o n ( the remainder being bound to non-dia IysabIe mater i a l ). The data show tha t the presence of horse serum can g r e a t l y a f f e c t the b ind ing of acronyc ine t o c e l l s , perhaps by c o n t r o l l i n g the concen t ra t i on of unbound acronyc i ne. The re lease of bound acronyc ine from L5I78Y c e l l s was a l s o ana lyzed. I 4 C e l l s were incubated with C -acronyc ine, and then c o l l e c t e d by c e n t r i f u g a t i o n and the supernatant removed. The c e l l s were then r e -suspended in d rug - f ree medium at 37° ; a t i n t e r v a l s a f t e r resuspension 14 the c e l l suspensions were r e cen t r i f u ged and the r e t en t i on of C-acronyc ine by the c e l l s analyzed by rad ioas say ing the c e l l p e l l e t s and the supernatants . It was found t h a t the re lease of bound acronyc ine from the c e l l s was very r a p i d , being complete w i t h i n the minimum per iod of time w i t h i n which a cen t r i f u ga t i on a n a l y s i s could be done ( c_. 2 min ). 81 Di scus s ion - Sect ion I Svoboda has shown t h a t acronyc ine at doses of 28 to 48 mg/kg i n h i b i t s the growth of a v a r i e t y of exper imental tumors in mice, i n c l ud i ng the L5178Y leukemia ( 30 ). He a I so reported t ha t the a l k a l o i d at a concent ra t i on of 25 yg/ml caused ex tens i ve l y s i s in HeLa and Chinese Hamster lung c u l t u r e s in v i t D O , but t ha t i t had " no apparent e^tetog ica I e f f e c t s " over a 24 hour per iod at a concent ra t i on of 10 yg/ml. The cu r r en t i n v e s t i g a t i o n has shown t h a t acronyc ine i n h i b i t s the growth i n v i t r o of two c e l l l i ne s growing as suspension c u l t u r e s , the L5I78Y mouse lymphocytic leukemia and the IRC ra t monocytic leukemia ( f i g u r e 8 ). Over a 24 hour p e r i o d , the growth of L5I78Y c u l t u r e s was i n h i b i t e d by a l k a l o i d concent ra t i ons as low as I yg/ml ( f i g u r e s 17, 19 ), although concent ra t i on s in the range of 3 to 6 yg/ml were necessary to cause more than a s l i g h t i n h i b i t i o n in the ra te of popu la t ion growth ( f i g u r e 8 ). TherlRC c e l l l i n e was more r e s i s t a n t to the drug, and an acronyc ine concen t ra t i on of 12 yg/ml was necessary t o achieve a leve l of growth i n h i b i t i o n s i m i l a r to t h a t seen at a concent ra t i on of 6 yg/ml in the L5I78Y c u l t u r e s . Dr. N. Auersperg of the Cancer Research Centre, U . B . C , has r e cen t l y i n ve s t i g a ted the e f f e c t s of acronyc ine on a number of c e l l l i ne s growing as monolayer c u l t u r e s ( 82 ) : the human carcinoma c e l l l i n e C-4iEI, the melanoma l i n e HFH-18, and l i n e SV/HT, a l i n e der ived from an SV-40 v i r u s induced hamster tumor. While most L5I78Y c e l l s in a c u l t u r e t r e a t ed with acronyc ine at a concent ra t i on of 6 yg/ml f o r 24 hours are dead at the end of t h i s p e r i o d , the c e l l l i ne s s tud ied by Dr. Auersperg su rv i ved f o r three days a t drug concent ra t ions of up to 24 yg/ml. E f f e c t s of the drug on the c e l l s became ev ident on ly a f t e r incubat ion per iods of 24 hours or more. When the two c e l l l i n e s 82 i n ve s t i g a ted in t h i s t h e s i s and the three l i ne s i n ve s t i g a ted by Dr. Auersperg are compared, there appears t o be an inverse c o r r e l a t i o n between generat ion time and the s e n s i t i v i t y of the c e l l s t o ac ronyc ine . Thus L5I78Y c e l l s ( generat ion time in c u l t u r e of 8-9 hours ) were more s e n s i t i v e than IRC c e l l s ( generat ion time 12-14 hours ). With in the s e r i e s of three monolayer c u l t u r e s the SV/HT ( generat ion time 15-17 hours ) was most s e n s i t i v e and the C-4I I ( generat ion t ime 24-48 hours ) was the leas t s e n s i t i v e . Th is c o r r e l a t i o n suggests t ha t acronyc ine may be p a r t i c u l a r l y c y t o t o x i c to r a p i d l y r e p l i c a t i n g c e l l s . In both the IRC and L5I78Y c e l l l i ne s ( as wel l as the SV/HT l i n e i n ve s t i ga ted by Auersperg ( 82 ) ) , acronyc ine induced the format ion of b i nuc lea ted c e l l s . Both nuc le i of b inuc lea ted c e l l s appeared to be c y to l og i ca I Iy normal, as noted in t h i s i n v e s t i g a t i o n in smears and p r e l im i na r y e l e c t r o n micrographs, and in the more d e t a i l e d u I t r a s t r u c t u r a I s tud ie s of Tan ( 81 ), and Tan and Auersperg ( 82 ). Th is suggests t h a t the two nuc le i of the b inuc lea ted c e l l s do not a r i s e as a r e s u l t of f ragmentat ion of a s i n g l e normal interphase nucleus. The f a c t t h a t c o l c h i c i n e could prevent the format ion of b i nuc lea ted c e l l s in L5I78Y c u l t u r e s t r e a t ed with acronyc ine ( f i g u r e 10 ) suggests t ha t the two nuc le i in the b inuc lea ted c e l l s a r i s e as a r e s u l t of normal nuc lear d i v i s i o n a t the end of the c e l l c y c l e , r a ther than by fu s i on of two mononucIeated c e l l s . B inuc lea ted c e l l s would thus a r i s e as a r e s u l t of a d i s tu rbance in the process of c y t o k i n e s i s , whereby a p ropor t i on of the t r e a t ed c e l l s f a i l t o d i v i d e normal ly i n to two daughter c e l l s . This suggest ion was supported by the r e s u l t s of s tud ie s using p a r t i a l l y synchronized popu la t ions of IRC c e l l s ( f i g u r e s II and 12 ) which i nd i ca ted t ha t b inuc lea ted c e l l s appeared on ly at about the time of m i t o s i s in t r ea ted 83 cu I t u r e s . The e f f e c t of acronyc ine on both c y t o k i n e s i s and the i n h i b i t i o n of popu la t ion growth was not immediate, and at lower acronyc ine concent ra t i ons ( L5I78Y, 3 yg/ml ; IRC, 6 yg/ml ) might be delayed f o r over a c e l l generat ion t ime. S ince the ce s sa t i on of popu la t ion growth in t r ea ted c u l t u r e s co i nc i ded with the appearance in the c u l t u r e s of b i nuc lea ted c e l l s , the f a i l u r e of c y t o k i n e s i s may play a r o l e in t h i s c e s s a t i o n . It cannot be excluded however t ha t there may a l s o be some l y s i s of c e l l s which would c o n t r i b u t e to the growth i n h i b i t i o n . The f a t e of the b inuc lea ted c e l l s was not i n ve s t i g a ted in the cu r ren t study. C e l l s t r ea ted with c y t o c h a l a s i n B - a mold metabo l i te s t r uc tu ra I Iy unreIated to acronyc ine which a l s o causes b inuc lea ted c e l l s ( 84 ) - cont inue t o proceed through the c e l l c y c l e and undergo nuc lear d i v i s i o n and may e ven tua l l y conta in 4 or even 8 n u c l e i . Acronycine however seems to be more t o x i c than c y t o c h a l a s i n B, and c e l l s with more than two nuc le i have not been observed in the cu r ren t i n v e s t i g a t i o n s ; on cont inued treatment with ac ronyc ine , a l l c e l l s e ven tua l l y l y se . Whether o r not b inuc lea ted c e l l s are more s u s c e p t i b l e to l y s i s than mononucIeated c e l l s i s not a t the moment known ; t h i s quest ion would have to be i n ve s t i ga ted using methods such as t ime - l ap se cinematography which a l low i n d i v i d u a l c e l l s to be foI I owed. Experiments with p a r t i a l l y synchronized popu lat ions of L5I78Y and IRC c e l l s i n d i c a t e t h a t acronyc ine may somewhat extend the time between the complet ion of m i t o s i s ( as marked by the appearance of m i t o t i c f i g u r e s and the increase in c e l l popu la t ion ) and the i n i t i a t i o n of DNA synthes i s in the S phase. Thus in the experiment in f i g u r e 12, m i t o s i s takes p lace at the same time in t r e a t ed and con t ro l c u l t u r e s . However 84 the pa t te rn of S-phase DNA synthes i s in the acronyc ine t r ea ted c u l t u r e then lags approximately 1.5 hours behind t ha t of the con t ro l c u l t u r e . S ince the o v e r a l l pa t te rn of DNA synthes i s in the S phase i s s i m i l a r in t r e a t ed and con t ro l c u l t u r e s , the data suggest t h a t the extens ion of the Gj per iod does not desynchronize the c u l t u r e s and t h a t the ex tent of the delay of the i n i t i a t i o n of the S phase i s probably the same in a l l c e l l s pass ing the G|/S boundary. The p rec i s e time in the M t o S i n t e r v a l at which t h i s delay occurs and i t s mechanism could not be determined from the experiments in t h i s study. However, i t may be noted t ha t i t has been reported t h a t agents which i n h i b i t RNA syn thes i s a f f e c t the i n i t i a t i o n of the S phase more than i t s c on t i nua t i on ( 18, 85 ). A d i s turbance in RNA syn thes i s would thus be capable of e x p l a i n i n g t h i s de lay . Although acronyc ine at r e l a t i v e l y low concent ra t i ons ( L5I78Y, 3 yg/ml ; IRC, 6 yg/ml ) somewhat extended the G per iod of the c e l l s , i t d id not i r r e v e r s i b l y b lock the progress of c e l l s through the c e l l c y c l e at any po i n t . The delayed e f f e c t s of acronyc ine on popu la t ion growth in asynchronous c u l t u r e s may thus be e xp l a i n ab l e as the r e s u l t of acronyc ine having r e l a t i v e l y l i t t l e e f f e c t on the passage of c e l l s through the c e l l c y c l e , wh i le having delayed e f f e c t s on the process of c e l l d i v i s i o n . It may be noted t h a t these delayed e f f e c t s are seen with low concent ra t i ons of the drug. At h igher drug concent ra t i ons ( i e . L5I78Y, 6 yg/ml ; IRC, 12 yg/ml ) acronyc ine may have r a p i d , although probably not immediate e f f e c t s on c e l l d i v i s i o n . Thus, a's shown in f i g u r e 10, w i t h i n 2 t o 4 hours of the a d d i t i o n of acronyc ine at a concent ra t i on of 6 yg/ml popu la t ion growth in L5I78Y c u l t u r e s i s i n h i b i t e d and b inuc lea ted c e l l s appear. A l o w - r e s o l u t i o n - e l e c t r o n microscope examination of L5I78Y c e l l s 85 t r ea ted with acronyc ine at a concent ra t i on of 6 yg/ml f o r 6 hours showed evidence of mi tochondr ia l s w e l l i n g in t r ea ted c e l l s . As ide from b inuc lea ted c e l l s , no o ther c y t o l o g i c a l changes were seen in t h i s l im i t ed study. More r e c e n t l y , Tan ( 8 1 ), and Tan and Auersperg ( 82 ) have reported the r e s u l t s of more d e t a i l e d i n v e s t i g a t i o n s i n to the time course of changes induced in the u l t r a s t r u c t u r e of L5I78Y c e l l s t r ea ted wi th the same concen t ra t i on of acronyc i ne. V.No u I t r a s t r u c t u r a I changes were seen in the nuc le i of t r ea ted c e l l s , i n c l ud i ng the nuc le i of b i nuc lea ted c e l l s . There was some i nconc lu s i ve evidence of c e l l s w e l l i n g and of a reduct ion in the frequency of m i c r o v i l l i , but no change was noted in the frequency with which p i n o c y t o t i c a c t i v i t y was seen at the plasma membrane. The major changes-;which occurred on acronyc ine treatment were the p rog res s i ve s w e l l i n g and p a r t i a l d i s r u p t i o n of both mitochondr ia and Golg i bod ies . The mi tochondr ia l s w e l l i n g observed by Tan ( 81 ) was s i m i l a r to t ha t observed in the cu r ren t study, and appeared t o be p rog re s s i ve . A f t e r one hour of t reatment, there was l i t t l e d e f i n i t e evidence of any a l t e r a t i o n in the u l t r a s t r u c t u r e of L5I78Y c e l l s , but a f t e r 2 hours there was a d e f i n i t e s w e l l i n g of the o r gane l l e s w i th some d i s r u p t i o n ( breakage ) of c r i s t a e . The degree of s w e l l i n g increased as the c u l t u r e s were incubated with the drug f o r longer pe r i od s . The e f f e c t of acronyc ine on Golg i bodies was even more pronounced than i t s e f f e c t on mi tochondr ia . These o r gane l l e s in con t ro l c e l l s were not .. prominent, but a f t e r one hour of treatment with acronyc ine ( 6 yg/ml ) they became d i l a t e d , with occas iona l membrane breakage. On f u r t h e r incubat ion the Golg i became even more swo l l en , u n t i l a f t e r severa l hours they resembled a c o l l e c t i o n of s phe r i c a l v e s i c l e s , r a ther than the c l a s s i c a l " s tack of co ins " ar ray which i s u sua l l y seen. Tan has noted 86 t ha t i t i s we l l known t ha t the appearance of Golg i bodies can depend markedly on the metabo l i c s t a t e of the c e l l ( 86 ). For t h i s reason he suggested t ha t the changes in t h i s o r gane l l e could not c o n c l u s i v e l y be c a l l e d " damage " but might a l s o r e s u l t from a change in the normal f unc t i on i n g of the Golg i ( f o r example h y p e r a c t i v i t y ). Acronycine at a concent ra t i on of 6 yg/ml causes r e a d i l y v i s i b l e changes in the mitochondr ia of L5I78Y c e l l s a f t e r incubat ion per iods as shor t as two hours ( 81 ). When however the consumption of oxygen by L5I78Y c u l t u r e s was i n ve s t i ga ted using oxygen e l e c t r ode techn iques , acronyc ine at a concent ra t i on of 6 or 12 yg/ml had l i t t l e e f f e c t on oxygen consumption, even in c u l t u r e s which had been exposed t o the drug f o r up t o four hours ( f i g u r e 16 ). Th is lack of marked changes in oxygen consumption in c u l t u r e s in which many c e l l s conta in swol len mitochondr ia might be due t o damaged mitochondr ia s t i l l having normal l e ve l s of oxygen eonsumption. Swollen mitochondr ia might s t i l l however be metaboIicaI Iy d e f i c i e n t ( f o r example with respect to ATP product ion ). Th i s problem i s one which cou ld bear f u r t h e r i n v e s t i g a t i o n , ; perhaps us ing app rop r i a te I pad ioact i ve Iy - I abe I Ied subs t ra te s t o i n ve s t i g a te mi tochondr ia l f unc t i on in the whole c e l l . Tan has noted ( 8 1 ) t ha t the degree of mi tochondr ia l change v a r i ed in i n t e n s i t y among L5I78Y c e l l s a f t e r the same per iod of acronyc ine t reatment, although w i t h i n an i n d i v i d u a l c e l l there was always an equal degree of s w e l l i n g in a l l m i tochondr ia . This iheteroge.neity can be seen in f i g u r e 15, micrograph b_, in which a c e l l adjacent to one with swol len mitochondr ia e x h i b i t s normal mi tochondr ia l morphology. Tan a l s o noted t ha t on continued. incubat ion with ac ronyc ine , a l l c e l l s e ven tua l l y show mi tochondr ia l damage. It i s po s s i b l e t h a t the i n i t i a I >\heterogeneity of • 87 the a c t i o n of acronyc ine on the mitochondr ia of i n d i v i d u a l c e l l s may represent a s e l e c t i v e e f f e c t of the a l k a l o i d on the mitochondr ia of c e l l s a t d i f f e r e n t phases of the c e l l c y c l e . Bosmann has shown t h a t the mitochondr ia of L5I78Y c e l l s undergo c y c l i c a l metabo l ic a c t i v i t y and probably d i v i d e in synchrony with the c e l l c y c l e ( 87 ). If acronyc ine s e l e c t i v e l y damaged mitochondr ia a t some p a r t i c u l a r stage of the c e l l c y c l e t h i s could e xp l a i n why not a l l the c e l l s of an asynchronous popu la t ion i n i t i a l l y show s igns of mi tochondr ia l damage. I 4 I n ve s t i ga t i on s in the cu r ren t study using C-acronycine showed t h a t the a l k a l o i d was r a p i d l y taken up and re leased by L5I78Y c e l l s . Acronycine i s h i gh l y f l u o r e s c e n t , and examination under a f l uo rescence microscope of c e l l s incubated wi th the drug i nd i ca ted t ha t the compound was s e l e c t i v e l y l o c a l i z e d in patches or granules in the cytoplasm ; l i t t l e appeared t o be a s soc ia ted wi th the nucleus. Because of the l im i t ed r e s o l u t i o n , i t was not po s s i b l e t o unambiguously i d e n t i f y the cy top la smic s t r u c t u r e s t o which the drug was bound, although the s i z e and l o ca t i on of the p a r t i c u l a t e mate r i a l was c on s i s t en t with a l o c a l i z a t i o n of acronyc ine in e i t h e r mitochondr ia or f a t d r o p l e t s . The amount of b ind ing of acronyc ine t o L5I78Y c e l l s as wel l as the c y t o t o x i c i t y of the drug were both a f f e c t e d t>y the amount of horse serum used to supplement the medium in which the c e l l s were grown. Higher serum concent ra t i on s were c o r r e l a t e d with a lower degree of b ind ing of the drug to c e l l s ( f i g u r e 20 ) and a reduced a b i l i t y of the drug to i n h i b i t the growth of L5I78Y c u l t u r e s ( f i g u r e 17 ). S ince e q u i l i b r i u m d i a l y s i s experiments i nd i ca ted t h a t acronyc ine was bound in large amounts by non-dia IysabIe components of horse serum, the most l i k e l y exp lana t i on f o r these r e s u l t s i s t ha t only acronyc ine which i s not bound t o serum 88 components i s a v a i l a b l e to c e l l s . 14 A Scatchard p l o t of the b ind ing of C-acronycine to non-dia IysabIe serum components gave a s t r a i g h t l i n e ( f i g u r e 19 ). S ince the s lope of a Scatchard p l o t i s d i r e c t l y r e l a t ed t o ifihe b ind ing constant of whatever a s s o c i a t i o n r eac t i on i s being measured ( 83 ), t h i s i s suggest ive t ha t b ind ing s i t e s of a s i n g l e a f f i n i t y account f o r the ma jo r i t y of the b ind ing w i t h i n the range of acronyc ine concent ra t ions used. The exact nature of the non-dia IysabIe mater i a l in horse serum which binds acronyc ine was not i d e n t i f i e d in the cu r ren t i n v e s t i g a t i o n . It seems l i k e l y however t ha t in view of the wel l documented a f f i n i t y of the albumin f r a c t i o n of serum f o r o rgan i c compounds ( 88 ), and the f a c t t ha t 14 p u r i f i e d bovine serum albumin bound app rec i ab le amounts of C -acronyc ine, t ha t the albumin of the horse serum was re spons ib le f o r the b ind ing of ac ronyc ine . RESULTS - SECTION 2 E f f e c t of acronyc ine on the i nco rpo ra t i on of nuc leos ides i n to n u c l e i c  ac i ds: P r e l im i na r y s tud ie s by the author using L5I78Y c e l l s i nd i ca ted t ha t I 4 acronyc ine had l i t t l e e f f e c t on the i nco rpo ra t i on of C-thymidine i n to DNA, but t ha t i t markedly i n h i b i t e d the i n co rpo ra t i on of ^H-u r id ine i n to RNA ( 4 1 ). Th is suggested t ha t acronyc ine might po s s i b l y d i s t u r b RNA synthes i s. The e f f e c t s of va r ious concent ra t i on s of acronyc ine on the i nco rpo ra t i on of r a d i o a c t i v e l y l a b e l l e d thymidine and u r i d i n e in to the DNA and RNA of both L5I78Y and IRC c e l l s i s shown in f i g u r e 21. The c e l l s were preincubated with the drug f o r 30 minutes. The r a d i o a c t i v e precur sor was then added 89 O * 8 12 0 Q 4 8 12 acronyc ine c oncen t r a t i on ( ug/ml ) F i gu re 21 E f f e c t of ac ronyc ine on the i n co rpo r a t i o n of u r i d i n e and thymid ine i n t o the RNA and DNA of L5I78Y and IRC c e l l s , C u l t u r e s were pre incubated w i th v a r i ou s concen t r a t i on s of ac ronyc ine f o r 30 minutes, then pulsed f o r 45 minutes w i th e i t h e r 3 H - 6 - u r i d i n e ( 0.096 yM ) o r 1 4 C - t h y m i d i n e ( 1.6 pM ). The i n co rpo r a t i o n of r a d i o a c t i v i t y i n t o ma te r i a l d i g e s t a b l e by RNase ( 3 H - u r i d i n e l a b e l l e d c e l l s ) o r i n to t o t a l a c i d - i n s o I ub I e mate r i a l 14 ( C- thymid ine l a b e l l e d c e l l s ) was then determined as desc r ibed in the methods s e c t i o n . The r e s u l t s are expressed as a percentage i n h i b i t i o n r e l a t i v e t o c o n t r o l va lues of i n c o r p o r a t i o n . I nd i v idua l po i n t s are the mean of data from th ree to f i v e exper iments . 90 and the incubat ion cont inued f o r a f u r t h e r 45 minutes. The c e l l s were I 4 c o l l e c t e d on f i l t e r s and the i n co rpo ra t i on of C-thymidine i n to a c i d -i n s o l ub l e mate r i a l and of "^H-uridine in to mate r i a l d i ge s t ab l e by RNase was determined as descr ibed in the methods s e c t i o n . It may be seen t ha t acronyc ine i n h i b i t s the i nco rpo ra t i on of both u r i d i n e and thymidine i n to the n u c l e i c ac id s of both c e l l l i n e s . The i n co rpo ra t i on of u r i d i n e i n to RNA was cons ide rab ly more a f f e c t e d than the i nco rpo ra t i on of thymidine i n to DNA, e s p e c i a l l y at lower drug concen t r a t i on s . Other experiments ' e s t a b l i s h e d t h a t t h i s i n h i b i t i o n of p recur sor i n co rpo ra t i on occurred very r a p i d l y . When "^H-uridine was added to an L5I78Y c u l t u r e a t the same time as acronyc ine and the i n co rpo ra t i on of u r i d i n e i n to RNA dur ing a 10 minute pulse was measured, the extent of the i n h i b i t i o n of u r i d i n e i n c o r -porationwwascc.. f;50$. Th is degree of i n h i b i t i o n was i d e n t i c a l to t ha t seen in L5I78Y c u l t u r e s which had been pre incubated f o r up to two hours with the same concent ra t i on of acronyc ine before a 10 minute pulse of u r i d i ne. The i n co rpo ra t i on of c y t i d i n e i n to RNA was i n h i b i t e d to a s i m i l a r ex tent as t ha t of u r i d i n e . Other experiments showed t ha t the i n co rpo ra t i on of guanosine i n to n u c l e i c ac ids was a l s o i n h i b i t e d . I n te rac t i on of acronyc ine with DNA in v i t r o : As noted in the i n t r o d u c t i o n , a number of compounds are capable of i n h i b i t i n g RNA and/or DNA s yn thes i s by i n t e r a c t i n g wi th c e l l u l a r DNA and thereby i n t e r f e r i n g with i t s template a c t i v i t y f o r n u c l e i c a c i d polymerases ( 42-44 ). These compounds inc lude a number of c l i n i c a l l y usefu l an t i neo -p l a s t i c agents, as wel l as c e r t a i n a c r i d i n e d e r i v a t i v e s such as p r o f l a v i n e . Acronycine i s an N-methyl acr idone which i s chemica l l y re I a t e d ' t o a c r i d i n e s ( see f i g u r e s 2 and 4 f o r s t r u c t u r a l formulae ) and i t seemed 91 po s s i b l e t ha t i t a l s o might i n t e r a c t with DNA and thereby i n h i b i t the i n co rpo ra t i on of nuc leos ides i n to n u c l e i c a c i d s . The a b i l i t y of acronyc ine t o i n t e r a c t with DNA in v i t r o was t he re f o re i n ve s t i ga ted using two techniques which are commonly employed in de tec t i ng DNA-drug i n t e r a c t i o n . i ) E f f e c t of DNA on the u l t r a v i o l e t and v i s i b l e spectrum of ac ronyc ine. When compounds which have w e l l - d e f i n e d absorpt ion spect ra bind to DNA or o ther macromolecules, there are f r equen t l y changes in the spectrum of the compound. These changes are produced as a r e s u l t of changes in the immediate phys i ca l environment of the compound. These s h i f t s have been of cons ide rab le use in de tec t i n g and s tudy ing the i n t e r a c t i o n between DNA and a v a r i e t y of drugs ( 53-59 ). The spec t ra of acronyc ine and of a c r i d i n e orange were determined in the presence and absence of DNA. The l a t t e r compound i s a known DNA-b ind ing drug which was chosen as a con t ro l ( f o r fo rmula , see f i g u r e 4, s t r u c t u r e f_ ). F igure 22 shows t ha t there were cons ide rab le changes in the spectrum of a c r i d i n e orange when i t was incubated with DNA, i n c l ud i ng both a decrease in absorbance at wavelengths below approximately 300 nm, and a s h i f t in the wavelength of the absorpt ion peak which e x i s t s between 450 and 500 nm. These changes were c on s i s t en t with the data of other workers using a c r i d i n e orange ( 56 ), and i nd i c a t e the phy s i ca l i n t e r a c t i o n between the drug and the n u c l e i c a c i d . In c o n t r a s t , the spectrum of acronyc ine was completely una l tered when the drug was incubated with DNA under s i m i l a r c ond i t i on s ( f i g u r e 22 ). i i ) E f f e c t of acronyc ine on the me l t ing of DNA. A second procedure commonly used to detect DNA-drug i n t e r a c t i o n i s to examine the e f f e c t of the drug on the me l t ing of DNA in v i t r o . When double-stranded DNA in s o l u t i o n i s heated, the two st rands separate or 92 c ZJ >-in c CD X J (0 u u c i_ o < o.&H 0.4-0.8H 0.4H a c r i d i n e orange ac ronyc ine w i th and w i thout DNA 500 600 Wavelength ( nm ) F i gu re 22 E f f e c t of DNA on the abso rp t i on spec t ra of a c r i d i n e orange I and ac ronyc i ne . S o l u t i on s of a c r i d i n e orange o r ac ronyc ine ( 20 uM ) were prepared in phosphate b u f f e r ( 10 mM, pH 7.0 ) or in b u f f e r p lus c a l f thymus DNA ( 100 yM wi th re spect to DNA phosphorus ). The spec t ra were recorded aga i n s t blanks of b u f f e r , o r b u f f e r p lus DNA, as a p p r o p r i a t e . 93 " m e l t " ( 89 ). The u l t r a v i o l e t absorbance of the separated strands i s g rea te r than t ha t of the o r i g i n a l double-stranded mate r i a l - t h i s phenomenon i s known as " hyperchromic i ty " . The mel t ing of the DNA can thus be fo l lowed by moni tor ing the u l t r a v i o l e t absorbance of the s o l u t i o n of DNA as i t i s heated ( 89 ). The b ind ing of compounds t o DNA normally r a i s e s the thermal s t a b i l i t y of the double h e l i x , and thus r a i s e s i t s me l t i ng temperature ( 52, 5.4, 59 ). The e f f e c t of a c r i d i n e orange and acronyc ine on the me l t ing of c a l f thymus DNA i s shown in f i g u r e 23. A c r i d i n e orange a l t e r e d the me l t i ng temperature* of the n u c l e i c a c i d from 56.8° to 7 3 . 6 ° . In c o n t r a s t , acronyc ine had no de tec tab le e f f e c t on the me l t ing temperature of the DNA. E f f e c t of acronyc ine on RNA synthes i s in vjjhrg_: Although there was no evidence from spect rophotometr ic and mel t ing temperature s t ud i e s t ha t acronyc ine was complexing with DNA, a l im i t ed i n t e r a c t i o n - undetectable by these phy s i ca l methods - could not be exc luded. In t h i s case acronyc ine might s t i l l i n t e r f e r e with the template a c t i v i t y of DNA and thereby a f f e c t RNA or DNA s yn the s i s . The e f f e c t of acronyc ine on the template a c t i v i t y of DNA was t he r e f o r e d i r e c t l y examined * The r e l a t i v e l y g rea te r hyperchromic i ty of the DNA in the presence of a c r i d i n e orange i s due t o the re lease of bound drug as the two strands of the DNA h e l i x separate on hea t i ng . The f ree a c r i d i n e orange absorbs u l t r a v i o l e t l i g h t more s t r ong l y a t 260 nm than the bound drug ( see f i g u r e 22 ). Th is increase i s not compensated f o r by the spectrophotometer b lank, and thus adds to the hyperchromic i ty produced by the DNA i t s e l f . 94 Temperature ( °C ) F i gu re 23 E f f e c t of ac ronyc ine and a c r i d i n e orange on the me l t i n g of c a l f thymus DNA. Ca l f thymus DNA ( 50 uM w i th re spec t t o DNA phosphorus ) e i t h e r a lone o r p lus ac ronyc ine o r a c r i d i n e orange ( 10 uM ) was d i s s o l v ed in water c on t a i n i n g 3 mM NaCl , 0.3 mM Na c i t r a t e ( pH 7.0 ) and 5 % dimethyl s u l f o x i d e . The s o l u t i o n s were heated in qua r t z cuvets in the thermoreguIated chamber of a G i l f o r d model 2000 spectrophotometer a t a r a te of approx imate ly 1° C per minute. The u l t r a v i o l e t absorbance of the s o l u t i o n s was monitored a t 260 nm. The me l t i ng temperature was taken as the temperature a t which ha l f the t o t a l hyperchromic i t y was ob ta i ned . 95 in v i t r o us ing an RNA s yn the s i z i n g system composed of c a l f thymus DNA as a template, E_. col i RNA polymerase, and the four nuc leos ide t r i -phosphates, of which the ATP was r a d i o a c t i v e ( see methods s e c t i on ). S ince the assay was being used to i n ve s t i g a t e the e f f e c t of acronyc ine on the template a c t i v i t y of DNA, an i n v e s t i g a t i o n was f i r s t made of the r e l a t i o n s h i p between template concent ra t i on and RNA synthes i s in v i t r o ( f i g u r e 24 ). The graph of DNA template concent ra t i on vs. RNA synthes i s shows a broad p lateau ranging from a concent ra t i on of DNA of 20 yM t o 200 yM ( expressed on the bas i s of DNA phosphorus ). At concent ra t i ons of DNA lower than 20 yM, the amount of r a d i o a c t i v i t y incorporated in to a c i d - i n so I ub I e mate r i a l dropped r a p i d l y with decreas ing DNA concent ra t i on u n t i l w i th no template present there was no i n c o r p o r a t i o n . Th is behaviour i s e xp l a i n ab l e on the bas i s t h a t both template and enzyme are necessary f o r the s ynthes i s of RNA : at high DNA concent ra t i ons there i s excess template, and the amount of s yn thes i s i s l im i t ed by the amount of enzyme, wh i l e below 20 yM DNA there i s i n s u f f i c i e n t template f o r the amount of enzyme, and the a v a i l a b i l i t y of the DNA becomes l i m i t i n g in the r e a c t i o n . For assays of drug e f f e c t s on DNA template a c t i v i t y , assays were performed e i t h e r a t 40 yM DNA ( a t which concent ra t i on RNA synthes i s in c o n t r o l s i s l im i t ed by the amount of enzyme ) o r a t 4 yM DNA, ( at which concent ra t i on template i s l i m i t i n g ). F igure 25 i nd i c a te s t ha t eth id ium bromide ( f o r fo rmula , see f i g u r e 4, s t r u c t u r e e ), a well-known i n t e r c a l a t i n g drug ( 55 ), i n h i b i t e d RNA s yn thes i s in v i t r o by over 80 % ( curve a ). Acronyc ine, even at a concent ra t i on of 40 yM ( £. 13 yg/ml ) had no e f f e c t on the i nco rpo ra t i on of r a d i o a c t i v i t y i n to ac i d - i n so I ub I e mate r i a l ; t h i s was t rue i r r e s p e c t i v e of whether ' th i s. process in coritro fs'-was l i m i t e d by.the amount of enzyme 96 ; 3 H CL !_ o u c £ CL o x2H to I >l U) CD -C +-c >-Ul < cr Enzyme I imited assays DNA I imited assays 10 20 -1 r~ 40 60 - 1 — 100 I— 200 DNA-P concen t r a t i o n ( yM ) F i gu re 24 R e l a t i o n s h i p between DNA template concen t r a t i on and the s yn the s i s of RNA in v i t r o . I 4 The i n co rpo r a t i o n of r a d i o a c t i v i t y from C-ATP i n to a c i d i n s o l u b l e ma te r i a l was determined as desc r ibed in the methods s e c t i o n . The concen t r a t i on s of DNA a t which the e f f e c t s of drugs on RNA s yn the s i s in v i t r o were i n ve s t i g a t ed are i nd i ca ted w i th arrows. " DNA-P " r e f e r s t o the c oncen t r a t i on of DNA on the bas i s of DNA phosphorus con ten t . 97 acronyc Ine ethidium bromide 0 10 20 30 40 Drug concentration ( uM ) F igure 25 E f f e c t of ac ronyc ine and e th id ium bromide on RNA s yn the s i s i n v i t r o . The a c t i v i t y of E. c o l i RNA polymerase was determined as desc r ibed in the methods s e c t i o n . Data are the mean of th ree de te rm ina t i on s , expressed as a percentage of the amount of r a d i o a c t i v i t y incorporated i n t o the a c i d - i n s o I ub I e ma te r i a l of the c o n t r o l s . a) | e f f e c t of e th id ium bromide :DNA concen t r a t i on 40 yM w i th re spect t o DNA phosphorus ( a t which concen t r a t i on RNA s yn the s i s in the c o n t r o l s i s l i m i t e d by the enzyme a c t i v i t y ( see f i g u r e 24 ) ). b) e f f e c t of ac ronyc ine : DNA concen t r a t i on 40 yM ( RNA s yn thes i s in c o n t r o l s i s enzyme- l im i ted ). c) e f f e c t of ac ronyc ine : DNA concen t r a t i on 4 yM ( RNA s yn thes i s in c o n t r o l s i s l i m i t e d by the DNA template a c t i v i t y ). 98 ( curve b ) or by the amount of DNA ( curve c ). E f f e c t of acronyc ine on the i n co rpo ra t i on of e x t r a c e l l u l a r nuc leos ides  i n to the a c i d - s o l u b l e mate r i a l of L5I78Y c e l l s : At about t h i s t ime, other work in t h i s laboratory had shown tha t i ) acronyc ine had no s e l e c t i v e e f f e c t on the l a b e l l i n g of va r ious subspecies of RNA by 3 H - u r i d i n e ( 147 ) - t h i s i s in con t r a s t t o many i n h i b i t o r s of RNA s yn the s i s , which, although they may ac t by d i f f e r i n g mechanisms, tend to s e l e c t i v e l y i n h i b i t the synthes i s of ribosomal RNA ( 43, 90 ). i i ) even though i t markedly i n h i b i t e d the i nco rpo ra t i on of u r i d i n e in to I 4 RNA, acronyc ine had l i t t l e e f f e c t on the l a b e l l i n g of p r o te i n by C-Ieuci ne. The above data , together with the f a i l u r e to show by phy s i ca l methods any i n t e r a c t i o n between acronyc ine and DNA o r any e f f e c t of the drug on RNA syn thes i s in v i t r o , suggested t h a t the a b i l i t y - o f .acronycine to i n h i b i t the l a b e l l i n g of n u c l e i c ac id s by e x t r a c e l l u l a r r a d i o a c t i v e nuc leos ides might not re s ide in an i n h i b i t i o n of n u c l e i c a c i d s yn thes i s per se. As reviewed in the i n t r o d u c t i o n , the biochemical pathways from e x t r a c e l l u l a r nuc leos ides t o n u c l e i c ac ids invo lve many s teps . Some of these are o u t l i n e d in f i g u r e 3 f o r u r i d i n e . An i n h i b i t i o n of u r i d i n e t neo rpo ra t i on i n to RNA need not occur at the polymerase l e v e l , but could r e s u l t from an i n t e r f e rence with any one of the steps leading from ur i d i ne t o RNA. It seemed po s s i b l e t ha t acronyc ine might i n h i b i t the i nco rpo ra t i on of e x t r a c e l l u l a r nuc leos ides i n to n u c l e i c ac ids by i n h i b i t i n g t h e i r ent ry i n to i n t r a c e l l u l a r nuc l eo t i de poo l s . In view of t h i s p o s s i b i l i t y , the e f f e c t of the a l k a l o i d on the i n co rpo ra t i on of u r i d i n e and thymidine 99 i n to i n t r a c e l l u l a r a c i d - s o l u b l e mate r i a l was i n v e s t i g a t e d , using procedures which are descr ibed in the methods s e c t i o n . B r i e f l y , c e l l s were c o l l e c t e d by c e n t r i f u g a t i o n , and washed with BSS f o r the determinat ion of the t o t a l c e I i - a s s o c i a t e d r a d i o a c t i v i t y ( a c i d - s o l u b l e p lus a c i d -i n s o l ub l e mate r i a l ). R e p l i c a t e c u l t u r e s were washed with BSS fo l lowed by TCA f o r the determinat ion of r a d i o a c t i v i t y in a c i d - i n s o l u b l e mater ia l a lone. The amount of a c i d - s o l u b l e r a d i o a c t i v i t y was then c a l c u l a t e d by s u b t r a c t i n g the amount of ac id - i n so I ub Ie r a d i o a c t i v i t y from the amount of t o t a l r a d i o a c t i v i t y . The e f f e c t of acronyc ine on the l a b e l l i n g of a c i d - s o l u b l e and ac i d - i n so I ub I e mate r i a l of L5I78Y c e l l s i s shown in f i g u r e 26. It may be seen t h a t acronyc ine not only reduced the l a b e l l i n g of a c i d - i n s o l u b l e mate r i a l by u r i d i n e , but a l s o reduced the l a b e l l i n g of a c i d - s o l u b l e m a t e r i a l . The extent of t h i s i n h i b i t i o n ( expressed as a percentage of the amount of r a d i o a c t i v i t y incorporated in to a c i d - s o l u b l e or a c i d -i n s o l ub l e mate r i a l in the absence of acronyc ine ) was almost i d e n t i c a l f o r each of these c e l l u l a r f r a c t i o n s . S i m i l a r r e s u l t s were found when the I 4 i n co rpo ra t i on of C-thymidine in to the a c i d - s o l u b l e and ac i d - i n so I ub I e mate r i a l of L5I78Y c e l l s was i n v e s t i g a t e d . E f f e c t of acronyc ine on the t r a n s f e r of r a d i o a c t i v i t y from p r e l a b e l l e d  i n t r a c e l l u l a r a c i d - s o l u b l e pools i n to n u c l e i c a c i d s : The above data i n d i c a t i n g tha t acronyc ine depresses the l a b e l l i n g of the a c i d - s o l u b l e mate r i a l of c e l l s by e x t r a c e l l u l a r nuc leos ides suggests t ha t the drug may i n h i b i t the l a b e l l i n g of n u c l e i c ac ids by i n t e r f e r i n g with p recur sor u t i l i z a t i o n , r a ther than with n u c l e i c a c i d s yn thes i s per se. In o rder to i n v e s t i g a t e d i r ec t I y the p o s s i b i l i t y t ha t acronyc ine might have no e f f e c t on n u c l e i c a c i d po l ymer i za t i on 100 ac ronyc ine concen t r a t i on ( yg/ml ) F igure 26 E f f e c t of ac ronyc ine on the i n co rpo r a t i o n of u r i d i n e i n t o a c i d - s o l u b l e and a c i d - i n s o I ub I e mate r i a l of L5I78Y ceI Is. R e p l i c a t e c u l t u r e s were incubated f o r 45 minutes with 3 H - 5 - u r i d i n e ( f i n a l c oncen t r a t i on 0.056 yM ) s t a r t i n g 30 minutes a f t e r the a d d i t i o n of ac ronyc ine . The i n co rpo r a t i o n of r a d i o a c t i v i t y i n to a c i d - s o l u b l e and a c i d - i n s o l u b I e ma te r i a l was then determined as desc r ibed in the methods s e c t i o n . 101 processes, experiments were done using a technique which a l lows the monitor ing of the i n co rpo ra t i on of i ntraceI IuIar r a d i o a c t i v e precursors i n to n u c l e i c a c i d s . Such a technique avoids amb igu i t i e s due to po s s i b l e drug e f f e c t s on the entry of precursors i n to the c e l l , o r on t h e i r a c t i v a t i o n to the nuc leos ide t r i phosphate i n s i de the c e l l . S c h o l t i s s e k ( 91 ) reported t ha t when ch i ck embryo c e l l s were incubated at reduced temperatures with r a d i o a c t i v e nuc leo s ide s , the c e l l s incorporated the nuc leos ides i n to a c i d - s o l u b l e nuc l eo t i de poo l s , withoudj i n co rpo ra t i ng any label i n to n u c l e i c a c i d s . Chromatographic a n a l y s i s of the a c i d - s o l u b l e mater ia l of c e l l s incubated at reduced temperatures with e x t r a c e l l u l a r nuc leos ides i nd i ca ted t ha t most incorporated r a d i o a c t i v i t y had been phosphoryIated t o the nuc leos ide t r i phosphate form ( 91 ). S i m i l a r f i n d i n g s were reported by Plagemann f o r Nov ikof f hepatoma c e l l s ( 92 ). This author a l s o reported t ha t on removing the e x t r a c e l l u l a r p recur sor and warming the c e l l s to 37° , a s u b s t a n t i a l p ropor t i on of the i n t r a c e l l u l a r r a d i o a c t i v i t y was incorporated i n to ac i d - i n so I ub I e m a t e r i a l . These techniques thus prov ide a method f o r accumulat ing s u b s t a n t i a l q u a n t i t i e s of l a b e l l e d i n t r a c e l l u l a r nuc leo t ide s in the absence of n u c l e i c a c i d s yn the s i s . A drug may then be added, and i t s e f f e c t on n u c l e i c a c i d po l ymer i za t i on processes examined in i s o l a t i o n by i n v e s t i g a t i n g i t s e f f e c t on the t r a n s f e r of r a d i o a c t i v i t y from the p r e l a b e l l e d pool i n to n u c l e i c a c i d s . The i n f l uence of temperature on the i nco rpo ra t i on of r a d i o a c t i v i t y from e x t r a c e l l u l a r u r i d i n e and thymidine in to t o t a l and ac i d - i n so I ub I e mater i a l of L5I78Y c e l l s i s shown in f i g u r e s 27 and 28. For both nuc leo s ide s , as the temperature was reduced the amount of i n co rpo ra t i on i n to ac i d - i n so I ub I e mate r i a l dropped much more r a p i d l y than the amount of 102 i n co rpo ra t i on i n to t o t a l c e l l u l a r m a t e r i a l . In p a r t i c u l a r , i t may be noted t ha t a t temperatures below approximately 10° there was s t i l l s u b s t an t i a l i n co rpo ra t i on of r a d i o a c t i v i t y i n to a c i d - s o l u b l e mate r i a l ( t o t a l minus ac id - i n so I ub Ie ) but e s s e n t i a l l y no i n co rpo ra t i on i n to ac id i n s o l ub l e m a t e r i a l . In the case of u r i d i n e , the nature of the r a d i o a c t i v i present in the a c i d - s o l u b l e pool formed when L5I78Y c e l l s were incubated with the nuc leos ide at 7° was i n ve s t i g a ted by two-dimensional t h i n layer chromatography ( t a b l e I ). Most of the a c i d - s o l u b l e r a d i o a c t i v i t y was a s soc ia ted with the U'FP chromatography s tandard, with l e s se r amounts wi th UDP. In agreement with the r e s u l t s of o ther workers ( 91, 142 ) l i t t l e or no r a d i o a c t i v i t y was incorporated i n to UDP-sugars at the reduced temperature. F igure 29 shows an experiment in which the a c i d - s o l u b l e mate r i a l of L5I78Y c e l l s was p r e l a b e l l e d with "^H-ur idine, and then acronyc ine or act inomycin D added. C e l l s were incubated with r a d i o a c t i v e u r i d i n e f o r 60 minutes a t 7° ; the label was incorporated almost e x c l u s i v e l y i n to the a c i d - s o l u b l e m a t e r i a l . On r a i s i n g the temperature to 37° , about 30 % of the r a d i o a c t i v i t y in t h i s a c i d - s o l u b l e pool was t r a n s f e r r e d i n to the ac i d - i n so I ub I e f r a c t i o n . As expected of a potent i n h i b i t o r of RNA s yn the s i s , act inomycin D almost completely i n h i b i t e d the t r a n s f e r of r a d i o a c t i v i t y from the p r e l a b e l l e d pool to RNA. Acronycine however, even at a concen t ra t i on of 12 yg/ml, had no i n h i b i t o r y e f f e c t on the i n co rpo ra t i on of i n t r a c e l l u l a r nuc leo t ide s i n to ac i d - i n so I ub I e m a t e r i a l . .._ in .-na'p JCO~' i ' • i r e . / j \ ' ' t y "of a * The f a i l u r e of L5I78Y c e l l s to t r a n s f e r more than 30 % of the t o t a l i n t r a c e l l u l a r r a d i o a c t i v i t y i n to a o i d - i n s o l u b l e mater ia l may be due to the ex i s t ence in the c e l l of a second u r i d i n e nuc l eo t i de pool which i s not r e a d i l y a v a i l a b l e f o r n u c l e i c a c i d s yn thes i s ( 92 ) . 150-CL £ Ul *|ioo-x m o o o C L 1_ o o = 50-0 103 ' • 4 *. t o t a l r a d i o a c t i v i t y • \ A \ a c i d - i n s o I u b l e r a d i o a c t i v i t y 40 -1 1 i 2 " t -30 20 10 Temperature ( °C ) F igure 27 Incorporat ion of J H - u r i d i n e i n t o t o t a l and a c i d - i n s o I u b I e mate r i a l of L5I78Y c e l l s a t va r i ou s temperatures. R e p l i c a t e c u l t u r e s a t each temperature were incubated f o r 30 minutes w i th H -5 -u r i d i ne ( f i n a l c oncen t r a t i on 0.038 uM ). At the end of t h i s t ime , the i n c o r p o r a t i o n of r a d i o a c t i v i t y i n t o t o t a l and a c i d - i n s o I u b l e ma te r i a l was determined as desc r ibed in the methods s e c t i o n . 104 15CH CD CL ro e CL XI x i o 100-c O ro s_ o CL L_ o u CD 5 50 i ft A \ \ t o t a l r a d i o a c t i v i t y •ac i d - inso Iub Ie \ r a d i o a c t i v i t y ft. 40 - i 1 r~ 30 20 10 Temperature ( °C ) -©-Frgure 28 I ncorpora t ion of 3 H- thymid ine i n to t o t a l - and a c i d - i n s o l u b l e ma te r i a l of L5I78Y c e l l s a t va r i ous temperatures. R e p l i c a t e c u l t u r e s a t each temperature were incubated f o r 30 minutes w i th ^H-thymidine ( 0.047 uM ). At the end of t h i s p e r i o d , the i n co rpo r a t i on of r a d i o a c t i v i t y i n t o t o t a l and a c i d - i n s o I ub I e mate r i a l was determined as desc r ibed in the methods s e c t i o n . 105 Table I D i s t r i b u t i o n of r a d i o a c t i v i t y w i t h i n the a c i d - s o l u b l e mate r i a l of L5I78Y c e l l s incubated with 3 H - u r i d i n e at 7 ° . Reference Percentage of r a d i o a c t i v i t y compounds recovered with reference compounds Uri d i ne < I UMP < I UDP 12 UD'P-g I ucose - I UDP-N-acety I-g I ucosami ne < -I UTP 84 C y t i d i n e < I CMP and CDP-cho I i ne--- < I CDP < I CTP 2 — CMP and CDP-chol ine are not reso lved Eedubs were incubated with 3 H - 5 - u r i d i n e ( 0.068 uM ) f o r I hour. A c i d - s o l u b l e mate r i a l was i s o l a t e d and a l i q u o t s were:mixed with chromatography standards and chromatographed on c e l l u l o s e t h i n layer chromatography sheets as descr ibed in the methods s e c t i o n . 106 F i gu re 29 E f f e c t of ac ronyc ine and act inomyc in D on the l a b e l l i n g of a c i d - i n s o l u b l e m a t e r i a l a t 37° from an a c i d - s o l u b l e pool p r e l a b e l l e d w i th ^ H - u r i d i n e . A bulk c u l t u r e of L5I78Y c e l l s was incubated a t 7° wi th "^H-5-ur idine ( f i n a l c oncen t r a t i on 0.056 yM ) f o r 60 minutes. The c e l l s were then washed f r e e of e x t r a c e l l u l a r r a d i o a c t i v e u r i d i n e by resuspens ion in p r e c u r s o r - f r e e medium at 7 ° . Acronyc ine o r act inomyc in D s o l u t i o n o r medium was added t o a l i q u o t s of the c u l t u r e , which were then incubated f i r s t a t 7° f o r 15 minutes and then at 3 7° . Tota l and a c i d - i n s o I ub I e r a d i o a c t i v i t y were measured a t the i n d i c a t e d t imes as desc r ibed in the methods s e c t i o n . 107 washing A c r 0 -0 20 40 60 80 100 I N C U B A T I O N T I M E ( M I N ) F igure 30 E f f e c t of ac ronyc ine on the l a b e l l i n g of a c i d - i n s o I ub I e mate r i a l a t 37° from an a c i d - s o l u b l e pool p r e l a b e l l e d 14 w i th C - thymid ine. A bulk c u l t u r e of L5I78Y c e l l s was incubated a t 7° w i th 14 C-thymidine ( 1.3 yM ) f o r 60 minutes. The c e l l s were then washed f r e e of e x t r a c e l l u l a r r a d i o a c t i v e thymid ine by resuspens ion in p recu r so r f r e e medium a t 7 ° . Acronyc ine s o l u t i o n or medium was added t o a l i q u o t s of the c u l t u r e , which were then incubated f i r s t . a t 7° f o r 15 minutes , and then at 3 7° . Tota l and a c i d - i n s o I ub I e r a d i o a c t i v i t y were measured at the i n d i c a t e d t imes as desc r ibed in the methods s e c t i o n . 108 This i s in sharp con t r a s t t o the a b i l i t y of acronyc ine to markedly i n h i b i t the i nco rpo ra t i on of extrace I IuIar u r i d i n e i n to ac i d - i n so I ub I e mater i a l w i t h i n minutes of i t s a d d i t i o n t o a c u l t u r e . As the incubat ion at 37° cont inued the t o t a l i n t r a c e l l u l a r r a d i o a c t i v i t y in the c u l t u r e s s l i g h t l y d e c l i n e d . Th is loss of r a d i o a c t i v i t y was the same f o r t r ea ted and con t ro l c u l t u r e s , suggest ing t ha t the a l k a l o i d had not induced the leakage of l a b e l l e d nuc leo t i de s from the c e l l s . I 4 The r e s u l t s of a s i m i l a r p r e l a b e l l i n g experiment using C-thymidine instead of ^H-u r id ine are g iven in f i g u r e 30. When L5I78Y c e l l s were I 4 p r e l a b e l l e d by incubat ion with C-thymidine at 7° f o r I hour, the label was incorporated almost e x c l u s i v e l y i n to the a c i d - s o l u b l e m a t e r i a l . Wi th in 30 minutes of r a i s i n g the temperature to 37° , c. 80 % of the a c i d -s o l ub l e r a d i o a c t i v i t y in the con t ro l c e l l s was incorporated i n to a c i d -i n s o l ub l e m a t e r i a l . Acronyc ine d id not i n h i b i t the l a b e l l i n g of the DNA from the p r e l a b e l l e d precur sor p oo l , even at the high concent ra t i on of 12 ug/ml, nor d id i t induce the leakage of r a d i o a c t i v i t y from the ceI Is. E f f e c t of acronyc ine on the d i s t r i b u t i o n of r a d i o a c t i v i t y w i t h i n the  a c i d - s o l u b l e pool of c e l l s l a b e l l e d with ^H-uridi.ne: In view of the p o s s i b i l i t y t ha t acronyc ine might i n h i b i t the i n co rpo ra t i on of u r i d i n e i n to a c i d - i n so I ub I e mate r i a l by d i s t u r b i n g the i n t r a c e l l u l a r phosphory la t ion steps between UMP and UTP, an examination was made of the e f f e c t of the a l k a l o i d on the d i s t r i b u t i o n of r a d i o a c t i v i t y w i t h i n the a c i d - s o l u b l e pool of c e l l s l a b e l l e d with ^H -u r i d i ne . C e l l s were incubated with the precursor f o r 45 minutes in the presence of va r ious concen t ra t i on s . o f ac ronyc ine , then c h i l l e d to 0° and c o l l e c t e d by c e n t r i f u g a t i o n . The a c i d - s o l u b l e pools of the c e l l samples were 109 ex t r ac ted and analyzed by two-dimensional t h i n layer chromatography as descr ibed in the methods s e c t i o n . Table II shows the d i s t r i b u t i o n of r a d i o a c t i v i t y w i t h i n the a c i d - s o l u b l e mate r i a l of t r e a t ed and con t ro l c u l t u r e s of L5I78Y c e l l s . Very l i t t l e r a d i o a c t i v i t y was a s soc ia ted wi th the u r i d i n e , c y t i d i n e , CMP, o r CDP-chol ine reference compounds. Of the t o t a l r a d i o a c t i v i t y recovered with the chromatography standards the ma jo r i t y was present as UTP ( 52.-57 % ), with l e s se r amounts in UDP ( 9 % ), UDP-glucose ( 6-8 % ), UDP-N-acetyI-gIucosamine ( 9-10 % ), and UMP ( 3-4 % ). In c on t r a s t t o the r e s u l t s of Plageman with hepatoma c e l l s ( 96 ), in the L5I78Y c e l l l i n e s u b s t a n t i a l amounts of r a d i o a c t i v i t y were found in c y t i d i n e n u c l e o t i d e s , mainly as CTP ( 11-12 % ). Although acronyc ine g r e a t l y reduced the amount of r a d i o a c t i v i t y incorporated in to a c i d - s o l u b l e mate r i a l of L5178Y c e l l s ( by 45 l a n d 84 % a t 3 and 12 pg/ml r e s p e c t i v e l y ), the d i s t r i b u t i o n of r a d i o a c t i v i t y among the nuc leo t i de s was e s s e n t i a l l y unchanged. This suggests t ha t the drug d id not i n h i b i t e i t h e r the phosphory la t ion of u r i d i n e nuc leo t ide s w i t h i n the c e l l , o r t h e i r convers ion t o c y t i d i n e nuc l eo t i de s . K i n e t i c s of the i n h i b i t i o n by acronyc ine of the i nco rpo ra t i on of u r i d i n e  and thymidine i n to t o t a l c e l l u l a r mate r i a l : The degree to which acronyc ine i n h i b i t s the i n co rpo ra t i on of nuc leos ides i n to ac i d - i n so I ub I e and a c i d - s o l u b l e mate r i a l i s the same ( as shown f o r example in f i g u r e 26 ). It t he r e f o r e appears v a l i d to use the i n h i b i t i o n of the i n co rpo ra t i on of nuc leos ides i n to the t o t a l of a c i d - s o l u b l e p lus a c i d - i n so I ub I e mate r i a l as a measure of t h i s e f f e c t of the drug. For convenience, t h i s i n co rpo ra t i on ( which represents the t o t a l accumulat ion by c e l l s of nuc leos ides as non -d i f f u s i n g Before c e l l s are analyzed f o r e i t h e r a c i d - i n so I ub I e or t o t a l r a d i o a c t i v i t y I 10 Table II E f f e c t of acronyc ine on the d i s t r i b u t i o n of r a d i o a c t i v i t y w i t h i n the a c i d - s o l u b l e mater ia l of L5I78Y c e l l s incubated wi th 3 H - u r i d i n e . Reference Percentage of r a d i o a c t i v i t y a compounds recovered with reference compounds contro1 aero.., 3'ryg/rri 1 aero. 12 ug/ml Uri d i ne < 1 < 1 < 1 UMP 3 4 4 UDP 9 9 9 UDP-g1ucose 8 8 6 UDP-N-acety1-g1ucosam i ne 9 10 9 UTP 54 52 57 Cy t i d i ne < 1 < 1 < 1 CMP plus CDP-cho1ine - < 1 < 1 < 1 CDP 4 4 3 CTP 12 12 1 1 — The t o t a l a c i d - s o l u b l e r a d i o a c t i v i t y recovered from 4 x 10 6 ce 1 1 s was 3.8 x 10^ dpm, 2.1 x 10 dpm, and 0.6 x 10 dpm f o r the c u l t u r e s incubated with 0, 3, and 12 ug/ml of acronyc ine r e s p e c t i v e l y . — CMP and CDP-cho1ine are not r e so l ved . C e l l s were incubated with ^H-5-ur i d i ne ( 0.08 yM ) f o r 45 minutes s t a r t i n g 30 minutes a f t e r the a d d i t i o n of ac ronyc ine . A c i d - s o l u b l e ma te r i a l was i s o l a t e d and a l i q u o t s were mixed with chromatography standards and chromatographed on c e l l u l o s e t h i n layer chromatography sheets as descr ibed in the methods s e c t i o n . I 11 metabo l i te s ) w i l l he rea f te r be r e f e r r ed t o in t h i s t h e s i s as nuc leo -s ide " uptake " . The term " uptake " w i l l a l s o be used in t h i s t h e s i s to desc r ibe the t o t a l accumulat ion by c e l l s of o ther compounds as n o n - d i f f u s i n g metabo l i t e s . As a p r e l im i na r y to i n v e s t i g a t i o n s of the k i n e t i c s of the e f f e c t of acronyc ine on the uptake of u r i d i n e and thymid ine, experiments were done t o examine the v a l i d i t y of using the amount of nuc leos ide incorporated in a set t ime i n t e r v a l as a measure of the ra te of nuc leos ide uptake. F igure 31 demonstrates the r e l a t i o n s h i p between the i n co rpo ra t i on of "^H-uridine i n to t o t a l c e l l u l a r mate r i a l and t ime. The extent of the i n co rpo ra t i on was a near ly l i n e a r f unc t i on of the length of the incubat ion pe r i od . At the end of a 60 minute^per iod, the c e l l s had taken up approximately 15 % of the u r i d i n e in the medium. The s l ggh t curvature observable in the p l o t i s most probably due to t h i s ' d ep l e t i on in s ub s t r a t e . It may be noted t ha t the ra te of i n co rpo ra t i on does not drop as r a p i d l y as would be expected from the dep le t i on of subs t ra te - t h i s i s most probably due t o the increase in c e l l number dur ing the incubat ion which would ac t as a compensating f a c t o r . A 30 minute incubat ion per iod wi th u r i d i n e was chosen f o r the e s t ima t i on of the ra te of u r i d i n e uptake. While t h i s procedure may po s s i b l y s l i g h t l y underest imate the i n i t i a l ra te of u r i d i n e uptake, the f a c t t ha t the graph of u r i d i n e uptake vs. t ime can be ex t r apo l a ted l i n e a r l y through the o r i g i n suggests such an e r r o r i s l i k e l y very s m a l l . S i m i l a r r e s u l t s I 4 were obta ined when the i n co rpo ra t i on of C-thymidine i n to t o t a l c e l l u l a r i t i s f i r s t necessary to wash them with BSS in order to remove unincorporated e x t r a c e l l u l a r r a d i o a c t i v i t y ( see methods s ec t i on ). The term " n o n - d i f f u s i n g " as used here r e f e r s to mate r i a l which i s r e ta ined by c e l l s dur ing t h i s washing procedure. F igure 31 R e l a t i o n s h i p between the i n co rpo r a t i on of u r i d i n e i n to the t o t a l c e l l u l a r ma te r i a l of L5I78Y c e l l s and t ime . C e l l s were incubated w i th 3 H - 5 - u r i d i n e ( 0.004 yM ) f o r the i nd i ca ted pe r i od s , and the i n co rpo r a t i on of r a d i o a c t i v i t y i n to the t o t a l c e l l u l a r ma te r i a l determined as desc r ibed in the methods s e c t i o n . I 13 mate r i a l was examined as a f unc t i on of the length of the incubat ion pe r i od . The r e l a t i o n s h i p between the i n h i b i t i o n of u r i d i n e uptake by acronyc ine and the concent ra t i on of u r i d i n e i s shown in f i g u r e 32. The data are g iven as a Lineweaver-Burk p l o t ( r e c i p r o c a l of the ra te of r eac t i on vs. the r e c i p r o c a l of the subs t ra te concent ra t i on ). The accumulat ion of u r i d i n e as n o n - d i f f u s i n g metabo l i te s by L5I78Y c e l l s obeyed s imple MichaeIis-_Menten k i n e t i c s . Acronycine behaved as a s imple compet i t i ve i n h i b i t o r of u r i d i n e uptake, r a i s i n g the of the r eac t i on but not a l t e r i n q the V . Thus as the concent ra t i on of e x t r a c e l l u l a r 3 max u r i d i n e was r a i s e d , the degree of i n h i b i t i o n of u r i d i n e uptake dropped u n t i l a t 1/ ( u r i d i n e c oncen t r a t i on ) ^ 0 , ( i n f i n i t e u r i d i n e concent ra t i on ) there was no i n h i b i t i o n of uptake ( V was una l tered ). S i m i l a r r max r e s u l t s were obta ined f o r the i n h i b i t i o n by acronyc ine of the accumulat ion of r a d i o a c t i v e thymidine as n o n - d i f f u s i n g metabo l i te s ( f i g u r e 33 ). - P i 1 ' , . _ ' • ' r ? .. +he i , ' o i l ~ u t ' ! •' '.3 Re l a t i on sh i p between the i n h i b i t i o n o f u r i d i n e uptake by acronyc ine  and the concent ra t i on of serum in c u l t u r e media : Experiments in s e c t i on I of t h i s t h e s i s have i nd i ca ted t h a t : i ) acronyc ine i s bound to one or more non-dia IysabIe components of the horse serum used to supplement F i s c h e r ' s medium i i ) the b ind ing of acronyc ine to L5I78Y c e l l s , and the g r o w t h - i n h i b i t o r y p r ope r t i e s of the a l k a l o i d in L5I78Y c u l t u r e s are a f f e c t e d by the concen t ra t i on of horse serum used to supplement the medium in which the c e l l s are grown. For these reasons, the r e l a t i o n s h i p between serum The term compet i t i ve i n h i b i t o r as used in t h i s t h e s i s r e f e r s s t r i c t l y to the ki net i cs of the i n h i b i t i o n , and i s not meant t o imply any p a r t i c u l a r molecu lar mechan i sm of i n h i b i t i o n . Th is po in t w i l l be f u r t h e r d e a l t with in the d i s cu s s i on at the end of s e c t i on 3 of the r e s u l t s . F igure 32 K i n e t i c s of the i n h i b i t i o n of u r i d i n e uptake by ac ronyc ine . ! L5I78Y c u l t u r e s were incubated wi th ^H -5 -u r i d i ne f o r 30 minutes in the presence of va ry ing concen t ra t i on s of ac ronyc ine . At the end of t h i s t ime , the i n co rpo ra t i on of r a d i o a c t i v i t y i n t o t o t a l c e l l u l a r ma te r i a l was determined as desc r ibed in the methods s e c t i o n . ( thymidine concen t r a t i on ) ( 1 0 x M ) F igure 33 K i n e t i c s of the i n h i b i t i o n of thymid ine uptake by ac ronyc ine . I 4 L5I78Y c u l t u r e s were incubated wi th C-thymidine f o r 30 minutes in the presence of va ry ing concen t ra t i on s of ac ronyc ine . At the end of t h i s t ime , the i n co rpo r a t i on of r a d i o a c t i v i t y i n t o t o t a l c e l l u l a r mate r i a l was determined as desc r ibed in the methods s e c t i o n . I 16 concent ra t i on and the e f f e c t of acronyc ine on u r i d i n e uptake in L5I78Y c e l l s was a l s o i n v e s t i g a t e d . F igure 34 shows the i n h i b i t i o n of u r i d i n e uptake at two acronyc ine concent ra t i ons in the presence of va r ious concent ra t ions of horse serum. It may be seen from the data t abu l a ted at the bottom of f i g u r e 34 t ha t the uptake of u r i d i n e was somewhat a f f e c t e d by the concent ra t i on of horse serum ; wh i l e the amount of uptake was r e l a t i v e l y independent of serum concent ra t i on in the range of 5 to 20 % serum, uptake was s i g n i f i c a n t l y less at serum concent ra t i on s of 0 and I %. The degree of i n h i b i t i o n of u r i d i n e uptake by acronyc ine va r i ed markedly w i th the concen t ra t i on of horse serum present . At the concent ra t i on of serum normal ly present in L5I78Y c u l t u r e s ( 10 % ) the uptake of u r i d i n e was i n h i b i t e d c_. 50 % by acronyc ine at a concent ra t i on of 3 yg/ml, and £. 85$% by acronyc ine at a concent ra t i on of 12 yg/ml. At h igher serum concen t r a t i on s , the degree of i n h i b i t i o n o f u r i d i n e uptake was lower, and at serum concent ra t i ons below normal l e ve l s the degree of i n h i b i t i o n of uptake was h igher , reaching a va lue of 99 % at an acronyc ine concent ra t i on of 12 yg/ml in the absence of serum. R e v e r s i b i l i t y of the i n h i b i t i o n of u r i d i n e uptake : It has been i nd i ca ted in s e c t i on I of t h i s t h e s i s t ha t on washing I 4 L5I78Y c e l l s w i th f re sh medium, ceI I -assoc iated C-acronyc ine i s r e a d i l y removed. The r e v e r s i b i l i t y of the e f f e c t of acronyc ine on u r i d i n e uptake was t he re f o re examined ( f i g u r e 35 ). C e l l s were incubated with acronyc ine ( 0, 3, or 12 yg/ml ) f o r I hour. The uptake of u r i d i n e by c e l l s in the presence of acronyc ine was measured by incubat ing a l i q u o t s of the c u l t u r e s w i th 3 H - u r i d i n e f o r a 30 minute per iod beginning 15 minutes, a f t e r the a d d i t i o n of the drug. At the end of the I hour incubat ion I 17 acronyc ine concen t r a t i on ( yg/ml ) F i gu re 34 In f luence of horse serum concen t r a t i on on the i n h i b i t i o n by acronyc ine of u r i d i n e uptake. L5I78Y c u l t u r e s were incubated w i th ^H -5 -u r i d i ne ( 0.004 yM ) f o r 30 minutes in the presence of va ry ing concen t r a t i on s of ac ronyc ine and horse serum, as i n d i c a t e d . At the end of t h i s t ime , the i n co rpo r a t i o n of r a d i o a c t i v i t y i n t o t o t a l c e l l u l a r ma te r i a l was determined as desc r ibed in the methods s e c t i o n . Data are expressed as a percentage i n h i b i t i o n r e l a t i v e t o c o n t r o l va lues a t each concen t r a t i on of horse serum in the absence of acronyc ine - these are t abu l a t ed below. concen t r a t i on of horse serum u r i d i n e uptake in c o n t r o l s ( dpm/30 m i n / m i l l i o n c e l l s ) 20 % 8.30 x I 0 4 15 % 8.24 " 10 % ( normal cone. ) 8.46 " 5 % 7.92 " 1 % 7.40 1 1 0 % 4.52 I 18 F igure 35 Recovery of u r i d i n e uptake on removal of ac ronyc i ne . A bulk c u l t u r e of L5I78Y c e l l s was incubated w i th acronyc ine ( 0, 3, o r 12 yg/ml ) f o r I hour. At the end of t h i s t ime , the c e l l s were c o l l e c t e d by c e n t r i f u g a t i o n , washed 2 t imes in f r e sh medium, and resuspended in f re sh medium. At i n t e r v a l s , a l i q u o t s of c e l l s were removed and incubated wi th 3 H - 5 - u r i d i n e ( 0.004 yM ) f o r 30 minutes, and the i n co rpo ra t i on of r a d i o a c t i v i t y i n t o t o t a l c e l l u l a r mate r i a l determined as desc r ibed in the methods s e c t i o n . I 19 p e r i o d , the c e l l s in the c u l t u r e s were c o l l e c t e d by c e n t r i f u g a t i o n , washed, and resuspended in f re sh medium. The uptake of u r i d i n e in a 30 minute per iod was then measured at i n t e r v a l s . F igure 35 shows t ha t the i n h i b i t i o n of u r i d i n e uptake observable in the presence of acronyc ine was r e a d i l y r e v e r s i b l e on removing the drug by resuspending the c e l l s in f re sh medium ; u r i d i n e uptake returned t o con t ro l l e ve l s w i t h i n 30 minutes of the removal of the drug. E f f e c t of acronyc ine on the uptake of compounds o ther than nuc leos ides : In view of the a b i l i t y of acronyc ine t o s t r ong l y i n h i b i t the uptake of e x t r a c e l l u l a r nuc leo s ide s , the e f f e c t of the drug on the uptake of o ther compounds was examined. The e f f e c t of acronyc ine on the i nco rpo ra t i on of c h o l i n e and i n o s i t o l i n to the t o t a l c e l l u l a r mate r i a l of L5I78Y c e l l s i s shown in t a b l e III . In c on t r a s t to nuc leo s ide s , these compounds are normal ly present in the t i s s u e c u l t u r e medium in which the c e l l s grow. Acronyc ine at a concent ra t i on of 3 yg/ml ( a concent ra t i on which a f f e c t s c e l l growth and which has marked e f f e c t s on the uptake of nuc leos ides ) had no s t a t i s t i c a l l y s i g n i f i c a n t e f f e c t on the uptake of e i t h e r c ho l i n e o r i n o s i t o l . At a concent ra t i on of 12 yg/ml, acronyc ine i n h i b i t e d the uptake of cho l i ne by 28 % and of i n o s i t o l by 12 % - these d i f f e r e n c e s were s t a t i s t i c a l l y s i g n i f i c a n t . The e f f e c t of acronyc ine on the uptake of the g lucose analog, 2-deoxygIucose, i s shown in t a b l e IV. Th is compound i s apparent ly t ran spor ted in to mammalian c e l l s and phos-phory I ated by the same mechanisms as g lucose ( 118 ), but i s not f u r t h e r u t i l i z e d . In c on t r a s t t o the e f f e c t s of acronyc ine on the uptake of nuc leo s ide s , c h o l i n e , and i n o s i t o l , the uptake of deoxyglucose by L5I78Y c e l l s was s l i g h t l y s t imu la ted by the a l k a l o i d a t a l l concent ra t ions t e s t e d . Table III E f f e c t of acronyc ine on the i nco rpo ra t i on of c ho l i n e and i n o s i t o l i n to t o t a l c e l l u l a r mate r i a l of L5I78Y ceI Is. Acronyci ne concen t ra t i on ( yg/ml ) Incorporat ion of precursors i n to t o t a l c e l l u l a r mate r i a l ( 10 ^ x dpm/30 m in/m i l l i o n c e l l s ± S.D. ) cho I i ne i nos i t o I 3 12 43.05 ± 0.84 41 .57 ± 0.57 - — 3 I . 09 ± 0.50 - ° ~ 8.37 ± 0 . 1 6 8.22 ± 0.14 -7.34 ± 0.15 -— D i f f e r ence from con t ro l not s i g n i f i c a n t ( p = 0.05 ) using Student!s t t e s t ^Tuaenr'r r -•2sr — D i f f e r ence from con t ro l i s s i g n i f i c a n t ( p = 0.01 ) using S tudent ' s t t e s t H-cho l ine ( 0.87 yM ) o r H - i n o s i t o l ( 0.29 yM ) was.added to L5I78Y c u l t u r e s 15 minutes a f t e r the a d d i t i o n of ac ronyc ine . The c u l t u r e s were incubated f o r 30 minutes, then c h i l l e d and the i nco rpo ra t i on of r a d i o a c t i v i t y i n to t o t a l c e l l u l a r mate r i a l determined as descr ibed in the methods s e c t i o n . Data are the mean ± S.D. of three dete rminat ions . 121 Table IV E f f e c t of acronyc ine on the i nco rpo ra t i on of deoxygIucose in to the t o t a l e e l l u l a r mate r i a l of L5I78Y eel Is. Acronycine Incorporat ion of deoxygIucose in to t o t a l c e l l u l a r concent ra t i on mate r i a l ( 10 3 x dpm/30 m in/m i l l i o n c e l l s ± S.D. ) ( yg/ml ) i ncorporat i on percent of con t ro l 0 35.02 ± 0.80 100 % 3 40.41 ± 0.78 115 ? 6 42.41 ± 0.97 121 % 9 42.14 ± 1.32 120 % 12 39.59 ± 1.30 1 13 % 2-deoxygIucose- H ( 0.15 yM ) was added to L5I78Y c u l t u r e s 15 minutes a f t e r the a d d i t i o n of ac ronyc ine . The c u l t u r e s were incubated f o r 30 minutes, then c h i l l e d and the i nco rpo ra t i on of r a d i o a c t i v i t y i n to t o t a l c e l l u l a r mater ia l determined as descr ibed in the methods s e c t i o n . Data are the mean ± S.D. of f i v e dete rminat ions . A l l d i f f e r e n c e s from the con t r o l va lues are s t a t i s t i c a l l y s i g n i f i c a n t ( p = 0.01, using S tudent ' s t t e s t ). 122 D i scuss ion - Sec t ion 2 Acronycine was found to markedly i n h i b i t the i nco rpo ra t i on of u r i d i n e in to the RNA of both L5I78Y and IRC ceI Is ( f i g u r e 21 ). The i n c o r -po ra t i on of r a d i o a c t i v e thymidine i n to DNA was a l s o a f f e c t e d by the drug, but to a much le s se r ex ten t . Experiments in s e c t i on I of t h i s t h e s i s i nd i ca ted t h a t acronyc ine at a concent ra t i on of 6 yg/ml had no e f f e c t on the ra te of l a b e l l i n g of DNA by thymidine dur ing the S phase of a p a r t i a l l y synchronized IRC c u l t u r e . However, the experiments in s e c t i on 2 of t h i s t h e s i s with asynchronous c u l t u r e s of t h i s c e l l l i n e a t the same a l k a l o i d concent ra t i on i nd i ca ted t ha t acronyc ine i n h i b i t e d the i n co rpo ra t i on of thymidine i n to ac i d - i nso I ub I e mater i a l by c_. 20 % ( f i g u r e 21 ). Th is apparent d i screpancy may be at l ea s t p a r t l y the r e s u l t of the f a c t t ha t acronyc ine can extend the G| phase of c e l l s ( s e c t i on I, f i g u r e s II and ;i!2 ) without otherwise a f f e c t i n g the progress of c e l l s through the S phase of the c e l l c y c l e . Thus the i n h i b i t i o n of thymidine i n co rpo ra t i on seen in asynchronous c u l t u r e s of IRC c e l l s at an a l k a l o i d concent ra t i on of 6 yg/ml may s imply be the r e s u l t of an acronyc ine- induced reduct ion in the number of c e l l s in the S phase of the c e l l c y c l e dur ing the time of l a b e l l i n g , ra ther than an i n h i b i t i o n of the ra te of thymidine i n co rpo ra t i on i n to the DNA of i n d i v i d u a l c e l l s in the S phase. There was a p a r a l l e l between the i n h i b i t i o n of u r i d i n e i nco rpo ra t i on in to RNA and the g r o w t h - i n h i b i t o r y p r ope r t i e s of ac ronyc ine . C e l l popu la t ion growth in L5I78Y and IRC c u l t u r e s ( s e c t i on I, f i g u r e 8 ) was i n h i b i t e d on ly a t drug concent ra t i ons which were s u f f i c i e n t to markedly i n h i b i t the i n co rpo ra t i on of u r i d i n e in to RNA ( s e c t i on 2, f i g u r e 21 ). Furthermore, the r e l a t i v e s e n s i t i v i t i e s of the two c e l l l i ne s w i th respect t o the 123 i n h i b i t i o n of u r i d i n e i nco rpo ra t i on i n to RNA ( s e c t i on 2 ) p a r a l l e l e d the r e l a t i v e s e n s i t i v i t i e s of the c e l l l i ne s with respect t o the i n h i b i t i o n of popu la t ion growth ( s e c t i on I) - in each case the L5I78Y c e l l l i n e was the more s e n s i t i v e of the two. In view of t h i s apparent c o r r e l a t i o n i t seemed l i k e l y a t t h i s po i n t in the exper imental work t ha t the c y t o t o x i c p r ope r t i e s of the a l k a l o i d might stem from an a b i l i t y t o i n t e r f e r e with RNA s yn the s i s . Such an i n h i b i t i o n might d i r e c t l y account f o r the a b i l i t y of acronyc ine to delay the i n i t i a t i o n of the S phase in synchronized IRC c u l t u r e s ( as noted in the d i s cu s s i on to s ec t i on I ). Ac ronyc ine, an a c r i d i n e d e r i v a t i v e , bears some s t r u c t u r a l s i m i l a r i t i e s to a number of compounds which i n h i b i t n u c l e i c ac id s yn thes i s by complexing wi th DNA and thus reducing i t s a c t i v i t y as a template f o r DNA or RNA synthes i s ( see i n t r o d u c t i o n , f i g u r e 4 f o r s t r u c t u r a l formulae of some of these compounds ). However, when acronyc ine was examined in v i t r o in systems which are useful f o r de tec t i n g DNA-drug i n t e r a c t i o n , there was no evidence t ha t the a l k a l o i d i n t e r ac ted with DNA. Thus there was no change in the absorpt ion spectrum of acronyc ine when i t was incubated with DNA ( f i g u r e ' 2 2 ) and the me l t ing temperature of the l a t t e r was unaf fected by the a l k a l o i d ( f i g u r e 23 ). Furthermore, acronyc ine had no e f f e c t on the sy.n theses c©;f RNA in v i t r o from the nuc leos ide t r i phosphates using a system - E_. co I i RNA polymerase and c a l f thymus DNA - which i s s e n s i t i v e to changes in template a c t i v i t y induced by DNA-drug i n t e r a c t i o n s ( f i g u r e 25 ). None of these observat ions o f f e r s any support f o r the suggest ion t ha t acronyc ine i n h i b i t s n u c l e i c a c i d s yn thes i s by i n t e r a c t i n g with DNA and thereby i n h i b i t i n g i t s template a c t i v i t y . 124 The r e s u l t s of the E_. co I i RNA polymerase experiment do not r u l e out the p o s s i b i l i t y t ha t acronyc ine might a f f e c t one or more n u c l e i c ac id polymerases of L5I78Y c e l l s - such an e f f e c t might go undetected in an assay system using a b a c t e r i a l .polymerase. Strong evidence t h a t , in whole c e l l s , acronyc ine does not d i r e c t l y i n h i b i t DNA or RNA s yn thes i s per se, e i t h e r by a f f e c t i n g n u c l e i c a c i d polymerases or o the rw i se , i s provided by experiments in which e f f e c t s of the a l k a l o i d on the entry and a c t i v a t i o n of nuc leos ides can be d i f f e r e n t i a t e d from e f f e c t s on the u t i l i z a t i o n of these a c t i v a t e d precursor s f o r n u c l e i c ac id s yn the s i s . It has been found, as have others with d i f f e r e n t c e l l l i ne s ( 91, 92 ) t ha t when L5I78Y c e l l s are incubated at a reduced temperature with r a d i o a c t i v e u r i d i n e or thymid ine, the n u c l e i c a c i d p recur sor pools can be l a b e l l e d without a s i g n i f i c a n t amount of the label appearing in the n u c l e i c a c i d s . In the case of u r i d i n e , the nature of the r a d i o a c t i v i t y appearing in the a c i d - s o l u b l e mate r i a l of c e l l s l a b e l l e d at 7° wi th the nuc leos ide was i n ve s t i g a ted by chromato-graphy. In agreement with the r e s u l t s of others ( 91, 142 ), most of the incorporated r a d i o a c t i v i t y was in the form of UTP, with l i t t l e or no nuc leo t i de sugars being formed ( t a b l e I ) ) . When L5I78Y c e l l s which had been p r e l a b e l l e d at 7° in t h i s manner were returned to p r e c u r s o r - f r e e medium and warmed t o 37° , s ub s t an t i a l amounts of the r a d i o a c t i v i t y in the i n t r a c e l l u l a r nuc leo t i de pool was t r a n s f e r r e d in to n u c l e i c ac id s ( f i g u r e s 29 and 30 ). When s tud ied in t h i s manner, both RNA and DNA synthes i s were completely unaf fected by acronyc ine even at a concent ra t i on of 12 yg/ml.. The f a i l u r e of acronyc ine t o i n h i b i t the t r a n s f e r of r a d i o a c t i v i t y from a p r e l a b e l l e d i ntraceI IuIar nuc l eo t i de pool i n to n u c l e i c ac id s suggests t ha t the i n h i b i t i o n by 125 acronyc ine of the i nco rpo ra t i on of extrace I 1uIar u r i d i n e o r thymidine i n to the l a t t e r i s not due to any i n h i b i t i o n of the n u c l e i c a c i d p o l y -mer i za t i on process i t s e l f . The i n h i b i t i o n must t he re f o re be due t o a d i s turbance of a t l ea s t one of the steps in the pathway from e x t r a c e l l u l a r nuc leos ides t o the nuc leos ide t r i pho spha te s . Support ing t h i s suggest ion i s the f i n d i n g t h a t acronyc ine markedly i n h i b i t e d the i n co rpo ra t i on of e x t r a c e l l u l a r u r i d i n e in to i n t r a c e l l u l a r a c i d - s o l u b l e mate r i a l ( f i g u r e 26 ). Th is i n h i b i t i o n of the l a b e l l i n g of the i n t r a c e l l u l a r nuc leo t i de pool appeared s u f f i c i e n t to account e n t i r e l y f o r the reduced i n c o r -po ra t i on of e x t r a c e l l u l a r u r i d i n e i n to RNA. Although acronyc ine markedly reduced the l a b e l l i n g of the a c i d -s o l ub l e mate r i a l of L5I78Y c e l l s by e x t r a c e l l u l a r u r i d i n e , i t d id not a l t e r the r e l a t i v e amounts of the var ious i n t r a c e l l u l a r phosphoryIated d e r i v a t i v e s ( t a b l e I P ) . This suggests t ha t the nuc leo t i de k inases invo lved in the phosphory lat ion of UMP to h igher phosphoryIated d e r i v a t i v e s were not a f f e c t e d by ac ronyc ine . Since the phosphory la t ion r eac t i on s r equ i re ATP, and s i nce i t has been shown.that a dep l e t i on of c e l l u l a r ATP supp l i e s markedly i n h i b i t s these i n t r a c e l l u l a r phosphory lat ion r eac t i on s ( 104 ), the data a l s o suggest t ha t the immediate energy s upp l i e s of the c e l l were not a f f e c t e d by acronyc ine. It was a l s o found t ha t acronyc ine d id not induce leakage of r a d i o a c t i v e 'muc<heo;lii des from the c e l l s ( f i g u r e s 29 and 30 ). Th is suggests t h a t the a l k a l o i d had ne i t he r i n t e r f e r e d with the f unc t i on of the plasma membrane with respect to i t s a b i l i t y t o r e t a i n nuc leos ide phosphates, nor had induced the i n t r a c e l l u l a r re lease of l y t i c enzymes ( e-.igb from lysosomes ) such as nuc leo t ida ses or phosphates which might l i b e r a t e nuc leos ides or bases which would then be l o s t when the c e l l s were washed with BSS. The above 126 f i nd i n g s suggest t ha t the reduced l a b e l l i n g of the nuc leo t i de pools by nuc leos ides in the presence of acronyc ine may be due mainly or e n t i r e l y t o an i n t e r f e rence with the entry of the e x t r a c e l l u l a r nuc leo -s ides i n to c e l l u l a r metabolism.' This imp l ie s t ha tcac ronyc i ne i n h i b i t s e i t h e r the passage of u r i d i n e through the plasma membrane, or i t s i n i t i a l phosphory la t ion to UMP. Experiments designed to i n v e s t i g a t e which of these mechanisms i s involved in the i n h i b i t i o n of u r i d i n e uptake by acronyc ine are descr ibed in s e c t i on 3 of t h i s t h e s i s . I n i t i a l i n v e s t i g a t i o n s of the e f f e c t of acronyc ine on the i n co rpo ra t i on of u r i d i n e and thymidine i n to the DNA and RNA of L5I78Y and IRC c e l l s i nd i ca ted t ha t the i n co rpo ra t i on of u r i d i n e i n to RNA was much more a f f e c t e d than t ha t of thymidine in to DNA ( f i g u r e 21 ). I 4 In these exper iments, C-thymidine had been used at much h igher concen-t r a t i o n s than "^H-uridine because i t had a lower s p e c i f i c a c t i v i t y ( e .g . ' f i g u r e 21 : u r i d i n e c o n c e n t r a t i o n , 0.096 yM ; thymidine c o n c e n t r a t i o n , 1.6 yM ). Subsequent k i n e t i c experiments ( f i g u re s 32 and 33 ) showed tha t the i n h i b i t i o n by acronyc ine of the i nco rpo ra t i on of both u r i d i n e and thymidine in to t o t a l c e l l u l a r mate r i a l was of a compet i t i ve nature, being less at h igher nuc leos ide concen t r a t i on s . It thus appears t ha t the apparent s e l e c t i v e e f f e c t of acronyc ine on the i n co rpo ra t i on of u r i d i n e in to RNA i s an a r t i f a c t . When the e f f e c t of acronyc ine on thymidine uptake i s determined at low nuc leos ide concentrat ions^, the i n h i b i t i o n of i t s i n co rpo ra t i on i s as g reat as t ha t of u r i d i n e . Thus the data of f i g u r e 33 i n d i c a t e t ha t a t a thymidine concen t ra t i on of 0.1 yM ( " 10 " on the ho r i z on t a l a x i s of f i g u r e 33 ), acronyc ine at a concent ra t i on of 3 yg/ml i n h i b i t s thymidine uptake by 45 % ( compared wi th an i n h i b i t i o n of c. 10 % seen in f i g u r e 21 ). 127 The degree t o which acronyc ine i n h i b i t e d the i nco rpo ra t i on of u r i d i n e in to the t o t a l c e l l u l a r mate r i a l of L5I78Y c u l t u r e s was s t r ong l y a f f e c t e d by the concent ra t i on of horse serum in the medium ( f i g u r e 34 ). The inverse r e l a t i o n s h i p between the amount of horse serum present and the i n h i b i t i o n of nuc leos ide uptake i s in agreement with the data of s e c t i on I of t h i s t h e s i s , which i nd i ca ted t h a t both the degree of I 4 b ind ing of C-acronyc ine to L5I78Y c e l l s and the c y t o t o x i c i t y of the drug were i n ve r se l y r e l a t ed t o the concent ra t i on of horse serum present in the e x t r a c e l l u l a r f l u i d . The s imp le s t exp lana t i on f o r the data i s t ha t both c y t o t o x i c i t y and the i n h i b i t i o n of u r i d i n e uptake are a f unc t i on of the b ind ing of acronyc ine to c e l l s . Th is b ind ing i s in tu rn a f unc t i on of the concen t ra t i on of unbound acronyc ine in the medium - a parameter which i s a f f e c t e d by the concent ra t i on of horse serum. In a d d i t i o n t o showing the e f f e c t of horse serum concent ra t i on on the i n h i b i t i o n of u r i d i n e uptake by ac ronyc ine , the data of f i g u r e 34 a l s o i nd i c a t e t ha t in con t ro l c u l t u r e s in the absence of ac ronyc ine , the uptake of u r i d i n e i s a f f e c t e d by the concent ra t i on of serum i t s e l f . This dependence of u r i d i n e uptake on serum may be r e l a t e d to the r e s u l t s of Hare and co-workers ( 93 ) who have found t ha t u r i d i n e uptake by c e l l s i s sharp ly reduced when serum i s removed, but can be res tored t o con t ro l va lues upon the r e a d d i t i o n of serum. This e f f e c t i s thought to be mediated by an i n t e r a c t i o n of some serum component with the c e l l s u r f a ce . As shown in s e c t i on I of t h i s t h e s i s , acronyc ine can i n t e r a c t w i th at l ea s t one serum component. It can be suggested t he r e f o r e tha t acronyc ine might a l s o i n t e r a c t with the serum component re spons ib le f o r s t i m u l a t i n g u r i d i n e uptake - t h i s i n t e r a c t i o n could a f f e c t the a b i l i t y of the serum component to a c t i v a t e u r i d i n e uptake in c e l l s . A mechanism 128 of t h i s nature cannot however account f o r the i n h i b i t i o n of u r i d i n e uptake by ac ronyc ine , as the a l k a l o i d i s capable of i n h i b i t i n g u r i d i n e uptake in the complete absence of serum ( f i g u r e 34 ), and indeed i s most e f f e c t i v e under t h i s c o n d i t i o n . 14 Experiments in s e c t i on I of t h i s t h e s i s i nd i ca ted t ha t C-acronycine was r a p i d l y re leased from c e l l s on resuspending them in d rug - f ree medium. This r e v e r s i b i l i t y of the b ind ing of acronyc ine to L5I78Y c e l l s was p a r a l l e l e d by a r e v e r s i b i l i t y of the i n h i b i t i o n of u r i d i n e uptake caused by the drug. Thus when acronyc ine was removed from L5I78Y c u l t u r e s , u r i d i n e uptake returned to conitrol l e ve l s w i t h i n the f i r s t 30 minutes a f t e r the washing of the c e l l s ( f i g u r e 35 ). The data i n d i c a t e t ha t the e f f e c t of acronyc ine on uridmne uptake i s only seen in the ac tua l presence of the drug, and t ha t the i n h i b i t i o n of u r i d i n e uptake thus seems u n l i k e l y to be the r e s u l t of any permanent damage to the nuc leos ide uptake mechanism. Cho l ine and i n o s i t o l p a r t i c i p a t e in the format ion of c e r t a i n e s s e n t i a l l i p i d s which are incorporated i n to c e l l u l a r membranes. Acronycine was found to only s l i g h t l y i n h i b i t the uptake of these compounds ( t a b l e III ). There was no s t a t i s t i c a l l y s i g n i f i c a n t i n h i b i t i o n a t an acronyc ine concent ra t i on of 3 ug/ml, and the i n h i b i t i o n was not marked even at the high drug concent ra t i on of 12 ug/ml. It seems u n l i k e l y t he r e f o r e t ha t an i n h i b i t i o n of the i n co rpo ra t i on of c ho l i ne or i n o s i t o l could account f o r the rap id e f f e c t s of acronyc ine on the s t r u c t u r e of Golg i bod ies , and the l a t e r e f f e c t s of the a l k a l o i d on mi tochondr ia l morphology. Because c ho l i n e and i n o s i t o l are e s s e n t i a l f o r c e l l growth they are inc luded in r e l a t i v e l y high concent ra t i ons in F i s c h e r ' s medium ( c. 8 and II yM r e s p e c t i v e l y ). For t h i s reason, the t o t a l concent ra t i on 129 of cho l i ne or i n o s i t o l in the uptake experiments ( r a d i o a c t i v e mate r i a l p lus non - r ad i oac t i ve mate r i a l from the medium ) i s h igher than the concent ra t i on of nuc leos ides used in uptake exper iments. If acronyc ine were to prove to be a compet i t i ve i n h i b i t o r of the uptake of c ho l i n e and i n o s i t o l ( as i t i s of the uptake of nuc leos ides ), then the r e l a t i v e l y low i n h i b i t i o n of t h e i r uptake may be more a > r e f l e c t i o n of t h e i r high concent ra t i on in uptake exper iments, r a the r than of the s e n s i t i v i t y of t h e i r uptake mechanisms to ac ronyc ine . A d d i t i o n a l experiments on the uptake of r a d i o a c t i v e cho l i ne and i n o s i t o l a t lower concent ra t i ons would be necessary t o reso lve t h i s p o i n t . In c on t r a s t t o the i n h i b i t i o n of the i nco rpo ra t i on of nuc leo s ide s , c h o l i n e , and i n o s i t o l , the i n co rpo ra t i on of r a d i o a c t i v e deoxyglucose in to t o t a l c e l l u l a r mate r i a l was s l i g h t l y s t imu la ted by acronyc ine ( t a b l e IV ). Kessel and Dodd have reported tha t the drug Per sant in ( which i n h i b i t s the uptake of a v a r i e t y of compounds i n c l ud i ng u r i d i n e ) a l s o s l i g h t l y s t imu la te s the uptake of a g lucose ana log, in t h i s case 3-0-methyI g lucose ( 94 ). They suggest t ha t t h i s s t i m u l a t i o n can be exp la ined on the bas i s of a p r e f e r e n t i a l i n h i b i t i o n of the e x i t of the g lucose analog from the c e l l s . A s i m i l a r mechanism may be ope ra t i ve in the case of the s t i m u l a t i o n of deoxyglucose uptake by ac ronyc ine . RESULTS - SECTION 3 - INTRODUCTION The r e s u l t s of s e c t i on 2 of t h i s t h e s i s have demonstrated t ha t acronyc ine i s a compet i t i ve i n h i b i t o r 6if t'h'e ih'66RpoRation of u r i d i n e i n to n u c l e i c a c i d precursor poo l s , and consequently i n to n u c l e i c a c i d s . Acronycine d id not a f f e c t the d i s t r i b u t i o n of r a d i o a c t i v i t y w i t h i n the 130 a c i d - s o l u b l e pool of L5I78Y c e l l s incubated with u r i d i n e , nor d id i t induce the leakage of r a d i o a c t i v i t y from c e l l s l a b e l l e d with u r i d i n e . It was i n f e r r ed t h a t the a l k a l o i d must a f f e c t e i t h e r the passage of u r i d i n e through the plasma membrane, or i t s phosphory lat ion to UMP. Experiments to determine which of these po s s i b I i I t i e s represents the mechanism by which acronyc ine i n h i b i t s the i n co rpo ra t i on of u r i d i n e i n to n u c l e i c a c i d p recur sor pools are descr ibed in t h i s s e c t i on of the t h e s i s . Before d i s cu s s i ng the r a t i o n a l e of experiments i n v e s t i g a t i n g the mechanism of u r i d i n e uptake by L5I78Y c e l l s and the nature of the mechanism by which acronyc ine t h i s uptake, the f i nd i n g s of others on the nature of fh§ mechani sm,, bymwhy oh n nuc leosjdes. are a ecu mud §^.§du N/u cj& I I Sc as non-d i f f u s i n g metabo l i te s w i l l be b r i e f l y reviewed. Mechanism of nuc leos ide uptake by c e l l s : As noted in the main i n t r oduc t i on t o t h i s t h e s i s , c e l l s of a v a r i e t y of organisms are capable of u t i l i z i n g e x t r a c e l l u l a r nuc leo s ide s , but not nuc l eo t i de s . Use can be made of t h i s c a p a b i l i t y to i n v e s t i g a t e n u c l e i c a c i d s ynthes i s by incubat ing c e l l s with r a d i o a c t i v e nuc leo s ide s , and f o l l o w i n g the i nco rpo ra t i on of r a d i o a c t i v i t y i n to t o t a l a c i d -i n s o l ub l e mate r i a l or i n to s p e c i f i c n u c l e i c a c i d s . Although changes in the ra te of s yn thes i s of n u c l e i c ac id s w i l l be r e f l e c t e d in changes in the ra te of t h i s i n c o r p o r a t i o n , a change in the ra te of i n co rpo ra t i on does not n e c e s s a r i l y mean t ha t a change in the ra te of s yn thes i s has taken p l a ce . La te l y there has been i nc rea s ing r e c o g n i t i o n " o f the f a c t t ha t the ra te of i n co rpo ra t i on of e x t r a c e l l u l a r nuc leos ides i n to n u c l e i c ac id s i s dependent not only on the ra te of synthes i s of the n u c l e i c a c i d s , but a l s o on the ra te at which the e x t r a c e l l u l a r nuc leos ides en te r i n to the n u c l e i c a c i d p recur sor pools ( 95-99 ). The r e cogn i t i on of t h i s 131 p o t e n t i a l de fec t in i n v e s t i g a t i o n s of n u c l e i c ac id s ynthes i s using e x t r a c e l l u l a r r a d i o a c t i v e nuc leos ides has in recent years r e s u l t ed in a number of s tud ie s s p e c i f i c a l l y aimed at determining the mechanism of ent ry of e x t r a c e l l u l a r nuc leos ides i n to n u c l e i c a c i d precursor poo l s . Because of the ex tens i ve use of r a d i o a c t i v e u r i d i n e f o r the i n v e s t i g a t i o n of RNA s yn the s i s , many of these s tud ie s have concentrated on the mechanism of ent ry of t h i s nuc leo s ide . In gene ra l , a r e l a t i v e l y c on s i s t en t p i c t u r e has come from these s t u d i e s . A gene ra l i zed diagram of the mechanism of ent ry of u r i d i n e in to i n t r a c e l l u l a r metabolism i s g iven in f i g u r e 36. Nucleos ides are thought t o enter the c e l l by one of two mechanisms, ( i ) i t h e . s i m p Ie d i f f u s i o n of the nuc leos ide through the plasma membrane and ( i i ) the passage of the nuc leos ide through the plasma membrane with the a i d of one or more s p e c i f i c membrane-Iocated t r an spo r t systems. Ne i the r of these mechanisms in i t s e l f r e s u l t s in the concent ra t i on of a nuc leos ide w i t h i n a ceI I . The bulk of the plasma membrane i s not h i gh ly permeable to nuc leos ides . C e l l s however u sua l l y possess c a r r i e r -med i a t ed t r an spo r t systems f o r nuc leo s ide s , which a i d the passage of the nuc leos ide through the otherwise irepatriwe I y impermeable membrane ( mammalian c e l l s , 100-108 ; c h i c k embryo c e l l s , 98, 109 ; sea urch in eggs, 97, 110 ; red blood c e l l s , I I I -114 ; b a c t e r i a , 115, 116 ). Such t r an spo r t systems behave in an enzyme-l i k e fa sh ion in t h a t they show subs t ra te s p e c i f i c i t y , are s a tu ra ted ' by high l e ve l s of sub s t ra te and f o l l ow MichaeI is-Menten k i n e t i c s , and can be i n h i b i t e d c o m p e t i t i v e l y by s t r u c t u r a l l y r e l a t e d compounds. The t r an spo r t systems are not capable in themselves of concen t r a t i ve t r an spo r t -they f a c i l i t a t e the passage of nuc leos ides through the plasma membrane p lasma membrane ur ld i ne saturatable , . enzymeH ike • transport system ATP ADP ur id i ne-ur idine kinase UMP • o t h e r n u c l e o t i d e s , n u c l e i c a c i d s c y t o p I asm s i m p l e d i f f u s i o n n o n - d i f f u s i n g m e t a b o l i t e s ( r e t a i n e d on wa sh i ng e e l Is w i t h BSS ') F i g u r e 36 Mechanism of t h e a c c u m u l a t i o n o f u r i d i n e as n o n - d i f f u s i n g m e t a b o I i t e s by e e l I s . 133 ( thus tending t o speed the e q u i l i z a t i o n of the i n t e rna l and ex te rna l concent ra t i ons of nuc leos ides ) but are not capable of producing a h igher i n t r a c e l l u l a r than e x t r a c e l l u l a r concent ra t i on ( 1 0 4 , 106, 112 ). This i f o r m of membrane t r an spo r t has been v a r i o u s l y g iven the names : f a c i l i t a t e d d i f f u s i o n , e q u i l i z i n g t r a n s p o r t , e q u i l i z i n g s e l e c t i v e t r a n s p o r t , f a c i l i t a t e d t r a n s f e r , and a s s i s t ed d i f f u s i o n ( I 17 ). As the term " f a c i l i t a t e d d i f f u s i o n " i s most commonly used to descr ibe t r an spo r t of t h i s nature, i t s use w i l l be adhered to in t h i s d i s c u s s i o n . A f a c i l i t a t e d d i f f u s i o n mechanism can t r an spo r t sub s t ra te in e i t h e r d i r e c t i o n across the plasma membrane. This cannot be demonstrated in most c e l l s , s i nce they r a p i d l y metabol i ze nuc leos ides - i t i s not t he re f o re po s s i b l e to accumulate an i n t r a c e l l u l a r pool of nuc leos ide whose e f f l u x can then be i n v e s t i g a t e d . It has been demonstrated however t ha t e r y th rocy te s do not m e t a b i l i z e nuc leos ides such as u r i d i n e , and t ha t an i n t r a c e l l u l a r pool of f r ee nuc leos ide can be formed in these c e l l s by incubat ing them with e x t r a c e l l u l a r nuc leos ides ( 112 ). Paterson and co-workers have shown tha t when such c e l l s are washed f r ee of e x t r a c e l l u l a r nuc leo s ide s , the e f f l u x of the i n t r a c e l l u l a r nuc leos ide can be fo l lowed ( 112 ). In t h i s type of c e l l i t i s a l s o po s s i b l e t o demonstrate two a d d i t i o n a l i n t e r e s t i n g phenomena which occur as a d i r e c t r e s u l t of the nature of the f a c i l i t a t e d d i f f u s i o n mechanism ( I I l - l 1 3 ).-These are i ) " a c c e l e r a t i v e exchange d i f f u s i o n " which occurs when the ra te of e f f l u x of a compound i s increased by the inward t r a n s f e r v i a the same c a r r i e r of the same or a r e l a t e d compound, and i i ) " counter t r an spo r t " , in which a compound, i n i t i a l l y present in equal concent ra t i ons i n s i de and ou t s i de the c e l l , i s d r i ven outward aga in s t i t s concent ra t i on g rad ien t by the inward f low of a second compound t ranspor ted by the same c a r r i e r . 134 In a d d i t i o n t o en te r i ng the c e l l by a s p e c i f i c membrane-Iocated c a r r i e r system, nuc leos ides can a l s o enter by s imple d i f f u s i o n through the plasma membrane ( f i g u r e 36 ), ( 100-104, 108 ). As a r e s u l t of i t s non-enzymatic nature, s imple d i f f u s i o n does not s a tu ra te with i nc reas ing sub s t ra te c oncen t r a t i o n , but r a the r , the rate of d i f f u s i o n i s a I'inear f unc t i on of the concent ra t i on of e x t r a c e l l u l a r nuc leo s ide . Simple d i f f u s i o n does not account f o r more than a very small f r a c t i o n of the t o t a l nuc leos ide en te r i ng the c e l l , except a t high nuc leos ide concent ra t i on s when the ra te of s imple d i f f u s i o n i s high and the entry of subs t ra te v i a f a c i I i t a t e d d i f f u s i o n ' i s l i m i t e d by s a t u r a t i o n of the membrane-located c a r r i e r system. Experiments in t h i s t h e s i s were done at u r i d i n e concent ra t i ons below those a t which s imple d i f f u s i o n has been reported to account f o r more than a small f r a c t i o n of u r i d i n e en te r i ng the c e l l . Although ne i t he r s imple nor f a c i l i t a t e d d i f f u s i o n i s capable of concent ra t i ng a nuc leos ide as such w i t h i n a c e l l , under cond i t i on s in which the t ranspor ted nuc leos ide i s metabol ized i n t r a c e l l u l a r ^ t o products which cannot cross the c e l l membrane i t i s po s s i b l e t o accumulate w i t h i n the c e l l high concent ra t i ons of nuc leos ide d e r i v a t i v e s . In e f f e c t , c e l l s are able t o concentrate the nuc leos ide g r e a t l y by t rapp ing i t in a n o n - d i f f u s i n g form. The constant removal of the nuc leos ide by metabo l i c t r an s fo rmat ion prevents the i n t r a c e l l u l a r concent ra t i on of the sub s t r a te from a t t a i n i n g the e x t r a c e l l u l a r c oncen t r a t i o n , and thus there w i l l be a cont inua l net f low of sub s t ra te i n to the c e l l . Although such a process can g r e a t l y concentrate w i t h i n the c e l l r a d i o a c t i v i t y from an e x t r a c e l l u l a r l a b e l l e d nuc leo s ide , i t i s important t o note t h a t in c on t r a s t to c l a s s i c a l a c t i v e t r a n s p o r t , the nuc leos ide as such i s not concent ra ted , only i t s d e r i v a t i v e i s accumulated. Such a mechanism f o r 135 the uptake of compounds from the e x t r a c e l l u l a r f l u i d i s a p p l i c a b l e not only to nuc leo s ide s , but has a l s o been proposed f o r the uptake of o ther compounds such as g lucose ( 118 ), and cho l i ne ( 100 ) by mamma Ii an ceI Is. In the case of nuc leos ides used to monitor n u c l e i c a c i d s y n t he s i s , the immediate metabo l ic f a t e a f t e r passage of the nuc leos ide through the plasma membrane appears t o be phosphory lat ion by nuc leos ide k inases to the nuc leos ide monophosphate. S ince c e l l s are impermeable to n u c l e o t i d e s , t h i s represents an e f f e c t i v e method of t r app ing the nuc leos ide w i t h i n the c e l l . The nuc leos ide kinases invo lved in t h i s process most probably do not e x i s t s p e c i f i c a l l y in order t o phosphoryI ate nuc leos ides from e x t r a c e l l u l a r sources, but are par t of i n t r a c e l l u l a r sa lvage pathways f o r degraded nuc leo t ide s ( 119 ). There have been suggest ions t h a t perhaps, in analogy t o the sugar t r a n s p o r t i n g systems of c e r t a i n b a c t e r i a ( 120, 121 ), the phosphory lat ion of a nuc leos ide might be d i r e c t l y coupled to i t s c a r r i e r -med i a t ed t r an spo r t through the plasma membrane ( 109, 122 ). There has however never been any d i r e c t evidence t ha t such coupled systems e x i s t f o r nuc leos ide uptake, and there are a number of l i ne s of evidence t h a t argue aga ins t i t . Thus e r y th rocy te s ( 112 ) and mutant " ki nase' 1 less " c e l l s ( 106 ) do not phosphoryI ate nuc leo s ide s , yet have f u l l y f u n c t i o na l t r an spo r t systems. In yeas t , u r i d i n e t r an spo r t and u r i d i n e phosphory la t ion are g e n e t i c a l l y d i s t i n c t , and can be independently l o s t by mutations ( 123 ). C e l l f r a c t i o n a t i o n s tud ie s with the Nov ikof f hepatoma c e l l l i n e have f a i l e d t o reveal any a s s o c i a t i o n between u r i d i n e phosphoryI a t i n g a c t i v i t y and the plasma membrane ( 100 ). Furthermore, when nuc leos ide phosphory la t ion in t h i s c e l l l i n e i s e l im i na ted by dep l e t i n g the c e l l s 136 of ATP by chemical man ipu la t i on , the c a r r i e r -med i a t ed t r an spo r t of nuc leos ides across the plasma membrane i s una l tered ( 104 ). Ra t i ona le of i n v e s t i g a t i o n s i n to the mechanism of nuc leos ide uptake : As d i scussed above, in c e l l types so f a r i n ve s t i g a ted the i n co rpo ra t i on of e x t r a c e l l u l a r nuc leos ides i n to i n t r a c e l l u l a r nuc leo t i de pools i s dependent on the sequent ia l opera t ion of two processes , namely the t r an spo r t of the nuc leos ide across the plasma membrane, and i t s subsequent phosphory la t ion to the nuc leos ide monophosphate ( f i g u r e 36 ). In a c e l l l i n e such as the L5I78Y which r e a d i l y phosphoryIates u r i d i n e , i t i s not p o s s i b l e in i n t a c t c e l l s t o e a s i l y study as separate processes the entry of a nuc leos ide in to the c e l l and i t s subsequent phosphory la t i on . However, there are two aspects of u r i d i n e uptake and metabolism t ha t may r e a d i l y be i n ve s t i g a ted - the o v e r a l l uptake of u r i d i n e by i n t a c t c e l l s , and the phosphory la t ion of u r i d i n e in v i t r o by c e l l e x t r a c t s . The behaviour of these two parameters under d i f f e r e n t cond i t i on s ( s ub s t ra te concent ra t ion ? presence of i n h i b i t o r s e t c . ) may shed cons ide rab le l i g h t on the f unc t i on i n g of the u r i d i n e uptake mechanism. i ) u r i d i n e uptake - As noted e a r l i e r in t h i s t h e s i s , the term " uptake " as used here r e f e r s to the t o t a l accumulat ion of a nuc leos ide by c e l l s , as n o n - d i f f u s i n g metabo l i te s ( both a c i d - s o l u b l e p lus a c i d -i n s o l ub l e ). The i n co rpo ra t i on of nuc leos ides i n to nuc l eo t i de pools i s an o b l i g a t o r y pa r t df the pathway by which they are incorporated i n to n u c l e i c a c i d s . For t h i s reason, the t o t a l accumulat ion of r a d i o a c t i v i t y from e x t r a c e l l u l a r l a b e l l e d nuc leos ides as n o n - d i f f u s i n g metabo l i te s g ives a measure of t h e i r ent ry i n to c e l l u l a r nuc l eo t i de poo l s . Th is uptake represents the r e s u l t of the combined aci i i oiii i of> u r i d i n e t r an spo r t and u r i d i n e phosphory la t i on . In the event t h a t u r i d i n e phosphory lat ion 137 and u r i d i n e t r an spo r t are separate steps and one of them occurs at a ra te less than t ha t of the o the r , t h i s o v e r a l l uptake process would be expected to have the enzymatic c h a r a c t e r i s t i c s ( e .g . k i n e t i c s , s e n s i t i v i t y to i n h i b i t o r s ) of the r a t e - I i m i t i n g s tep . If f o r example the t r an spo r t of u r i d i n e across the plasma membrane i s much f a s t e r than phosphory l a t i on , then u r i d i n e w i l l be able t o enter the c e l l f a s t e r than |i:t^eah:be ; phosphory I a ted , and the i n t r a c e l l u l a r concent ra t i on of u r i d i n e w i l l approach the e x t r a c e l l u l a r c oncen t r a t i on . The ra te of the o v e r a l l uptake r eac t i on ( convers ion of e x t r a c e l l u l a r u r i d i n e t o i n t r a c e l l u l a r phosphoryIated d e r i v a t i v e s ) wi I I then be l i m i t e d by the ra te at which the phosphory la t ion r eac t i on proceeds. In t h i s case, the o v e r a l l uptake process wbu:ld be expected to have the same c h a r a c t e r i s t i c s as those of the phosphory la t ion r eac t i on ( e.g. the K and V of the r r 7 3 m max overaI I uptake process should be those of the phosphory lat ion r e a c t i o n , which i s r a t e - I i m i t i n g ). If on the o ther hand t r an spo r t i s much s lower than phospho ry l a t i on , i n t r a c e l l u l a r u r i d i n e w i l l be phosphoryIated wi th such e f f i c i e n c y t ha t the i n t r a c e l l u l a r concent ra t i on of t h i s nuc leos ide w i l l approach zero . In t h i s case, the rate of uptake w i l l be l i m i t e d by the ra te a t which u r i d i n e i s t r an spor ted across the plasma membrane. In e i t h e r case, any change in the ra te of the r a t e - l i m i t i n g r e a c t i on ifludue ^ toof orxexamp lenaifiiilinih i b i t i n g -"drug ! ) w i d) I r correspond i ng I y a f f e c t the ra te o f , t h e o v e r a l l uptake process , wh i l e changes in the non - r a t e - I i m i t i n g step would be expected to have l i t t l e or no e f f e c t on the ra te of the o v e r a l l r e a c t i o n . As shown p rev i ou s l y in s e c t i o n 2 of t h i s t h e s i s , the o v e r a l l uptake of u r i d i n e a t the concent ra t i ons used in the present i n v e s t i g a t i o n i s a s a t u r a t ab l e process , and obeys s imple MichaeI is-Menten k i n e t i c s . This 138 i s suggest ive t ha t a s i n g l e , enzymatic process ( e i t h e r u r i d i n e t r an spo r t or u r i d i n e phosphory la t ion ) c o n t r o l s the ra te a t which u r i d i n e enter s the c e l l and i s converted t o UMP. It i s p o s s i b l e to study the c h a r a c t e r -i s t i c s of t h i s r a t e - I i m i t i n g process even in the absence of p r ec i s e knowledge of which par t of the uptake mechanism ( t r an spo r t o r phos-pho r y l a t i on ) determines these c h a r a c t e r i s t i c s . i i ) u r i d i n e phosphory la t ion in v i t r o - The c h a r a c t e r i s t i c s of u r i d i n e phosphory la t ion can be s tud ied independent ly, in v i t r o , by homogenizing c e l l s t o remove the pe rmeab i l i t y b a r r i e r of the plasma membrane. Although t h i s removes the process from i t s normal i n t r a c e l l u l a r environment and introduces the p o s s i b i l i t y t ha t u r i d i n e phosphory lat ion may behave d i f f e r e n t l y than in i n t a c t c e l l s , such s tud i e s neverthe les s are usefu l as a guide to the c h a r a c t e r i s t i c s of u r i d i n e kinase in the eel I . The c h a r a c t e r i s t i c s of the o v e r a l l uptake of u r i d i n e by c e l l s may be compared with those of the phosphory lat ion of u r i d i n e in v i t r o t o reveal the r o l e played by t r an spo r t and phosphory lat ion in determining the ra te of accumulat ion of extrace I IuIar u r i d i n e as n o n - d i f f u s i n g metabo l i t e s . Thus Plagemann and Roth have i n ve s t i g a ted u r i d i n e uptake and u r i d i n e phosphory la t ion by Nov ikof f hepatoma eel Is C .100 ). No evidence could be found in t h i s c e l l l i ne t ha t u r i d i n e phosphory la t ion was a membrane-associated event. The o v e r a l l uptake of u r i d i n e by hepatoma c e l l s and the phosphory lat ion of u r i d i n e by e x t r a c t s of c e l l s d i f f e r e d markedly in t h e i r k i n e t i c s and in t h e i r s e n s i t i v i t i e s to a range of i n h i b i t o r s . As d i scussed above, i f phosphory la t ion was the r a t e - I i m i t i n g step in the o v e r a l l uptake of u r i d i n e , then the c h a r a c t e r -i s t i c s of u r i d i n e uptake would be expected to be s i m i l a r to those of 139 phosphory la t i on . S ince the c h a r a c t e r i s t i c s of these two processes were d i ss i mi Iar, i t was i n f e r r ed t ha t the phosphory lat ion of u r i d i n e was not the r a t e - I i m i t i n g step in u r i d i n e uptake, and t he re f o re ( by e l i m i n a t i o n ) the t r an spo r t of u r i d i n e through the plasma membrane must be the r a t e - I i m i t i n g s tep . Th is conc lu s i on has very r e cen t l y been confirmed in s tud ie s in the same c e l l l i n e which a l lowed the t r an spo r t r e a c t i on to be i n ve s t i g a ted in i s o l a t i o n by e l i m i n a t i n g the phosphory lat ion r eac t i on by dep l e t i n g the c e l l s of ATP by chemical manipu lat ions ( 104 ). The c h a r a c t e r i s t i c s of u r i d i n e t r an spo r t in these c e l l s resembled those of the o v e r a l l accumulat ion of u r i d i n e as non -d i f f u s i n g metabo l i te s by untreated c e l l s , support ing the suggest ion t ha t the permeation of the nuc leos ide through the plasma membrane i s normally the r a t e - I i m i t i n g step in i t s uptake. I n ve s t i ga t i on s s i m i l a r t o those of Plagemann but somewhat more l i m i t e d were undertaken with L5I78Y c e l l s in o rder t o a s c e r t a i n i f the general conc lu s ions reached by t h a t i n v e s t i g a t o r with Nov ikof f hepatoma c e l l s were a p p l i c a b l e t o t h i s c e l l l i n e . In both the i n v e s t -i ga t i on s of Plagemann and those descr ibed in the cu r ren t r epo r t , the e s s e n t i a l method was to comparre the c h a r a c t e r i s t i c s of u r i d i n e uptake by whole c e l l s with those of u r i d i n e phosphory lat ion in c e l l e x t r a c t s , in order t o determine i f the c h a r a c t e r i s t i c s of u r i d i n e phosphory lat ion were c on s i s t en t with t h i s step being the r a t e - I i m i t i n g step in the uptake of t h i s nuc leos ide . RESULTS - SECTION 3 K i n e t i c s of the accumulat ion of u r i d i n e as n o n - d i f f u s i n g metabo l i te s  by L5I78Y c e l l s and of u r i d i n e phosphory la t ion by c e l l homogenates: 140 As descr ibed e a r l i e r in t h i s t h e s i s , the accumulat ion of u r i d i n e as non -d i f f u s i n g metabo l i te s by L5I78Y c e l l s obeyed M i c h a e l i s -Menten k i n e t i c s . Over a number of exper iments, the K of the o v e r a l l m uptake process in c e l l s taken from l o g a r i t h m i c a l l y growing c u l t u r e s ranged from 4 to 5 yM, and the V. of the process ranged from 70 to 80 pmoIes/min/mi I I ion c e l l s . U r i d i ne k inase a c t i v i t y was i n ve s t i g a ted i n ' c e l l e x t r a c t s as descr ibed in the methods s e c t i o n . C e l l s were homogenized, and debr i s and p a r t i c u l a t e mate r i a l removed by c e n t r i f u g a t i o n . Kinase a c t i v i t y was found on ly in the s o l ub l e po r t i on of the homogenate, i : e . the supernatant ; no u r i d i n e phosphoryI a t i n g a c t i v i t y was detected in the p a r t i c u l a t e f r a c t i o n ( which inc luded membrane fragments ). F igure 37 shows a Lineweaver-Burk p l o t of the ra te of u r i d i n e phosphory lat ion vs. u r i d i n e c oncen t r a t i o n . The r eac t i on obeyed s imple MichaeI is-Menton k i n e t i c s , w i th a K of 48 yM and a V of 1,050 pmoles/min/mi I I ion ' m max ' r c e l l s . In o ther exper iments, the values of K and V ranqed from r m max 3 90 to 60 yM, and from 1000 to 2000 pmoles/min/mi I I ion c e l l s r e s p e c t i v e l y . These values of K and V f o r u r i d i n e phosphory lat ion are both m max r r ' approximately an order of magnitude g rea te r than those of the corresponding parameters f o r u r i d i n e uptake by i n t a c t L5I78Y c e l l s . E f f e c t of temperature on u r i d i n e uptake and u r i d i n e phosphory la t ion : F igure 38 demonstrates the i n f l uence of temperature on the uptake of u r i d i n e by L5I78Y c e l l s , and on the phosphory lat ion of u r i d i n e by c e l l e x t r a c t s . The data are g iven as an Arrhen ius p l o t ( the logar i thm of the ra te of the r eac t i on vs. the r e c i p r o c a l of the abso lute temperature ). The accumulat ion of u r i d i n e as n o n - d i f f u s i n g metabo l i te s by i n t a c t L5I78Y c e l l s was ra ther s t r ong l y temperature dependent, 141 F igure 37 K i n e t i c s of the phosphory la t i on of u r i d i n e by an e x t r a c t of L5I78Y eel Is. 5 x 10^ L5I78Y c e l l s were homogenized in 2.0 ml b u f f e r , and p a r t i c u l a t e ma te r i a l was removed by c e n t r i f u g a t i o n . The phosphory la t i on of u r i d i n e by a l i q u o t s of t h i s e x t r a c t was determined as desc r ibed in the methods s e c t i o n . 142 1.5-l.oH CD CD + - C C L O ZI — g 0.5-— > -— O Z3 CL cn 4 - O O -£z CL CD 4- CD 2 .E 0.2-T 3 CD — > !-CD !_ — O CD o.H 0.06-Temperature ( °C ) 40 3,0 2,0 10 0^  I F up take— A 3.4 ~T~ 3.6 10"^  x ( abso lu te temperature ) ' F i gure 38 Inf luence of temperature on u r i d i n e uptake by L5I78Y c e l l s and on u r i d i n e phosphory la t ion by c e l l e x t r a c t s . The uptake of " H - 5 - u r i d i n e ( 0.004 uM ) in a 30 minute pe r iod i n t o the t o t a l c e l l u l a r ma te r i a l of L5I78Y c e l l s , and the phosphory la t i on of H -5 -u r i d i ne ( 0.4 yM ) a t pH 9.0 were determined a t va r i ou s temperatures as desc r ibed in the methods s e c t i o n . The data are expressed as a f r a c t i o n of the ra te of uptake or phosphory la t i on a t 3 7 ° , and are the mean of two de te rm ina t i on s . 143 proceeding a t 0° a t less than 10 % of the rate at 37° . In c o n t r a s t , the phosphory la t ion of u r i d i n e by c e l l e x t r a c t s was r e l a t i v e l y i n -s e n s i t i v e to changes in temperature, proceeding" at near ly the same rate at 0° as at 37° . The Arrhenius p l o t of u r i d i n e uptake by L5I78Y c e l l s was b i p h a s i c , showing a t r a n s i t i o n in s lope ( and thus in the apparent a c t i v a t i o n energy of u r i d i n e uptake ) at about 20° . The experiment in f i g u r e 28 was done at a low u r i d i n e concent ra t i on f o r convenience. Other experiments at h igher u r i d i n e concent ra t i ons showed e s s e n t i a l l y the same pa t te rn of temperature dependence of u r i d i n e uptake ( i n c l ud i ng the same t r a n s i t i o n in s lope ). A s i m i l a r pa t te rn was a l s o obta ined when the ^ m a x of u r i d i n e uptake at va r ious temperatures was p l o t t e d accord ing t o the method of A r rhen iu s . E f f e c t of acronyc ine on the phosphory la t ion of u r i d i n e in v i t r o : Experiments reported e a r l i e r in t h i s t h e s i s have shown tha t acronyc ine i s a compet i t i ve i n h i b i t o r of u r i d i n e uptake by i n t a c t L5I78Y c e l l s . As d i scussed at the end of s e c t i on 2, the data suggest t ha t acronyc ine i n h i b i t s e i t h e r the t r an spo r t of u r i d i n e through the plasma membrane, or i t s i n t r a c e l l u l a r phosphory la t ion to UMP. Although the e f f e c t of acronyc ine on the t r an spo r t of u r i d i n e cannot d i r e c t l y be examined, i t s e f f e c t on phosphory la t ion can be i n ve s t i g a ted in ceI I e x t r a c t s . The e f f e c t of acronyc ine on the phosphory lat ion of u r i d i n e in v i t r o under va r ious exper imental cond i t i on s i s shown in t a b l e V. At pH 9.0, acronyc ine at the h ighest concen t ra t i on used ( 40 yM, or £ . 1 3 yg/ml ) caused a s l i g h t but s t a t i s t i c a l l y s i g n i f i c a n t i n h i b i t i o n of u r i d i n e phosphory la t ion a t each of two sub s t ra te concen t r a t i on s . At the more p h y s i o l o g i c a l pH of 7.5, the a l k a l o i d had no s t a t i s t i c a l l y s i g n i f i c a n t 144 Table V E f f e c t of acronyc ine on the phosphory lat ion of u r i d i n e by e x t r a c t s of L5I78Y ceI Is. P pH f i n a l cone, of u r i d i n e phosphory lat ion — 3 H - 5 - u r i d i ne contro1 acronyc i ne acronyc i ne 20 yM 40 yM ( 6.4 yg/ml ) ( 12.8 yg/ml ) 9. 0 0. ,4 yM 150.7 ± 3. 4 146.2 ± 4.9 - 135.0 ± 2.0 -9. 0 0. .004 yM 1.682 ± 0. 046 _ 1.524 ± 0.043 -b h 7. 5 0. ,4 yM 121.7 ± 7. 0 114.6 ± 2.8 114.4 ± 2.8 -— Data are expressed in pmoles of u r i d i n e phosphoryIated/15 m in/m i l l i o n c e l l s and are the mean of f i v e determinat ions ± S.D. — D i f f e r e n c e from con t ro l not s i g n i f i c a n t ( p = 0.05, using S tudent ' s t tests - ) ) — D i f fe rence from con t r o l nis s i g n i f i c a n t ( p = 0.01, using S tudent ' s t t e s t ). The phosphory lat ion of 3 H - 5 - u r i d i n e by e x t r a c t s of L5I78Y c e l l s was determined as descr ibed in the methods s e c t i o n . 145 e f f e c t at any concent ra t i on on the phosphory lat ion of u r i d i n e . The magnitude of the maximum i n h i b i t i o n of u r i d i n e phosphory lat ion caused by acronyc ine ( c_. 10 % a t an acronyc ine concent ra t i on of c. 13 yg/ml ) i s very small compared wi th the i n h i b i t i o n of u r i d i n e uptake seen i n who Ie ceI Is. Inf luence of membrane fragments on the i n h i b i t i o n of u r i d i n e phosphory lat ion  by acronyc ine : S c h o l t i s s e k has i n ve s t i g a ted the e f f e c t of the drug Pe r san t i n on u r i d i n e uptake and uridiiine phosphory la t ion by ch i ck embryo c e l l s . Th i s compound was found to be a potent i n h i b i t o r of u r i d i n e uptake in i n t a c t c e l l s ( 109 ), but had no e f f e c t on the phosphory lat ion of u r i d i n e in c e l l e x t r a c t s . In o rder to e x p l a i n these f i n d i n g s , S c h o l t i s s e k suggested t ha t under con'd ittonsr-norma I I y e x i s t i n g in ch i ck embryo c e l l s , u r i d i n e kinase was ab le to r e v e r s i b l y a s s oc i a te wi th the plasma membrane. He proposed t h a t Pe r san t i n i n t e r f e r e d wi th u r i d i n e uptake by i n h i b i t i n g the a c t i v i t y of t h i s membrane-associated kinase,aarid t ha t the non membrane-associated kinase was i n s e n s i t i v e to the drug. The reason t he r e f o r e t ha t an i n h i b i t i o n of u r i d i n e phosphory lat ion c o u l d n ' t be demonstrated in v i t r o was t ha t under these cond i t i on s the assay measured only the a c t i v i t y of non membrane-associated enzyme ( pos tu la ted to be i n s e n s i t i v e to the drug ). Subsequent work has suggested t ha t Pe r san t i n i n h i b i t s the t r an spo r t of u r i d i n e across the plasma membrane ( 94, 100, 103, 108 ) ( a p o s s i b i l i t y not cons idered by S c h o l t i s s e k ). However, a s i m i l a r mechanism t o t ha t proposed by S c h o l t i s s e k could s t i l l be invoked to e x p l a i n the i n s e n s i t i v i t y of u r i d i n e phosphory la t ion in v i t r o to ac ronyc ine . As d i scussed immediately above, there seemed a p o s s i b i l i t y t ha t 146 the removal of the plasma membrane from the ceI I homogenate p r i o r to assay ing u r i d i n e phosphory lat ion might r e s u l t in the removal of an important modify ing f a c t o r . For t h i s reason, the e f f e c t of acronyc ine on the phosphory la t ion of u r i d i n e in the presence of membrane fragments was examined ( t a b l e VI ). L5I78Y c e l l s were homogenized, and the homogenate d i v i ded i n to two p o r t i o n s . Debr i s , i n c l ud i ng membrane fragments, n u c l e i , and cy top la smic o r gane l l e s ( a I I of which were v i s i b l e by phase con t r a s t microscopy ) were removed from one po r t i on by c e n t r i f u g a t i o n , wh i l e the o ther po r t i on was used d i r e c t l y in assays without the removal of p a r t i c u l a t e m a t e r i a l . The amount of phosphory lat ion was near ly the same in the two p repa ra t i on s . The s l i g h t l y g rea te r a c t i v i t y of the deb r i s pcon ta i n i ng homogenate may s imply r e f l e c t the f a c t t ha t t h i s preparationwwas subjected to fewer manipu lat ions before the assay, or may be due to u r i d i n e k inase a c t i v i t y present in non-d i s rupted c e l l s which normal ly would be removed by c e n t r i f u g a t i o n . Acronycine i n h i b i t e d the phosphory la t ion of u r i d i n e by both the p a r t i c u I a t e - f r e e c e l l e x t r a c t and the e x t r a c t con ta i n i ng membrane fragments by e s s e n t i a l l y the same minor ex tent ( c_. 5 % ). E f f e c t of temperature on u r i d i n e uptake in the presence of ac ronyc ine: It has been p rev i ou s l y shown ( f i g u r e 38 ) t ha t an Arrhen ius p l o t of the i n f l uence of temperature on the uptake of u r i d i n e by L5I78Y c e l l s i s b i p h a s i c , and has a s teeper s lope ( and thus i nd i c a te s a g rea te r a c t i v a t i o n energy ) a t temperatures below about 20° . F igure 39 shows the i n f l uence of temperature on the uptake of u r i d i n e in the presence of an i n h i b i t i n g concent ra t i on of ac ronyc ine . As may be seen in comparison wi th f i g u r e 38, acronyc ine d id not change the ba s i c nature of the Arrhen ius p l o t ; in p a r t i c u l a r i t may be noted t h a t the t r a n s i t i o n 147 Table VI E f f e c t of acronyc ine on the phosphory lat ion of u r i d i n e by e x t r a c t s of L5I78Y c e l l s : e f f e c t of membrane fragments. Nature of eel e x t r a c t Concent rat ion of U r i d i ne phosphory la t ion acronyc ine ( pmoles/15 m in/m i l l i o n c e l l s c l ea red by 0 yM c e n t r i f uga t ion c l ea red by 40 yM ( 12.8 yg/ml ) c e n t r i f uga t ion debr i s present 0 yM 124.3 ± 2.0 I 17.3 ± 1.4 136.5 ± 1.4 debr i s present 40 yM ( 12.8 yg/ml ) : '.n ' 13018 ± 2:3 The phosphory la t ion of H -5 -u r id ine ( 0.35 yM ) was determined at pH 9.0 as descr ibed in the methods s e c t i o n . Where noted, c e l l homogenates were not c l ea red of p a r t i c u l a r mater ia l by c e n t r i f u g a t i o n ( as descr ibed in the t e x t ). Data are the mean of four determinat ions ± S.D. A l l d i f f e r e n c e s are s t a t i s t i c a l l y s i g n i f i c a n t ( p = 0.01, using S tudent ' s t t e s t ) 148 1.CH 0 ro 4-ZJ 0.54 i _ 0) 4-(O !_ 0) > CD 0.2H o.H 0.06H l"emperature ( °C ) 4 0 30 _J 20 _J 10 i -aero. cone. = 0 ug/ml -aero. cone. = I ug/ml - r -3.2 3.4 3.6 0 3 - i 10 x ( abso lu te temperature ) F i gu re 39 Inf luence of temperature on u r i d i n e uptake by L5I78Y c e l l s in the presence of ac ronyc ine . The uptake of H -5 -u r i d i ne ( 0.004 yM ) in a 30 minute pe r iod i n t o the t o t a l c e l l u l a r ma te r i a l of L5I78Y c e l l s was determined a t va r i ou s temperatures as desc r ibed in the methods s e c t i o n . The data are expressed as a f r a c t i o n of the ra te of u r i d i n e uptake in the absence of acronyc ine a t 37° , and are the mean of two de te rm ina t i on s . 149 in s lope of the p l o t a t about 20° was r e t a i n e d . Th is t r a n s i t i o n was a l s o seen a t h igher acronyc ine concen t r a t i on s . D i scus s ion - Sect ion 3 Nature of u r i d i n e uptake in L5I78Y c e l l s : As descr ibed in the i n t r oduc t i on to t h i s s e c t i o n of the r e s u l t s , s tud ie s in a number of c e l l l i ne s other than the L5I78Y c e l l l i n e have i nd i ca ted t h a t u r i d i n e k inase i s not a s soc ia ted with the plasma membrane, and t ha t the phosphory lat ion of u r i d i n e i s a r eac t i on separate from i t s t r a n spo r t across the plasma membrane. Thus Rlagemann and Roth ( 100 ) have i n ve s t i g a ted u r i d i n e k inase a c t i v i t y in va r ious f r a c t i o n s of Nov ikof f hepatoma c e l l s , and have found no evidence of any a s s o c i a t i o n between u r i d i n e phosphoryI a t i n g a c t i v i t y and the plasma membrane. S i m i l a r l y , in the cu r ren t s tudy, u r i d i n e phosphory lat ion in e x t r a c t s of L5I78Y c e l l s was found t o be a s soc ia ted on ly w i th the s o l ub l e po r t i on of ahee l l homogenate. No u r i d i n e phosphory Iat ing a c t i v i t y cou ld be found in the p a r t i c u l a t e f r a c t i o n of the homogenate, which inc luded membrane fragments, and when membrane fragments were inc luded in the u r i d i n e k inase assay the ra te of u r i d i n e phosphory lat ion was not g r e a t l y d i f f e r e n t from t h a t of the s o l ub l e po r t i on of the homogenate alone ( t a b l e VI ). These r e s u l t s suggest t ha t in L5I78Y c e l l s as w e l l , u r i d i n e phosphory la t ion i s a process d i s t i n c t from the mechanism by which u r i d i n e penetrates the plasma membrane. The accumulat ion of u r i d i n e as n o n - d i f f u s i n g metabo l i te s by L5I78Y c e l l s obeys s imple MichaeI i s -Menten k i n e t i c s ( f i g u r e 32 ). Th is i s suggest ive t ha t a s i ng Ie ,enzymat i c , o r enzyme- l ike process ( which a p r i o r i could be e i t h e r u r i d i n e t r an spo r t o r u r i d i n e phosphory lat ion ) c o n t r o l s the ra te a t which u r i d i n e i s taken up and re ta ined; by L5178Y 150 c e l l s . There are two arguments based on the data presented in t h i s s e c t i on of the t h e s i s which i n d i c a t e t ha t in L5I78Y c e l l s t h i s c o n t r o l l i n g process i s the t r an spo r t of u r i d i n e across the plasma membrane: I) If u r i d i n e phosphory la t ion was the step which c o n t r o l s the ra te of u r i d i n e uptake by L5I78Y c e l l s ( or in o ther words'swas r a t e -l i m i t i n g in u r i d i n e uptake ), then the enzymatic c h a r a c t e r i s t i c s of u r i d i n e uptake would be expected to be s imi I an, i f not i d e n t i c a l , t o those of u r i d i n e phosphory la t i on . The data which have been presented in t h i s s e c t i on of the t h e s i s i n d i c a t e however t h a t in two important respects the c h a r a c t e r i s t i c s of u r i d i n e phosphory la t ion are not s i m i l a r to those of the accumulat ion of u r i d i n e as n o n - d i f f u s i n g metabo l i te s by L5I78Y ce l l s . - F i r s t l y , the k i n e t i c s of the two processes are markedly d i f f e r e n t . Thus the of u r i d i n e uptake by L5I78Y c e l l s was 4 t o 5 yM, wh i l e t ha t of phosphory la t ion was 40 t o 60 yM, and the V of uptake was 70 t o 80 pmoIes/min/mi I I ion c e l l s wh i l e t h a t of max r r phosphory la t ion was 1000 t o 2000 pmo I es/mi n/mi I I ion ce l l s - . Secondly, the i n f l uence of temperature on the ra te of these two reac t i on s was very d i f f e r e n t ( f i g u r e 38 ). U r i d i n e uptake by L5I78Y c e l l s was h i gh l y temperature dependent, and when the ra te of uptake was p l o t t e d as a f unc t i on of temperature accord ing to the method of Ar rhen ius the data gave a b i pha s i c l i n e a r p lot.srl,nwcontrast^othe • phosphory lat ion of u r i d i n e by cetlrlrex-trracts was r e l a t i v e I yn temperature ( Insens i t i ve, --and when the data were p l o t t e d accord ing to the method of Arrhenius they gave a smooth curve. The above-mentioned d i f f e r e n c e s between the c h a r a c t e r i s t i c s of u r i d i n e phosphory lat ion in v i t r o and those of u r i d i n e uptake by L5I78Y c e l l s s t r ong l y suggest tha t in whole c e l l s u r i d i n e phosphory lat ion 151 cannot be the r a t e - I i m i t i n g process in the uptake of t h i s nuc leos ide . By-e I imi nat ion t h e r e f o r e , the t r an spo r t of u r i d i n e through the plasma membrane must be the r a t e - I i m i t i n g step in u r i d i n e uptake. 2) A second argument which i nd i c a t e s t h a t the t r an spo r t of u r i d i n e through the plasma membrane i s normal ly the r a t e - I i m i t i n g step in i t s uptake by L5I78Y c e l l s i s based on the shape of the Arrhen ius p l o t of u r i d i n e uptake ( f i g u r e 38 ). The graph of the logar i thm of the ra te of u r i d i n e uptake vs. the r e c i p r o c a l of the abso lute temperature shows a t r a n s i t i o n in s lope at about 2 0 ° , i n d i c a t i n g a marked change in the apparent a c t i v a t i o n energy of u r i d i n e uptake at t h i s temperature. There i s cons ide rab le evidence from experiments using a v a r i e t y of phy s i ca l techniques t ha t the l i p i d bi layer of membranes can undergo r e v e r s i b l e phase t r a n s i t i o n s ( X-ray d i f f r a c t i o n , 124, 125 ; d i f f e r e n t i a l scanning c a l o r i m e t r y , 126, 127 ; f l u o r e s c e n t membrane probes, 125-128 ; s p i n - l a b e l membrane probes, 128 ; e l e c t r o n microscopy, 129 ; and t u r b i d i m e t r y , 130 ). At h igher temperatures, the l i p i d s are in a r e l a t i v e l y f l u i d s t a t e , wh i l e a t lower temperatures the motion of the hydrocarbon chains of the l i p i d s i s r e s t r i c t e d . When the a c t i v i t y of t r a n s p o r t c a r r i e r s or o ther membrane-associated enzymes i s p l o t t e d accord ing to the method of A r rhen iu s , the graph i s f r equen t l y b i p h a s i c , w i th changes in the s lope of the p l o t a t a s i n g l e w e l l - d e f i n e d temperature ( 131-137 ). These changes in s lope i n d i c a t e an increased a c t i v a t i o n energy of the r ea c t i on ( g rea te r temperature dependence ) a t lower temperatures. These changes in a c t i v a t i o n energy can be c o r r e l a t e d with phase changes in the membrane. For example, the f a t t y - a c i d compos i t ion of the membranes of b a c t e r i a can be manipulated to g i ve membranes con ta in i ng a h igher o r lower p ropor t i on of sa tu ra ted f a t t y 152 a c i d s . When t h i s i s done, the temperature a t which the t r a n s i t i o n in the s lope of the Arrhen ius p l o t of membrane-associated enzymes occurs i s a l t e r e d ( 135-137 ). Higher p ropor t i ons of unsaturated f a t t y - a c i d s are a s soc ia ted with phase changes a t lower temperatures ( as detected by phy s i ca l means ) and with Arrhenius p l o t t r a n s i t i o n s a t lower temperatures. Not a l l membrane-associated enzymes y i e l d Arrhen ius p l o t s of a b i pha s i c nature ( 138 ). It has however been argued on t h e o r e t i c a l grounds t ha t the presence of such a t r a n s i t i o n n e c e s s a r i l y impl ies t h a t a phase change of some form i s t a k i n g p lace at the t r a n s i t i o n temperature ( 139 ). When the uptake of nuc leos ides has been examined as a f unc t i on of temperature, in some cases but not in o thers the Arrhen ius p l o t s have been found to be b i pha s i c ( 101, 140 ). The t r a n s i t i o n in s lope of the Arrhen ius p l o t of the uptake of u r i d i n e by L5I78Y eel Is i s s imi l a r to those seen in known membrane-associated enzymes. This i s suggest ive t h a t the r a t e - I i m i t i n g step in u r i d i n e uptake in the L5I78Y c e l l l i n e i s membrane-associated. In view of the evidence t ha t u r i d i n e phos-pho r y l a t i o n in L5I78Y c e l l s i s not a membrane-associated event, t h i s imp l ie s t ha t the t r an spo r t of u r i d i n e across the plasma membrane i s normal ly the r a t e - I i m i t i n g step in i t s uptake. Nature of the i n h i b i t i o n of u r i d i n e uptake by acronyc ine : As d i scussed above the data of t h i s s e c t i o n of the t h e s i s suggest t h a t u r i d i n e phosphory la t ion i s a process d i s t i n c t from the t r an spo r t of t h i s nuc leos ide through the plasma membrane. The accumulat ion by L5I78Y c e l l s of e x t r a c e l l u l a r u r i d i n e as n o n - d i f f u s i n g metabo l i te s must t he r e f o r e be the r e s u l t of the sequent ia l opera t ion of these two s teps . The data furthermore i n d i c a t e t h a t of these s tep s , the t r an spo r t of u r i d i n e through the plasma membrane i s most l i k e l y normally to be the 153 r a t e - l i m i t i n g process in the uptake of t h i s nuc leos ide . These con -c l u s i o n s are in agreement with those of Plagemann and Roth, who have s tud ied u r i d i n e uptake by Nov ikof f hepatoma c e l l s ( 100 ). As a p re l im ina r y to the f o l l o w i n g d i s cu s s i on on the nature of the mechanism by which acronyc ine i n h i b i t s u r i d i n e uptake by L5I78Y c e l l s , i t i s important to emphasize a bas i c p r i n c i p l e . This i s t ha t i r r e s p e c t i v e or whether acronyc ine i n h i b i t s u r i d i n e t r an spo r t or u r i d i n e phosphory la t i on , whichever step has been a f f e c t e d by the a l k a l o i d w i l l in drug--treated c e l l s be the r a t e - I i m i t i n g step in the o v e r a l l uptake process. For t h i s reason, any data which i d e n t i f i e s the step which i s r a t e - I i m i t i n g in u r i d i n e uptake in acronyc ine t r ea ted c e l l s a l s o immediately i d e n t i f i e s the step in the uptake process which i s i n t e r f e r e d wi th by the a I ka lo i d. The f o l l o w i n g three arguments suggest t h a t acronyc ine i n h i b i t s , the t r an spo r t of u r i d i n e acrSIs the plasma membrane - the l a s t two of As d iscussed above, in the absence of acronyc ine the t r an spo r t of u r i d i n e through the plasma membrane appears t o be the r a t e - I i m i t i n g step in u r i d i ne uptake by L5I78Y c e l l s . A p r i o r i , when acronyc ine i s added to c e l l s an i n h i b i t i o n of u r i d i n e uptake could occur in two ways : i ) acronyc ine could i n t e r f e r e with the t r an spo r t of u r i d i n e through the plasma membrane ( thus f u r t h e r reducing the v e l o c i t y of t r a n s p o r t , which in d rug - f ree c e l l s i s a l ready r a t e - I i m i t i n g ), o r i i ) acronyc ine cou ld i n t e r f e r e with the phosphory lat ion of u r i d i n e to such an extent t ha t in the presence of the drug t h i s step then becomes r a t e - I i m i t i n g . It may be noted t ha t even i f both t r an spo r t and phosphory lat ion are i n t e r f e r e d w i t h , only one of the above mechanisms can a c t u a l l y be re spons ib le f o r the i n h i b i t i o n of the o v e r a l l uptake process - t h i s i s because i t i s imposs ib le to have two " r a t e - I i m i t i n g " s teps . 154 these arguments u t i l i z e the p r i n c i p l e j u s t d i scussed : I) Acronycine had l i t t l e e f f e c t on the phosphory lat ion of u r i d i n e in v i t r o , i r r e s p e c t i v e of whether or not membrane fragments were present in the assay ( t a b l e s V and VI ). Thus at the h ighest a l k a l o i d concen t ra t i on used ( c. 13 ug/ml ), acronyc ine i n h i b i t e d u r i d i n e phosphory la t ion by a maximum of 5 to 10 %. Th is i n h i b i t i o n i s very small when i t i s compared with the i n h i b i t i o n by acronyc ine of u r i d i n e uptake by c e l l s . Thus the data of f i g u r e 34 show tha t acronyc ine at a concent ra t i on of 12 ug/ml i n h i b i t e d u r i d i n e uptake by 85 % in the presence of 10 % horse serum and by 99 % in the absence of horse serum ; i t may be noted t ha t t h i s l a t t e r c ond i t i o n i s t ha t which i s most d i r e c t l y comparable to assays of u r i d i n e phosphory la t ion in v i t r o , which do not con ta in serum ( which lowers the e f f e c t i v e a l k a l o i d concent ra t i on ). S i m i l a r l y , acronyc ine at a concent ra t i on of 3 ug/ml i n h i b i t e d u r i d i n e uptake by L5I78Y c e l l s in the absence of serum by 80 %, wh i l e even at double t h i s concent ra t i on i t had no s t a t i s t i c a l l y s i g n i f i c a n t e f f e c t on u r i d i n e phosphory la t ion in v i t r o . A Although i t cannot be excluded t h a t the phosphory lat ion of u r i d i n e may be more s e n s i t i v e t o acronyc ine in the c e l l than in v i t r o , i t appears u n l i k e l y t h a t an i n h i b i t i o n of u r i d i n e phosphory lat ion could account f o r the s t rong i n h i b i t i o n of u r i d i n e uptake. Th is i s e s p e c i a l l y t r ue in the l i g h t of the evidence which suggests t ha t phosphory lat ion i s u n l i k e l y to be normally the r a t e - I i m i t i n g step in u r i d i n e uptake. Because of t h i s , anyth ing o ther than a very marked i n h i b i t i o n of the phosphory lat ion r ea c t i on would not be expected to have an app rec i ab le e f f e c t on the o v e r a l l u r i d i n e uptake r a t e . S ince i t does not seem po s s i b l e t ha t an i n h i b i t i o n of u r i d i n e phosphory la t ion by acronyc ine can account f o r the 155 i n h i b i t i o n of u r i d i n e uptake b y . c e l l s , the a l k a l o i d must, by e l i m i n a t i o n , i n h i b i t the o ther step in the uptake process , namely the t r an spo r t of the nuc leos ide across the plasma membrane. 2) A second l i n e of reasoning which i nd i ca te s t h a t acronyc ine i n h i b i t s the t r an spo r t of u r i d i n e across the pIasmaomembrane can be developed from the k i n e t i c s of the i n h i b i t i o n of u r i d i n e uptake by ac ronyc ine . S ince acronyc ine behaves k i n e t i c a l l y as a compet i t i ve i n h i b i t o r of u r i d i n e uptake ( s e c t i o n 2, f i q u r e 32 ), the V of u r i d i n e r 3 max uptake i s i d e n t i c a l in the presence and absence of ac ronyc ine . This correspondence suggests t ha t in both ( i ) c e l l s in which u r i d i n e uptake i s i n h i b i t e d by the drug, and ( i i ) con t ro l c e l l s in the absence of the drug, the rate of u r i d i n e uptake i s c o n t r o l l e d by the same enzymatic process. As d i scussed e a r l i e r , i t appears t ha t in untreated c e l l s u r i d i n e uptake i s l i m i t e d by the rate at which the nuc leos ide i s t r an spor ted across the plasma membrane. It f o l l ows t he re f o re tha t a l s o in c e l l s i n . which u r i d i n e uptake i s i n h i b i t e d by ac ronyc ine , the t r an spo r t of u r i d i n e across the plasma membrane i s the r a t e - I i m i t i n g step in i t s uptake. This imp l ie s t ha t acronyc ine must i n h i b i t u r i d i n e t r a n s p o r t . 3) As shown in f i g u r e 38, when the i n f l uence of temperature on the uptake of u r i d i n e was p l o t t e d accord ing t o the method of A r rhen iu s , a The c o n t r o l l i n g or r a t e - I i m i t i n g process i s r e spons ib le f o r the V of the uptake process. If a d i f f e r e n t enzymatic process c o n t r o l l e d max the ra te of u r i d i n e uptake in t r e a t e d c e l l s than in con t r o l c e l l s , then i t would be h iqh l y u n l i k e l y t ha t the V of u r i d i n e uptake would be 3 max r i d e n t i c a l under these two c o n d i t i o n s . 156 t r a n s i t i o n in the s lope of the p l o t and thus in the apparent a c t i v a t i o n energy of u r i d i n e uptake was seen at £. 20° . As d iscussed e a r l i e r , the most l i k e l y exp lana t i on f o r t h i s t r a n s i t i o n i s . t h a t i t r e s u l t s from a phase change in the plasma membrane. When c e l l s were incubated with acronyc ine and the uptake of u r i d i n e examined as a f unc t i on of temperature, the Arrhen ius p l o t of the data a l s o had a t r a n s i t i o n in s lope ( f i g u r e 39 ). Th is t r a n s i t i o n suggests t h a t the r a t e - I i m i t i n g step in c e l l s in which u r i d i n e uptake has been i n h i b i t e d by acronyc ine i s s t i l l the t r an spo r t of u r i d i n e across the plasma membrane. This in tu rn imp l ie s t h a t acronyc ine must i n h i b i t u r i d i n e t r a n s p o r t . The th ree l i ne s of reasoning presented above suggest t h a t acronyc ine i n h i b i t s the t r an spo r t of u r i d i n e across the plasma membrane. Such an i n h i b i t i o n i s capable of e x p l a i n i n g the a b i l i t y of acronyc ine to i n h i b i t the i n co rpo ra t i on of e x t r a c e l l u l a r u r i d i n e both in to i n t r a c e l l u l a r a c i d -s o l u b l e nuc leo t i de poo l s , and in to n u c l e i c a c i d s . The i n h i b i t i o n by acronyc ine of the i nco rpo ra t i on of o the r nuc leos ides probably a l s o r e s u l t s from an i n h i b i t i o n of t h e i r t r a n spo r t across the plasma membrane, as may the s l i g h t i n h i b i t i o n of the i n co rpo ra t i on of c ho l i n e and i n o s i t o l i n to t o t a l c e l l u l a r m a t e r i a l . Mechanism by which acronyc ine i n h i b i t s the t r an spo r t of u r i d i n e through  the plasma membrane : It i s not po s s i b l e from the in format ion p re sen t l y a v a i l a b l e t o determine the p r e c i s e nature of the mechanism by which acronyc ine i n h i b i t s the t r an spo r t of u r i d i n e through the plasma membrane. It appears h i gh ly u n l i k e l y however t ha t acronyc ine s imply complexes wi th e x t r a c e l l u l a r u r i d i n e and thus r e s t r i c t s i t s a v a i l a b i l i t y t o the t r an spo r t system, s i nce in t h i s case acronyc ine would be expected t o very s t r ong l y 157 i n h i b i t the ac t i v i ty of u r i d i ne ki nase _i_n_ v i t r o by a s i m i l a r mechan i sm. The s imp le s t exp lana t i on f o r the data i s t ha t acronyc ine a f f e c t s the a b i l i t y of the membrane-associated t r an spo r t system to f a c i l i t a t e the entry of t h i s nuc leos ide i n to the c e l l . As ide from nuc leos ides and nuc leos ide analogs ( 95, 98, 100, 103, 106, 107, I I I , 115 ), and i n h i b i t o r s such as N-ethyI ma Ie imide, H g + + ions , and PCMB which i n a c t i v a t e enzymes by r e l a t i v e l y wel l understood mechanisms ( 103, 114-116 ), a number of o ther compounds have been reported to i n h i b i t the uptake of nuc leo s ide s . These compounds, which s t r ong l y resemble acronyc ine in t h e i r a c t i o n on nuc leos ide uptake, inc lude Pe r s an t i n ( 94, 100, 103, 108, 109 ), phenethyl a l coho l ( 92, 100, 103 ), c o l c h i c i n e and c o l c h i c i n e d e r i v a t i v e s ( 141 ), and c y t o c h a l a s i n B ( 142 ) ( f i g u r e 40, s t r u c t u r e s b_ t o e_ ) . These compounds are a l l compet i t i ve i n h i b i t o r s of nuc leos ide uptake, and in some cases have a l s o been shown t o i n h i b i t the uptake of o ther compounds as w e l l . The c l a s s i c a l form of a compet i t i ve i n h i b i t o r of an enzymatic process i s a compound which i s a s t r u c t u r a l analog of the normal s ub s t r a t e , and which competes with i t f o r the a c t i v e s i t e of the enzyme ( 143 ). Th is form of i n h i b i t i o n appears to be themmechanism by which nuc leos ides and nuc leos ide analogs i n h i b i t the uptake of o ther nuc leos ides . In eont ra s t to these compounds however, acronyc ine and the o ther compet i t i ve i n h i b i t o r s of nuc leos ide t r an spo r t shown in f i g u r e 40 bear no obvious s t r u c t u r a l s i m i l a r i t y to u r i d i n e or o ther nuc leo s ide s . In s p i t e of t h i s , some of these compounds ( i n c l ud i n g acronyc ine ) are on a molar bas i s more powerful i n h i b i t o r s of nuc leos ide uptake than many nuc leos ides or nuc leos ide analogs themselves. It appears u n l i k e l y t he r e f o r e t h a t these compounds and acronyc ine c o m p e t i t i v e l y i n h i b i t nuc leos ide t r an spo r t by 158 d Pe r s an t i n e c o l c h i c i n e F i gu re 40 S t r u c t u r a l formulae of c ompe t i t i v e i n h i b i t o r s compounds which are of nuc leos ide t r a n s p o r t ,|5Q s imply a c t i n g as s t r u c t u r a l analogs of the normal sub s t ra te and competing with i t f o r the a c t i v e s i t e per se of the membrane-associated nuc leos ide t r an spo r t system. In a d i s cu s s i on of the mechanisms by which a compound can be a compet i t i ve i n h i b i t o r of an enzyme, Webb, and Dixon and Webb ( 143, 144 ) note t h a t although s t r u c t u r a l analogs of a subs t ra te are the most common form of compet i t i ve i n h i b i t o r s of enzymes, o ther mechanisms of i n h i b i t i o n are p o s s i b l e . The term " compet i t i ve i n h i b i t o r " p roper l y r e f e r s not tto) the mechanism of i n h i b i t i o n , but to the e f f e c t s of the i n h i b i t o r on the k i n e t i c s of the enzymatic r e a c t i o n . A compet i t i ve i n h i b i t o r may be def ined as one which ra ises ' the K of the r ea c t i on m ( the value of subs t ra te concent ra t i on necessary to g ive a v e l o c i t y of ha l f the maximal v e l o c i t y of the enzyme when i t i s sa tu ra ted with sub s t ra te ) but does not chanqe the maximal v e l o c i t y ( V ) i t s e l f . 3 ' max Dixon and Webb note t h a t such an e f f e c t can a l s o r e s u l t from the i n t e r a c t i o n of the i n h i b i t o r with the enzyme, a t a s i t e other than the a c t i v e s i t e so as t o reduce the a f f i n i t y of the enzyme f o r the s ub s t r a t e . Acronyc ine i s a l i p o p h i l i c compound, and thus might reasonably be expected t o i n t e r a c t with l i p i d s and/or hydrophobic reg ions of p r o t e i n s . There i s evidence f o r ex tens i ve hydrophobic regions in membrane p ro te i n s ( 145 ), and there i s no reason to suppose t ha t t h i s f ea tu re would not be present in membrane-associated nuc leos ide t r an spo r t systems. Specu la -t i v e l y i f hydrophobic reg ions ( e i t h e r l i p i d or p r o t e i n in nature ) were present adjacent to the sub s t ra te b ind ing s i t e of the c a r r i e r re spons ib le f o r u r i d i n e t r a n s p o r t , acronyc ine might bind to these regions and r e s t r i c t the access of u r i d i n e to the s i t e by s imple s t e r i c h inderance. 160 S ince a l l the compet i t i ve i n h i b i t o r s of nuc leos ide uptake shown in f i g u r e 40 are t o some degree l i p o p h i l i c , a s i m i l a r mechanism might a l s o account f o r t h e i r a b i l i t y to i n h i b i t nuc leos ide t r a n s p o r t . In t h e i r i n v e s t i g a t i o n s of the mechanismsby which c o l c h i c i n e and c o l c h i c i n e d e r i v a t i v e s i n h i b i t nuc leos ide i n co rpo ra t i on in severa l l i ne s of mammalian c e l l s , M ize l and Wilson ( 141 ) have a l s o pos tu la ted t h a t a d i r e c t i n t e r a c t i o n of the i n h i b i t i n g compound with hydrophobic regions of the nuc leos ide t r an spo r t system may be i nvo l ved . The f i nd i n g s of Nott ( 146 ), who has i n ve s t i g a ted the b i o l o g i c a l a c t i v i t y of a s e r i e s of d e r i v a t i v e s of the drug Pe r san t i n ( f i g u r e 40, s t r u c t u r e d_ ), a l s o suggest t ha t the l i p o p h i l i c nature of the compounds under;d i scuss ion mayihave some bear ing on the nature of the mechanism by which they i n h i b i t nuc leos ide uptake. This compound i s used as a v a s o d i l a t o r in the treatment of heart d i s o r d e r s , and i s thought to ac t by i n h i b i t i n g the t r an spo r t of adenosine in to heart c e l l s ( ( _ l 4 6 ). Among the d e r i v a t i v e s of Pe r s an t i n t e s t ed by Not t , those which were the most l i p o p h i l i c a l s o had the most b i o l o g i c a l a c t i v i t y . The l i p o p h i l i c nature of the compounds under d i s cu s s i on most probably i s not the so le determinant of t h e i r b i o l o g i c a l a c t i v i t y - o ther m©;lieca(har fea tu re s could p lay a cons ide rab le r o l e in determining whether or not a g iven l i p o p h i l i c compound i s a l s o an i n h i b i t o r of t r a n spo r t processes. In examining however the d i v e r s i t y of the molecu la r s t r u c t u r e s e x h i b i t e d by the compounds shown in f i g u r e 40, i t i s d i f f i c u l t to see what such determining featu res might be. 761 GENERAL DISCUSSION AND CONCLUSIONS Svoboda and co-workers have shown ( 30, 37 ) t ha t the a l k a l o i d acronyc ine has cons ide rab le ant i tumor a c t i v i t y aga in s t a v a r i e t y of neoplasms in exper imental an imals . Th is t h e s i s has descr ibed some of the e f f e c t s of acronyc ine on the b iochemist ry and b io logy of two tumor l i ne s in c u l t u r e , the L5I78Y mouse and the IRC r a t leukemias. Sec t ion I of the r e s u l t s desc r ibes the g r o w t h - i n h i b i t o r y e f f e c t s of acronyc ine on L5I78Y and IRC c u l t u r e s , and some of the c y t o l o g i c a l changes seen in t r ea ted c e l l s . The i n h i b i t i o n by acronyc ine of the growth of both the L5I78Y and IRC c e l l l i ne s was accompanied by the appearance in the c u l t u r e s of b i nuc lea ted c e l l s . These appeared to r e s u l t from an i n t e r f e rence by the a l k a l o i d with the process of c y t o k i n e s i s ( c e l l s epara t i on ) a t the end of c e l l d i v i s i o n . Although acronyc ine appeared to have a s e l e c t i v e t o x i c i t y to c e l l s in the process of d i v i s i o n ( as shown by the i n h i b i t i o n of c y t o k i n e s i s ), i t d id not appear otherwise to have any s t r i k i n g e f f e c t s on the progress of c e l l s through the c e l l c y c l e . There were some i n d i c a t i o n s t ha t the a l k a l o i d could extend the G| phase, but t h i s e f f e c t d id not seem t o be detr imenta l t o the subsequent progress of c e l l s towards m i t o s i s . Acronycine t r ea ted c e l l s showed cons ide rab le mi tochondr ia l s w e l l i n g - t h i s u I t r a s t r u c t u r a I e f f e c t was not however accompanied by any marked changes in the oxygen consumption bf ceI I. cu I t u re s . I 4 C-acronyc ine was r e a d i l y bound to and re leased from L5I78Y c e l l s ( r e s u l t s , s e c t i on I ). The b ind ing of the a l k a l o i d to c e l l s , as we l l as the i n h i b i t i o n by acronyc ine of popu la t ion growth ( s e c t i on I, f i g u r e 17 ) and of nuc leos ide i nco rpo ra t i on ( s e c t i on 2, f i g u r e 34 ) were s t r ong l y 162 d imin i shed by i nc reas ing the amount of horse serum present in the I 4 t i s s u e c u l t u r e medium. C-acronycine was found t o bind e x t en s i v e l y t o one o r more non-dia IysabIe components of horse serum, and the r e s u l t s are c on s i s t en t with the suggest ion t ha t acronyc ine bound in t h i s way i s not a v a i l a b l e f o r i n t e r a c t i o n wi th c e l l s . In the cu r ren t i n v e s t i g a t i o n s , the most s t r i k i n g biochemical e f f e c t of acronyc ine on tumor c e l l s in c u l t u r e was a marked i n h i b i t i o n of the i n co rpo ra t i on of e x t r a c e l l u l a r nuc leos ides ( u r i d i n e , thymid ine , c y t i d i n e , guanosine ) i n to c e l l u l a r mate r i a l ( r e s u l t s , s e c t i on 2 ). In view of the po s s i b l e r e l a t i o n s h i p between t h i s e f f e c t and the growth i n h i b i t o r y p r ope r t i e s of the a l k a l o i d , a s e r i e s of i n v e s t i g a t i o n s was made t o determine the c h a r a c t e r i s t i c s and the mechanism of t h i s i n h i b i t i o n ( r e s u l t s , s e c t i on s 2 and 3 ). For the most p a r t , these i n v e s t i g a t i o n s concentrated on the e f f e c t s of acronyc ine on the u t i l i z a t i o n by L5I78Y c e l l s of e x t r a c e l l u l a r u r i d i n e . The i nco rpo ra t i on of e x t r a c e l l u l a r u r i d i n e i n to i n t r a c e l l u l a r metabolism i s diagrammed in f i g u r e 3, in the i n t r oduc t i o n to t h i s t h e s i s . Acronyc ine, an a c r i d i n e d e r i v a t i v e , bears some s t r u c t u r a l s i m i l a r i t y to a number of compounds which can i n t e r a c t with DNA. These compounds can i n h i b i t the i nco rpo ra t i on of e x t r a c e l l u l a r nuc leos ides i n to a c i d -i n s o l ub l e mate r i a l by r e s t r i c t i n g the template a c t i v i t y of DNA, and thus i n t e r f e r i n g with n u c l e i c ac id s ynthes i s at the polymerase l e v e l . Howeverm when acronyc ine was examined in v i t r o in systems which are useful f o r de tec t i n g DNA-drug i n t e r a c t i o n ( r e s u l t s , s e c t i on 2 ), there was no evidence t ha t the a l k a l o i d complexed with DNA. Furthermore, acronyc ine had no e f f e c t on the synthes i s of RNA in v i t r o using a system - E. co I i RNA polymerase and c a l f thymus DNA - which is s e n s i t i v e 163 to changes in template a c t i v i t y induced by DNA-drug i n t e r a c t i o n s . These observat ions t he r e f o r e o f f e r no support f o r the suggest ion t ha t a c r o -nycine i n t e r a c t s with DNA and a l t e r s i t s template a c t i v i t y . The quest ion of whether or not acronyc ine had any e f f e c t on n u c l e i c ac id synthes i s in tumor c e l l s was f u r t h e r examined in c e l l s in which the i n t r a c e l l u l a r nuc l eo t i de pools had been p r e l a b e l l e d with r a d i o a c t i v e nuc leos ides ( s e c t i on 2 ). It was found tha t when L5I78Y c e l l s were incubated with e x t r a c e l l u l a r r a d i o a c t i v e u r i d i n e o r thymidine at 7 ° , the i n t r a c e l l u l a r nuc leo t i de pools could be l a b e l l e d without any r a d i o a c t i v i t y appearing in the n u c l e i c a c i d s . On warming the c e l l s t o 37° , r a d i o a c t i v i t y from t h i s p r e l a b e l l e d pool was t r a n s f e r r e d in to n u c l e i c a c i d s . Acronycine had no e f f e c t on t h i s t r a n s f e r , suggest ing t ha t the a l k a l o i d does not i n h i b i t n u c l e i c a c i d s yn thes i s per se, e i t h e r by an i n t e r a c t i o n of the a l k a l o i d wi th DNA or o therw i se . It f o l l ows t he re f o re t h a t the i n h i b i t i o n by acronyc ine of the i n co rpo ra t i on of e x t r a c e l l u l a r nuc leos ides i n to n u c l e i c ac id s must r e s u l t from an i n t e r f e rence by the drug wi th some other step in the u t i l i z a t i o n of such precur sor s ( f igure 3 ). Acronycine was tound to markedly i n h i b i t the i nco rpo ra t i on of e x t r a c e l l u l a r u r i d i n e and thymidine i n to i n t r a c e l l u l a r a c i d - s o l u b l e mater ia l ( r e s u l t s , s e c t i on 2 ). This i n h i b i t i o n of the l a b e l l i n g of the- i n t r a -c e l l u l a r n u c l e i c a c i d p recur sor pools appeared s u f f i c i e n t to account e n t i r e l y f o r the reduced i n co rpo ra t i on of e x t r a c e l l u l a r u r i d i n e and thymidine i n to n u c l e i c a c i d s . Although acronyc ine markedly reduced the l a b e l l i n g of the a c i d - s o l u b l e mate r i a l of L5I78Y c e l l s by e x t r a c e l l u l a r r a d i o a c t i v e u r i d i n e , i t d id not a l t e r the r e l a t i v e amounts of the va r ious i n t r a c e l l u l a r phosphoryIated d e r i v a t i v e s ( s e c t i o n 2 ). The V64 a l k a l o i d a l s o d id not i n t e r f e r e with the f unc t i on of the plasma membrane wi th respect to i t s a b i l i t y t o r e t a i n nuc leo t i de s w i t h i n the c e l l ( a s i nd i ca ted by the lack of e f f e c t of acronyc ine on the r e t en t i on of l a b e l l e d nuc leo t i de s dur ing the p r e l a b e l l i n g experiments ). These f i nd i n g s t he r e f o r e suggested t ha t the reduced l a b e l l i n g of the nuc l eo t i de pools of L5I78Y c e l l s by u r i d i n e in the presence of acronyc ine may be due mainly i f not e n t i r e l y to an i n h i b i t i o n of the entry of the nuc leos ide i n to the c e l l and/or i t s i n i t i a l phosphory la t ion ( to UMP ). These steps in the u t i l i z a t i o n of e x t r a c e l l u l a r u r i d i n e are diagrammed in f i g u r e 36, in the i n t r oduc t i on to s e c t i on 3 of t h i s t h e s i s . Sect ion 3 of the t h e s i s desc r ibes i n v e s t i g a t i o n s i n t o the means by which acronyc ine i n h i b i t s the entry of e x t r a c e l l u l a r u r i d i n e in to i n t r a c e l l u l a r nuc leos ide poo l s . An examination was f i r s t made of the nature of the u r i d i n e uptake process in untreated L5I78Y c e l l s . The t r an spo r t of u r i d i n e through the plasma membrane appeared to be a process d i s t i n c t from i t s subsequent phosphory la t ion to UMP. A comparison of the k i n e t i c s and temperature dependence of u r i d i n e uptake by whole c e l l s and u r i d i n e phosphory la t ion in v i t p o by c e l l e x t r a c t s suggested t ha t the t r an spo r t of u r i d i n e through the plasma membrane was normal ly the r a t e -l i m i t i n g step in i t s uptake by c e l l s . These conc lu s ions are in agreement wi th those of o ther workers ( 100, 108 ) using c e l l s o ther than the L5I78Y leukemia. Acronycine had very l i t t l e e f f e c t on the phosphory la t ion of u r i d i n e i n\/vi t r o . Furthermore, arguments based on the k i n e t i c s and the temperature dependence of u r i d i n e uptake in the presence of acronyc ine suggested t ha t in d rug - t reated c e l l s the t r an spo r t of u r i d i n e across the plasma membrane was r a t e - I i m i t i n g in i t s uptake. The data t he r e f o r e i n d i c a t e t ha t acronyc ine i n t e r f e r e s with the entry of e x t r a c e l l u l a r u r i d i n e 165 i n to i n t r a c e l l u l a r nuc leo t i de pools by i n h i b i t i n g the t r an spo r t of the nuc leos ide across the plasma membrane. It may be noted tha t these arguments ( which are presented in f u l l in the d i s cu s s i on a t the end of s e c t i o n 3 of the r e s u l t s ) do not r u l e out a s imultaneous i n h i b i t i o n of u r i d i n e phosphory la t ion w i t h i n the c e l l ; they do however suggest t ha t such an i n h i b i t i o n , i f p resent , i s not re spons ib le f o r the i n h i b i t i o n of u r id ineuuptake by ac ronyc ine . Mechanism by which acronyc ine i n h i b i t s the growth of tumor c e l l s : It i s not po s s i b l e with the in format ion at present a v a i l a b l e to make many f i r m conc lu s ions on the nature of the mechanism by which acronyc ine i n h i b i t s the growth of tumor c e l l s . An i n h i b i t i o n of nuc leos ide uptake cannot account f o r the growth i n h i b i t o r y p r ope r t i e s of the a l k a l o i d , as the c e l l s have no requirement f o r extrace I IuIar nuc leo s ide s , and the normal growth medium f o r L5I78Y andiIRC c e l l s does not even inc lude these compounds. Acronyc ine, even at the high concent ra t i on of 12 yg/ml, had on ly a small e f f e c t on the uptake of cho l i ne o r i n o s i t o l - compounds which are normally present in the medium ( r e s u l t s , s e c t i o n 2 ). When deoxyglucose was used to i n ve s t i g a t e the e f f e c t of acronyc ine on g lucose uptake mechanisms, there was n©oinh ib i t ion of the uptake of t h i s model compound, but even a s l i g h t s t i m u l a t i o n . An i nh i b i tionco'f the uptake of c h o l i n e , i n o s i t o l , o r g lucose does not seem t he re f o r e l i k e l y t o be ab le to account f o r the g r o w t h - i n h i b i t o r y p r ope r t i e s of ac ronyc ine . It cannot be excluded however t ha t acronycine. may i n h i b i t the uptake of some other e s s e n t i a l n u t r i e n t from the e x t r a c e l l u l a r f l u i d . Many, i f not a l l , of the b i o l o g i c a l e f f e c t s of acronyc ine reported in t h i s t h e s i s o r by other workers can be e xp l a i ned , a t l ea s t s p e c u l a t i v e l y , 166 as r e s u l t i n g from e f f e c t s of the a l k a l o i d on the f unc t i on i n g of b i o l o g i c a l membranes. Some of these e f f e c t s are noted here : a) the i n h i b i t i o n by acronyc ine of the i nco rpo ra t i on of nuc leos ides i n t o a c i d - s o l u b l e and ac i d - i n so I ub I e m a t e r i a l : As p rev i ou s l y d i s cus sed, t h i s e f f e c t appearsrto r e s u l t s from an i nterferenee.. by .-acronyci ne with the t r an spo r t of nuc leos ides across the plasma membrane. b) the i n h i b i t i o n by acronyc ine of c y t o k i n e s i s : As wi I I be d i scussed in g rea te r d e t a i l below, t h i s e f f e c t of the drug i s very s i m i l a r in some ways to t h a t of the compound c y t o c h a l a s i n B. It has r e cen t l y been suggested t h a t the e f f e c t s of thms l a t t e r compound on c y t o k i n e s i s are best exp la ined as r e s u l t i n g from a d i r e c t a c t i o n of the agent on the plasma membrane ( 142, 148 -150 ) . c) s w e l l i n g of mitochondr ia and Golg i bodies : The accumulat ion of f l u i d by these membranous o r gane l l e s could po s s i b l y be the r e s u l t of an osmotic imbalance caused by an i n t e r f e r ence by acronyc ine with membrane ion t r an spo r t systems. d) the cen t r a l nervous system depressant a c t i v i t y of acronyc ine : As noted in the i n t r o d u c t i o n , i n v e s t i g a t i o n s by the workers of E l i L i l l y and Co. have i nd i ca ted t ha t acronyc ine depresses the cen t r a l nervous system a c t i v i t y of exper imental amiimals, and produces symptoms s i m i l a r to those of known hypnot ics ( 32 ). It i s we I I e s t a b l i s h e d t ha t drugs which a f f e c t the f unc t i on i n g of the nervous system may do so by i n t e r -a c t i n g with the membranes of nerve c e l l s ( 151 ). A s i m i l a r i n t e r a c t i o n by acronyc ine might be the bas i s of i t s depressant a c t i v i t y . It should be emphasized t h a t , with the except ion of the e f f e c t of acronyc ine on nuc leos ide uptake, the above exp lanat ions f o r the e f f e c t s of the a l k a l o i d are a t the moment on ly s p e c u l a t i v e . Other exp lanat i on s 167 f o r the phenomena noted are e n t i r e l y p o s s i b l e . Furthermore, i t should be noted t ha t not a l l membrane func t i on s of the c e l l are n e c e s s a r i l y a f f e c t e d by ac ronyc ine . Thus acronyc ine does not appear to a f f e c t the s u c e e s s f u p r e f o r m a t i o n of the nuc lear membrane a f t e r m i t o s i s , even in c e l l s in which c y t o k i n e s i s has been i n h i b i t e d . The f a c t remains however t ha t most of the known b i o l o g i c a l and biochemical e f f e c t s of acronyc ine are most e a s i l y exp la ined on the bas i s of changes in membrane f u n c t i o n . Th i s suggests t h a t the g r o w t h - i n h i b i t o r y p r ope r t i e s of acronyc ine may a l s o stem, e i t h e r d i r e c t l y or i n d i r e c t l y , from an e f f e c t of the a l k a l o i d on c e l l membranes, although c y t o t o x i c e f f e c t s based on other mechanisms cannot at the moment be exc luded. Fur ther research i s c l e a r l y needed to determine the mechanism by which acronyc ine i n h i b i t s the growth of tumor ceI Is. Imp l i ca t ions of the i n v e s t i g a t i o n s f o r the use of acronyc ine in the  treatment of human neoplasms : The r e s u l t s obta ined in the i n v e s t i g a t i o n s descr ibed in t h i s t h e s i s may have important i m p l i c a t i o n s when con s i de r i ng the use of acronyc ine in combination wi th other dnugs f o r the treatment of cancer. The a b i l i t y of a c e l l t o take up an an t i c ance r drug i s one of the major determinants of the s e n s i t i v i t y of t ha t c e l l t o the drug. Thus mutations whi.ch a f f e c t the t r an spo r t of a drug in to a c e l l are one of the major mechanisms by which n e o p l a s t i c c e l l s develop r e s i s t ance to ant i tumor agents ( 7 ). Warnick e t a I. have shown tha t c e r t a i n potent i n h i b i t o r s of nuc leos ide t r an spo r t can p r o tec t L5I78Y c e l l s in c u l t u r e from the a n t i p r o l i f e r a t i v e e f f e c t s of a n t i n e o p l a s t i c nuc leos ide analogs ( 152 ). S ince the cu r ren t i n v e s t i g a t i o n s show tha t acronyc ine i n h i b i t s the uptake of a number of n a t u r a l l y - o c c u r r i n g nuc leo s ide s , the a l k a l o i d i>; 8 may po s s i b l y a l s o i n h i b i t the uptake of a n t i n e o p l a s t i c nuc leos ides such as 5 - f I u o r o -deoxyu r i d i ne . Th is could p r o t e c t the n e o p l a s t i c c e l l from the e f f e c t s of the l a t t e r drug. It might be po inted out however t ha t i t cannot s imply be assumed tha t an i n t e r f e rence by acronyc ine with the t r an spo r t of o ther a n t i n e o p l a s t i c drugs i s n e c e s s a r i l y d e l e t e r i o u s to combination chemotherapy. It i s not inconceiveabIe t ha t acronyc ine might i n h i b i t the t r an spo r t of another agent more s t r ong l y i n t o normal c e l l s than in to tumor c e l l s . In t h i s case, acronyc ine might actuaI Iy increase the t h e r a p e u t i c e f f e c t i v e n e s s of the other agent by reducing i t s t o x i c i t y to norma I ceI Is. The r e s u l t s of the i n v e s t i g a t i o n s in t h i s t h e s i s have suggested t h a t acronyc ine i s bound in app rec i ab le amounts by serum components, . and t ha t serum-bound drug i s unava i l ab l e f o r i n t e r a c t i o n with c e l l s . Th is b ind ing may play an important r o l e in the pharmacology of acronyc ine in c l i n i c a l use, as most of the acronyc ine in the c i r c u l a t o r y system of a p a t i e n t i s expected to be bound t o plasma components. Other drugs used in t r e a t i n g the cancer p a t i e n t could po s s i b l y b indoto the same serum component s i t e s as acronyc ine and d i s p l a c e i t . Th is could increase the e f f e c t i v e concen t ra t i on s - o f the a l k a l o i d in the body, and thus i t s t o x i c i t y to both tumor and normal c e l l s . P o t e n t i a l f o r the employment of acronyc ine in i n v e s t i g a t i o n s of c e l l  b i oIogy: Acronyc ine, in a d d i t i o n to showing some promise as an an t i c ance r drug, a l s o has p o t e n t i a l f o r i n v e s t i g a t i o n s of c e l l b i o l o g y . In t h i s connect ion , the a b i l i t y of acronyc ine to i n t e r f e r e with c y t o k i n e s i s and thus cause the formation of b inuc lea ted c e l l s i s of p a r t i c u l a r i n t e r e s t . This a c t i on of acronyc ine i s s u p e r f i c i a l l y very s i m i l a r to 169 t ha t of the i n t e n s i v e l y i n ve s t i g a ted agent, c y t o cha l a s i n B ( 84 ). Ea r l y i n v e s t i g a t i o n s i n to the mechanism of a c t i on of c y t o c h a l a s i n B suggested t ha t i t i n t e r f e r e d wi th c y t o k i n e s i s by d i s r u p t i n g the system of m ic ro f i l ament s which appears t o be re spons ib le f o r the c o n s t r i c t i o n of the c e l l a t c y t o k i n e s i s ( 153 ). La te l y however, i t has been suggested t ha t the a c t i o n of c y t o c h a l a s i n B on c y t o k i n e s i s i s b e t t e r exp la ined by a d i r e c t a c t i o n of the compound on the plasma membrane ( 142, 148— 150 ). A s i m i l a r mechanism ( d i r e c t i n t e r a c t i o n with the plasma membrane ) i s a l s o thoughtTto account f o r the a b i l i t y of c y t o cha l a s i n B to i n h i b i t the t r an spo r t of nuc leos ides ( 142 ) and glucose and glucosamine ( 150 ) i n to c e l l s . Thus acronyc ine and c y t o c h a l a s i n B appear qu i t e s i m i l a r in t ha t they are both l i p o p h i l i c compounds which can i n h i b i t t r a n spo r t processes and can i n t e r f e r e wi th c y t o k i n e s i s . There are however some i n t e r e s t i n g d i f f e r e n c e s in the a c t i on of the two compounds on d i v i d i n g c e l l s . Acronycine i s much less e f f i c i e n t in inducing b inuc lea ted c e l l s than i s c y t o cha l a s i n B . Thus wh i l e a maximum of 30 t o 40 % of the ceI Is in a c u l t u r e t r ea ted with acronyc ine have been observed in the cu r ren t study t o become b i nuc l ea t ed , c y t o cha l a s i n B i s capable of caus ing a l l c e l l s in a c u l t u r e to become b inuc lea ted ( 84 ). This d i f f e r e n c e could e i t h e r r e f l e c t a bas i c d i f f e r e n c e in the a c t i on of the two compounds on d i v i d i n g c e l l s , or could merely r e s u l t from a g rea te r t o x i c i t y of acronyc ine to c e l l s which have not reached c y t o k i n e s i s . A l so in c on t r a s t t o c y t o c h a l a s i n B , acronyc ine does not seem to ac t immediately on c y t o k i n e s i s , but on ly a f t e r a contact per iod ranging from a minimum of 2 t o -4 hours up t o one and one-ha l f c e l l generat ion t imes . Further s tud ie s aimed at determining the r e l a t i o n s h i p between the s i m i l a r i t i e s and d i f f e r e n c e s of the e f f e c t s of these two compounds on 170 d i v i d i n g c e l l s might shed cons ide rab le l i g h t on the poor ly understood phenomenon of c y t o k i n e s i s . Another property of acronyc ine which bears f u r t h e r i n v e s t i g a t i o n i s i t s e f f e c t s on Golg i bodies. Tan ( 81 ) and Tan and Auersperg ( 82 ) have found t ha t the morphology of the Golg i bodies of L5I78Y c e l l s i s a f f e c t e d w i t h i n one hour of the a d d i t i o n of the drug to c e l l c u l t u r e s . Fur ther i n v e s t i g a t i o n s are needed to determine the nature of the u I t r a s t r u c t u r a I changes seen in the e l e c t r o n microscope -i . e . do the changes represent damage, or are they merely the r e s u l t of h y p e r a c t i v i t y of t h i s o r g a n e l l e . S ince i t i s wel l e s t a b l i s h e d t h a t the Golg i apparatus i s a source of new membrane f o r the c e l l per iphery ( 154 ), damage to t h i s o r gane l l e might b lock the movement of newly-synthes i zed membrane components to the plasma membrane. Such a b lock might markedly a f f e c t c e l l f unc t i on s which are dependent on the plasma membrane such as c e l l v i a b i l i t y , the i n t e r a c t i o n of c e l l s with the environment, o r even c y t o k i n e s i s . An i n t e r f e rence wi th the f unc t i on of the Golg i apparatus thus would not only be i n t e r e s t i n g in terms of i t s e f f e c t s on the c e l l , but might prove to be a useful t oo l in i n v e s t i g a t i o n s of membrane synthes i s and/or tu rnover . 171 BIBLIOGRAPHY I) Farber , S.L.K., Diamond, R.D., Mercer, R.F., S y l v e s t e r , R.F., and Wo l f f , J . A . , New Eng. J ' . Med. 238: 787, (1948). 2) Gi lman, A., Amer. J . 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