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The effect of vinblastine on the incorporation of C14-formate into the acid-soluble fraction of thymus… Jones, Richard Gareth Wyn 1963

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Jl  THE EFFECT OF VINBLASTINE ON THE INCORPORATION OF C^-FORMATE INTO THE ACID-SOLUBLE FRACTION OF THYMUS CELL SUSPENSIONS•  by  Richard Gareth Wyn Jones B . S c . The U n i v e r s i t y of Wales 1961 A THESIS SUBMITTED IN, PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Biochemistry We accept t h i s t h e s i s as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA May, 1963.  I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study.  I f u r t h e r agree that per-  m i s s i o n f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head o f my.Department or by h i s r e p r e s e n t a t i v e s . I t i s understood" that copying, or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n .  Department o f  Biochemistry  The U n i v e r s i t y of B r i t i s h ColumbianVancouver 8, Canada. Date  A p r i l 26th, 1963.  ABSTRACT In e a r l i e r studies by B e e r the a l k a d l o i d v i n b l a s t i n e , 1  VLB, has been shown- to cause an almost complete i n h i b i t i o n i n the incorporation  of C^-formate i n t o the deoxyribonucleic  acid (DNA) of r a t bone marrow i n vivo.»  Also the i n c o r -  poration of the same isotope i n t o the DNA of suspensions of r a t bone marrow and thymus c e l l s i n v i t r o was s u b s t a n t i a l l y 2  depressed w i t h i n one hour by the presence of the a l k a l o i d . In t h i s work, the e f f e c t of VLB on the incorporation of C ^-formate i n t o the purine and pyiimidine bases i s o l a t e d 1  from the hydrolysed acid-soluble preparations was  f r a c t i o n of thymus c e l l  studied.  A method f o r the separation of the constituents of t h i s . f r a c t i o n was standardized and some of the major u l t r a v i o l e t absorbing and r a d i o - a c t i v e  compounds were i d e n t i -  fied. In general, VLB d i d not cause a 14 incorporation  of C  marked depression i n the  -formate i n t o the bases i s o l a t e d from  the hydrolysed acid-soluble  f r a c t i o n of e i t h e r washed or  unwashed c e l l suspensions. I t was therefore considered probable that v i n b l a s t i n e d i d not i n h i b i t the a c t i v a t i o n of 1-carbon u n i t s by t e t r a h y d r o f o l i c acid or t h e i r t r a n s f e r . Also the r e s u l t s indicated that the de novo synthesis of the purine and pyrimidine compounds was not i n h i b i t e d by 1 Beer, G.T., Canadian Cancer Conference 4, 355 (1961) Academic Press, Inc., New York. 2  Richards, J.F., and Beer, C.T., personal communication.  the a l k a l o i d .  I t i s t e n t a t i v e l y suggested that VLB  i n h i b i t s a step i n the incoorporation of the mononucleotides i n t o the n u c l e i c a c i d s . Comparison of the compounds i s o l a t e d from the hydrolysed a c i d - s o l u b l e f r a c t i o n s of washed and unwashed c e l l preparations showed that considerably l e s s hypoxanthine and xanthine were recovered from the former. VLB was found to markedly increase the t o t a l r a d i o - a c t i v i t y of serine i n washed c e l l preparations i n the t h i r d hour of incubation but t h i s e f f e c t was not pronounced i n unwashed c e l l preparations.  The t o t a l count  of adenine decreased i n the t h i r d hour of washed c e l l i n c u bations i n the presence of VLB.  Neither the e f f e c t on  serine nor on adenine i s considered to be d i r e c t l y r e l a t e d to the i n h i b i t i o n of DNA synthesis as the l a t t e r i s apparent w i t h i n only 1 hour's incubation.  Washing appears to increase  the s e n s i t i v i t y of c e r t a i n biochemical changes i n the c e l l . I t i s t e n t a t i v e l y suggested that the appearances of changes i n the t o t a l r a d i o - a c t i v i t y of adenine may be r e l a t e d to the l o s s of a substrate on washing.  ACKNOWLEDGEMENTS  The author wishes t o express h i s g r a t i t u d e to D r . C,T. Beer f o r h i s encouragement and c r i t i c i s m d u r i n g t h i s worko  Thanks are a l s o extended to D r .  J . F . R i c h a r d s f o r h i s guidance and p e r m i s s i o n to use many o f h i s r e s u l t s and a l s o to D r . R.L. Noble f o r the use o f the f a c i l i t i e s  In h i s laboratory.  The a s s i s t a n c e o f Dr. W.A.. Webber i n the n i n h y d r i n colorIme t r i e d e t e r m i n a t i o n s i s g r a t e f u l l y acknowledged. The author i s g r a t e f u l f o r the a s s i s t a n c e o f M r s . M. W i l s o n , Miss J . Leonard and Mrs. J . Gold i n the p r e p a r a t i o n and t y p i n g o f t h i s t h e s i s .  TABLE OP CONTENTS  INTRODUCTION  1  EXPERIMENTAL I.  ....  9  STUDY AND DEVELOPMENT OP METHODS ....... A.  8  9  BASIC METHODS  lo Materials  9  2. B i o l o g i c a l and chemical procedure f o r i s o l a t i o n and h y d r o l y s i s o f ' a c i d - s o l u b l e f r a c t i o n ' o f thymus c e l l p r e p a r a t i o n s and i o n exchange s e p a r a t i o n o f l i b e r a t e d bases •• 10 3© Chromatographic Techniques ..................  13  Ij.. U l t r a - v i o l e t Spectroscopy  15  5. R a d i o - a c t i v i t y D e t e c t i o n and D e t e r m i n a t i o n •.  l£  6. I s o t o p i c D i l u t i o n Technique  17  •  B. STANDARDISATION OP METHODS 1. I n t r o d u c t i o n  18 ••  18  2. M a t e r i a l s  18  3. Procedure and R e s u l t s  18  G. USE OP CHARCOAL TO DESALT SAMPLES... 1. I n t r o d u c t i o n  •  2. Methods 3* R e s u l t s  22 22 23  and D i s c u s s i o n  I I . RESULTS A. I n d e n t i f i c a t i o n o f the Compounds found i n the h y d r o l y s e d a c i d - s o l u b l e f r a c t i o n o f unwashed thymus c e l l p r e p a r a t i o n s and the e f f e c t o f v i n b l a s t i n e on Cllj—formate i n c o r p o r a t i o n i n t o these compounds  21\. 29  29  B. The e f f e c t o f washing on thymus c e l l suspensions and on the a c t i o n o f v i n b l a s t i n e on the i n c o r p o r a t i o n o f clM—formate i n t o the a c i d - s o l u b l e f r a c t i o n o f those p r e p a r a t i o n s • • • • • • •.«••••.•••». • • C. The examination o f u n i d e n t i f i e d r a d i o - a c t i v e compounds • ....... D. Comparison o f (a) the i n c o r p o r a t i o n o f C ^ - f o r m a t e i n washed and unwashed c e l l suspensions (b) The s e n s i t i v i t y o f the two types - o f c e l l p r e p a r a t i o n to v i n b l a s t i n e ............ DISCUSSION Appendix SUMMARY BIBLIOGRAPHY  .  TABLES  1  Recovery o f p u r i n e and p y r i m i d i n e bases from i o n exchange c o l u m n s . o . . . . . . . . . . o . . . . . . . . ( t o 0  Page face)••20  2a b  Recovery o f mononucleotides Recovery o f adenine and guanine c o n t a i n i n g compounds ...... • • • •«(to face)••27  3  Recovery o f the products o f NAD I; j h y d r o l y s i a and x a n t h i n e and hygoxanthine from i o n exchange columns....o........• ©.(to  face)..32  E f f e c t o f v i n b l a s t i n e on i n c o r p o r a t i o n o f formate i n t o a c i d - s o l u b l e f r a c t i o n o f unwashed c e l l s o .•••oo.©••(to  face)•.36  l\.  5  E f f e c t o f v i n b l a s t i n e on i n c o r p o r a t i o n o f Glhformate i n t o the n u c l e i c a c i d s o f unwashed c e l l p r e p a r a t i o n s • • • © • • • ..• © • • • • ©. • o © (to f a c e ) • .37 0  6  E f f e c t o f v i n b l a s t i n e on G-h~ formate incor« p o r a t i o n i n t o a c i d - s o l u b l e fractions*, t o t a l radio-activity.............•.©••• • • • o « ( t o face)©.39  7  Comparison o f e f f e c t o f v i n b l a s t i n e on G'H|*» f o r mate I n c o r p o r a t i o n i n t o the a c i d s o l u b l e f r a c t i o n o f b o t h types o f c e l l p r e p a r a t i o n . ( t o  8  face)..53  E f f e c t o f v i n b l a s t i n e on C ^ - formate i n c o r p o r a t i o n i n t o the n u c l e i c a c i d e o f washed c e l l preparationso ( t o face)..514-  Figures Page 1*  Formulae o f v i n b l a s t i n e and v i n c r i s t i n e (to f a c e ) ...o.......  2 . DNA b i o s y n t h e t i c pathways 3.  (to face)  7  o . . . . . . . . . .  Ion-exchange s e p a r a t i o n o f p u r i n e and p y r i m i d i n e bases (to face)  3  •  19  !}.• Recovery o f ATP from c h a r c o a l column (to f a c e )  2I4.  5» Ion exchange s e p a r a t i o n o f bases o b t a i n e d by h y d r o l y s i n g a c i d s o l u b l e f r a c t i o n o f unwashed thymus c e l l suspension, ( t o face) ...........  30  6. S e p a r a t i o n o f h y d r o l y s i s products o f NAD (to face)  31  7. S e p a r a t i o n on Dowex $0 IT" o f bases o b t a i n e d from a c i d - s o l u b l e f r a c t i o n p r e - t r e a t e d w i t h c h a r c o a l , (to face) 1  . o . . . . . * . » .  8. E f f e c t o f VLB on t o t a l n o n - v o l a t i l e radio-activity i n acid-soluble f r a c t i o n o f unwashed thymus c e l l s (to f a c e ) .......o... 9..Comparison o f h y d r o l y s e d a c i d s o l u b l e f r a c t i o n s o f washed and unwashed thymus c e l l s (to face) 10.  E f f e c t o f VLB and time o f i n c u b a t i o n on p u r i n e and s e r i n e s y n t h e s i s by washed thymus c e l l s ( t o f a c e ) ...........  11.  E f f e c t o f VLB on G-^-formate I n c o r p o r a t i o n i n t o the ' a c i d - s o l u b l e f r a c t i o n o f washed thymus c e l l s . (to follow)  3^4-  38  I4.2  1  12.  1  I s o l a t i o n o f s e r i n e on Dowex 50 ET*" ........... (to f a c e )  I44 lj.8  E f f e c t o f VLB on i n c o r p o r a t i o n o f C ^formate i n t o DNA o f washed thymus c e l l s (to f a c e ) 1  B i o s y n t h e s i s o f A d e n y l i c and G u a n y l i c a c i d s from I n o s i n i c a c i d ( t o f a c e ) ••  - 1 -  INTRODUCTION I s o l a t i o n of Vinca A l k a l o i d s . I n t e r e s t i n the b i o l o g i c a l p r o p e r t i e s the s u b - t r o p i c a l p e r i w i n k l e V i n c a Rosea L i n n ,  of extracts of a r o s e from  reports  t h a t the n a t i v e p o p u l a t i o n o f the West I n d i e s used a t e a made from the l e a v e s o f t h i s p l a n t , i n the t r e a t m e n t o f d i a b e t e s mellitus.  However, the p r o p e r t i e s  these r e p o r t s  a t t r i b u t e d to the p l a n t by  have n o t been s u b s t a n t i a t e d by a c l o s e r  scientific  examination. D e t a i l e d s t u d i e s ftere made, i n d e p e n d e n t l y , by Noble (1) a t the U n i v e r s i t y o f Western O n t a r i o and by a group a t the E l i L i l l y P h a r m a c e u t i c a l Company ( 2 ) .  N e i t h e r group c o u l d produce  any a l t e r a t i o n i n the b l o o d sugar o f normal o r d i a b e t i c or rabbits  by o r a l a d m i n i s t r a t i o n o f p l a n t e x t r a c t s .  rats,  However,  Noble found t h a t the i n j e c t i o n o f p l a n t e x t r a c t s caused r a t s  to  d i e I n f i v e t o seven days from a f u l m i n a t i n g i n f e c t i o n , w h i c h was a s s o c i a t e d w i t h a r e d u c t i o n i n c i r c u l a t i n g l e u k o c y t e s a depression  o f the bone marrow.  The d e s t r u c t i o n o f  granulocytes  was p a r t i c u l a r l y s t r i k i n g .  I t was f e l t t h a t f u r t h e r i n v e s t i g a t i o n o f the  and  the  leukopenic  a c t i v i t y o f the e x t r a c t s m i g h t l e a d to the i s o l a t i o n o f a compound p o t e n t i a l l y u s e f u l i n the t r e a t m e n t o f l e u k a e m i a The c h e m i c a l I s o l a t i o n o f the t o x i c m a t e r i a l was  (3).  undertaken,  u s i n g the d e p r e s s i o n o f the w h i t e b l o o d c e l l count o f a normal r a t as an a s s a y . The a c t i v e p r i n c i p l e was found to be b a s i c and crude e x t r a c t s gave t y p i c a l a l k a l o i d a l r e a c t i o n s  with  Dragendorff1s  reagent and w i t h a l k a l o i d p r e c i p i t a t i n g reagents, such as p i c r i c a c i d , and  silicotungstic acid.  The  crude e x t r a c t s  f r a c t i o n a t e d by e x t r a c t i o n w i t h o r g a n i c s o l v e n t s media o f d i f f e r e n t pH v a l u e s . medium at pH t i o n o f the  The  from aqueous  e x t r a c t i o n o f the  7 by-benzene r e s u l t e d i n a c o n s i d e r a b l e 'active p r i n c i p l e ' .  t h e r p u r i f i e d by  were  aqueous purifica-  T h i s benzene e x t r a c t was  chromatography on alumina.  fur-  Eventually,  Beer  i s o l a t e d an a c t i v e a l k a l o i d as the c r y s t a l l i n e sulphate This  compound which was  named V i n c a l e u k o b l a s t i n e  (VLB),  was  found to i n h i b i t the growth of c e r t a i n experimental tumours, as w e l l as c a u s i n g l e u k o p e n i a and The  d e p r e s s i n g the bone marrow.  group at the E l i L i l l y Research L a b o r a t o r i e s  (2).  crude f r a c t i o n s o f the p l a n t f o r anti-tumour a c t i v i t y ?-l$3k Leukaemia, implanted i n t o DBA/2 mice, was  tested  found to  be  markedly a f f e c t e d by c e r t a i n p a r t i a l l y p u r i f i e d e x t r a c t s . U s i n g anti-tumour a c t i v i t y as a b i o a s s a y , l e u r o s i n e , an l o i d c l o s e l y resembling v i n c a l e u k o b l a s t i n e , (£)•  The  was  i n h i b i t o r y action of vincaleukoblastine  mental tumours, p a r t i c u l a r l y leukaemias, was  alka-  then i s o l a t e d on  experi-  a l s o confirmed  by  t h i s group. E a r l y i n t h e i r i n v e s t i g a t i o n s Johnson et a l (6) that  found  c e r t a i n p a r t i a l l y p u r i f i e d f r a c t i o n s f r e e from v i n c a l e u k o -  b l a s t i n e and  leurosine,  could produce i n d e f i n i t e s u r v i v o r s ,  on  i n j e c t i o n i n t o mice impregnated w i t h P-l£3lv leukaemia.  This  suggested the presence o f o t h e r ' a c t i v e ' compounds and  a fur-  t h e r s e a r c h l e d to the i s o l a t i o n o f two i e u r o c r i s t i n e and  leurosidine  (7).  other active a l k a l o i d s ,  FIGURE 1 FORMULAE OF VINBLASTINE AND LEUROCRISTINE  \  VINBLASTINE  R - CH  LEUROCRISTINE  R = CHO  3  - 3 The tive  four  dimeric  mainly  from  structure. physical  leukoblastine portion  of  a l k a l o i d  the  the  monomers  b i o l o g i c a l or  and  active  Gorman  and  measurements,  l e u r o c r i s t i n e  VLB m o l e c u l e  catheranthine,  resembles dual  b i o l o g i c a l l y  while  monomeric nor  Is  a c t i v i t y  of  the  Neuss the  shown very  the  a l k a l o i d  equimolar  alkaloids  type  structures i n  Figure  indoline  shown  by  for  the  vinca-  The  closely  Neither  the  monomers  the  Indole  monomer!c  portion  the  d i s t i n c -  proposed,  1.  to  vindoline. of  a  i n d i v i -  have  vincaleukoblastine  l e u r o c r i s t i n e . The  the  American  following  names  Medical for  the  Association  (A.M.A.)  The  (VLB)  Leurosine  Vinleurosine  (VLS)  Leurocristine  V i n c r i s t i n e  (VCR)  Leurosidine  Vinrosidine  (VRD)  adopted  by  the  A.M.A.,  the  Vinca  are  used  i n  the  remainder  of  thesis.  The normal  BDF  Properties. action  tissue  vinblastine  mour  nomenclature.  Vinblastine  B i o l o g i c a l  IRC  adopted  Vincaleukoblastine  names,  this  has  alkaloids: A.M.A..  i n  have  (8)  s i m i l a r  mixtures  have  has  of been  i n h i b i t s  hybrid  leukaemia  mice, i n  studied.  the the  growth AK  F i s c h e r  i n  Swiss  mice.  control  group  was  The  used  alkaloids  as  Noble of  rats  and  index  of  neoplastic  a l  (9)  L-1210  i n  also  prolongation an  et  the  leukaemia  on  Inbred the  of  have  shown  leukaemia  anti-tumour  the  Ascites  r e l a t i v e  that  carried  AKR m i c e ,  E h r l i c h  l i f e  and  to  t u a  a c t i v i t y .  Usually multiple  doses of the  a l k a l o i d were g i v e n , s t a r t i n g  21\. hours a f t e r tumour i m p l a n t a t i o n . against was  delayed two  delayed 72  VLB  to three days, although the growth of the P.l53^  s t i l l markedly i n h i b i t e d even i f treatment  hours a f t e r tumour i m p l a n t a t i o n (9)»  v i n c r i s t i n e was exhibited  mours  action of  the m a j o r i t y of tumours decreased r a p i d l y when treatment  leukaemia was  it  However, the  found to be  The  was  activity  s i m i l a r to t h a t of v i n b l a s t i n e  greater a c t i v i t y against  well established  of  but  tu-  (10). I n g e n e r a l , the f o u r a c t i v e a l k a l o i d s have a comparable  range o f anti-tumour p r o p e r t i e s .  However, although  vinblastine  r e a d i l y caused l e u k o p e n i a , the o t h e r a c t i v e a l k a l o i d s caused t h i s form o f t o x i c i t y a t very h i g h doses. c a u s i n g a d e p r e s s i o n of the  only  As w e l l  bone marrow, v i n b l a s t i n e was  been no  s u g g e s t i o n t h a t VLB  cortex function.  Johnson (12) o f J 96  s e c r e t i o n o f the  has  c e l l s , derived  by  gluco-cortieoids.  The  a s p a r t i c acids  co-enzyme A.  inhibition  and  s i m i l a r to the  Gutts (ll}.) r e p o r t e d t h a t by  the  administration  Padawar (l£)  has  was  partially  reversed  D u r i n g t h i s work Johnson  served t h a t v i n b l a s t i n e caused m i t o t i c a r r e s t . a r r e s t was  growth  from p e r i p h e r a l b l o o d o f human monocytic  glutamic and  tryptophan and  involution  r e p o r t e d the i n h i b i t i o n o f the  leukaemia, i n t i s s u e c u l t u r e by VLB. r e v e r s e d by  i n t e r f e r e s with adrenal  Thus, I t i s not probable t h a t the  i s caused by i n c r e a s e d  found  (11).  to cause the i n v o l u t i o n o f the thymus gland o f r a t s There has  as  c-mitosis  caused by  The  metaphase  colchicine  the metaphase a r r e s t c o u l d be  of glutamic a c i d and  ob-  tryptophan.  (13)•  delayed Recently  r e p o r t e d t h a t v i n b l a s t i n e a l t e r s the morphology  o f mast c e l l s i n a manner almost i d e n t i c a l to c o l c h i c i n e . C l i n i c a l Action of Vinblastine  and V i n c r i s t i n e .  At present v i n b l a s t i n e sulphate and v i n c r i s t i n e sulphate are undergoing c l i n i c a l e v a l u a t i o n  (16, 17)•  Vinblastine i s  most u s e f u l i n the treatment o f Hodgkin's d i s e a s e and c h o r i o epithelioma.  V i n c r i s t i n e , although a l s o used i n the treatment  o f these d i s e a s e s ,  I s p a r t i c u l a r l y u s e f u l i n the chemotherapy  o f c h i l d leukaemia.  A number o f o t h e r tumours have a l s o been  t r e a t e d by b o t h a l k a l o i d s but w i t h l e s s s u c c e s s .  Also  other  d i f f e r e n c e s i n the c l i n i c a l e f f e c t s o f the two a l k a l o i d s have been found.  I n view o f the c l o s e s i m i l a r i t y i n structure.* o f  these compounds, i t i s extremely i n t e r e s t i n g t h a t there has been no  evidence o f p a t i e n t s  loids  (10).  developing cross-resistance  to the a l k a -  These o b s e r v a t i o n s have been i n t e r p r e t e d as evidence  of d i f f e r e n t modes o f a c t i o n . The  t o x i c e f f e c t s o f the two a l k a l o i d s a l s o d i f f e r des-  p i t e the s i m i l a r i t y o f t h e i r chemical s t r u c t u r e s .  The c l i n i c a l  dose o f v i n c r i s t i n e (0.02 - 0.05 mg./Kg.) i s l i m i t e d by neuromuscular t o x i c i t y  (10).  Vinblastine  only  causes t r a n s i e n t  d i s o r i e n t a t i o n a t very h i g h dose l e v e l s and l e u k o p e n i a i s the dose l i m i t i n g f a c t o r (10).  The u s u a l dose o f v i n b l a s t i n e i s  0.10 to 0.20 mg./Kg. Biochemical The  Studies. b i o l o g i c a l o b s e r v a t i o n s o u t l i n e d above, l e d to more  d e t a i l e d b i o c h e m i c a l i n v e s t i g a t i o n s on the mode o f a c t i o n o f vinblastine.  Beer (18) s t u d i e d  the e f f e c t o f the a l k a l o i d on  the r e g e n e r a t i o n o f the l i v e r o f p a r t i a l l y hepatectomised r a t s .  A dose o f the a l k a l o i d which produced pronounced  leukopenia  by the t h i r d o r f o u r t h day, d i d not slow the r a t e o f regenerat i o n o f the l i v e r , o r the i n c o r p o r a t i o n o f G ^ - f o r m a t e i n t o the deoxyribonucleic  a c i d (DNA) o r r i b o n u c l e i c a c i d (RNA) o f the  liver tissue. The  i n v i v o i n c o r p o r a t i o n o f G ^-formate Into the 1  n u c l e i c a c i d s o f 10 day E h r l i c h A s c i t e s c e l l s was a l s o not s i g n i f i c a n t l y a f f e c t e d by v i n b l a s t i n e ( 1 8 ) . S i m i l a r l y , R i c h a r d s e t a l (19) found no e f f e c t on the i n c o r p o r a t i o n o f the same p r e c u r s o r c e l l s incubated  i n t o the n u c l e i c a c i d s o f E h r l i c h A s c i t e s  i n vitro.  Comparable r e s u l t s were r e p o r t e d  by Johnson e t a l ( 1 0 ) ,  who found no i n h i b i t i o n o f C ^--formate o r g l y c i n e - 2 -  incor-  1  p o r a t i o n i n t o the n u c l e i c a c i d s o f S-180 A s c i t e s c e l l s e i t h e r i n vivo or i n v i t r o . I n c o n t r a s t to the o b s e r v a t i o n s c e l l s , a very l a r g e d e p r e s s i o n  on l i v e r and a s c i t e s  I n the i n c o r p o r a t i o n o f G-k-  forraate i n t o r a t bone marrow was found i n v i v o a f t e r i n t r a p e r i t o n e a l i n j e c t i o n o f 0.6 mg./Kg. o f the a l k a l o i d ( 1 8 ) . The  i n c o r p o r a t i o n o f the i s o t o p e i n t o DNA and RNA was found to  be depressed by 90$ and $0% r e s p e c t i v e l y i n the presence o f VLB.  R i c h a r d s et a l (19) u s i n g washed suspensions o f r a t bone  marrow c e l l s incubated  i n Robinson's medium a t 37°  VLB, a t 5 0 & per ml., caused a d e p r e s s i o n  C. found  o f approximately  i n the C ^ - f o r m a t e i n c o r p o r a t i o n i n t o the n u c l e i c a c i d s . The  e f f e c t o f the a l k a l o i d on the b i o s y n t h e s i s  n u c l e i c a c i d s i n other  tissue preparations  In view o f the b i o l o g i c a l o b s e r v a t i o n  was a l s o  o f the  studied.  t h a t v i n b l a s t i n e caused  FIGURE 2 DNA BIOSYNTHETIC PATHWAYS,  Carbamyl ,.  Aspartate  GI-it a mine  PRPP  phosphate  J i i  T  f o r m a te  Carbamyl  glycine  aspartate  DNA  RNA i s synthesized from the r i b o n u c l e o s i d e triphosphates of adenine, guanine, c y t o s i n e and u r a c i l .  - 7 -  the i n v o l u t i o n o f the thymus gland of r a t s , t h i s t i s s u e was investigated.  Using suspensions of r a t thymus c e l l s incubated  i n Robinson's medium, the presence of v i n b l a s t i n e caused a 60% depression of the i n c o r p o r a t i o n of C^-formate i n t o the DNA, and a \\.0% depression of the i n c o r p o r a t i o n i n t o the RNA ( 1 9 ) .  The  i n c o r p o r a t i o n of C^-formate i n t o the n u c l e i c acids of thymus c e l l preparations was also found to be p a r t i a l l y i n h i b i t e d by v i n c r i s t i n e and v i n l e u r o s i n e . Present  Investigations. Although VLB decreased the i n c o r p o r a t i o n of C^-formate  i n t o the DNA and RNA of r a t thymus c e l l suspensions,  there was  no evidence to i n d i c a t e which stage i n the b i o s y n t h e t i c processes, as shown i n Figure 2, was influenced by the drug.  In  an attempt to gain further i n f o r m a t i o n , an i n v e s t i g a t i o n i s being c a r r i e d out on the effect of v i n b l a s t i n e on the n u c l e o t i d e precursors of the n u c l e i c acids which are found i n the ' a c i d soluble f r a c t i o n ' of the c e l l p r e p a r a t i o n s . t h i s study i s described i n t h i s  The f i r s t part of  thesis.  -formate was again chosen as the r a d i o - a c t i v e precursor as i t offered a w e l l defined and economic m a t e r i a l f o r studying some o f the more important b i o s y n t h e t i c pathways. I t i s known to be incorporated v i a the tetrahydro f o l i c a c i d a c t i v a t e i n g systems i n t o the C£ and GQ p o s i t i o n s of the purine r i n g (20) and s p e c i f i c a l l y i n t o the methyl group of t h y m i d y l i c a c i d (21). The conversion of i n o s i n i c a c i d , the f i r s t purine n u c l e o t i d e to be synthesised, to the other purines takes place at the monon u c l e o t i d e l e v e l (22) and the triphosphate ribose and deoxy-  - 8r i b o s e d e r i v a t i v e s of adenine and guanine are then incorporated i n t o RNA (23) and DNA r e s p e c t i v e l y ( 2 b J .  On the other hand,  t h y m i d y l i c a c i d , a f t e r conversion to the t r i p h o s p h a t e , i s s p e c i f i c a l l y incorporated i n t o DNA  (2i|.)»  In t h i s p r e l i m i n a r y i n v e s t i g a t i o n the ' a c i d - s o l u b l e f r a c t i o n ' was hydrolysed to l i b e r a t e the purine and pyrimidine bases, which were then p u r i f i e d and i s o l a t e d by i o n exchange and paper chromatography. were then measured.  The s p e c i f i c a c t i v i t i e s of the bases  The DNA and RNA of the tissue, i n the same  incubations were i s o l a t e d by D r . R i c h a r d s u s i n g a m o d i f i c a t i o n 1  of the technique of Hecht and P o t t e r  (2f?)«  The r e s u l t s obtained  by D r . Richards w i l l be r e f e r r e d to i n the t e x t .  Dr. J . P . R i c h a r d s , Department o f Biochemistry, U n i v e r s i t y of B r i t i s h Columbia.  - 9 EXPERIMENTAL I.  STUDY AND DEVELOPMENT OP METHODS.  A . BASIC METHODS. 1. M a t e r i a l s . Animals: Female. Wis t a r r a t s (120-11+0 grams) from the colony at the U n i v e r s i t y o f B r i t i s h Columbia were used i n a l l experiments.  The animals were fed Master's Fox Chow ad l i b i t u m .  Incubation Medium; The incubation medium, which was a s l i g h t m o d i f i c a t i o n o f Robinson's medium consisted o f : 0,151}. M Sodium C h l o r i d e  -  232 m l .  0.151+ M Potassium Chloride  -  8 ml.  0.151+ M Magnesium Sulphate  -  2 ml.  0.110 M Calcium Chloride  -  6 ml.  The s o l u t i o n was brought to pH 7*1+ by the a d d i t i o n o f 3 to 5 drops o f sodium hydroxide s o l u t i o n (O.OlWand 0.1 N) and 12 m l . of 0.67 M phosphate b u f f e r (pH 7.1+) were then added. 1  Immedi-  a t e l y before use 0.1+ m l . of a 10$ glucose s o l u t i o n was added to 19.6 m l . o f buffered medium. R a d i o - a c t i v e Formate: Sodium C^-formate was obtained from Merck and Company L i m i t e d ,  One m i l l i c u r i e ,  (11.1 mg.) of  sodium formate, was d i s s o l v e d i n 50 m l . o f water to give a standard formate s o l u t i o n containing 20 micro c u r i e s per m l . V i n b l a s t i n e : V i n b l a s t i n e sulphate was a g i f t from E l i L i l l y and Company.  2.85 mg. o f v i n b l a s t i n e sulphate were  d i s s o l v e d i n 1+ m l . of incubation medium.  A l i q u o t s of the s o l u -  t i o n were added to the i n c u b a t i o n mixtures, as i n d i c a t e d i n the IP.B.Hawk, B . L . Oser, W.H. Summerson, P r a c t i c a l P h y s i o l o g i c a l Chemistry, p . 6 3 6 , Maple Press C o . , 1 9 4 7 .  c o n c e n t r a t i o n o f 5G- X p e r m l . o f v i n -  t e x t , to g i v e a f i n a l blastine. 2.  B i o l o g i c a l and chemical procedure f o r the h y d r o l y s i s o f the ' a c i d - s o l u b l e f r a c t i o n  1  o f thymus c e l l p r e p a r a t i o n s  and the Ion exchange s e p a r a t i o n o f the l i b e r a t e d  bases.  P r e p a r a t i o n o f t i s s u e suspensions; A l l m a n i p u l a t i o n s i n the p r e p a r a t i o n o f the c e l l suspensions were c a r r i e d out between 0° C and  G«  The r a t s were a n a e s t h e t i s e d w i t h ether and de-  capitated.  The thymus glands were q u i c k l y removed and p l a c e d on  moist c h i l l e d f i l t e r paper.  The glands, u s u a l l y f o u r o r f i v e  thymuses a t a time, were minced w i t h s c i s s o r s i n approximately 5 m l . o f i n c u b a t i o n medium and f o r c e d through a s y r i n g e w i t h an aperture o f 2 mm.  diameter.  The crude suspension was  then  f o r c e d s u c c e s s i v e l y through a s y r i n g e w i t h an a p e r t u r e o f 1 ram. and a s y r i n g e w i t h a s i z e 18 needle a t t a c h e d . pension was then f i l t e r e d  through Ij. l a y e r s o f m u s l i n to remove  the f i b r o u s m a t e r i a l and the f i l t r a t e w i t h c h i l l e d medium.  a d j u s t e d to a known volume  U s u a l l y a suspension o f 25 m l . was o b t a i n e d  from 20 thymus g l a n d s .  The wet weight o f c e l l s  suspension was measured by weighing  c e r t a i n experiments,  i n 1 ml, o f the  the wet r e s i d u e f o l l o w i n g  c e n t r i f u g a t i o n a t approximately 800g f o r In  The smooth sus-  5  minutes.  as noted i n the t e x t , the c e l l  suspension was washed by c e n t r i f u g i n g a t l,100g f o r 6 minutes at  0° C i n an angle head U n i v e r s a l R e f r i g e r a t e d C e n t r i f u g e .  supernatant was decanted medium.  and the c e l l s  Any l a r g e clumps o f c e l l s  through 2 l a y e r s o f m u s l i n .  resuspended  were removed by  The  i n incubation filtering  The washed c e l l suspension was r e -  - 11 a d j u s t e d to the o r i g i n a l volume and the wet weight o f c e l l s i n 1 m l . again measured as d e s c r i b e d above© Incubation  conditions;  Incubations  were u s u a l l y done  i n 50 m l . Erlenmeyer f l a s k s f i t t e d w i t h c o t t o n wool p l u g s . The  i n c u b a t i o n mixture u s u a l l y  contained:-  Thymus c e l l suspension  -  Sodium C ^"-formate s o l u t i o n 1  V i n b l a s t i n e sulphate  1 ml. O.I4. m l .  s o l u t i o n (0.2 ml.) was added to c e r t a i n  f l a s k s to g i v e a f i n a l c o n c e n t r a t i o n o f $0 % p e r m l . t u r e was made up to a t o t a l volume o f 2.5 m l . w i t h medium.  The contents  were i n c u b a t e d  T h i s mix-  incubation  i n a i r u s u a l l y f o r 3 hours,  at 37° C a t a shaking r a t e o f 120 c y c l e s p e r minute and a d i s placement o f 5«5 era. i n an Eberbach C o r p o r a t i o n water b a t h . departures  from these  Any  c o n d i t i o n s are mentioned i n the t e x t .  P r e p a r a t i o n and H y d r o l y s i s o f 'Acid S o l u b l e  fraction';  A f t e r i n c u b a t i o n , the f l a s k s were c h i l l e d i n i c e and the contents (2.5 ml.) t r a n s f e r r e d , w i t h one washing (1 m l . ) , c e n t r i f u g e tube.  to a lj.0 m l .  H a l f a volume o f 2.1 N p e r c h l o r i c a c i d was  added to each sample.  A f t e r s t a n d i n g i n the c o l d f o r 8 to 10  minutes, the mixture was c e n t r i f u g e d at l,200g f o r 15 minutes. The  c e l l s were resuspended i n one volume o f 0.7 N p e r c h l o r i c  a c i d , allowed The  to stand f o r 6-8 minutes and again c e n t r i f u g e d .  supernatant  p o r t i o n s were combined to g i v e the ' a c i d s o l u b l e  f r a c t i o n ' and the r e s i d u e was r e t a i n e d by Dr. Richards determination The  f o r the  o f DNA and RNA.  a c i d s o l u b l e f r a c t i o n was a d j u s t e d to pH I4..5 - 5»0  w i t h potassium hydroxide  s o l u t i o n (5 N and 1 N) and the r e s u l t -  - 12 i n g mixture  allowed  to stand f o r s e v e r a l hours a t da 0° C to  a l l o w complete p r e c i p i t a t i o n o f potassium c e n t r i f u g a t i o n , the supernatant a 5 ml» washing.  perchlorate.  After  was decanted and combined w i t h  The combined s o l u t i o n s were made up t o a known  volume, u s u a l l y 15 m l . and a s m a l l p o r t i o n , approximately onet e n t h o f the t o t a l , was s e t a s i d e as a r e f e r e n c e sample and f o r r a d i o - a c t i v i t y determinations. dryness  The remainder was evaporated to  a t 70° C w i t h a c u r r e n t o f a i r and the r e s i d u e hydro-  l y s e d by h e a t i n g i n a b o i l i n g water bath f o r one hour w i t h 2 ml, o f 12% p e r c h l o r i c a c i d .  A f t e r c o o l i n g , the excess p e r c h l o r i c  a c i d was again removed as the potassium described.  The supernatant  s a l t as p r e v i o u s l y  p l u s two washings  :bf 2 m l , o f  water was a d j u s t e d to a known volume, u s u a l l y 15 m l , counter  A small  sample was again s e t a s i d e and the remainder o f the  'hydrolysed a c i d - s o l u b l e f r a c t i o n ' a p p l i e d to a Dowex 50 H " 4  i o n exchange column, which was developed gradient.  The d e t a i l s o f the p r e p a r a t i o n o f the column and the  g r a d i e n t e l u t i o n technique The continuous  with a h y d r o c h l o r i c acid  are g i v e n i n the next s e c t i o n .  e f f l u e n t from the column was r u n through a GME 2651F u l t r a - v i o l e t a b s o r p t i o n meter attached to a R e c t i l i n e a r  r e c o r d i n g m i l l i a m e t e r , which gave a continuous u l t r a - v i o l e t a b s o r p t i o n between 265  and 280 mu.  r e c o r d o f the Individual frac-  tions  (i+.O - ij.,5 ml.) o f the e f f l u e n t were c o l l e c t e d on an auto-  matic  fraction collector.  U s u a l l y the r a d i o - a c t i v i t y o f a l i q u o t s  o f each f r a c t i o n was determined and the m a t e r i a l i n the f r a c t i o n s absorbing u l t r a - v i o l e t l i g h t were f u r t h e r p u r i f i e d by paper chromatography.  The procedure f o l l o w e d i n i n d i v i d u a l experiments  - 13 i s d e s c r i b e d i n the t e x t . 3.  Chromatographic  Techniques.  Ion exchange chromatography; Dowex 50 H  +  (200-1+00 mesh,  Q% c r o s s - l i n k e d ) columns were prepared by f i r s t washing the r e s i n free of 'fines'  by s u c c e s s i v e l y suspending the r e s i n i n  water and d e c a n t i n g the more s l o w l y sedimenting p a r t i c l e s . s l u r r y o f the r e s i n was a column 18  cm.  washed w i t h 150  then poured onto a bed o f sand to form  i n l e n g t h and 1 cm.  i n diameter.  The r e s i n  was  ml. o f 1+ N h y d r o c h l o r i c a c i d f o l l o w e d by  d i s t i l l e d water u n t i l the e f f l u e n t was f r e e from  A  approximately pH 1+.5  and  chloride.  'Hydrolysed a c i d - s o l u b l e f r a c t i o n s ' were chromatographed on these columns.  The e l u t i o n c o n d i t i o n s employed, were o r i -  g i n a l l y d e s c r i b e d by Wells and W i n z l e r (26) t i o n o f the o r i g i n a l method o f Conn (27). g r a d i e n t was  and were a m o d i f i c a A concentration  developed w i t h 1+ N h y d r o c h l o r i c a c i d  (500  ml.)  e n t e r i n g a mixing b o t t l e f i l l e d i n i t i a l l y w i t h water (500 which i t s e l f f e d the i o n exchange column.  ml.),  The e l u a t e from the  column was monitored f o r u l t r a - v i o l e t a b s o r p t i o n and  fractions  c o l l e c t e d as p r e v i o u s l y d e s c r i b e d . Paper Chromatography;  Whatman No.  1 paper was  used i n  chromatograms developed by the descending technique u s i n g s o l vent systems  I to V.  System I  I s o p r o p a n o l - HCl (29)  Isopropyl aleohol Concentrated HCl.. Water  Butanol-Ammonia (29)  Butanol 86 m l . Water llj. ml. A pe^tri d i s h o f ammonia (S.Gr.0.88) was p l a c e d i n the bottom o f the tank.  \System I I  The f o l l o w i n g s o l v e n t systems were used: 130 33 37  ml* ml* ml.  - 14 System I I I  System  System  Butanol-Acetic Acid  IV  (28)  Butanol Acetic acid (glacial) Water  I s o b u t y r i c Acid-Ammonia (29)  V  Isopropanol-Ammonia (28)  400 ml 100 ml. 100 ml.  Isobutyric Acid Water 25% NH OH solution  0.4 ml.  Isopropanol Ammonia Water  80 ml. 10 ml. 10 ml.  400 •ml. 203 ml.  Ion exchange paper chromatography pH 3.1 c i t r a t e b u f f e r (ascending).  Knight (30) described a method f o r the separation of  amino acids on i o n exchange paper developed with pH 3.1 b u f f e r (31).  citrate  This method was examined and i t was found that  good d e f i n i t i o n of the areas of reference compounds was u s i n g Grade SA 2,  Amberlite 120 Na  obtained  ion exchange paper i f 0,6%  of Tween 80 were added to the b u f f e r , and the reference compounds were applied t o the pre-wetted paper. Usually when using t h i s method, the solvent f r o n t was allowed to more approximately  8 cm. up the paper.  was then l i g h t l y d r i e d and the samples applied.  The  origin  The paper was  then returned to the chromatography j a r and the b u f f e r allowed to ascend f o r a f u r t h e r 20 to 25  cm.  The u l t r a - v i o l e t absorbing areas and the r a d i o - a c t i v e areas on the chromatograms were detected by i n s p e c t i o n with a M i n e r a l i t e short wave-length mercury lamp and by 1  radio-autography.  The p o s i t i o n s of any reference compounds applied were v i s u a l i s e d by spraying with e i t h e r ninhydrin-butanol or Dragendorff s 1  re-  agent (56). I f f u r t h e r studies were to be made these areas were cut wave-length of l i g h t 254  m.u.  - 15 out and e l u t e d w i t h 0.5  m l . o f 0.1  N hydrochloric acid.  In the  q u a n t i t a t i v e d e t e r m i n a t i o n of u l t r a - v i o l e t s p e c t r a , b l a n k of  equal dimensions  and comparable p o s i t i o n were a l s o cut out  and e l u t e d as d e s c r i b e d . ence c u v e t t e o f the ij..  Ultra-violet  areas  These samples were used i n the r e f e r -  spectrophotometer,  Spectroscopy.  The u l t r a - v i o l e t s p e c t r a and the maximum a b s o r p t i o n o f samples were measured i n a Cary 1 1 R e c o r d i n g  Spectrophotometer.  The s p e c t r a o f samples e l u t e d from paper chromatograms were measured a g a i n s t the e l u a t e s from corresponding b l a n k areas the chromatogram.  The  on  s p e c t r a o f o t h e r samples were measured  a g a i n s t r e f e r e n c e samples o f the corresponding s o l v e n t concentrat i o n and 5.  pH.  R a d i o - a c t i v i t y D e t e c t i o n and  Determination.  P l a t e Counting. Samples, 0.5 m l . c o n t a i n i n g m i n e r a l a c i d were p l a t e d on p l a t i n u m p l a n c h e t t e s and d r i e d to g i v e an even d i s t r i b u t i o n of residue.  The r a d i o - a c t i v i t y was  determined  a Nuclear Chicago window-less gas flow counter and a t l e a s t to  three thousand  counts r e c o r d e d .  the g e i g e r counter was  s e t mid-way along the p l a t e a u and  L i q u i d S c i n t i l l a t i o n Counting;  meter.  two  The h i g h v o l t a g e supply to  background count o f each p l a n c h e t t e was  determined  in  determined  the  before  use.  R a d i o - a c t i v i t y was  also  In a Packard T r i - C a r b l i q u i d s c i n t i l l a t i o n s p e c t r o -  M i n e r a l a c i d s e v e r e l y quenches the s c i n t i l l a t i o n p r o c e s s ,  and hyamlne s o l u t i o n was  t h e r e f o r e added to n e u t r a l i s e  the  h y d r o c h l o r i c a c i d present i n f r a c t i o n s c o l l e c t e d from the i o n exchange chromatogram.  - 16 One l i t r e of S c i n t i l l a t i o n 600 400 100 4  f l u i d contained:  ml. Toluene ml. Ethyl alcohol mg. l , 4 , b i s - 2 - (phenyl oxazolyl)-benzene grams Diphenyl oxazole  The counting v i a l u s u a l l y contained 0.2 ml. of the r a d i o - a c t i v e sample, 9 ml. of S c i n t i l l a t i o n i n methanol.  f l u i d and 1 ml. of 1M hyamine  Using t h i s f l u i d , the concentration of hydro-  c h l o r i c acid d i d not a f f e c t the count.  The r a t i o of counts  i n the flow-gas counter to those i n the s c i n t i l l a t i o n method was 1.7:1.  The s p e c i f i c a c t i v i t i e s  of the bases are expressed  as counts per minute per micromole, a l l counts being r e f e r r e d to the values obtained i n the flow-gas counter. Radio-autography:  The presence of r a d i o - a c t i v i t y i n  c e r t a i n areas of chromatograms was revealed by radio-autography. Kodak Medical No-Screen F.0034 BE X-ray f i l m was placed i n cont a c t with the chromatograms f o r periods varying from 3 to 10 days depending on the amount of r a d i o - a c t i v i t y present.  The  exposed f i l m s were then developed, f i x e d and washed. Determination of 'non-volatile' counts a f t e r the removal of C —formate: 1  the  A sample (0.2 ml.), f o r examplepf  'acid-soluble f r a c t i o n " , was placed i n a v i a l and evaporated  to dryness i n a warm (40° C) water bath.  0.1. ml. of 98% formic  acid was added and the sample re-evaporated to dryness. The f i n a l traces of formic acid were removed by blowing a gentle stream of a i r into the v i a l .  This procedure was repeated three  times and the v i a l was then placed i n an oven at 100° C f o r 10 minutes.  Following t h i s treatment the residue was dissolved  i n 0.2 ml. of water and 1 ml. of hyamine s o l u t i o n and 9 ml. of  -  s c i n t i l l a t i o n f l u i d added.  17  -  The sample was  counted i n the  l i q u i d s c i n t i l l a t i o n spectrometer. 6,  Isotopic Dilution  Technique.,  The f o l l o w i n g procedure was r a d i o - a c t i v i t y of a particular  used to measure the t o t a l  compound i n a complex m i x t u r e .  A known amount o f a n o n - r a d i o - a c t i v e was  sample o f the compound  added i n l a r g e excess to the r a d i o - a c t i v e m i x t u r e .  compound was  then i s o l a t e d  change chromatography,  and p u r i f i e d by paper o r i o n ex-  as a p p r o p r i a t e .  amount o f the p u r i f i e d m a t e r i a l was a c t i v i t y determined.  The  Hence, one may  An a c c u r a t e l y known  then taken and i t s r a d i o c a l c u l a t e the t o t a l r a d i o -  a c t i v i t y o f the compound i n the o r i g i n a l m i x t u r e . By t h i s method the t o t a l r a d i o - a c t i v i t y contaminated thymine column was measured.  i n the i n i t i a l  'peak  The same method was  1  o f the g r o s s l y  on the i o n exchange employed to c a l c u l a t e  the t o t a l r a d i o - a c t i v i t y o f compounds g i v i n g r i s e to adenine and guanine on h y d r o l y s i s i n a small unhydrolysed sample.  reference  - 18 B . STANDARDISATION OP METHODS 1 » Introduction The b a s i c methods used i n t h i s i n v e s t i g a t i o n have been outlined*  However, before applying these procedures to the  study of the e f f e c t of v i n b l a s t i n e on G^-formate i n c o r p o r a t i o n i n t o the ' a c i d - s o l u b l e f r a c t i o n ' , the recoveries of added r e ference compounds at c e r t a i n points i n these procedures were checked. The recoveries of standard amounts of the bases a f t e r e l u t i o n from the i o n exchange r e s i n were s t u d i e d , and the p o s i t i o n s at which the i n d i v i d u a l bases were eluted from the ion exchange column were also e s t a b l i s h e d .  Also the  efficiency  of the h y d r o l y t i c procedure and the l o s s o c c u r r i n g during the removal of potassium perchlorate were checked by the use of reference samples of the bases and the mononucleotides. 2.  Materials Approximately 0 . 0 2 M s o l u t i o n s of u r a c i l , thymine,  cytosine and adenine were prepared i n 0 . 1 N h y d r o c h l o r i c a c i d . A 0 . 0 2 M s o l u t i o n of guanine was prepared i n 1 . 0 N h y d r o c h l o r i c acid.  The u l t r a - v i o l e t spectra of these s o l u t i o n s were measured  and the concentrations c a l c u l a t e d using the s p e c t r a l data i n the literature (32). Standard s o l u t i o n s of the mononucleotides of the above bases i n 2 N h y d r o c h l o r i c a c i d were also prepared. 3«  Procedure and Results The e l u t i o n p o s i t i o n s and the percentage recoveries of  added adenine, guanine, c y t o s i n e , u r a c i l and thymine from the  FIGURE 3  ION-EXCHANGE SEPARATION OF PURINE AND PYRIMIDINE BASES  (DOWEX 50 H ; GRADIENT ELUTION, H 0 — 2  4N HCl)  m <  >•  —i—:  —r -  0  -  2 0  40  60  ~I— 8 0  — I —  —I  . too  FRACTION  120  —I  140  NO.  URACIL and THYMINE B  CYTIDYLIC ACID CYTOSINE  D  GUANINE  E  ADENINE  —I  160  —I  180  - 19 s u l p h o n i c a c i d i o n exchange  chromatographic system were i n v e s t i -  gated. Samples (0.5 ml.) o f each o f the s t a n d a r d s o l u t i o n s o f the bases were a p p l i e d to an i o n exchange  column and e l u t e d as p r e -  viously described with a hydrochloric acid gradient.  The  effluent  was monitored f o r u l t r a - v i o l e t a b s o r b i n g m a t e r i a l s and then collected i n  ml* f r a c t i o n s .  Pour u l t r a - v i o l e t a b s o r b i n g  r e g i o n s were o b t a i n e d , as shown i n F i g u r e 3» and the f r a c t i o n s comprising each o f these regions were combined a known volume.  P o r t i o n s o f the combined  and a d j u s t e d to  samples were evaporated  to  dryness at 60° C i n a stream o f a i r and the r e s i d u e d i s s o l v e d  in  a s m a l l volume o f 0 . 1 N h y d r o c h l o r i c a c i d and a p p l i e d to paper  chromatograms, which were developed i n s o l v e n t system I .  The  u l t r a - v i o l e t s p e c t r a o f o t h e r p o r t i o n s o f the samples were measured i n the Cary spectrophotometer. The r e s u l t s showed, i n agreement of ion  w i t h the p u b l i s h e d d a t a  Conn ( 2 7 ) and o f W a l l ( 3 3 ) , that the f i r s t exchange  chromatography  peak o b t a i n e d by  contained both u r a c i l and  w h i l e adenine, guanine and c y t o s i n e gave i n d i v i d u a l  thymine,  ultra-violet  absorbing peaks, as shown i n F i g u r e 3« The percentage r e c o v e r i e s o f c y t o s i n e , adenine and guanine and o f the combined u r a c i l and thymine, were c a l c u l a t e d from the u l t r a - v i o l e t a b s o r p t i o n maxima o f the combined p r i s i n g those peaks, and are g i v e n i n Table 1 .  f r a c t i o n s comI t w i l l be seen  t h a t good r e c o v e r i e s were o b t a i n e d * In of  a d d i t i o n , the r e c o v e r i e s o f the bases i n the presence  s a l t were examined.  Also known amounts o f the mononucleotides  TABLE 1 Recovery of purine and pyrrolidine bases from i o n exchange columns. Resin:  Dowex 50 H , 200-400 mesh, 8% cross l i n k e d . +  Gradient E l u t i o n :  500 ml. water to 500 ml. 4N H C l .  MATERIALS APPLIED COMPOUNDS RECOVERED  Free Bases  %  Free bases following treatment w i t h HCIOA 7 • 10  Mononucleotides following treatment w i t h HCIO4 %.  Adenine  92  81  77  Guanine  88  83  82  U r a c i l plus Thymine  85  82  77  Cytosine  92  82  73  1 Sample of adenine, guanine, c y t o s i n e , u r a c i l and thymine. 2 Sample of the mononucleotides of the foregoing bases. 3 Recoveries c a l c u l a t e d from o p t i c a l density a t 265 imx and average values of E x t i n c t i o n C o e f f i c i e n t s of thymine and u r a c i l .  20  -  were f i r s t hydrolysed with p e r c h l o r i c acid and the l i b e r a t e d bases separated on Dowex 50 H determined.  Thus one was  the h y d r o l y t i c process.  +  r e s i n and t h e i r recoveries  able to measure the e f f i c i e n c y of 0.5  ml. samples of the standard s o l u -  t i o n s of the purine and pyrimidine bases were added to 1 of t i s s u e suspension containing 130 mg. c e l l s , to which 6.5  ml.  wet weight of thymus  ml. of incubation medium were also added  to.give t o t a l sample of 10 ml.  A s i m i l a r sample, containing  0.5 ml. of each of the standard mononucleotide s o l u t i o n s ,  was  also prepared. Both samples were evaporated to dryness and the residues hydrolysed with 2 ml. of 72% p e r c h l o r i c acid f o r 1 hour at 100°  C.  A f t e r the removal of excess p e r c h l o r i c a c i d , as i t s potassium s a l t , the samples were i n d i v i d u a l l y chromatographed on Dowex 50 H  +  ion exchange columns using the hydrochloric  acid  gradient.  In both eases the eluate was monitored f o r u l t r a - v i o l e t absorbi n g material and f r a c t i o n s c o l l e c t e d .  The f r a c t i o n s comprising  the i n d i v i d u a l peaks were pooled i n the manner described i n the previous experiment.  Aliquots were applied to paper chromato-  grams which were developed using solvent system I .  The  ultra-  v i o l e t spectra of other portions of the combined f r a c t i o n s were measured and the percentage recoveries of the bases were c a l c u l a ted from the s p e c t r a l data.  These recoveries, shown i n Table I ,  were found to be c o n s i s t e n t l y s a t i s f a c t o r y . In a d d i t i o n to the four u l t r a - v i o l e t peaks obtained by monitoring the eluate from the ion exchange chromatograms of the free bases, an a d d i t i o n a l peak was found i n the run to which the  hydrolysed  21  -  sample o f the mononucleotides had been added.  peak, as shown i n F i g u r e 3»  was  This  e l u t e d i n the r e g i o n o f f r a c t i o n  No.  3Q«  and  i t s paper chromatographic behaviour u s i n g s o l v e n t system I I  The  u l t r a - v i o l e t absorbing  i n d i c a t e d that i t was c y t i d y l i c a c i d was  spectrum o f t h i s  cytidylic acid.  material  Furthermore, added  e l u t e d from the i o n exchange column i n the  same p o s i t i o n as the unknown m a t e r i a l . that t h i s m a t e r i a l was  I t was  therefore  apparent  r e s i d u a l , unhydrolysed c y t i d y l i c a c i d ,  which i s known to be q u i t e r e s i s t a n t to p e r c h l o r i c a c i d  h y d r o l y s i s (3i|)» Much l e s s o f t h i s compound s u r v i v e d h y d r o l y s i s i f care was  taken to remove most o f the r e s i d u a l s a l t s from the  sample.  A l s o the presence o f a large- q u a n t i t y o f s a l t s i s a disadvantage i n the i o n exchange chromatography of the n u c l e o t i d e s unhydrolysed  'acid-soluble f r a c t i o n ' .  I n a d d i t i o n i t would  an advantage to remove the excess C ^ - f o r m a t e before raphy. and  Therefore,  both i n order  be  chromatog-  to s o l v e the immediate problem  as a p o s s i b l e technique i n f u t u r e work, the use  to d e - s a l t the  i n the  ' a c i d - s o l u b l e fraction'was  of  investigated.  charcoal  - 22 -  •  C . USE OP CHARCOAL TO DESALT SAMPLES. 1,  Introduction C e r t a i n a c t i v a t e d charcoals r e a d i l y adsorb aromatic  and h e t e r o c y c l i c compounds and have been frequently used to i s o l a t e and separate mixtures of biochemical and chemical compounds of t h i s nature (35)•  The use of c h a r c o a l , has f r e -  quently been proposed for d e - s a l t i n g aqueous s o l u t i o n s of nucleotides and other components of the ' a c i d - s o l u b l e f r a c t i o n ' of t i s s u e e x t r a c t s .  However, the f r i a b i l i t y of those charcoal  samples w i t h s u f f i c i e n t l y h i g h adsorptive capacity has l e d to low flow r a t e s , plugging of columns and poor r e c o v e r i e s .  This  has s e r i o u s l y hindered the use of charcoal f o r t h i s purpose. Recent reports from B a r t l e t t (36),  Wu and Wilson  and s e v e r a l other workers (38, 39) suggest that these c u l t i e s have l a r g e l y been overcome.  (37)  diffi-  However, there were marked  differences i n the methods employed by the i n d i v i d u a l groups, p a r t i c u l a r l y i n the quantity of charcoal used to adsorb a given amount of n u c l e o t i d e . Thus, Schweiger and Bremer (38) adsorbed 0.2 micromoles of n u c l e o t i d e onto 100 mg. of charcoal ( N o r i t A) which had been prewashed w i t h ethylenediamine t e t r a - a c e t i c  a c i d (EDTA) s o l u t i o n  and with p y r i d i n e In aqueous ethanol (10:^5^5 by volume). The adsorbed nucleotides were then eluted w i t h EDTA s o l u t i o n and p y r i d i n e i n aqueous e t h a n o l .  The method was stated to give  e x c e l l e n t recoveries of the n u c l e o t i d e s . On the other hand, Tsuboi and P r i c e (39) adsorbed  2.5  micromoles of nucleotides onto 8 mg. of N o r i t A , which had  - 23  -  p r e v i o u s l y o n l y been washed w i t h d i l u t e a c i d . n u c l e o t i d e s was ethanol  accomplished w i t h a mixture o f p y r i d i n e l w a t e r $  (10:Ji5:li5), but no EDTA s o l u t i o n was  c o v e r i e s were again The  E l u t i o n o f the  used.  Good r e -  reported.  r a t i o s o f weight o f c h a r c o a l to micromoles o f  n u c l e o t i d e adsorbed used by the two  groups were the most  treme values noted i n the l i t e r a t u r e .  ex-  I n p a r t i c u l a r Schweiger  et a l used a f a r g r e a t e r amount o f c h a r c o a l per micromole o f n u c l e o t i d e than o t h e r workers.  However, because o f t h e i r  c e l l e n t r e c o v e r i e s , the technique was  ex-  proposed by Schweiger e t a l  i n v e s t i g a t e d i n order to f i n d a s u i t a b l e method o f d e - s a l t i n g  the a c i d - s o l u b l e f r a c t i o n . The  r e c o v e r y o f adenosine t r i - p h o s p h a t e from the  column, u s i n g t h i s method, was  charcoal  found to be e x c e l l e n t but  the  volume o f e l u a t e r e q u i r e d to e l u t e even a s m a l l percentage o f the bases was  very l a r g e .  o f the bases was  achieved  However almost q u a n t i t a t i v e e l u t i o n from a s m a l l c h a r c o a l column, not  pre t r e a t e d w i t h p y r i d i n e rwater: ethanol s o l u t i o n . A l t h o u g h t h i s column was  not found to be a s u i t a b l e method  o f d e - s a l t i n g the whole a c i d - s o l u b l e f r a c t i o n , i t proved to be a convenient  method f o r d e - s a l t i n g and p a r t i a l l y p u r i f y i n g the  thymine from the i o n exchange chromatogram. 2.  Methods. P r e p a r a t i o n o f c h a r c o a l columns. The  obtained  reported  (36)  from Darco Department, A t l a s Powder Co.,  New  c h a r c o a l , Darco G60^  which was  to have  York.  FIGURE 4  I  RECOVERY OF ATP FROM CHARCOAL COLUMN.  50 0 -  400  300  -  20 0  100  I  A  -r , , , 2  |  4  A  6  -i—i  C  B  F R A C T I O N  e  i IO  NO.  —i—r 12  i  14  i  D E ( 2 4-5M L )  A  2:3 mixture o f EDTA and p y r i d i n e , water, e t h a n o l (10:45:45).  B  HC1 (0.01N).  C  ATP sample a p p l i e d .  D  S o l u t i o n A*.  E  Solution B .  *  D e t a i l s o f s o l u t i o n s g i v e n i n text.  -2kalmost i d e n t i c a l p r o p e r t i e s to N o r i t A* was periments.  I t was  used i n a l l ex-  suspended i n water s e v e r a l times and on  o c c a s i o n the f i n e f r a c t i o n was suspension o f c h a r c o a l was  removed by d e c a n t a t i o n .  cm,; In diameter was  allowed  o f powdered g l a s s supported by  A c h a r c o a l column o f 2 cm.  g l a s s wool p l u g .  The  then poured i n t o a column and  to sediment onto a shallow bed  each  u s u a l l y allowed  1.6  i n l e n g t h and  to form*, however the  a  size  o f each column w i l l be g i v e n i n the t e x t . 3«  Results  and  Discussion  Absorption  and  e l u t i o n o f the p u r i n e and  bases and o f Adenosine ATP  (6  micromoles) was  (2 cm. rxjf 1.6 had  cm.)  pyrimidine  Tri-phosphate. absorbed onto a c h a r c o a l column  c o n t a i n i n g 600-700 mg.  o f c h a r c o a l which  been p r e t r e a t e d w i t h p y r i d i n e :water:ethanol ( l O : ! ^ : ! ^ ) •  A f t e r washing w i t h 0.01  N h y d r o c h l o r i c a c i d , the sample  e l u t e d , as shown i n F i g u r e I4. w i t h 25 t u r e (5sl  ml, o f s o l u t i o n A, a mix-  by volume) o f EDTA s o l u t i o n (0.27  water, e t h a n o l  M)  and p y r i d i n e ,  s o l u t i o n , f o l l o w e d by s o l u t i o n B,  by volume o f the same s o l u t i o n s .  The  was  e l u a t e was  a 2:3  mixture  collected i n  2lj..5 ml. p o r t i o n s and the t o t a l phosphorus content o f each f r a c t i o n determined by the method o f A l l e n (ij.0). Considerably column t h a t was  more phosphorus was  a p p l i e d as ATP.  s o l u t i o n contained  I t was  recovered  from the  found t h a t the EDTA  a l a r g e amount of phosphorus and  i f the  r e s u l t s were c o r r e c t e d f o r t h i s contaminant, the recovery ATP  was  found to be 102%.  T h i s agrees w e l l w i t h  the  of  recoveries  r e p o r t e d by Schweiger e t a l . The  recovery  o f the bases from a c h a r c o a l column was  also  - 25 -  I n v e s t i g a t e d , since they are known to be more s t r o n g l y adsorbed than the nucleotides ( 3 7 ) • The s i z e of the column was increased to maintain the r a t i o of weight of charcoal to micromoles of purines or pyrimidines absorbed, used by Schweiger et a l .  How-  ever, l e s s than \\.0% of the bases were recovered i n over 65 m l . of e l u a t e .  Therefore the method was thought to be u n s u i t a b l e  f o r d e - s a l t i n g the quantity of nucleotides and bases i n the acid-soluble fraction. However, i t was found, when the bases were being adsorbed by the c h a r c o a l , that there was simultaneously a release of p y r i d i n e , which was presumably d i s p l a c e d by these bases.  Since  Schweiger and Bremer who pre-washed t h e i r charcoal w i t h p y r i d i n e used a f a r l a r g e r quantity of charcoal to absorb a given amount of n u c l e o t i d e than other workers, i t seemed l i k e l y that the a b i l i t y of the charcoal to absorb the n u c l e o t i d e was markedly decreased by the p y r i d i n e pretreatment. Therefore the use of charcoal not p r e v i o u s l y exposed to p y r i d i n e was s t u d i e d .  I t was found that 70% of the bases were  eluted from a column ( 2 , 5 cm. x 1.0 cm,.) c o n t a i n i n g a p p r o x i mately 750 mg. of charcoal i n the f i r s t 37«5 m l . of p y r i d i n e i n aqueous ethanol e l u a t e ; 85$ of the bases were recovered a f t e r 115 m l . of eluate had been passed through the column.  The l a s t  60 m l . contained a h i g h p r o p o r t i o n of guanine, which suggested that guanine was more f i r m l y bound to the charcoal than the other  bases. The o v e r a l l recovery of the added bases u s i n g t h i s  quantity of c h a r c o a l , not pretreated w i t h p y r i d i n e , was found  to be s a t i s f a c t o r y and i t was f e l t that t h i s method offered a good system f o r d e - s a l t i n g the acid-soluble f r a c t i o n of incubated thymus c e l l s . De-salting the Acid-soluble f r a c t i o n of thymus c e l l suspensions. As s a t i s f a c t o r y recoveries of added bases were found from charcoal columns which had not been pretreated with p y r i d i n e i n aqueous ethanol s o l u t i o n , t h i s method was used f o r d e - s a l t i n g the acid-soluble f r a c t i o n of unwashed thymus c e l l suspensions. Two  3 hour incubations were set up containing 10 ml.  of a suspension of unwashed c e l l s and 1.8 microcuries per ml. of C"^-formate i n a t o t a l volume of 11 ml. i n a 50 ml. f l a s k . V i n b l a s t i n e (50& per ml.) was added to one f l a s k .  After incu-  b a t i o n , the acid soluble f r a c t i o n s were prepared as previously described and made up to 25 ml.  4 ml. reference samples were  set aside and the remainder applied to charcoal columns (2.5 x 1.0  cm.).  cm.  The flow rates of the columns dropped immediately  the samples were applied and f i n a l l y almost stopped.  However  the absorption of the sample and the washing of the column w i t h 4 ml. of 0.1 N hydrochloric acid and 2 ml. of water were f i n a l l y accomplished. I t was impossible to elute the absorbed m a t e r i a l from the charcoal columns i n s i t u because of the low flow r a t e s . Therefore the charcoal was t r a n s f e r r e d to a 250 ml. centrifuge b o t t l e and extracted s i x times with 25 ml. portions of a mixture of pyridine:water:ethanol  (10:45s45).  The combined e x t r a c t s  were evaporated to dryness and the residue hydrolysed usual manner.  i n the  A f t e r removal of excess perchlorate, both super-  TABLE 2a Recovery of bases from samples of mononucleotides after p e r c h l o r i c a c i d h y d r o l y s i s and i o n exchange separation.  COMPOUND RECOVERED  % RECOVERY Adenylic and Guanylic Acids added to Reference Sample I Reference Sample I I  Adenine  89  86  Guanine  98  95  1 Mononucleotides, a d e n y l i c and guanylic a c i d s , were added to reference samples of the a c i d - s o l u b l e f r a c t i o n s taken before charcoal chromatography, as described i n the t e x t . TABLE 2b Recovery of guanine and adenine c o n t a i n i n g compounds from charcoal columns. QUANTITY OF RADIOACTIVITY RECOVERED (c.p.m. i n 21 m l . ) Reference sample Main sample^ without following charcoal treatment charcoal treatment 1  COMPOUND RECOVERED  Recovery from charcoal. % of reference sample  Adenine Sample I  205,800  47,000  23  Sample I I  234,400  58,000  25  Sample I  29,300  3,650  12.5  Sample I I  28,900  5,280  18  Guanine  1 c a l c u l a t e d by i s o t o p i c a n a l y s i s . 2 c a l c u l a t e d by summing the r a d i o a c t i v i t y of each base obtained by i o n exchange chromatography.  -  27  -  n a t a n t f r a c t i o n s were chromatographed  on Dowex £ 0 H*  u s i n g the h y d r o c h l o r i c a c i d g r a d i e n t . t i o n o f the e l u a t e was i|..6 ml. c o l l e c t e d .  columns  The u l t r a - v i o l e t absorp-  c o n t i n u o u s l y r e c o r d e d and f r a c t i o n s o f  A l i q u o t s o f these f r a c t i o n s were counted  i n the l i q u i d s c i n t i l l a t i o n spectrometer. The amounts o f r a d i o - a c t i v i t y r e c o v e r e d as adenine and guanine were measured by summing the i n d i v i d u a l counts o f the f r a c t i o n s c o r r e s p o n d i n g to those compounds. c l o g g i n g o f the c h a r c o a l columns  However, the  and the r e s u l t a n t  transfers  c o u l d have r e s u l t e d i n poor r e c o v e r i e s o f the compounds g i v i n g r i s e to the bases on h y d r o l y s i s . pounds i n the r e f e r e n c e samples were measured by i s o t o p i c  The amounts o f these comtaken b e f o r e c h a r c o a l treatment  dilution.  Known amounts o f a d e n y l i c and g u a n y l i c a c i d s were added to the r e f e r e n c e samples, and, f o l l o w i n g h y d r o l y s i s , the bases were r e c o v e r e d by i o n exchange the r e c o v e r e d bases was  chromatography.  e s t a b l i s h e d by paper  The q u a n t i t y o f each base r e c o v e r e d was u l t r a - v i o l e t spectrum o f the combined  The p u r i t y o f chromatography.  c a l c u l a t e d from the  f r a c t i o n s corresponding  to the base and found to be e x c e l l e n t , see T a b l e 2 a . The r a d i o - a c t i v i t y i n the combined  f r a c t i o n s was  also  measured and, u s i n g the percentage r e c o v e r i e s o f the added bases, the t o t a l r a d i o - a c t i v i t y o f the adenine and guanine compounds i n the r e f e r e n c e sample were c a l c u l a t e d . The t o t a l r a d i o - a c t i v i t y o f the adenine and guanine compounds i n the r e f e r e n c e samples was  compared w i t h the t o t a l  counts o f these compounds o b t a i n e d by the h y d r o l y s i s and i o n  - 28 exchange chromatography of the main charcoal-treated sample. The recoveries from charcoal, shown i n Table 2b, were very poor.  The use of t h i s method f o r d e - s a l t i n g the a c i d - s o l u b l e  f r a c t i o n was, t h e r e f o r e , abandoned, although i t was found to be very u s e f u l i n d e - s a l t i n g the thymine peak, obtained by i o n exchange chromatography p r i o r to f u r t h e r p u r i f i c a t i o n . Use of charcoal f o r d e - s a l t i n g thymine-containing fractions. As was shown e a r l i e r (see Figure 3)»  the f i r s t u l t r a -  v i o l e t absorbing peak obtained by the i o n exchange chromatography of a mixture of purine and pyrmidine bases contained thymine and u r a c i l .  However, the high s a l t content i n these  f r a c t i o n s s e r i o u s l y i n t e r f e r e d with the subsequent p u r i f i c a t i o n of the thymine, by paper chromatography.  I t was t h e r e f o r e , a  great advantage to use the charcoal columns to d e - s a l t the thymine-containing f r a c t i o n s before paper chromatography. The f r a c t i o n s , comprising the f i r s t r a d i o - a c t i v e and u l t r a - v i o l e t absorbing- peak, were evaporated to small volume (10 mis.) and were applied to a charcoal column (0.7 cm. x  1.0  cm.), which had not-been pretreated with p y r i d i n e . A f t e r absorpt i o n of the sample, the column was washed with 15 ml. of 0.01 hydrochloric acid.  N  The bases were eluted with 20 ml. of a mixture  of pyridine:water:ethanol (10:45:45).  The solvent was removed  by evaporation to dryness and,after treatment with 2 ml. of 1$ ammonia solution^re-evaporated. The residue, which was  completely  f r e e of s a l t , was applied to paper chromatograms which were developed with solvent system I I . Well defined and c l e a r l y separated areas corresponding to thymine and u r a c i l were obtained.  -  29  -  I I RESULTS A . IDENTIFICATION OF THE COMPOUNDS FOUND IN THE HYDROLYSED ACID-SOLUBLE FRACTION OF UNWASHED THYMUS CELL PREPARATIONS AND THE EFFECT OF VINBLASTINE ON C^-FORMATE INCORPORATION INTO THOSE COMPOUNDS. (1)  I d e n t i f i c a t i o n of u l t r a - v i o l e t absorbing m a t e r i a l s obtained on i o n exchange chromatography of the hydrolysed acid-soluble fraction. Before examining the effects of v i n b l a s t i n e on the i n c o r -  p o r a t i o n of C"^-formate i n t o the a c i d - s o l u b l e f r a c t i o n of unwashed thymus c e l l p r e p a r a t i o n s ,  the i d e n t i t y of the compounds  obtained by the i o n exchange chromatography of the h y d r o l y s i s products of t h i s f r a c t i o n was  Investigated.  A suspension (10 ml©) of unwashed thymus c e l l s , c o n t a i n i n g 150 mg. wet weight of c e l l s per m l . , was prepared as described above.  The suspension was immediately a c i d i f i e d w i t h U4. m l . of  5 N perchloric acid.  A f t e r standing i n the c o l d f o r 8 minutes,  excess perchlorate was removed from the mixture by p r e c i p i t a t i o n as the potassium s a l t .  The supernatant was evaporated to dryness  and the residue hydrolysed w i t h p e r c h l o r i c a c i d under the u s u a l conditions.  Excess perchlorate was again removed and the super-  natant was chromatographed on a column of Dowex 50  sulphonlc  a c i d r e s i n using a gradient of water (500 m l . ) to I4. N hydroc h l o r i c a c i d (500 m l . ) .  The e f f l u e n t was monitored f o r u l t r a -  v i o l e t absorbing m a t e r i a l s and then c o l l e c t e d I n ij.,5 m l . f r a c tions.  The e l u t i o n p a t t e r n of the u l t r a - v i o l e t absorbing  FIGURE 5 ION EXCHANGE SEPARATION OF BASES OBTAINED BY HYDRO'LYSING 'ACID SOLUBLE FRACTION  1  OF UNWASHED THYMUS  CELL SUSPENSION.;  FRACTION NO.  A  URACIL and THYMINE  B  CYTIDYLIC AG \  C  XANTHINE  D  HYPOXANTHINE  E  CYTOSINE  F  'NICOTINIC ACID'  G  GUANINE  H  ADENINE  ' •.•  - 30 -  m a t e r i a l s from the i o n exchange chromatogram i s shown i n Figure 5» The i d e n t i t i e s of the compounds i n these u l t r a - v i o l e t absorbing peaks were then s t u d i e d .  The spectra of the m a t e r i a l  i n each of these regions were determined i n the Cary spectrophotometer.  The f r a c t i o n i n each region showing maximum absorp-  t i o n was adjusted to pH 6 w i t h ammonia and evaporated to dryness. The residues were d i s s o l v e d i n a small volume of 0 . 1 N hydroc h l o r i c a c i d and a p p l i e d to paper chromatograms which were developed i n system 1. The Rf values and u l t r a - v i o l e t spectra o f these f r a c t i o n s showed that the u l t r a - v i o l e t absorpting peaks obtained by i o n exchange chromatography, corresponded to the f o l l o w i n g compounds:Peak Peak Peak Peak Peak  A B E G H  -  Thymine and U r a c i l C y t i d y l l c Acid Cytosine Guanine Adenine  These r e s u l t s agree w i t h the data reported i n Figure 3» The u l t r a - v i o l e t absorption spectra and the Rf values on paper chromatograms developed i n solvent systems I and I I , supported the conclusion that Peak C was xanthine and s i m i l a r l y peak D was i d e n t i f i e d as hypoxanthine.  A l s o added xanthine and  hypoxanthine appeared on Ion exchange chromatography i n the f r a c t i o n s corresponding to Peaks C and D r e s p e c t i v e l y .  The recovery  of added xanthine and hypoxanthine, f o l l o w i n g p e r c h l o r i c a c i d h y d r o l y s i s and i o n exchange chromatography as p r e v i o u s l y d e s c r i b e d , was i n v e s t i g a t e d .  The f r a c t i o n s comprising the i n d i v i d u a l peaks  obtained by i o n exchange chromatography were combined and adjusted  FIGURE 6 SEPARATION OF HYDROLYSIS PRODUCTS OF NAD ON DOWEX 50 H . +  (a). NAD hydrolysate'r  - 31 -  to a known volume.  The u l t r a - v i o l e t absorption d e n s i t i e s of  portions o f the pooled samples were determined.  The percentage  r e c o v e r i e s , shown i n Table 3» were c a l c u l a t e d , and again found to be very good. Another peak,  M  F , shown i n F i g u r e 5» appeared between n  cytosine and guanine on i o n exchange chromatography.  The u l t r a -  v i o l e t spectrum of the f r a c t i o n corresponding to the peak of t h i s m a t e r i a l was determined i n the Gary spectrophotometer and was found to be very s i m i l a r , but not i d e n t i c a l , to that of pyridine.  This suggested that t h i s peak was derived from the  h y d r o l y s i s o f a p y r i d i n e - c o n t a i n i n g compound such as the n i c o t i n i c a c i d co-enzymes.  This hypothesis was confirmed by the  i o n exchange chromatography o f the products of p e r c h l o r i c a c i d h y d r o l y s i s of nicotinamide adenine d i n u c l e o t i d e .  The i o n ex-  change chromatogram as expected gave an adenine peak, and also u l t r a - v i o l e t m a t e r i a l corresponding i n e l u t i o n p o s i t i o n to Peak F , as i s shown i n Figure 6 ( a ) .  The experiment was repeated  w i t h the a d d i t i o n o f cytosine and guanine, and the unknown m a t e r i a l was e l u t e d , as may be seen i n Figure 6(b) between these two bases, i . e . i n the p o s i t i o n of F i n Figure 5. Furthermore, samples of both Peak F and the unknown m a t e r i a l from the h y d r o l y s i s of NAD were a p p l i e d to paper chromatograms and developed i n solvent system I I I .  N i c o t i n i c acid  and nicotinamide were also applied to the same chromatograms and both unknown samples gave u l t r a - v i o l e t absopting areas corresponding to both n i c o t i n i c a c i d and n i c o t i n a m i d e .  The  density of the u l t r a - v i o l e t absorbing m a t e r i a l was greater l h '  TABLE 3  Recovery of the products of the h y d r o l y s i s of nicotinamide adenine d i n u c l e o t i d e and o f xanthine and hypoxanthine from i o n exchange columns. Resin: Dowex 50 H , 200-400 mesh, 8% c r o s s - l i n k e d . Gradient E l u t i o n : (500 ml.) H 0 to (500 mo.) 4N HC1. +  2  MATERIALS APPLIED COMPOUNDS RECOVERED  Adenine 2N i c o t i n i c acid  NAD f o l l o w i n g HCIO4 treatment  Free bases f o l l o w i n g HCIO4 treatment %  85 89  Xanthine  85  Hypoxanthine  82  1 Sample composed of xanthine and hypoxanthine. 2 N i c o t i n i c a c i d peak a l s o shown to contain nicotinamide, as i n d i c a t e d i n the t e x t .  - 32 the n i c o t i n i c a c i d areas than i n the nicotinamide areas of both the sample derived from Peak P and that from the unknown peak from NAD h y d r o l y s i s .  This suggested that both contained mainly  n i c o t i n i c a c i d and some, estimated at 30$» n i c o t i n a m i d e .  Also,  the p o s i t i o n of added n i c o t i n i c a c i d on i o n exchange chromatography as shown i n Figure 6(c) was found to correspond to that of Peak F .  Hence i t was considered that Peak F was composed  l a r g e l y of n i c o t i n i c a c i d . The q u a n t i t a t i v e recovery of adenine and o f the combined n i c o t i n i c a c i d and nicotinamide component from the h y d r o l y s i s of NAD was estimated.  The f r a c t i o n s , obtained by the i o n ex-  change chromatography, corresponding to adenine and n i c o t i n i c a c i d (Figure 6a) were pooled and t h e i r u l t r a - v i o l e t spectra determined.  The percentage recoveries c a l c u l a t e d from these  measurements, using spectra data from the l i t e r a t u r e (111), are shown i n Table 3»  These suggest that NAD gives adenine almost  q u a n t i t a t i v e l y on h y d r o l y s i s and also the p y r i d i n e p o r t i o n i s not degraded. (2) E f f e c t of VLB on C ^ - f o r m a t e i n c o r p o r a t i o n i n t o the A c i d - s o l u b l e fraction"! The s t a n d a r d i s a t i o n of the i o n exchange chromatographic system has been d e s c r i b e d , and i n the l a s t s e c t i o n the bases separated by t h i s system from the hydrolysed a c i d - s o l u b l e f r a c t i o n of unwashed thymus c e l l s were I d e n t i f i e d .  Good recoveries  of the bases were also obtained a f t e r h y d r o l y s i s and i o n exchange chromatography, (Tables 1 and 3)» The effect of VLB on the i n c o r p o r a t i o n of C * -formate 1  1  i n t o the ' a c i d - s o l u b l e f r a c t i o n ' of incubated unwashed thymus  - 33 -  c e l l s was therefore  studied.  A suspension of unwashed c e l l s  (130 mg. wet weight of t i s s u e per ml..) was prepared as deTwo i n c u b a t i o n f l a s k s were prepared c o n t a i n i n g 1 m l .  scribed.  of the unwashed c e l l suspension and 1 m l . o f t i o n (20 m i c r o c u r i e s ) .  formate  solu-  V i n b l a s t i n e sulphate s o l u t i o n ( 0 . 5 m l . )  was added to one f l a s k to give a f i n a l concentration of 50 per m l . o f v i n b l a s t i n e .  15  Both f l a s k s were incubated at 37° 0  f o r 3 hours at a shaking r a t e of lij.0 cycles per minute. A f t e r i n c u b a t i o n , the a c i d s o l u b l e f r a c t i o n s  (25 m l . )  from both f l a s k s were prepared, and I4. m l . reference samples aside.  set  As described i n the s e c t i o n on charcoal chromatography,  the a p p l i c a t i o n of the remainder o f both o f the a c i d - s o l u b l e f r a c t i o n s to charcoal column caused the columns almost to c l o g . The amount o f r a d i o - a c t i v i t y corresponding to adenine and guanine d e r i v a t i v e s i n the reference samples were measured by I s o t o p i c dilution.  From t h i s data, as described i n the previous s e c t i o n ,  the percentage recoveries of the compounds from the charcoal columns were c a l c u l a t e d and were found to be very low (Table 2 ) . The adsorbed p o r t i o n of the ' a c i d s o l u b l e f r a c t i o n ' was eluted from the charcoal w i t h p y r i d i n e , ethanol, water mixture (10:1+5:1^5) •  A f t e r the removal of the p y r i d i n e solvent by r e -  peated evaporation to dryness, the residue was hydrolysed w i t h perchloric acid.  The excess perchlorate was removed i n the  u s u a l manner and the supernatants (25 m l . ) were a p p l i e d to Dowex i o n exchange columns, and eluted w i t h the h y d r o c h l o r i c a c i d concentration g r a d i e n t .  The density of the u l t r a - v i o l e t absorp-  t i o n of the e f f l u e n t was continuously recorded and f r a c t i o n s o f  FIGURE 7 SEPARATION ON DOWEX 50 H  +  OF BASES OBTAINED  FROM 'ACID SOLUBLE FRACTION' PRETREATED WITH CHARCOAL.  U . V . AND RADIOACTIVITY ELUTION PATTERNS  ~i— 50  .100  FRACTION ' NO.  FRACTION NO.  35  ;  - 31+ 1±.5  ml. c o l l e c t e d .  r a d i o - a c t i v i t y o f 0.2  The  o f the i n d i v i d u a l f r a c t i o n s o f both runs was liquid scintillation  ml.  samples  measured i n the  counter.  The r a d i o - a c t i v i t y and u l t r a - v i o l e t a b s o r p t i o n o f the h y d r o l y s e d  curves  a c i d - s o l u b l e f r a c t i o n o f the c o n t r o l incuba-  t i o n are shown i n F i g u r e 7 and n e i t h e r d i f f e r e d from the corresponding  curves  obtained  significantly  from the p r e p a r a t i o n to  which v i n b l a s t i n e had been added. As  may  be seen i n t h i s f i g u r e , a l a r g e u l t r a - v i o l e t  absorbing peak was which completely  found between f r a c t i o n numbers 105  and  120  encompassed the r e g i o n p r e v i o u s l y shown to be  occupied by the degradation  products  and o v e r l a p p i n g the guanine r e g i o n *  o f the p y r i d i n e n u c l e o t i d e s , T h i s m a t e r i a l , was  found to  have an u l t r a - v i o l e t a b s o r p t i o n spectrum i d e n t i c a l to p y r i d i n e and to be v o l a t i l e i n a l k a l i n e media.  T h i s peak t h e r e f o r e appears  to be r e s i d u a l p y r i d i n e h y d r o c h l o r i d e , almost c e r t a i n l y  carried  over from the p y r i d i n e used i n the c h a r c o a l chromatography. This contaminating f r a c t i o n s by e v a p o r a t i n g  p y r i d i n e was  removed from the a f f e c t e d  them to dryness and  then r e - d r y i n g the  r e s i d u e s a f t e r the a d d i t i o n o f a s m a l l amount o f ammonia s o l u tion.  The u l t r a - v i o l e t a b s o r p t i o n s p e c t r a o f the n o n - v o l a t i l e  r e s i d u e o f these f r a c t i o n s were then determined i n o r d e r to d e f i n e the p o s i t i o n o f guanine, b e f o r e s p e c i f i c a c t i v i t y  deter-  minations. The xanthine,  s p e c i f i c a c t i v i t i e s o f adenine, hypoxanthine i n those f r a c t i o n s o f the r e s p e c t i v e peaks  and  having  - 35 -  maximum r a d i o - a c t i v i t y were determined a f t e r measuring t h e i r u l t r a - v i o l e t spectra.  Previous experiments had shown that  the 'thymine peak' also contained u r a c i l , which precluded directs i measurement of i t s s p e c i f i c a c t i v i t y . In order to check the accuracy of these r e s u l t s and to o b t a i n the s p e c i f i c a c t i v i t i e s of the guanine and thymine, the combined f r a c t i o n s i n the thymine and guanine areas and the peak f r a c t i o n s of the adenine, hypoxanthine and xanthine areas were evaporated to dryness and chromatographed s u c c e s s i v e l y on solvent systems I and I I or I I I .  On the second paper chroma-  tographic system, the guanine areas overlapped w i t h other fluorescent areas and were, therefore,  cut out, eluted and r e -  chroma tographed u s i n g solvent system I V .  The guanine areas on  t h i s system were free from contaminants. The u l t r a - v i o l e t spectra and the r a d i o - a c t i v i t y of the i n d i v i d u a l p u r i f i e d bases were determined by the methods already described and the s p e c i f i c a c t i v i t i e s c a l c u l a t e d (Table bj • Although the s p e c i f i c a c t i v i t y of hypoxanthine and adenine changed l i t t l e , that of xanthine decreased tremendously a f t e r paper chromatography.  This suggested the presence of other  r a d i o - a c t i v e compounds i n those f r a c t i o n s corresponding to the xanthine r e g i o n obtained oh i o n exchange chromatogram.  Radio-  autographic examination of paper chromatograms of m a t e r i a l from t h i s peak confirmed t h i s .  The r e s u l t s of p r e l i m i n a r y studies  on these compounds and on Peak Y (as shown i n F i g u r e 7) are reported i n a l a t e r s e c t i o n of t h i s  thesis.  TABLE 4 14  E f f e c t of v i n b l a s t i n e on the incorporation of C -formate i n t o the a c i d s o l u b l e f r a c t i o n of unwashed thymus c e l l suspensions. SPECIFIC ACTIVITY (c.p.m. per micromole) 3 hour incubation Control VLB added  TOTAL RADIOACTIVITY  1  COMPOUNDS ISOLATED  E f f e c t of VLB 7 of c o n t r o l 0  Hypoxanthine  85,000  91,000  107  Adenine  95,000  94,000  99  Xanthine  49,000  50,000  102  Guanine"  28,000  29,000  104  Thymine  215,000  260,000  121  3 hour incubation Control VLB added  E f f e c t of VLB '% of c o n t r o l  44,650  39,200  89  5,580  5,510  99  /•"  1  The a c i d s o l u b l e f r a c t i o n had been recovered from charcoal and then hydrolyzed w i t h HCIO^ before ion-exchange separation.  2  T o t a l r a d i o a c t i v i t y of adenine and guanine compounds from reference sample taken before charcoal treatment, obtained by i s o t o p i c d i l u t i o n a n a l y s i s described i n t e x t .  /  - 36 -  In g e n e r a l , the s p e c i f i c a c t i v i t i e s of the bases i s o l a t e d from both the c o n t r o l and VLB-containing incubations were very s i m i l a r , although the s p e c i f i c a c t i v i t y of the thymine derived from the l a t t e r was about 20$ higher than the control.  Small changes,th.e' order of 10$,were not regarded as  s i g n i f i c a n t as there are errors of s e v e r a l percent i n the determination of u l t r a - v i o l e t absorption maxima of m a t e r i a l eluted from paper chromatograms and of the r a d i o - a c t i v i t y . Thus apart from the increase i n thymine s p e c i f i c a c t i v i t y , VLB had e s s e n t i a l l y no effect on the i n c o r p o r a t i o n of  -formate  i n t o these components of a c i d - s o l u b l e f r a c t i o n . These r e s u l t s were viewed w i t h some reservations because of the poor recoveries of the nucleotides and bases from the charcoal.  Although poor recoveries i n themselves would not i n -  v a l i d a t e the s p e c i f i c a c t i v i t i e s , some f r a c t i o n a t i o n of the nucleotides might have occurred which could mask s i g n i f i c a n t differences caused by the a l k a l o i d . As described i n the s e c t i o n on charcoal chromatography, the r a d i o - a c t i v i t y present as both adenine and guanine d e r i v a t i v e s i n the reference samples was determined by i s o t o p i c d i l u tion analysis.  The t o t a l r a d i o - a c t i v i t y of adenine and guanine  d e r i v a t i v e s i n the a c i d - s o l u b l e f r a c t i o n s of both the c o n t r o l and v i n b l a s t i n e t r e a t e d incubations are shown i n Table if'. No marked differences were found, although the s m a l l depression i n the i n c o r p o r a t i o n i n t o adenine of v i n b l a s t i n e treated incubation was perhaps s i g n i f i c a n t as the i s o t o p i c d i l u t i o n method i s quite exact.  TABLE 5 ^"Effect of v i n b l a s t i n e on the i n c o r p o r a t i o n of C^-formate i n t o the n u c l e i c a c i d s of unwashed thymus c e l l preparations. E f f e c t on RNA and DNA. SPECIFIC ACTIVITY (c.p.m. per microgram phosphorus) COMPOUNDS ISOLATED  3 HOUR INCUBATIONS Control VLB added  E f f e c t of VLB 7o of c o n t r o l  DNA  272  159  .58.5  RNA  163  114  70  E f f e c t on Purine and Pyrimidine bases. SPECIFIC ACTIVITY (c.p.m. per micromole) COMPOUNDS ISOLATED  3 HOUR INCUBATIONS Control VLB added  E f f e c t of VLB 7o of c o n t r o l  Thymine  22,364  13,947  62  DNA - adenine  6,019  2,753  46  DNA - guanine  7,290  3,222  44  RNA - adenine  14,230-  10,245  72  RNA - guanine  9,850  7,585  77  1 Results reproduced w i t h the k i n d permission of Dr. J.F. Richards, Department of Biochemistry, U n i v e r s i t y of B r i t i s h Columbia.  -  These r e s u l t s t h e r e f o r e  37 -  substantiated  the c o n c l u s i o n  t h a t v i n b l a s t i n e d i d not s u b s t a n t i a l l y depress the i n c o r p o r a t i o n o f C ^ - f o r m a t e i n t o the a c i d - s o l u b l e f r a c t i o n o f unwashed 1  suspensions.  The r e s u l t s , o b t a i n e d by Dr. R i c h a r d s on examina-  t i o n o f the DNA and RNA o f the same i n c u b a t i o n , Table 5.  cell  are shown i n  As i n p r e v i o u s experiments, a $Q% d e p r e s s i o n  was found  i n the i n c o r p o r a t i o n o f G ^ - f o r m a t e i n t o the DNA o f the v i n blastine containing depression  incubations,  and there was a somewhat  o f i n c o r p o r a t i o n i n t o the RNA.  Whilst  — : ' " Ik i n v e s t i g a t i n g the e f f e c t o f v i n b l a s t i n e on G -  formate i n c o r p o r a t i o n i n t o the DNA and RNA o f thymus in vitro  smaller  cells  i t was found t h a t the i n c o r p o r a t i o n i n t o t h e DNA o f  a  the c o n t r o l i n c u b a t i o n was l i n e a r throughout a u s u a l l y 3 hour incubation period.  However, the r a t e o f i n c o r p o r a t i o n i n t o RNA  o f the same i n c u b a t i o n hours ( 1 8 ) .  decreased d u r i n g  the second and t h i r d  The i n c o r p o r a t i o n i n t o both n u c l e i c a c i d s was de-  p r e s s e d by the presence o f v i n b l a s t i n e i n the i n c u b a t i o n . I n view o f these r e s u l t s , the changes i n the r a t e s o f i n c o r p o r a t i o n o f C ^"-formate i n t o the ' a c i d - s o l u b l e f r a c t i o n ' 1  were i n v e s t i g a t e d over v a r i o u s p e r i o d s one  and a h a l f hours and three The  described.  incubation  o f time:- h a l f an hour,  hours.  f l a s k s were s e t up f o r each o f the times  Each contained  2 m l . o f unwashed c e l l  suspension  (380 mg. wet weight o f c e l l s per ml.), and 16 micro c u r i e s o f C^*formate (0,8 m l . ) *  V i n b l a s t i n e (50 * p e r ml.) was added to one  f l a s k and the f i n a l  volume adjusted  medium.  to 5.0 m l . w i t h  incubation  The f l a s k s were i n c u b a t e d , f o r the times i n d i c a t e d above,  at 3 7 ° 0 and a shaking r a t e o f IkO c y c l e s per minute.  FIGURE  EFFECT  O F V L B ON  IN 'ACID  SOLUBLE  TOTAL  8  NON-VOLATILE  FRACTION'  RADIOACTIVITY  OF UNWASHED  THYMUS  CELLS.  A: RADIOACTIVITY IN 'ACID SOLUBLE FRACTION' BEFORE HYDROLYSIS. B: RADIOACTIVITY IN 'ACID SOLUBLE FRACTION' AFTER  HYDROLYSIS.  - 38 A f t e r i n c u b a t i o n , each a c i d - s o l u b l e f r a c t i o n was i s o l a t e d i n the u s u a l manner and made up to a volume o f 18 m l . 2.1+ ml. r e f e r e n c e samples were s e t a s i d e and the remainder evaporated to dryness and h y d r o l y s e d . excess p e r c h l o r a t e , the h y d r o l y s e d a volume o f 15 m l .  s o l u t i o n s were adjusted to  A r e f e r e n c e sample (1.1+ ml.), was again s e t  a s i d e from each r u n . aliquots  A f t e r the removal o f  The n o n - v o l a t i l e r a d i o - a c t i v i t y o f  (0.2 ml.) o f the r e f e r e n c e samples taken, b e f o r e and  a f t e r h y d r o l y s i s , from each r u n were determined by the method previously The  described. counts o b t a i n e d ,  are p l o t t e d i n F i g u r e 8 and showed  t h a t the r a t e o f i n c o r p o r a t i o n i n t o both t h e a c i d - s o l u b l e 1  f r a c t i o n ' and the 'hydrolysed w i t h time.  acid-soluble fraction'  decreased  Furthermore, h y d r o l y s i s caused the l o s s o f a  s i g n i f i c a n t p r o p o r t i o n o f the counts f i x e d i n t o the ' a c i d - s o l uble f r a c t i o n ' .  I n a d d i t i o n , i n c o r p o r a t i o n i n t o the h y d r o l y s e d  and unhydrolysed  a c i d - s o l u b l e f r a c t i o n s o f the p r e p a r a t i o n con-  t a i n i n g v i n b l a s t i n e was s i g n i f i c a n t l y h i g h e r than i n t o the c o n t r o l s a f t e r three h o u r s ' i n c u b a t i o n . In o r d e r to i n v e s t i g a t e t h i s d i f f e r e n c e and t o confirm, i f p o s s i b l e , the o b s e r v a t i o n s made i n t h e p r e v i o u s the h y d r o l y s e d  experiments,  a c i d - s o l u b l e f r a c t i o n s from the 3 hour  incuba-  t i o n s were chromatographed on Dowex s u l p h o n i c a c i d r e s i n columns w i t h the u s u a l h y d r o c h l o r i c a c i d g r a d i e n t .  The u l t r a - v i o l e t  a b s o r p t i o n o f the e f f l u e n t was c o n t i n u o u s l y r e c o r d e d  and the  r a d i o - a c t i v i t y o f samples (0.2 ml.) o f t h e f r a c t i o n s c o l l e c t e d , was  measured i n the l i q u i d s c i n t i l l a t i o n  spectrometer.  TABLE 6 E f f e c t of v i n b l a s t i n e on C^-formate i n c o r p o r a t i o n i n t o the a c i d soluble f r a c t i o n on unwashed thymus c e l l suspensions.  TOTAL RADIOACTIVITY (c.p.m.) COMPOUNDS ISOLATED  3 HOUR INCUBATIONS Control VLB added  E f f e c t of VLB % of c o n t r o l  Adenine  14,470  14,200  98  Hypoxanthine  37,150  45,200  122  5,050  6,450  128  550,900  711,200  127  Thymine 'Substance "X"  1 R a d i o a c t i v i t y determined by i s o t o p i c d i l u t i o n a n a l y s i s . 2 Substance "X" was l a t e r shown to be s e r i n e .  - 39 The f r a c t i o n s comprising the f i r s t u l t r a - v i o l e t r a d i o - a c t i v i t y peak were combined thymine i n that combined  and  and the t o t a l count o f  sample measured by i s o t o p i c  dilution.  The t o t a l r a d i o - a c t i v i t y o f adenine and hypoxanthine was  measured  by summing the counts i n the i n d i v i d u a l f r a c t i o n s c o r r e s p o n d i n g to each base.  The r e s u l t s , which, i n the main confirmed the  p r e v i o u s c o n c l u s i o n , are shown i n Table 6 . t o t a l r a d i o - a c t i v i t y due to adenine was  E s s e n t i a l l y , the  the same i n both the  i n c u b a t i o n s , whereas the t o t a l r a d i o - a c t i v i t y due to both and hypoxanthine was  thymine  s i g n i f i c a n t l y h i g h e r i n the, v i n b l a s t i n e  containing incubation. I n a d d i t i o n a l a r g e r a d i o - a c t i v e peak which showed no u l t r a - v i o l e t a b s o r p t i o n was p o s i t i o n o f t h i s peak may i t y i n the peak 'X' was  found j u s t b e f o r e x a n t h i n e .  be seen i n F i g u r e 1 1 .  The  The  radio-activ-  about 25% g r e a t e r i n the sample d e r i v e d  from the i n c u b a t i o n to which VLB was  added.  t a i n e d a c o n s i d e r a b l e p r o p o r t i o n (50%)  As t h i s peak con-  o f counts i n the 'hydro-  l y s e d a c i d - s o l u b l e ' , t h i s change would p a r t i a l l y account f o r the d i f f e r e n c e s seen i n the t o t a l counts o f the h y d r o l y s e d a c i d s o l u b l e f r a c t i o n s o f the c o n t r o l and VLB c o n t a i n i n g i n c u b a t i o n s (Figure  8).  -  1+0 -  B . THE EFFECT OF WASHING ON THYMUS CELL SUSPENSIONS AND ON THE ACTION OF VINBLASTINE ON THE INCORPORATION OF C^-FORMATE INTO THE ACID-SOLUBLE FRACTION OF THOSE PREPARATIONS• In the experiments j u s t d e s c r i b e d , unwashed c e l l suspensions were used.  However, i t was known from the work  of Roof and Aub (1+2), that there are considerable differences i n the RNA content of unwashed suspensions o f thymus c e l l s and of the same c e l l s f o l l o w i n g c e n t r i f u g a t i o n and resuspension. I n the present work the differences i n quantity of n u c l e i c acids i n thymus c e l l s suspension prepared as described i n an e a r l i e r s e c t i o n were i n v e s t i g a t e d by D r . R i c h a r d s .  A l s o , the e f f e c t of  t h i s 'washing process on the metabolic s e n s i t i v i t y of the c e l l 1  to v i n b l a s t i n e was s t u d i e d . As a p r e l i m i n a r y step, the amount o f DNA, RNA and bases derived from the a c i d - s o l u b l e f r a c t i o n In washed and unwashed thymus c e l l suspensions were compared.  An unwashed c e l l  suspension (20 ml#) was prepared and 8 m l . of i t d i l u t e d to 12 m l . to form the unwashed sample.  The remainder (12 m l . ) was  centrifuged at l , 1 0 0 g f o r 6 minutes at 8° C ,  The supernatant  was discarded and the residue resuspending i n medium.  The  suspension was then f i l t e r e d through 2 l a y e r s of muslin and made up to 12 m l . to give the 'washed' sample.  - kl The preparation of the two suspensions i s o u t l i n e d below. Thymus glands  (10)  Disintegrated Suspension f i l t e r e d C e l l suspension (20 m l . )  8 mlo I d i l u t e d to 12 m l . w i t h buffer  12 m l . I l , 1 0 0 g f o r 6 minutes I Packed c e l l s suspended i n buffer. I f i l t e r e d through m u s l i n . I d i l u t e d to 12 m l . w i t h buffer.  Unwashed C e l l Suspension  Washed c e l l  suspension  The wet weight of c e l l s i n 1 m l . of each suspension was determined.  P e r c h l o r i c a c i d was added to the suspensions and the  supernatants r e t a i n e d as the ' a c i d - s o l u b l e f r a c t i o n ' .  The DNA  and RNA were extracted from the residue by D r . R i c h a r d s . a c i d - s o l u b l e f r a c t i o n s were adjusted  The  to pH k.5 w i t h potassium  hydroxide to remove excess perchlorate  and the supernatants  t r e a t e d i n the normal manner to o b t a i n the hydrolysed a c i d - s o l u b l e fractions.  Each f r a c t i o n was chromatographed on a Dowex 50  r e s i n column using the h y d r o c h l o r i c a c i d e l u t i o n g r a d i e n t .  The  u l t r a - v i o l e t absorption of the eluate was continuously recorded and k.5 m l . f r a c t i o n s were c o l l e c t e d .  The u l t r a - v i o l e t density  curves of the eluates are shown i n Figure 9 .  FIGURE 9 COMPARISON OF HYDROLYSED 'ACID SOLUBLE FRACTIONS' OF WASHED AND UNWASHED THYMUS CELLS.  U . V . DENSITY PATTERNS OF ELUENT FROM DOWEX 50 H . +  ~ 50 \  1 FRACTION  100/ NO.  '  "•  "I  So  . .  •  - k-2 -  The wet weight of c e l l s i n both suspensions was approximately the same, which i n d i c a t e d , i n view of the d i l u t i o n of the unwashed c e l l suspension, a l o s s of about 30% of wet weight of c e l l s during the preparation of the washed suspension.  A s i m i l a r l o s s of 25 - 30% olf wet weight of c e l l s  has been recorded i n other runs.  The r a t i o of DNA to wet weight  of c e l l s was found by D r . R i c h a r d s , to be the same i n both suspensions showing that there was a l o s s of DNA approximately equal to the l o s s i n wet weight of t i s s u e during the manipulations. However, the r a t i o of RNA tto wet weight of c e l l s was ;  39 - k f° Q  lower i n the washed c e l l suspension which suggested a p r e f e r e n t i a l l o s s of RNA during the washing process. I n s p e c t i o n of the e l u t i o n patterns of the  chromatograms  of the hydrolysed a c i d - s o l u b l e f r a c t i o n s , Figure 9, showed that l i t t l e hypoxanthine and xanthine were recovered from the washed c e l l suspension.  On the other hand, the amounts of adenine and  guanine r e l a t i v e to t i s s u e weight recovered from the a c i d s o l u b l e f r a c t i o n of both suspensions appear to be very s i m i l a r . In a d d i t i o n to the marked decrease i n the quantity of hypoxanthine and xanthine, there was s i g n i f i c a n t l y l e s s cytosine present i n the a c i d - s o l u b l e f r a c t i o n obtained from the washed c e l l preparation.  These r e s u l t s , on the RNA and DNA, agree w i t h those  obtained by Roof arid Aub  (I4.2).  I n a d d i t i o n the present work  has revealed marked differences i n the a c i d - s o l u b l e components. The e f f e c t of v i n b l a s t i n e on the i n c o r p o r a t i o n of formate i n t o washed thymus c e l l over a range of i n c u b a t i o n periods was therefore s t u d i e d .  A suspension of thymus c e l l s  was prepared i n the u s u a l manner.  The suspension (20 m l . ) was  -  1|3  -  centrifuged at l , 1 0 0 g f o r 6 minutes at 0° G and the supernatant d i s c a r d e d .  The packed c e l l s were resuspended i n medium  and, a f t e r f i l t e r i n g the suspension through 2 l a y e r s of m u s l i n , adjusted to a volume of 20 m l .  This suspension contained 130  mg. wet weight o f c e l l s per m l .  The \  i n c u b a t i o n f l a s k s were  prepared each c o n t a i n i n g 1 m l . of c e l l suspension and 8 microc u r i e s of C ^ - f o r m a t e (0.1+ml..).  V i n b l a s t i n e sulphate s o l u t i o n  ( 0 . 2 m l . ) was added to h a l f of the f l a s k s and the. volume i n a l l f l a s k s was then made up to 2 . 5 m l . w i t h medium.  The f l a s k s  were incubated at 3 7 ° C f o r 3 hours at a shaking r a t e o f li+0 c y c l e s per minute.  Two e x a c t l y s i m i l a r groups of 1+ f l a s k s were  prepared at the same time; one incubated f o r 2 hours and the other f o r h a l f an hour.  At the end o f these p e r i o d s , the con-  tents of the d u p l i c a t e f l a s k s were combined and t r e a t e d by the p r e v i o u s l y described procedures to give the p e r c h l o r i c acid hydrolysed a c i d - s o l u b l e f r a c t i o n s .  The excess p e r c h l o r a t e was  removed from the samples as the potassium s a l t and the supernatants a p p l i e d to i o n exchange columns and eluted w i t h the hydrochloric acid gradient.  The u l t r a - v i o l e t absorption of the  eluates was recorded and f r a c t i o n s c o l l e c t e d i n the usual manner. The r a d i o - a c t i v i t y of a l i q u o t s of the fractions was measured i n the l i q u i d s c i n t i l l a t i o n  counter.  As only a small quantity of t i s s u e was used i n each i n c u b a t i o n , i n s u f f i c i e n t of each base was present to give r e l i a b l e u l t r a - v i o l e t d e n s i t y measurements.  However the t o t a l  counts of adenine, guanine, hypoxanthine and of 'x» (Figure 11) were obtained by summing the counts i n the i n d i v i d u a l  fractions  FIGURE 10 EFFECT OF VLB AND TIME OF INCUBATION ON PURINE AND SERINE SYNTHESIS BY WASHED THYMUS CELLS.  - kh -  corresponding to each compound.  The r e s u l t s obtained are  shown g r a p h i c a l l y i n Figure 10,  The r a d i o - a c t i v i t y o f the  f r a c t i o n s from the i o n exchange columns f o r the 3 hour run i s plotted i n Figure 11.  The t o t a l r a d i o - a c t i v i t y i n the guanine  peaks from the i hour and 2 hour incubations was too small to be s i g n i f i c a n t .  However i n the f i n a l hour there was a  marked increase i n the t o t a l count of the guanine from the c o n t r o l i n c u b a t i o n i n a manner s i m i l a r to adenine. Marked differences were found i n the t o t a l r a d i o - a c t i v i t y present i n peak ' X ' ( l a t e r shown to be serine) and i n the peaks corresponding to adenine and guanine i n the 3 hour i n c u b a t i o n experiment.  These differences were almost e n t i r e l y due to  changes o c c u r r i n g i n the f i n a l hour (Figure 1 0 ) .  I t may be  observed that although the i n c o r p o r a t i o n o f isotope i n t o the adenine i n the Incubation to which v i n b l a s t i n e was added, had completely stopped i n the f i n a l hour, there was enhanced i n c o r p o r a t i o n i n t o the serine of the same r u n . I t was observed that the area under the adenine u l t r a v i o l e t absorption peak obtained from the i o n exchange column was smaller i n the 2 and 3 hour v i n b l a s t i n e c o n t a i n i n g incubations.  Thus the decrease i n the s p e c i f i c a c t i v i t y of adenine  i n these incubations would, perhaps, be l e s s than the depression i n t o t a l a c t i v i t y found. I n summary, although v i n b l a s t i n e had a pronounced  effect  on the i n c o r p o r a t i o n of formate i n t o the a c i d - s o l u b l e components of washed c e l l suspensions incubated for 3 hours, only small differences were found a f t e r a 2 hour i n c u b a t i o n p e r i o d .  FIGURE  11  EFFECT OF VLB ON C 1 4 -FORMATE INCORPORATION INTO THE 'ACID SOLUBLE FRACTION' OF WASHED THYMUS CELLS  RADIOACTIVITY PATTERN OF HYDROLYSEDFRACTION ON ELUTION FROM DOWEX 50 H - .  control • vlb  rC  • /  .  THYMINE URACIL  : i  o . J  XANTHINE  HYPO XANTHINE  11  GUANINE  X 50 F R A C T I O N  The i d e n t i t y o f X and Y i s  NO.  (SO  d i s c u s s e d i n the  text,  - k5 However, Dr. R i c h a r d s has shown t h a t the i n c o r p o r a t i o n o f formate i n t o D M of incubation, three hours. discussed  was a l r e a d y depressed by 60% a f t e r 2 hours  and that t h i s i n h i b i t i o n was m a i n t a i n e d f o r The s i g n i f i c a n c e o f these o b s e r v a t i o n s  later.  w i l l be  - k6 -  C. THE EXAMINATION OP UNIDENTIFIED RADIO-ACTIVE COMPOUNDS. Nature of r a d i o - a c t i v e m a t e r i a l i n Peak X . I t has been noted e a r l i e r that the 'hydrolysed a c i d s o l u b l e f r a c t i o n * of both washed and unwashed c e l l  preparations  contained a h i g h l y r a d i o - a c t i v e compound which was eluted from the i o n exchange column j u s t before xanthine (Figure 1 1 ) .  As  the r a d i o - a c t i v i t y due to t h i s 'compound' was considerably greater i n preparations  derived from incubations containing  v i n b l a s t i n e , i t was of considerable importance to i d e n t i f y t h i s material. The substance ' X ' d i d not appear i n the eluent from i o n exchange chromatograms of hydrolysed a c i d - s o l u b l e  fractions  which had p r e v i o u s l y been d e - s a l t e d on charcoal (Figure 7)» Also the f r a c t i o n s containing the unknown r a d i o - a c t i v e substance ' X ' had no u l t r a - v i o l e t absorption (Figure 1 1 ) .  Furthermore  ' X ' accounted for a h i g h p o r t i o n of the t o t a l r a d i o - a c t i v i t y i n the hydrolysed a c i d - s o l u b l e f r a c t i o n which suggested that i t was very r e a d i l y l a b e l l e d by C^--formate.  This evidence i n d i c a t e d  that the unknown m a t e r i a l was a small non-aromatic molecule, which r a p i d l y accepted a 1-carbon u n i t , p o s s i b l y serine or methionine.  I n a d d i t i o n the e a r l y work of Moore and S t e i n (k3)  using a sulphonic a c i d r e s i n column and h y d r o c h l o r i c a c i d as the e l u t a n t for the separation of amino acids showed that these amino acids were eluted from a sulphonic a c i d column i n a comparable p o s i t i o n to the unknown m a t e r i a l . Further information was obtained by paper chromatography. A p o r t i o n of peak ' X ' (Figure 11) was evaporated to dryness and  - 47 -  applied to a paper chromatogram to which methionine, g l y c i n e , serine and choline were also applied as reference compounds. The chromatogram was developed i n solvent system I I I .  Radio-  autography showed a s i n g l e spot (Rf.22) which, although w e l l removed from the other reference compounds, corresponded i n Rf. value to both glycine and s e r i n e , which run at very s i m i l a r r a t e s i n t h i s system. The i o n exchange paper chromatographic system, previously described, was able to d i s t i n g u i s h c l e a r l y between glycine and serine.  Therefore, the r a d i o - a c t i v e area was cut out of the  previous chromatogram and eluted (0.5 ml. of 0.1 N HCl).  The  eluate ( p a r t i a l l y evaporated) was applied together with" i n e r t serine to the'wetted i o n exchange paper and developed i n pH 3.1 c i t r a t e buffer. the same paper.  Reference g l y c i n e and serine were also run on The r a d i o - a c t i v e area and the reference i n e r t  s e r i n e , v i s u a l i s e d by ninhydrin, were found to coincide and i t therefore seemed very probable that the unknown compound was s e r i n e . I t was also important to discover whether the increased amounts of serine r a d i o - a c t i v i t y i s o l a t e d from the runs containing VLB were the r e s u l t of a biochemical r e a c t i o n of v i n b l a s t i n e or were merely a r e f l e c t i o n of minor d i f f e r e n c e s i n the i s o l a t i o n procedure.  I t was, therefore, p a r t i c u l a r l y important to e s t a b l i s h  the s t a b i l i t y of serine under the hot p e r c h l o r i c acid treatment used to l i b e r a t e the bases from the acid-soluble f r a c t i o n . A sample was prepared containing incubation medium (8.4 ml.), an a l i q u o t (4 ml.) of the 'serine' tube 49, VLB. containing run,  FIGURE 12 ISOLATION OF 'SERINE' ON DOWEX 50 H . +  ^COMPARISON OF ELUTION PATTERNS OF RADIOACTIVE AND NINHYDRIN-POSITIVE MATERIAL.  I  5  10  z  o  tu <t tc  -55. z  3  s  a.  u  - 2  4 0  20 FRACTION  JL  <  60  NO. I  RADIOACTIVITY  ; N I NHYDRIN  COLOUR »  F r a c t i o n s 48 and 49 were c o l l e c t e d as one sample. The values p l o t t e d here for each f r a c t i o n are equal to one-half of the t o t a l found i n the two f r a c t i o n s .  - k8 -  F i g u r e 11, and authentic i n e r t serine (2.16 m g . ) .  A portion  (0.2 m l . ) was removed f o r r a d i o - a c t i v i t y determination and 6 m l . . o f 2.1 N p e r c h l o r i c a c i d were added to the remainder of the sample.  The excess perchlorate was p r e c i p i t a t e d as the  potassium s a l t at pH k.5 i n the c o l d .  The supernatant and a  washing (5 m l . ) were combined, evaporated to dryness and hydrolysed under the usual c o n d i t i o n s .  Excess perchlorate  was again removed and the supernatant and a washing (2 m l . ) combined and made up to 10 m l .  A sample (0.2 m l . ) was again  removed and counted i n the l i q u i d s c i n t i l l a t i o n  spectrometer.  The remainder of the s o l u t i o n was a p p l i e d to a Dowex 50 H+ r e s i n column and eluted w i t h the h y d r o c h l o r i c a c i d g r a d i e n t . F r a c t i o n s (k.6 m l . ) were c o l l e c t e d and a l i q u o t s o f these f r a c t i o n s were taken f o r r a d i o - a c t i v i t y determination.  Other a l i -  quots were set aside f o r the c o l o r i m e t r i c e s t i m a t i o n of the amino a c i d concentration by the n i n h y d r i n method of Moore and S t e i n (31) s l i g h t l y modified by Webber ( k k ) .  Colorimetric  determinations were also c a r r i e d out on the s e r i n e s o l u t i o n . The r a d i o - a c t i v i t y and o p t i c a l density d i s t r i b u t i o n curves are shown i n Figure 12. The curves were found to c o i n c i d e , which r e i n f o r c e d the evidence i d e n t i f y i n g the unknown m a t e r i a l as s e r i n e .  Further-  more i t was c a l c u l a t e d from the r a d i o - a c t i v i t y measurements before and a f t e r h y d r o l y s i s , t h a t 82% of the added r a d i o - a c t i v i t y was present a f t e r h y d r o l y s i s and that 99.8% of these counts were recovered i n the serine peak on the i o n exchange column.  These  figures were substantiated by the c o l o r i m e t r i c determinations  - lj-9 which  showed Part  caused case i n  by  the  the  may  the  the two  small  of  r e s u l t  very  determine  i f  i n  the  of  was  a  evaporated usual 50  H*  i n  to  27  m l .  Both  acid  the  l i q u i d  of  as  to  the  to  the  t o t a l  r u n .  In  the  the  free  an  the  i t  of  incubation acid by  also  amino This  is  hydrolysis the  of  a l k a l o i d .  interest  acid  unhydrolysed  hydrolysed  Thus  hydrolysed  h y d r o l y s i s .  any  changes  count  mediated was  In  marked  p e r c h l o r i c  determining  to  acidi n  point  the was  acid-soluble  fraction  on  quantity  of  the  usual  radio-active  case.  and  a  3  prepared  m l .  l a t t e r  columns and  developed  f r a c t i o n  the  usual  The  former  chromatographed  applied  fractions  i n d i v i d u a l  s c i n t i l l a t i o n  and  was  were  i n  p o r t i o n .  hydrolysed  the  gradient  run  i t  been  steps.  the  change  f r a c t i o n  dryness,  column.  ml..)  and  acid-soluble  while  (0.2  prior  each  manner,  chloric  present  corresponding  system  a  a  chromatographing  isolated  into  to  the  serine.  have  containing  i d e n t i f i e d  fraction  divided  due  In  might  for  i n  serine,  by  An  from  observations  investigated  serine  account  increase  diluent  washing  these  f r a c t i o n  exchange  and  vinblastine  i s  acid-soluble  ion  the  added  serine  peak  variations  f r a c t i o n ,  and  the  of  not  serine  that  the  loss  would  the  of  view  of  p r e c i p i t a t i o n  probably,  In  soluble  small  loss  observed  the  recovery  86$  concluded  serine  but,  of  height  be  not  an  -  of  each  (li.5  spectrometer.  manner was i n  the  d i r e c t l y  to  with  usual  the  collected.  ml.)  were  was  a  Dowex hydro-  Samples  counted  i n  -  50  Total r a d i o - a c t i v i t y of serine  -  ( c o r r e c t e d to 3 0 ml, sample)  h y d r o l y s e d run = 1 7 3 , 2 5 0 c.p.m, unhydrolysed run = 1 8 7 , 5 0 0 c.p.m. Approximately 1 0 % more s e r i n e was sample than f r o m t h e  equivalent  r e c o v e r e d from the unhydrolysed  volume o f the h y d r o l y s e d r u n .  T h i s confirmed the figur'e's" "previously o b t a i n e d f o r the s t a b i l i t y o f s e r i n e to p e r c h l o r i c a c i d h y d r o l y s i s . Examination o f o t h e r u n i d e n t i f i e d compounds. Preliminary  work was  c a r r i e d out to i d e n t i f y the r a d i o -  a c t i v e components found i n the same f r a c t i o n s as xanthine i n the e f f l u e n t from the i o n exchange column.  The  radio-active  m a t e r i a l i n peak Y ( F i g u r e s k and 1 1 ) which had no absorption  was  also  Fractions  ultra-violet  investigated.  from these r e g i o n s were evaporated to dryness  and chromatographed on s e v e r a l systems.  The evidence suggested  t h a t g l y c i n e and some s e r i n e  the t a i l o f the main  peak) was  (presumably  p r e s e n t i n the x a n t h i n e peak.  Peak Y c o n t a i n e d two  r a d i o - a c t i v e compounds one o f which behaved l i k e methionine paper chromatography.  on  However these s t u d i e s have not y e t  p r o g r e s s e d to the p o i n t o f unambiguous I n d e n t i f i c a t i o n .  If  the e f f e c t of v i n b l a s t i n e on amino a c i d metabolism were to be investigated other separation  systems would be more  appropriate.  - 51 -  D. COMPARISON OP (a) THE INCORPORATION OP C^-PORMATE IN WASHED AND UNWASHED CELL SUSPENSIONS (b) THE SENSITIVITY OP THE TWO TYPES OP CELL PREPARATION TO VINBLASTINE. In view o f the d i f f e r e n c e s i n the RNA content and i n the c o n s t i t u e n t s of the a c i d - s o l u b l e f r a c t i o n s of washed and unwashed c e l l preparations (Figure 9 ) , i t was of i n t e r e s t t o compare w i t h i n a s i n g l e experiment the i n c o r p o r a t i o n of C^-formate i n t o these two p r e p a r a t i o n s .  I n a d d i t i o n there were i n d i c a t i o n s o f  d i f f e r e n c e s i n the s e n s i t i v i t y of these two c e l l suspensions to vinblastine.  I n p a r t i c u l a r , there was a 150$ increase i n the  t o t a l count of s e r i n e i n the washed c e l l i n c u b a t i o n c o n t a i n i n g the a l k a l o i d (Figure 1 0 ) , whereas only a 25$ increase was found i n an unwashed c e l l i n c u b a t i o n , Table 6 .  Because o f v a r i a t i o n s  which had been found i n the a c t i v i t y of d i f f e r e n t preparations of washed thymus c e l l s , these comparisons were made w i t h i n s i n g l e experiment. 50 m l . o f a suspension o f thymus c e l l s was prepared as p r e v i o u s l y described.  A 20 m l . p o r t i o n was d i l u t e d to 30 m l .  to give the 'unwashed c e l l suspension' while the other 30 m l . were c e n t r i f u g e d at l , 0 0 0 g f o r 8 minutes a t 0° C.  The superna-  t a n t was discarded and the packed c e l l s resuspended i n medium. After f i l t e r i n g  through 2 l a y e r s of muslin,'the washed c e l l  suspension' was adjusted to 30 ml. The wet weight o f c e l l s i n 1 m l . o f each o f these preparations was found t o be 100 mg. per m l . unwashed c e l l 10ii mg. per m l . washed c e l l  suspension suspension  - 52 S i g h t i n c u b a t i o n f l a s k s were s e t up, each c o n t a i n i n g 1 m l . o f unwashed c e l l suspension, 1+ ml.). and  1  the volume i n a l l o f the f l a s k s adjusted  t o 2.5 m l . w i t h  An i d e n t i c a l s e r i e s o f e i g h t f l a s k s were  up c o n t a i n i n g the washed c e l l suspension and both s e r i e s  were incubated for  (0.2  of C ^-formate  V i n b l a s t i n e (50 * P©r ml.) was added to f o u r o f the f l a s k s  i n c u b a t i o n medium. set  raicrocuries  a t 37° C a t a r a t e o f 120 c y c l e s per minute  3 hours. At the end o f t h i s p e r i o d , the contents o f each group  o f f o u r f l a s k s were combined and t r e a t e d w i t h p e r c h l o r i c a c i d to o b t a i n the a c i d - s o l u b l e f r a c t i o n .  The excess  perchlorate  was removed i n the u s u a l manner and the supernatant, a f t e r evaporation  to dryness, was h y d r o l y s e d  c h l o r i c a c i d f o r 1 hour a t 100° C. the excess p e r c h l o r a t e ,  w i t h 2 m l . o f 12% per-  After p r e c i p i t a t i o n of  the supernatant o f each r u n was  applied  to a Dowex 50 H* i o n exchange column and e l u t e d w i t h the u s u a l hydrochloric acid gradient. e l u a t e s was c o n t i n u o u s l y collected.  recorded  o f the  and I4..5 m l . f r a c t i o n s were  A l i q u o t s o f these f r a c t i o n s were counted i n the  liquid scintillation The  The u l t r a - v i o l e t a b s o r p t i o n  spectrometer.  t o t a l counts i n the hypoxanthine, s e r i n e and adenine  r e g i o n s were c a l c u l a t e d from these r e s u l t s and are shown i n Table 7»  I n order  to o b t a i n the s p e c i f i c a c t i v i t i e s o f a l l the  bases, the i n d i v i d u a l p u r i n e peaks were combined and graphed s u c c e s s i v e l y on s o l v e n t systems I and I I I .  ehroraatoThe thymine  samples were p u r i f i e d from the f i r s t r a d i o - a c t i v e and u l t r a v i o l e t absorbing  peak by c h a r c o a l treatment and paper chromatog-  raphy u s i n g s o l v e n t system I I as p r e v i o u s l y d e s c r i b e d  i n the  TABLE 7 Comparison of the effect of v i n b l a s t i n e on C -formate i n c o r p o r a t i o n i n t o a c i d s o l u b l e f r a c t i o n of washed and unwashed c e l l p r e p a r a t i o n s . E f f e c t of VLB on unwashed c e l l preparation  COMPOUNDS ISOLATED  SPECIFIC ACTIVITY (c.p.m. per micromole) 3 hour incubation Control VLB added  Hyp oxan thine  150,000  161,500  108  28,575  34,875  122  Adenine  164,000  124,000  76  11,025  12,375  112  Xanthine  65,500  74,500  113  Guanine  64,000  56,000  88 460,125  469,800  102  Effect of VLB % of c o n t r o l  Serine Thymine  327,000  328,000  TOTAL RADIOACTIVITY (c .p .m.) 3 hour i n c u b a t i o n Control VLB added  E f f e c t of VLB % of c o n t r o l  100  . E f f e c t of VLB on washed c e l l preparation Hypoxanthine  215,000  226,000  105  18,900  24,975  132  Adenine  130,000  115,000  88  17,482  12,712  73  Xanthine  174,000  161,000  93  Guanine  47,500  51,000  107 366,750  551,250  150  Serine Thymine  199,800  216,000  108  - 53 -  s e c t i o n on charcoal chromatography.  The u l t r a - v i o l e t absorption  and r a d i o - a c t i v i t y of the samples of the p u r i f i e d base were }  measured and the s p e c i f i c a c t i v i t i e s c a l c u l a t e d from these r e s u l t s are shown i n Table ?• The effects  of v i n b l a s t i n e on the i n c o r p o r a t i o n of  C^-formate i n t o the a c i d - s o l u b l e f r a c t i o n of the two c e l l prepar a t i o n s w i l l be considered f i r s t .  In general, i n the unwashed  preparations no marked differences were found i n the s p e c i f i c a c t i v i t i e s of the bases or of the t o t a l serine r a d i o - a c t i v i t y present i n the c o n t r o l and v i n b l a s t i n e containing i n c u b a t i o n s . These r e s u l t s therefore  l a r g e l y support the e a r l i e r observation  described i n Part A of the Results (Table k ) .  However although  i n the previous experiments the s p e c i f i c a c t i v i t y and t o t a l r a d i o - a c t i v i t y of adenine was depressed by v i n b l a s t i n e to the extent of 5-10%, i n t h i s experiment a depression of 2k% was found.  Nevertheless t h i s difference d i d not a l t e r the o v e r a l l  picture. Considering the washed c e l l i n c u b a t i o n s , i t was found that the serine r a d i o - a c t i v i t y was much greater i n the v i n blastine treated incubation.  On the other hand the  total  r a d i o - a c t i v i t y of adenine was l e s s i n the presence of v i n blastine.  Thus the r e s u l t s were s i m i l a r to the  observations  made i n the time study using washed c e l l s (Figure 1 0 ) , although the changes were greater i n the previous experiment.  In a d d i -  t i o n , i t may be noted that i n two other experiments, not  reported  i n d e t a i l i n t h i s t h e s i s , the t o t a l r a d i o - a c t i v i t y of serine was found to be between 65 and 70% higher i n the v i n b l a s t i n e con-  TABLE 8 •'•Effect of v i n b l a s t i n e on C -formate incorporation i n t o the n u c l e i c acids of washed and « unwashed c e l l preparations a f t e r 3 hour incubations. 14  PREPARATIONS  UNWASHED CELLS  WASHED CELLS  Specific Activity  E f f e c t of VLB  Specific Activity  E f f e c t of VLB  Control  VLB added  % of c o n t r o l .  Control  % of c o n t r o l  RNA  160.1  112.5  70.3  347.7  252.7  72.7  DNA  151.7  70.3  46.3  214.3  91.8  42.8  VLB added  1  Result reproduced w i t h the k i n d permission of Dr. J.F. Richards, Department of Biochemistry, U n i v e r s i t y of B r i t i s h Columbia.  2  S p e c i f i c A c t i v i t y counts per minute per microgram of phosphorus.  -  taining incubation.  5V-  Thus a l l the evidence, suggests that the  t o t a l r a d i o - a c t i v i t y o f s e r i n e i s increased by v i n b l a s t i n e much more i n washed c e l l incubations than i n unwashed c e l l i n c u b a t i o n s . D r . Richards has i n v e s t i g a t e d the i n c o r p o r a t i o n of C ^-1  forraate i n t o the RNA and DNA of the same incubations and the r e s u l t s are shown i n Table 8. The effect of v i n b l a s t i n e on Ik C  -formate i n c o r p o r a t i o n i n t o the n u c l e i c acids was the same  i n both types of c e l l preparation and i n both cases the dep r e s s i o n o f i n c o r p o r a t i o n i n t o DNA* about  was greater than  i n t o the RNA, about 3 0 $ . The r e s u l t s shown i n Table 1, also i n d i c a t e that there were c o n s i d e r a b l e . d i f f e r e n c e s i n the s p e c i f i c a c t i v i t i e s of the compounds i s o l a t e d from the c o n t r o l incubations of washed and unwashed c e l l p r e p a r a t i o n s .  The s p e c i f i c a c t i v i t i e s o f the  hypoxanthine and xanthine i s o l a t e d from the a c i d - s o l u b l e f r a c t i o n o f the washed c e l l i n c u b a t i o n were considerably higher than those derived from unwashed c e l l s .  A l s o i t was found (Figure  9) that xanthine and hypoxanthine were recovered i n f a r smaller q u a n t i t i e s from thewashed c e l l p r e p a r a t i o n .  There appears to  be a d i l u e n t pool of these two bases o r t h e i r n u c l e o t i d e s i n the unwashed c e l l suspension.  However the s p e c i f i c a c t i v i t y of  adenine from the washed run was not increased as a r e s u l t of the higher hypoxanthine a c t i v i t y , and was, indeed, s l i g h t l y lower than i t s counterpart from the unwashed c e l l suspension;:. E x a c t l y the same i n t e r - r e l a t i o n s h i p was found between xanthine and guanine i n the two c e l l  preparations.  In a d d i t i o n to these observations on the purine bases,  55 -.. the s p e c i f i c a c t i v i t y o f the thymine from the washed c e l l preparation was somewhat lower than i t s counterpart from the unwashed c e l l p r e p a r a t i o n . Prom the r e s u l t s obtained by D r . R i c h a r d s , Table 8 , I t w i l l be seen that the RNA s p e c i f i c a c t i v i t y was"considerably higher i n the washed c e l l p r e p a r a t i o n .  As RNA was removed  from the c e l l preparation during the washing procedure,  this  great d i f f e r e n c e might i n d i c a t e the presence o f an i n e r t pool of RNA i n the unwashed c e l l suspension.  DISCUSSION I t has been shown t h a t , i n v i v o , v i n b l a s t i n e causes an almost complete i n h i b i t i o n of the incorporation of C ^-formate 1  i n t o the deoxyribonucleic acid (DNA) of r a t bone marrow (18).  Also,  Richards et a l (19) found that i n v i t r o the presence of v i n b l a s t i n e caused a depression of 60% i n the incorporation of  G ^1  formate i n t o the DNA of r a t bone marrow and thymus c e l l suspensions.  Both i n vivo and i n v i t r o there was a s i g n i f i c a n t but  somwehat smaller depression of incorporation i n t o the r i b o n u c l e i c acids (RNA), (Tables 5 and 8 ) .  There was, however, no  evidence to i n d i c a t e at which stage i n the biosynthesis of the n u c l e i c acids the drug was a c t i n g , nor even that the observed i n h i b i t i o n was the r e s u l t of a d i r e c t interference by the a l k a l o i d with a s p e c i f i c step i n the b i o s y n t h e t i c route.  The pur-  pose of t h i s work, the f i r s t part of which i s reported i n the t h e s i s , i s to i n v e s t i g a t e the e f f e c t of v i n b l a s t i n e on the biosynthesis of the precursors of the n u c l e i c a c i d s , and, i f p o s s i b l e , t o locate the s i t e of a c t i o n of the a l k a l o i d . The numerous nucleotides, which are intermediates i n the formation of the deoxyribonucleic acids (DNA), and of the r i b o n u c l e i c acids (RNA), are found i n the 'acid-soluble f r a c t i o n ' of the c e l l . hydrolysed  In t h i s preliminary study, these compounds were  and t h e i r parent purine and pyrimidine bases i s o l a t e d . 14  The synthesis of these compounds was studies by the use of C  -  formate which, i t has been w e l l e s t a b l i s h e d , i s incorporated s p e c i f i c a l l y i n t o the  and Cg p o s i t i o n s of the purine r i n g of  the bases, adenine, guanine, xanthine and hypoxanthine and i n t o  FIGURE 13 EFFECT OF VLB ON INCORPORATION OF C  1 4  FORMATE INTO DNA OF WASHED THYMUS CELLS.  •  O  I INCUBATION  •  . PERIOD  •  2 ' ( . • • • (HOURS,) • \'  '  .  •  •CONTROL  •-  3  -•  VLB -  \  Results reproduced w i t h the k i n d permission o Dr. J . F . Richards, Department of Biochemistry U n i v e r s i t y of B r i t i s h Columbia.  - 57 the m e t h y l group on the  p o s i t i o n o f the p y r i m i d i n e r i n g o f  thymine (45-46). I n p r e s e n t i n v e s t i g a t i o n s ' , the e f f e c t s  o f v i n b l a s t i n e on  the i n c o r p o r a t i o n o f Cp-^-formate i n t o b o t h the n u c l e i c a c i d s and t h e i r s m a l l m o l e c u l a r weight p r e c u r s o r s i n the fraction1  o f thymus c e l l suspensions  t h e same e x p e r i m e n t . the e f f e c t  'acid-soluble  have been s t u d i e d w i t h i n  The q u a n t i t a t i v e r e l a t i o n s h i p between  o f v i n b l a s t i n e on the b i o s y n t h e s i s o f the n u c l e i c  a c i d s and o f t h e i r p r e c u r s o r s c o u l d t h e r e f o r e be  established.  The DNA and RNA o f t h e i n c u b a t e d thymus c e l l suspensions  were  i s o l a t e d and s t u d i e d by D r . J . F . R i c h a r d s and h i s r e s u l t s shown i n Tables 5 and 8 .  are  A l s o i t may be seen i n F i g u r e 13 t h a t  t h e r a t e o f i n c o r p o r a t i o n o f t h e i s o t o p e i n t o the DNA o f b o t h washed, and unwashed c e l l s u s p e n s i o n s , a t h r e e hour p e r i o d .  was found t o be almost l i n e a r over  There was a l s o an almost l i n e a r i n c o r p o r a -  t i o n o f C 1 ^ - f o r m a t e i n t o the DNA o f the c e l l s i n c u b a t e d i n the presence o f v i n b l a s t i n e , but the r a t e o f i n c o r p o r a t i o n o f  formate  i n t o the DNA was depressed by 50 t o 60% by the a l k a l o i d .  This  i n h i b i t i o n was found t o be the same i n b o t h washed and unwashed cell  preparations. The r a t e o f i n c o r p o r a t i o n o f formate i n t o the RNA de-  creased i n the second and t h i r d hours o f i n c u b a t i o n .  In addi-  t i o n , VLB d i d not depress the i n c o r p o r a t i o n of the i s o t o p e i n t o RNA n e a r l y as much as i n t o DNA.  Thus at the end o f one h o u r ' s  i n c u b a t i o n t h e r e was a d e p r e s s i o n o f 15% o f i n c o r p o r a t i o n i n t o RNA, w h i l e at the end o f 3 h o u r s , i t had o n l y reached 30-40%. From t h e r e s u l t s g i v e n i n Tables 4 , 6, and 7 , i t may be  - -8 seem t h a t the presence o f the a l k a l o i d d i d not cause a marked d e p r e s s i o n i n the i n c o r p o r a t i o n o f C ^ - f o r m a t e i n t o the bases 1  i s o l a t e d from the ' a c i d - s o l u b l e f r a c t i o n ' o f unwashed thymus c e l l suspensions even a f t e r three hours' i n c u b a t i o n . the a l k a l o i d  In f a c t ,  caused a s l i g h t i n c r e a s e i n the i n c o r p o r a t i o n o f  the i s o t o p e i n t o  thymine and hypoxanthine.  Thus, the r e s u l t s  o b t a i n e d i n the experiments w i t h unwashed c e l l s suggest? t h a t the v a r i o u s processes into  the p u r i n e s  blastine.  l e a d i n g to the i n c o r p o r a t i o n o f formate  and p y r i m i d i n e s  a r e not i n h i b i t e d by v i n -  Because i n the same c e l l p r e p a r a t i o n s  there was a  marked d e p r e s s i o n o f the i n c o r p o r a t i o n o f the i s o t o p e i n t o the DNA,  i t appears q u i t e p o s s i b l e t h a t the a l k a l o i d i s a c t i v e a t  some stage  above the de novo s y n t h e s i s o f the p u r i n e and  pyriraidine  compounds.  T h i s p o i n t w i l l be d i s c u s s e d i n g r e a t e r  detail later. Although the a l k a l o i d a p p a r e n t l y d i d n o t i n h i b i t the formation  o f the n u c l e i c a c i d p r e c u r s o r s i n unwashed thymus  c e l l p r e p a r a t i o n s , the presence o f v i n b l a s t i n e caused a p r o nounced d e p r e s s i o n and  i n the t o t a l r a d i o - a c t i v i t y  o f the adenine  guanine i s o l a t e d from the a c i d - s o l u b l e f r a c t i o n o f washed  thymus c e l l p r e p a r a t i o n s  ( F i g u r e 10 and Table 7 ) «  However,  the r e l a t i v e d e p r e s s i o n o f t h e s p e c i f i c a c t i v i t i e s appeared to be somewhat l e s s than o f the t o t a l r a d i o - a c t i v i t i e s . (as may be seen i n F i g u r e 10),  A l s o , ..  the pronounced d e p r e s s i o n  i n total  r a d i o - a c t i v i t y o n l y o c c u r r e d i f the c e l l s were i n c u b a t e d f o r three h o u r s .  S i n c e the a l k a l o i d  the i n c o r p o r a t i o n i n t o  caused a 50% d e p r e s s i o n i n  the DN£ o f t h e same suspension  i n only  •  /  • . • • ' . FIGURE 14 BIOSYNTHESIS OF ADENYLIC AND GUANYLIC ACIDS FROM INOSINIC ACID.  ;  •'  MOOC CM -CH< COOH  I  I CO  N  . vI , «. «  I''  MCT  C  IMP  /  CM succmas*  ^  a den(AMPS) yto succinic ixid MM  X  N  N  -i'Y> .C.  N  >l  M--RSP  AMP  \  C^ \. N-RSP  xanthosine - 5' phosphate (XMP)  V  N-RSP  OMP  V  one  hour's i n c u b a t i o n  (see F i g u r e  probable that the d e p r e s s i o n and  13),  i t would appear im-  o f i n c o r p o r a t i o n i n t o the adenine  guanine o f the ' a c i d - s o l u b l e f r a c t i o n ' i s d i r e c t l y r e l a t e d  to the a c t i o n o f the a l k a l o i d on DNA  synthesis.  I t i s g e n e r a l l y accepted t h a t a d e n y l i c and g u a n y l i c are b o t h s y n t h e s i s e d ( F i g u r e 11+) •  acids  (1+7)  i n the c e l l from i n o s i n i c a c i d  The route l e a d i n g to g u a n y l i c a c i d i n v o l v e s the  amination o f x a n t h y l i c a c i d by glutamine, w h i l e the s y n t h e s i s o f a d e n y l i c a c i d from i n o s i n i c a c i d r e q u i r e s a s p a r t i c a c i d , I n o s i n i c a c i d , shown i n F i g u r e t i d e to be s y n t h e s i s e d .  2,  i s the f i r s t p u r i n e  nucleo-  I t i s t h e r e f o r e s i g n i f i c a n t t h a t the  i n c o r p o r a t i o n o f C ^ - f o r m a t e i n t o hypoxanthine, which i s the p u r i n e base o f i n o s i n i c a c i d , i s n o t depressed by the presence o f v i n b l a s t i n e i n e i t h e r washed o r unwashed c e l l Thus the de novo s y n t h e s i s o f the p u r i n e to be i n h i b i t e d by VLB. preparations,  suspensions.  r i n g does n o t appear  However, p a r t i c u l a r l y i n washed  cell  the t o t a l r a d i o - a c t i v i t y o f adenine and guanine  are depressed i n the presence o f VLB.  I n p a r t these observa-  t i o n s t e n t a t i v e l y suggest the c o n v e r s i o n  o f i n o s i n i c a c i d to  a d e n y l i c and g u a n y l i c a c i d s i s depressed by t h e presence o f v i n blastine.  However, because t h i s e f f e c t o n l y appears i n t h e  f i n a l hour o f i n c u b a t i o n and i s more marked i n washed c e l l s than i n unwashed c e l l p r e p a r a t i o n s ,  i t i s not f e l t t h a t t h i s i s  d i r e c t l y r e l a t e d to the a c t i o n o f the a l k a l o i d on DNA In t h i s connection, t h a t glutamic  i t i s i n t e r e s t i n g t h a t Johnson (12)  and a s p a r t i c a c i d s p a r t i a l l y r e v e r s e d  o f the growth o f J 96  synthesis. found  the i n h i b i t i o n  c e l l s i n t i s s u e c u l t u r e by v i n b l a s t i n e .  -  60  -  He d i d n o t c o n s i d e r t h a t i n t e r f e r e n c e  w i t h the metabolism o f  these amino acids was the primary e f f e c t o f the a l k a l o i d . Formate i s i n c o r p o r a t e d N  1 0  into thymidylic  a c i d v i a the liK  methylene t e t r a h y d r o f o l i c a c i d i n t e r m e d i a t e and the b i o -  synthesis  of i n o s i n i c acid involves  carbon u n i t s from f o r m y l d e r i v a t i v e s (1+5, 1-1-8) •  Since neither  the t r a n s f e r o f two 1o f tetrahydro f o l i c  thymlnenor hypoxanthine  acid  biosynthesis  i s i n h i b i t e d by v i n b l a s t i n e , i t may be concluded t h a t the tetrahydro f o l i c  a c i d formate a c t i v a t i n g systems and the  t r a n s f e r o f the one-carbon fragments are not i n h i b i t e d . t h i s work was completed, Johnson (10) r e p o r t e d t h a t and  After  vinblastine  v i n c r i s t i n e , do not i n h i b i t e i t h e r f o l i c a c i d reductase o r  formyl t e t r a h y d r o f o l a t e  synthetase a c t i v i t y i n v i t r o .  a p r e v i o u s i n v e s t i g a t i o n Noble, Beer and Cutts (1+)  Also, i n  reported  t h a t the l e u c o p e n i c and the bone marrow d e p r e s s i n g a c t i v i t y o f v i n b l a s t i n e were n o t r e v e r s e d w i t h f o l i c a c i d , f o l i n i c thymine, c y t i d i n e , guanine o r V i t a m i n B-]_2» have o b t a i n e d c l i n i c a l  acid,  H e r t z e t a l (1+9)  remissions with v i n b l a s t i n e i n patients  w i t h c h o r i o e p i t h e l i o m a which had become r e s i s t a n t to the a n t i folic  drugs.  This  evidence again suggests t h a t the a c t i o n o f  v i n b l a s t i n e i s not r e l a t e d to a n t i - f o l i c a c i d a c t i v i t y .  These  o b s e r v a t i o n s were o f p a r t i c u l a r i n t e r e s t i n r e l a t i o n s h i p to reports  t h a t c e r t a i n f r a c t i o n s o f V i n c a Rosea e x t r a c t s  than those t h a t  contain  anti-folic activity  other  v i n b l a s t i n e o r v i n c r i s t i n e do possess  (50) •  The evidence presented here together  w i t h the b i o l o g i c a l and c l i n i c a l  evidence support the  t h a t the i n h i b i t i o n o f DNA s y n t h e s i s  conclusion  by v i n b l a s t i n e i s n o t the  - 61 r e s u l t o f a d i s t u r b a n c e i n a f o l i c a c i d mediated  reaction.  As the r e d u c t i o n o f the o n e - c a r b o n f r a g m e n t ,  attached  to t h e t e t r a h y d r o f o l i c a c i d , from the formate to the oxidation l e v e l requires  formaldehyde  reduced n i c o t i n a m i d e adenine d i n u c l e o -  t i d e phosphate (LL8), i t appears t h a t the p r o d u c t i o n o f NADPH I s n o t s u p p r e s s e d by the p r e s e n c e o f v i n b l a s t i n e . These c o n c l u s i o n s a r e s u p p o r t e d by the o b s e r v a t i o n  that  the t o t a l r a d i o - a c t i v i t y o f s e r i n e i n unwashed c e l l  incubations  was s l i g h t l y i n c r e a s e d  indicates  ( T a b l e s 6 and 7 ) .  This also  t h a t the s e r i n e a l d o l a s e r e a c t i o n (1±£, 1+6) i s u n i m p a i r e d . I n a d d i t i o n , the i n c r e a s e  i n the t o t a l r a d i o - a c t i v i t y o f  s e r i n e was f a r more pronounced ( a v e r a g i n g o v e r 70%) when v i n b l a s t i n e was added t o the washed c e l l s u s p e n s i o n s ( F i g u r e 10 and Table 7 ) . increase  However, i t may be seen i n F i g u r e 10 t h a t t h i s marked i n the t o t a l s e r i n e r a d i o - a c t i v i t y o c c u r r e d i n the  f i n a l hour o f i n c u b a t i o n .  Therefore,  s i n c e DNA s y n t h e s i s  is  i n h i b i t e d much e a r l i e r i n the i n c u b a t i o n p e r i o d ( F i g u r e 13) i s d e p r e s s e d by the same amount i n washed and unwashed incubations  ( T a b l e 8), I t i s u n l i k e l y t h a t the e f f e c t  and  cell of"the  a l k a l o i d on s e r i n e i s d i r e c t l y r e l a t e d t o the same s t e p w h i c h i s i n h i b i t e d i n the b i o s y n t h e s i s  o f DNA.  There may be s e v e r a l p o s s i b l e  reasons f o r t h e  i n the t o t a l r a d i o - a c t i v i t y o f s e r i n e .  increase  P o s s i b l y the f o r m a t i o n  o f s e r i n e v i a the s e r i n e a l d o l a s e r e a c t i o n i s s t i m u l a t e d . the* o t h e r hand the / u t i l i s a t i o n o f s e r i n e by s e r i n e  On  transaminase  o r the i n c o r p o r a t i o n o f the amino a c i d i n t o p r o t e i n o r i n t o p h o s p h o l i p i d s may be d e p r e s s e d .  T h i s c o u l d l e a d to an accumula-  - 62 t i o n of the amino a c i d .  -  However, i t i s d i f f i c u l t to c o r r e l a t e  these suggestions with the recent observation that transaminase a c t i v i t y i s sometimes increased i n the serum:: of v i n b l a s t i n e treated patients (10).  Another possible explanation may  that serine i s l o s t from the c e l l .  be  I f v i n b l a s t i n e increased  the permeability of the c e l l membrane to serine then the l o s s of serine i n t o the e x t r a - c e l l u l a r f l u i d could lead to an i n crease i n the t o t a l r a d i o - a c t i v i t y recovered.  I t i s noticeable,  Figure 10, that the s t i m u l a t i o n of the formate incorporation i n t o serine during" the' t h i r d hour of incubation of theppreparat i o n containing v i n b l a s t i n e corresponds to the period of complete i n h i b i t i o n of i n c o r p o r a t i o n of formate i n t o the adenine and guanine of the same preparation. between the two phenomena.  There may be a r e l a t i o n s h i p  One may  speculate, that there i s  a l o s s of the amino acids s e r i n e , a s p a r t i c acid and glutamine from the c e l l .  This would lead to an increase i n the t o t a l  r a d i o - a c t i v i t y of the serine but decrease the synthesis of adenylic and guanylie acids.  However as a s p a r t i c acid and  glutamine are also involved i n several other steps i n purine and pyrimidine b i o s y n t h e s i s , i t would be of great i n t e r e s t to study the question of leakage of compounds from the c e l l i n the presence of VLB.  I t should also be noted that these e f f e c t s  on serine may not be s p e c i f i c to t h i s amino a c i d as most other amino a c i d s , apart from methionine are not r a p i d l y l a b e l l e d by C^^-formate.  Any d i f f e r e n c e s i n t h e i r metabolism would not  therefore be revealed by the present methods. Although v i n b l a s t i n e does not appear to i n h i b i t the  - 63 de novo b i o s y n t h e s i s o f the p u r i n e n u c l e o t i d e s o r the i n c o r p o r a t i o n o f C ^ - f o r m a t e i n t o t h y m i d y l i c a c i d , the p r e s e n t data do not exclude  the p o s s i b i l i t y t h a t the subsequent metabolism o f  b •• these compounds i s a f f e c t e d by the a l k a l o i d .  The experimental  approach adopted i n t h i s p r e l i m i n a r y study i n v o l v e d the h y d r o l y s i s o f the ' a c i d - s o l u b l e f r a c t i o n ' and the r e c o v e r y o f the parent b a s e s . a compound, present  Thus, a s i g n i f i c a n t e f f e c t o f v i n b l a s t i n e on o n l y i n very s m a l l q u a n t i t y i n the ' a c i d -  s o l u b l e f r a c t i o n ' c o u l d escape d e t e c t i o n by t h i s procedure, Dr, R i c h a r d s  has found t h a t (Tables 5 and 8) the synthesis^  o f DNA i s i n h i b i t e d t o a g r e a t e r degree by v i n b l a s t i n e than i s t h a t o f RNA*  He a l s o found t h a t i n the DNA the s p e c i f i c a c t i v i t i e s o f  the p u r i n e bases a r e lowered to a g r e a t e r extent by the a l k a l o i d than the a c t i v i t y o f thymine. the h y p o t h e s i s  Thus one may t e n t a t i v e l y advance  t h a t the v i n b l a s t i n e may e x e r t i t s c y t o t o x i c  e f f e c t s by i n h i b i t i n g e i t h e r the b i o s y n t h e s i s o f the p u r i n e deoxyr i b o n u c l e o t i d e s o f t h e i r subsequent i n c o r p o r a t i o n i n t o DNA, ever, t h i s p r o p o s a l l e a v e s , unexplained, synthesis.  the d e p r e s s i o n o f RNA  I t i s o f i n t e r e s t t h a t , although P o t t e r e t a l (51)  found the expected p y r r o l i d i n e deoxynucleotides  i n c a l f thymus,  they c o u l d not show t h e presence o f t h e corresponding derivatives.  How-  They concluded  t h a t the deoxy p u r i n e  were p r e s e n t o n l y i n very s m a l l q u a n t i t y .  purine  nucleotides  I n t h i s case,  quite  l a r g e d i f f e r e n c e s I n the i n c o r p o r a t i o n o f r a d i o - a c t i v i t y i n t o the deoxy p u r i n e i n t e r m e d i a t e s might escape d e t e c t i o n a f t e r h y d r o l y s i s o f the t o t a l n u c l e o t i d e s t o the b a s e s .  - 64 The question of the t i s s u e s p e c i f i c i t y of the a l k a l o i d has been the object of much speculation.  I t was noted i n the  Introduction that c e r t a i n t i s s u e s are s e n s i t i v e to v i n b l a s t i n e while others e.g. l i v e r and gut are almost completely r e s i s t a n t (18). However, as f a r as i s known, the synthesis of DNA i n these t i s s u e s appears to f o l l o w the same pathway (52).  It i s  d i f f i c u l t to accept that a simple r e a c t i o n of a system common to a l l t i s s u e s should be the s i t e of a c t i o n of v i n b l a s t i n e unless the r e l a t i v e a c t i v i t y of the enzymes varied g r e a t l y from t i s s u e to t i s s u e (53).  The very rapid depression i n the bone  marrow (18) and the rapid regression of c e r t a i n lymphomas (16) i n the presence of v i n b l a s t i n e l e d to suggestions that  cell  l y s i s , as w e l l as m i t o t i c a r r e s t , might be a f a c t o r i n the a c t i o n of the a l k a l o i d .  But again c e l l l y s i s cannot i t s e l f be  the complete explanation as the depression of C^-formate i n t o the DNA and RNA of r a t bone marrow has been demonstrated i n v i v o . The r e s u l t s i n d i c a t e that none of the primary processes leading to the synthesis of the mononucleotides i s i n h i b i t e d by v i n b l a s t i n e .  This suggests that the s i t e of the biochemical  l e s i o n i n the DNA synthesis mononucleotides.  i s at a point subsequent to the  Addendum Apart from the e f f e c t s -of v i n b l a s t i n e on the biochemical r e a c t i o n s proceeding i n r a t thymus c e l l suspensions, d i f f e r e n c e s have been found i n the a c t i v i t y of the c o n t r o l incubations of washed and unwashed' c e l l s .  Thus, the s p e c i f i c a c t i v i t i e s  of RNA, xanthine and hypoxanthine  i n the washed c e l l prepara-  t i o n s are considerably higher than t h e i r counterparts i n unwashed preparations (Tables 7 and 8 ) . This d i f f e r e n c e could be explained by the presence of a d i l u e n t , m e t a b o l i c a l l y i n a c t i v e , pool of these compounds i n unwashed c e l l preparations. I t i s , t h e r e f o r e , i n t e r e s t i n g that these compounds are recovered i n much smaller quantity from the equivalent weight of washed c e l l s than of unwashed c e l l s (Figure 9 ) . These compounds may e x i s t as an i n e r t e x t r a - c e l l u l a r pool i n the l a t t e r suspension or be s e l e c t i v e l y leached out on washing.  I t should perhaps  be noted that there i s no c y t o l o g i c a l evidence of c e l l damage during the washing procedure (54). Despite the increased s p e c i f i c a c t i v i t i e s of xanthine and hypoxanthine  i n the washed c e l l preparation, the a c t i v i t i e s of  adenine and guanine are not greater i n t h i s preparation (Table 7 ) . Usually i n unwashed c e l l preparations the a c t i v i t i e s of hypoxanthine and adenine are very s i m i l a r (Tables 4 and 7)*, however i n washed c e l l s , the s p e c i f i c a c t i v i t y of adenine i s markedly lower than that of hypoxanthine.  This may be i n t e r p r e t e d as a  decrease i n the conversion of i n o s i n i c acid to adenylic acid i n the washed c e l l preparation.  T e n t a t i v e l y I t may be  suggested that a substrate required f o r t h i s conversion e.g. A s p a r t i c a c i d , i s l o s t on washing.  The same hypothesis may be  applied to the conversion of x a n t h y l i c acid to guanylic.  However  since glutamine and a s p a r t i c acid are required i n other iaspects of purine and pyrmmidine b i o s y n t h e s i s , i t i s not l i k e l y that t h i s i s a complete explanation.  I t might be noted that g l u t a -  mine and a s p a r t i c acid are sometimes added,to f o r t i f y  incuba-  t i o n mixtures and to stimulate the i n c o r p o r a t i o n of small molecular weight precursors i n t o the n u c l e i c acids  (35).  -  67  -  SUMMARY 1.  The  a l k a l o i d , v i n b l a s t i n e , had been shown to cause  a J?0$ d e p r e s s i o n i n the i n c o r p o r a t i o n o f C ^ ^ f o r m a t e i n t o the DNA  o f washed and unwashed thymus  preparations.  T h i s d e p r e s s i o n was  l e a s t 3 hours (19).  The  cell  maintained f o r at  e f f e c t o f v i n b l a s t i n e on  the b i o s y n t h e s i s o f the a c i d - s o l u b l e p r e c u r s o r s the n u c l e i c a c i d s has  of  t h e r e f o r e been s t u d i e d i n both  types o f p r e p a r a t i o n . 2.  The  i n v i t r o s y n t h e s i s o f the p r e c u r s o r s was  measured by  the i n c o r p o r a t i o n o f G ^ - f o r m a t e i n t o the p u r i n e p y r l m i d i n e bases i s o l a t e d from the h y d r o l y s e d s o l u b l e f r a c t i o n o f these p r e p a r a t i o n s .  and  acid-  Methods are  d e s c r i b e d f o r the s e p a r a t i o n o f the c o n s t i t u e n t s o f the h y d r o l y s e d  a c i d - s o l u b l e f r a c t i o n and  major u l t r a - v i o l e t absorbing  3.  some o f  the  and r a d i o - a c t i v e components  are  identified.  VLB  i s found to have no marked e f f e c t on the  incorpora-  t i o n o f C ^ - f o r m a t e i n t o the p u r i n e bases o r i n t o the thymine o f e i t h e r c e l l p r e p a r a t i o n , except t h a t the  total  count o f adenine i s c o n s i d e r a b l y depressed at the end 3 hour i n c u b a t i o n s o f washed c e l l s . concluded  of  I t i s , therefore,  t h a t the t e t r a h y d r o f o l i c a c i d a c t i v a t i n g  systems and  the t r a n s f e r o f 1-carbon u n i t s are unimpaired  by v i n b l a s t i n e . A l s o s i n c e the de novo s y n t h e s i s o f bases does not appear to be impaired,  the  i t is tentatively  suggested t h a t v i n b l a s t i n e i n h i b i t s a step i n  DNA  s y n t h e s i s a f t e r the formation o f the mononucleotides.  - 68 A comparison o f the compounds i s o l a t e d from the h y d r o l y s e d a c i d - s o l u b l e f r a c t i o n s o f washed and unwashed c e l l s  shows  t h a t c e r t a i n components, i n p a r t i c u l a r hypoxanthine and x a n t h i n e - c o n t a i n i n g compounds, are l o s t on washing. The  t o t a l r a d i o - a c t i v i t y o f s e r i n e i s i n c r e a s e d i n the  presence o f VLB, p a r t i c u l a r l y i n washed c e l l s , ,  The e f f e c t  o f VLB on the t o t a l r a d i o - a c t i v i t y o f s e r i n e o n l y becomes apparent i n the t h i r d hour o f incubation,, and i s t h e r e f o r e not c o n s i d e r e d to be d i r e c t l y r e l a t e d to the i n h i b i t i o n o f DNA s y n t h e s i s .  The d i f f e r e n c e s i n the b e h a v i o r o f washed  and unwashed c e l l s i n d i c a t e t h a t the method o f p r e p a r a t i o n o f c e l l s can s u b s t a n t i a l l y change the s e n s i t i v i t y .  I t Is  suggested t h a t the enhanced e f f e c t o f VLB on adenine and s e r i n e i n the f i n a l hour o f i n c u b a t i o n s o f washed  cell  p r e p a r a t i o n s may be r e l a t e d to the l o s s o f an e s s e n t i a l s u b s t r a t e on washing.  BIBLIOGRAPHY  Noble, R.L.., Canadian Cancer Conference |L, Academic P r e s s , I n c . New York,  333 (1961)  Johnson, I.S., Wright, H.F., and Svoboda, G.H.., J , Lab. C l i n , Med., J^., 8 3 0 ( 1 9 5 9 )  33 >87 (1955)  Beer, G.T.,  rd  Ann. Rept. B r i t . Empire Cancer Campaign  Noble, R.L.., Beer, C.T., and C u t t s , J.H., Ann. N.Y. Acad.  S c i . 2£, 882 (1958)  83k (1958)  Svoboda, G.H.., J • Pharra. S c i .  Johnson, I.S.., Svoboda, G.H., and Wright, H.P.,  P r o c . Am. A s s o c . Ca. Res.  Svoboda, G.H., L l o y d i a Neuss, NY, Gorman, "M...  331 (1962)  2k. 173 (1961) Boaz, H.E., and Cone, N.J..,  J . Am. Chem. Soc. .81L, 1509 (1962)  C u t t s , J.HY, Beer, C.T., and Noble, R.L. 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