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Possible effects of antimetabolites on virus replication McMillan, Jeanette Margot 1965

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POSSIBLE EFFECTS OF ANTIMETABOLITES  ON  VIRUS BEPLICATION  by JEANETTE MARGOT McMILLAN B.Sc,  The U n i v e r s i t y of B r i t i s h Columbia,  A THESIS SUBMITTED IN PARTIAL FULFILMENT THE REQUIREMENTS FOR THE DEGREE OF  i960  OF  MASTER OF SCIENCE i n the Department of B a c t e r i o l o g y and Immunology  We accept t h i s t h e s i s as conforming to the r e q u i r e d standard.  THE UNIVERSITY OF BRITISH COLUMBIA September, 1965  In p r e s e n t i n g the  fulfilment  of  requirements f o r an advanced degree at the U n i v e r s i t y  of  British  Columbia,  available  for  this  thesis  I agree that  in p a r t i a l  the L i b r a r y s h a l l  r e f e r e n c e and s t u d y .  I f u r t h e r agree that  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 purposes may be granted his  representatives*  cation of t h i s  thesis  without my w r i t t e n  Department o f  thesis  for  It  per-  scholarly  i s understood that copying o r p u b l i -  for financial  gain shall  not be allowed  permission.  B a c t e r i o l o g y and Immunology  September,  freely  by the Head o f my Department o r by  The U n i v e r s i t y o f B r i t i s h Vancouver 8, Canada  Date  make i t  Columbia  1963  i i .  Abstract  Because ing of  the  effect  both  was  RNA  of  and  undertaken  Newcastle  purine  observe and  The  ties  i n other  attempted made day  at  old  been  the  than  chick  These  the  to  an  RNA  the  to  of  the  the  project  and  a  on  DNA  virus  pyrimidine and  anal-  thioguanine,  chosen  because  inhibiting  virus  cells,  determine  for  growth  of  replication  antimetabolites  were  have  concern-  this  compounds were  protection  embryo  on  5-iodo-deoxyuridine,  Inhibition  outset  findings  viruses,  e f f e c t s of  chosen  shown  systems.  rather  animal  vaccinia viruses:  compounds  already  and  antimetabolites  the  antimetabolite.  have  opinions  DNA-containing  disease  they  12  varied  5 - f l t r o r o u r a c i l and  ogues  was  the  cellular  to  respectively.  a  of  proper-  replication although  tolerance  antimetabolites  was  some  of  the  used  in  effort 10  -  this  study.  The mined with  by the  tolerance  i n o c u l a t i n g the analogues  concentration to  of  survive  for  of  48  and  the  the  -  12  day  chick  sac  of  noting  the  s u r v i v a l time.  at  37°  which c  w  a  s  the  old  yolk  analogue  hours  10  developing  allowed the  was  dose  3  out  embryo  The of  deter-  maximal 4  employed  embryos in  the  tests.  Inoculation the  allantoic  of  the  embryos  with  the  v i r u s was  c a v i t y or  the  c h o r i o - a l l a n t o i c membrane.  v i a  Administration the if-  yolk  sac  hours  either  were the  f l u i d  assayed  and to  made v i a  virus  inoculation  48  hours  at  or  homogenized  -  37°C, t h e a l - ^  c h o r i o - a l l a n t o i c membranes  whether,there  virus-specific  was  material  any  decrease  i n  i n the presence  of  antimetabolites.  with  cultures  vaccinia virus  served  itory the  effects  on  RNA  metabolite l i c a t i o n  of  of  of  either  as  compared  appear  to  differ  The  vary  and  i n different  RNA  containing  DNA  or  was  found  to  no  have  to  i n the no  Disease  DNA  i n degree  ob-  inhibit chick  significant  w i t h - t r n t r eat ed  inhib-  mechanism  organisms. viruses and  viruses  and  manner  embryo. effect  of  themselves.  p a r t i a l l y  the  The on  anti-  the  rep-  virus.  observable differences  harvested from  i n their  enzymatic  vary  they  vaccinia virus  were  to  may  the  infected  antimetabolite  or  Newcastle  virus  with  r e p l i c a t i o n of  appeared  There  fibroblast cells  inhibit  5-Fluorouracil replication  treated  analogues  seems  may  among  chick  different viruses.  viruses,  inhibition  of  changes.  acid  inhibitions  Antimetabolites of  were  for noticeable  Nucleic  not  was  after  f o r 36  determine  Tissue  of  antimetabolite  immediately  incubation  p r o d u c t i o n of  the  the  later.  After lantoic  of  i n the  thioguanine treated  embryos.  t i t e r s  embryos  The p y r i m i d i n e analogue,  5-iodo-2'-deoxyuridine  ap-  peared to exert some i n h i b i t o r y e f f e c t on v a c c i n i a v i r u s i n tissue  culture.  SIGNED: Dr.J.E.Bismanis  V. Table  Part  of  Contents  I 1  Introdxtct i o n Methods  14  and M a t e r i a l s  I. II. III. IV. V. VI.  Antimetabolites  .  .  .  14  .  15  Eggs Red  Blood  Preparation Tests  with  Methods Virus  17  Cells of  Virus  l8  Suspensions  Antimetabolites  and V i r u s  of Assaying the Production of Specific Material  . . . .  20  . . . .  21  24  Results  I.  Tolerance of Chick Antimetabolites  II.  T e s t s on Newcastle with 5 - l o r o F  III.  IV.  V.  u  u  Tests on V a c c i n i a 5-Fluorouracil  r  Embryo  a  c  Disease i l  Virus  Discussion  Virus  24  Virus  with  Tests on Newcastle Disease with Thioguanine Tests on V a c c i n i a Thioguanine  24  f o r  27  29  Virus  with  . . . . . . . .  . . . . .  31  33  II  46  Introduction  48  Materials  49  Part  and Methods  vi.  Results  51  Pi scussion  •  54  Summary  . . . . .  56  Appendix;  .  58  .  .;•  1.  Chemical  2.  Formation of  3.  Known S i t e s  4.  Site  Bibliography  of  Structures  50  RNA a n d DNA of  A c t i o n of  A c t i o n of  60 Purine Analogues  P y r i m i d i n e Analogues  . . .  .  6l 62 63  Acknowledgement  The the  Department  Dr. agement  wishes  Dolman  f o rh i sh e l p f u l  and  t h e f o l l o w i n g members o f Immunology:  f o rh i s guidance,  i n the supervision C E .  t o thank  of Bacteriology  J.E. Bismanis  Dr. and  author  of this  interest and  study.  f o rpermission t o undertake suggestions  encour-  this  i n the early phases  project  of the  research. Dr. to  extend  J . J .Stock sincere  made p o s s i b l e  thanks  f o r their  the completion  Thanks ogy  a n d D r . D.C.B.  a r e due also  of the University  Duff;  t h e author  interest  of this  and help  which  work.  t o t h e Department  of California  wishes  of B a c t e r i o l -  f o r the use of  laboratory  f a c i l i t i e s .  Finally, scholarships ia  Sugar  t h e author  o f t h e P.E.O.  Refining  Company.  acknowledges  Sisterhood  most  g r a t e f u l l y the  and the B r i t i s h  Columb-  Part I  S t u d i e s on Newcastle  Disease V i r u s  V a c c i n i a V i r u s i n the Chick Embryo  and  Introduction The d i s e a s e s was ous  study of chemotherapy i n the  developed because of the p o s s i b l e  viral  r o l e of numer-  compounds i n i n t e r f e r i n g w i t h s p e c i a l metabolic pathways.  It i s e s s e n t i a l  that  compounds d i r e c t e d  v i r u s - i n d u c e d metabolic events do or do apy  treatment of  so to a l e s s e r degree.  not  s p e c i f i c a l l y against  damage normal host  T h i s i s the b a s i s  cells,  of chemother-  as proposed by E h r l i c h ; to cure i n f e c t i o u s d i s e a s e s  by  chemical agents without i n j u r y to the organism a f f e c t e d , the  a c t i o n of a chemotherapeutic agent must be  i.e.,  selective.  V i r a l d i s e a s e s i n animals pose a major problem f o r chemotherapeutic s t u d i e s .  Such i n v e s t i g a t i o n s  mount importance s i n c e v i r u s e s and  are  i n t r a c e l l u l a r parasites  t h e i r r e p l i c a t i o n i s c l o s e l y dependent on  of the host c e l l s . tween v i r u s and  the  metabolism  Because of t h i s i n t i m a t e r e l a t i o n s h i p  host, the use  tools  be-  of a n t i m e t a b o l i t e s as chemother-  a p e u t i c agents a g a i n s t v i r u s e s logues are h e l p f u l  are of para:-  i s limited.  in providing  i c aspects of host c e l l metabolism and  N e v e r t h e l e s s , ana-  insight  i n t o the  specif-  viral  synthesis.  (Todd,  1959)  Although a n t i m e t a b o l i t e s had study of v i r a l i t was viral  not  i n h i b i t i o n as  until  agent was  1962  that  e a r l y as  been employed i n 1947  the  (Thompson, 1947),  a therapeutically  found (Kaufman et.. a l . , 1962  useful  anti-  a, b; Kaufman,  - 2 -  1963).  T h i s d i s c o v e r y opened up new  the chemotherapy of v i r a l  infections.  analogues have been s t u d i e d properties  of v i r u s e s  nucleic acid  (SNA)  avenues i n the P u r i n e and  intensively,  resides  pyrimidine  s i n c e the  in their nucleic  or d e o x y r i b o n u c l e i c a c i d  study of  genetic  acids,  ribo-  (DNA), which con-  t a i n the p u r i n e and p y r i m i d i n e bases i n the form of n u c l e o tides. There i s an abundance of i n f o r m a t i v e l i t e r a t u r e on the  e f f e c t s of v a r i o u s compounds on v i r u s r e p l i c a t i o n both  i n v i t r o and ed the  in vivo.  As  e a r l y as 1947> Thompson  e f f e c t of sodium a c e t a t e and  growth of v a c c i n i a v i r u s , a DNA observed that  deduced that  the  (Thompson, 1947).  virus  of c h i c k  He  embryonic t i s s u e ,  the a c t i o n p r o b a b l y r e s u l t e d from the  pounds combining w i t h v i t a l systems and  sodium malonate on  these compounds p r e v e n t e d m u l t i p l i c a t i o n of vac-  c i n i a virus in tissue cultures he  investigat-  thiol  and  com  r  groups i n the t i s s u e enzyme  r e n d e r i n g them u n a v a i l a b l e  for v i r a l p r o l i f e r a t -  ion. This  same worker and h i s a s s o c i a t e s  f e c t s of a n t a g o n i s t s such as 5-oromouracil>  studied  the  5- i* <>uracil n  r  efand  2 , 6 - d i a m i n o p u r i n e on the m u l t i p l i c a t i o n of v a c c i n i a v i r u s i n tissue culture. but  5-Bromouracil and  5 - i t r o u r a c i l gave small n  s i g n i f i c a n t i n h i b i t i o n s , w h i l e 2,6-diaminopurine  found to e x h i b i t  was  s t r o n g i n h i b i t o r y e f f e c t s which were reversed  - 3 -  by  p u r i n e s and  1949,  nucleic  the  i n f e c t i v e a g e n t s on coworkers  A and The  comparative v i r u c i d a l a c t i v i t i e s two  unrelated  viruses  et a l . , 1955)*  (Groupe  vaccinia viruses  under the  v i r u s on  the  one  amined.  T h e s e two  t i n o m y c i n D at (ug./ml.) had  h a n d and  a DNA  no  e f f e c t on  a weak e f f e c t on  v i r u s oh  of  40  the  pioneer  was  of cell  and  tumour by p r e v e n t i n g sequently that investigated pyrimidines et  of  the  synthesis  chick  information  for  of  use  these  other,  the  antibiotic  of  was  RNA exac-  milliliter  a t 37°C when t h e  nucleic  acid  on  had viruses  e f f e c t s of  antimetabolites.  ef-  found  to  ascites  a c i d and  con-  Karnofsky  5-fluoro-substituted  pregnant  probably  the  metabolism  (FUDR) was  thymidylic  embryo and  and  an  25°C, and  rat  These experiments p r o v i d e d  preliminary the  the  ( D a n n e b e r g e t a l . , 1958).  toxicologic  a l . , 1958> i 9 6 0 ) .  viruses,  growth i n mice w i t h E h r l i c h  DNA  i n the  conditions.  micrograms per  5-Fluorouridine  virus  Groupe  influenza  investigations  f l u o r i n a t e d p y r i m i d i n e s on  inhibit  anti-  embryo.  f e c t s of  Danneberg.  of  two  i n f l u e n z a v i r u s at  chick  Among some o f  that  that  vaccinia virus  were c u l t i v a t e d i n t h e  done by  same e x p e r i m e n t a l  workers reported  a concentration  was  They s t u d i e d  b i o l o g i c a l d i s s i m i l a r i t y between the  only  (Thompson et a l . ,  1950). Work on  and  acid derivatives  s e r v e d as  an  (Karnofsky some o f impetus  the  - 4 -  Major c o n t r i b u t i o n s  i n the a r e a of s e l e c t i v e v i r u s  i n h i b i t i o n were made by Tamm ( i 9 6 0 ) .  He maintained that  t e c t i o n of c e l l s was a more important  index of chemotherapy  than was mere r e d u c t i o n difficult  of v i r u s y i e l d .  I t was a l s o the more  c r i t e r i o n to achieve i n s u c c e s s f u l  virus inhibition.  Up t o t h i s time no agents were known which were u s e f u l therapy of v i r a l By  pro-  i n the  diseases.  1961 (Kaplan and Ben-Porat, 1961)  investigations  began on the mechanism o f a c t i o n of a n t i m e t a b o l i t e s .  These  i n v e s t i g a t o r s proposed that 5 - f l u o r o u r a c i l (5-FU) i n h i b i t s the  synthesis  of DNA i n r a b b i t kidney c e l l s i n at l e a s t two  ways: (1) by p r e v e n t i n g the f o r m a t i o n of t h y m i d y l i c e s s e n t i a l p r e c u r s o r i n DNA s y n t h e s i s ;  a c i d , an  (2) 5-FU-containing RNA  c o n t r o l s t h e f o r m a t i o n of f a l s e DNA which i s then unable to replicate  itself. Another group of i n v e s t i g a t o r s  obtained s i g n i f i c a n t information  (Reich  et a l . , 1961)  on the e f f e c t of t h e a n t i -  b i o t i c a c t i n o m y c i n D on t h e s y n t h e s i s  of c e l l u l a r n u c l e i c >  a c i d and on v i r u s p r o d u c t i o n .  Actinomycin D i n h i b i t s mammal-  i a n c e l l u l a r RNA b i o s y n t h e s i s ,  and suppresses t h e r e p l i c a t i o n  of v a c c i n i a v i r u s , a DNA-containing v i r u s . DNA s y n t h e s i s  However, c e l l u l a r  i s not a f f e c t e d , nor i s t h e r e p l i c a t i o n of Men-  go v i r u s , an RNA v i r u s .  From these o b s e r v a t i o n s i t can be  i n f e r r e d that  the r e p l i c a t i o n of v i r a l RNA i s d i s t i n c t  from  RNA s y n t h e s i s  which i s c o n t r o l l e d by c e l l u l a r or v i r a l  DNA.  - 5 -  F u r t h e r s t u d i e s by these workers showed that ability  of some RNA  v i r u s e s to i n f e c t c e l l s and  the  replicate  w i t h i n them i s not i n h i b i t e d w i t h a c t i n o m y c i n D treatment. They demonstrated RNA  that more than 90 per cent of DNA-primed  s y n t h e s i s can be i n h i b i t e d by actinomycin D i n L  without  significant  cells,  i n t e r f e r e n c e w i t h the p r o d u c t i o n of i n -  fectious,RNA  virus  (Reich et a l . ,  by complexing  w i t h DNA  1962).  Actinomycin a c t s  and i n h i b i t i n g RNA-polymerase syn-  t h e t i c r e a c t i o n s (Goldberg, R e i c h et a l . , 1 9 6 2 ) . In  s t u d i e s of adenovirus m u l t i p l i c a t i o n and  proliferation,  i t was proposed  5-halogenated p y r i m i d i n e s was  that the b i o l o g i c a l  cell  e f f e c t of  most p r o b a b l y due to t h e i r i n -  t e r f e r e n c e w i t h the n a t u r a l bases of n u c l e i c a c i d s ( K j e l l e n , 1962).  The major breakthrough  came i n 1962 when the pyrim-  i d i n e a n t i m e t a b o l i t e , 5 - i o d o - 2 ' - d e o x y u r i d i n e (IUDR), found to be t h e r a p e u t i c a l l y a c t i v e as a n a n t i - v i r a l It was  agent.  shown to improve or cure r a b b i t c o r n e a l i n f e c t i o n  caused by herpes  simplex and v a c c i n i a v i r u s e s , both DNA-con-  t a i n i n g v i r u s e s (Kaufman, 1 9 6 3 ) , T h i s e f f e c t was  (Kaufman et a l . , 1962 a,  o b t a i n e d even a f t e r the i n f e c t i o n was  e s t a b l i s h e d and even when the d i s e a s e was 1963;  was  Kaufman and Maloney,  1963).  well  severe (Kaufman,  b.)  - 6 -  C l i n i c a l t r i a l s o f IUDR were u n d e r t a k e n by C a l a b r e s i and h i s a s s o c i a t e s but  ( C a l a b r e s i e t a l . , 1962; C a l a b r e s i ,  1963).  t h e r e was d i f f i c u l t y i n i t s s y n t h e s i s and i t was v e r y  r a p i d l y degraded in v i v o .  T h i s compound was not o r i g i n a l l y  used i n t h e s t u d y o f v i r a l d i s e a s e , researchers  but was chosen by t h e s e  as t h e most l i k e l y agent t o be u s e f u l i n t h e t h e r -  apy o f d i s e a s e  caused by DNA v i r u s e s because of i t s p a r t i c u l a r  metabolic s i t e of action.  I t i s b e l i e v e d t o i n h i b i t t h e phos-  p h o r y l a t i o n of t h y m i d i n e and t h e p o l y m e r i z a t i o n a c i d i n t o DNA.  of t h y m i d y l i c  The a c t i o n on t h e polymerase system i s no  doubt somewhat s p e c i f i c , s i n c e i t i s i n h i b i t o r y i n some t y p e s of c e l l s , but not i n o t h e r s (Delamore and P r u s o f f , The  1962).  a b i l i t y o f IUDR t o i n h i b i t DNA s y n t h e s i s and  t h u s t h e growth o f v a c c i n i a v i r u s i n v i v o was demonstrated by Calabresi  ( C a l a b r e s i et a l . , 1 9 6 2 ) .  the p y r i m i d i n e  base t h y m i d i n e .  IUDR i s an analogue o f  The degree o f s u p p r e s s i o n  of t h e r e s p o n s e of t h e v i r u s i n t h i s experiment was g r e a t e r w i t h i n c r e a s i n g doses o f IUDR, and furthermore, t h e r e was no evidence of drug t o x i c i t y .  These w o r k e r s i n o c u l a t e d  rabbits  i n t r a d e r m a l l y w i t h v a r y i n g d i l u t i o n s of v a c c i n i a v i r u s c u l t i v a t e d i n c h i c k embryo t i s s u e c u l t u r e . S t u d i e s have a l s o been c a r r i e d out on t h e i n v i t r o e f f e c t s of t h i s p y r i m i d i n e  analogue i n t i s s u e c u l t u r e (Kauf-  man and Maloney, 1 9 6 3 ; Loddo et a l . , 1 9 6 3 ) .  The l a t t e r group  - 7 -  established  that  IUDB and FUDR i n h i b i t herpes simplex v i r u s  growth both i n v i v o  and i n v i t r o .  Vaccinia virus replication  i n HeLa c e l l s i s i n h i b i t e d by IUDR, but the analogue has no i n h i b i t o r y a c t i v i t y against viruses,  these l a t t e r two The  p o l i o Type 1 and Coxsackie B-j ' being RNA-containing  formation of v a c c i n i a v i r u s p r o t e i n  i n the  ence of FUDR was observed by Shatkin, who noted that of v i r a l p r o t e i n  synthesis  i n Hela  FUDR was maximal f o r the f i r s t 4-6  cells  viruses. pres-  the  rate  i n the presence of  hours a f t e r i n f e c t i o n and  c o n t i n u e d a t a d i m i n i s h e d r a t e d u r i n g t h e l a t t e r 8-10 hours of t h e i n f e c t i o u s c y c l e In  1959*  (Shatkin,  1963).  Fairman r e p o r t e d that he observed no i n h i b -  i t i o n of v i r u s p r o d u c t i o n by 5-FU i n c e l l s i n f e c t e d with Rous Sarcoma v i r u s On  (RSV), an RNA-containing v i r u s  the c o n t r a r y ,  a t i o n of c h i c k  Gold6 and V i g i e r  (1963)  (Fairman,  report  that  1959)'  incub-  embryo f i b r o b l a s t s i n f e c t e d w i t h RSV i n t h e  presence of 5-FU immediately a f t e r i n f e c t i o n , i n h i b i t e d both cell  growth and v i r u s p r o d u c t i o n .  required  The c o n c e n t r a t i o n s of 5-FU  t o supress t h e growth o f i n f e c t e d c e l l s were lower  than f o r n o n - i n f e c t e d c o n t r o l s .  V i r u s p r o d u c t i o n was i n h i b -  i t e d o n l y when 5-FU was added t o the medium b e f o r e a c e r t a i n time.  There was no i n h i b i t i o n i f 5-FU was added, l a t e r than  3 days a f t e r i n f e c t i o n .  These authors p o s t u l a t e d  that  inhib-  i t i o n of v i r u s p r o d u c t i o n by 5-FU f o l l o w e d the replccement of  -  of  the pyrimidine  viral  RNA  rather  In which  when  5-FU-free  of  Kaufman  healthy  on  the treatment  growth  medium  was  virus  free  with  1959).  1963).  an  concentrations production  do  culture. days no  s i n c e RSV  production RSV  Results  can exist of  o f RSV  was  D  Sarcoma V i r u s ; Concentrations  while  with  RSV,  i s a moderate  host  yg/ml)  cell i n a  DNA.  virus,  provirus  (1963) Actinomy-  with  DNA.  reversibly  concentrations  so t h a t  they  of  death  by Temin  of actinomycin  cells  RNA,  interaction  i n c e l l s  higher  appeared  of cells  by complexing  (0.1  observed  viruses.  not cause  with  ex-  "cured."  contains  experiments  effect  not destroy  do  herpes  In their  were  involve  t h e work  of  relapse  RSV  infection  genome  of actinomycin  irreversibly. production  1963).  that  inhibitory  with  IUDR  are DNA-containing  interaction  of Rous  IUDR,  v i r u s e s , might  and v i r u s  a direct exerts  RNA  the host  and Rubin (Temin  of  4  was r e -  cultivated  c u l t u r e s and t h e c e l l s  simplex  suggested  other  infection  D  i n tissue  and Maloney,  and herpes  the  RSV  viruses  contrast with  production  5-FU,  with  subsequently  and Maloney  not with  suggest  were  i s i n sharp  but  it  cells  of virus  of contact  This  It  state  inhibition  medium.  (Kaufman  vaccinia  (Temin  RNA.  period  the previously diseased  i.e.  the antimetabolite i n non-  c u l t u r e s t r e a t e d f o r about  When g i v e n  cin  i n viral  limited  -  by  experiment  and v a c c i n i a  periment  in  a  uracil  the infected  in  simplex  than  Golde's  followed  versed  base  8  Low  inhibit inhib-  inhibitory  to  cannot p r o -  - 9 -  duce v i r u s .  A c o n c e n t r a t i o n of actinomycin  er than that r e q u i r e d t o g i v e 95 percent p r o d u c t i o n does not prevent  100 times  great-  i n h i b i t i o n o f RSV  s y n t h e s i s of NDV-an RNA v i r u s , so  that the e f f e c t of actinomycin on RSV p r o d u c t i o n i s p r o b a b l y mediated through DNA. Another group o f workers (Barry et a l . , 1962) i n v e s t i g a t e d the e f f e c t s of actinomycin  D on the r e p l i c a t i o n  of two myxoviruses; i n f l u e n z a v i r u s , and Newcastle d i s e a s e virus.  Although  both v i r u s e s c o n t a i n RNA, i n f l u e n z a v i r u s  r e p l i c a t i o n i s i n h i b i t e d by the a n t i m e t a b o l i t e w h i l e that of NDV i s not a f f e c t e d .  Since actinomycin  D b l o c k s the f u n c t i o n  of c e l l u l a r DNA i t appears that i n f l u e n z a v i r u s w i l l  repli-  c a t e o n l y i n c e l l s whose DNA f u n c t i o n i s unimpaired. Using 5-FU on both DNA and RNA c o n t a i n i n g v i r u s e s i n t i s s u e culture.;systems,  a group o f Japanese workers showed  that t h i s a n t i m e t a b o l i t e had no e f f e c t on the r e p l i c a t i o n of RNA-containing v i r u s e s such as p o l i o v i r u s types 1, It suppressed  the growth of v a c c i n i a v i r u s , w h i l e  2,  and 3.  adenovirus,  which a l s o c o n t a i n s DNA, showed no s e n s i t i v i t y t o t h i s compound. fToyoshima et a l . , zman  (1962),  1962).  high concentrations  v i r u s had the same s p e c i f i c  According  t o Munyon and S a l -  (10"^M) o f 5-FU s u b s t i t u t e d  i n f e c t i v i t y as the u n s u b s t i t u t e d  virus. Much l e s s i n f o r m a t i o n has been made a v a i l a b l e on t h e  - 10 -  e f f e c t s of p u r i n e a n t i m e t a b o l i t e s on v i r u s r e p l i c a t i o n ,  since  o n l y a few p u r i n e analogues have been found e f f e c t i v e a g a i n s t animal v i r u s e s .  Thioguanine (2-amino-6-mercaptopurine  or  6-  TG) has been found to be capable of p r o d u c i n g 50 p e r c e n t or more tumour growth i n h i b i t i o n of the RC carcinoma and Chester, 1958)*  (Tarnawoski  Other carcinomas a r e l e s s s e n s i t i v e than  the RC type to 6-mercaptopurine  and t h i o g u a n i n e .  S a r t o r e l l i and LePage (1958) observed that thioguanine i n h i b i t e d the a z a z e r i n e - i n d u c e d accumulation of formyl g l y c i n a m i d e r i b o n u c l e o t i d e (FGAR) (See Appendix). i n v e s t i g a t i o n s on 6-TG  their  c o r p o r a t i o n of 6-TG produced  i n normal  i n t o DNA  support the concept that the i n i s c l o s e l y l i n k e d to the t o x i c i t y  or n e o p l a s t i c c e l l s .  be i n c o r p o r a t e d i n t o RNA.  iest  and RNA  The analogue may  found i n the n u c l e o s i d e s  of s u s c e p t i b l e t i s s u e s .  i n the n u c l e a r RNA  also  In a s e r i e s of normal and n e o p l a s t -  i c t i s s u e s of the rodent, 6-TG was of both DNA  R e s u l t s of  and,  It appears  earl-  i n g e n e r a l , i s i n c o r p o r a t e d most  r a p i d l y where the turnover of RNA  i s fastest  (LePage and Jones,  1961). Working w i t h A s c i t e s c e l l mouse tumours,the group of i n v e s t i g a t o r s  (LePage and Jones, 1961)  latter  established  that s u s c e p t i b l e tumours showed maximum response to treatment i n i t i a t e d at any time d u r i n g the rapid-growth phase;  that i n  s u s c e p t i b l e tumours, a l a r g e p a r t of the i n c o r p o r a t i o n was i n  - 11  DNA,  and t h a t  three  treatments  p r o d u c e an e s s e n t i a l l y inhibitory properties into It  nucleic  appears  acids,  that  remain v i a b l e cate their  DNA.  thioguanine the  for  the  is  t i m e but  The t u m o u r -  incorporation  thioguanine  are unable  to  s u g g e s t e d as t h e mechanism by  susceptible a similar  tumours.  effect  It;is  (1949, 1950)  Thompson et  al.  aminopurine  (DAP) d e c r e a s e d  report growth  the of  repliwhich  likely  on v i r u s - i n f e c t e d  on p u r i n e a n t i m e t a b o l i t e s  to  DNA m a t e r i a l .  w h i c h make D N A - c o n t a i n i n g  This  In s t u d i e s  from i t s  specifically  a considerable  exerts  sufficient  inhibition.  6-TG r e s u l t e d  probably  inhibits  analogue  w i t h 6 - T G were  maximum tumour of  cells  -  that cells.  and r e p l i c a t i o n ,  finding  that  2,6-di-  vaccinia virus,  in  tis-  6 sue c u l t u r e , oratory  (Gifford  inopurine that  2,  cells  i n a c o n c e n t r a t i o n of et  al.,  ( 2 - A P ) was  respiration.  at  inactive  testicular  tissue  were  virus  2-amand  i n f e c t i o n of  i n h i b i t o r y to  lab-  HeLa cell  2, 6-DAP i n h i b i t e d m u l t -  type 2  culture with p a r t i a l  (MEFj)  reversal  i n monkey of  inhibition  guanine.  The o b s e r v e d  6-MP  that  Brown (1952) f o u n d t h a t  of p o l i o m y e l i t i s  purine  a s an i n h i b i t o r y a g e n t ,  on p o l i o v i r u s  concentrations  iplication  by a d e n i n e o r  From a n o t h e r  1954), t h e r e a r e r e p o r t s t h a t  6-DAP h a d no e f f e c t  except  5X10~"M.  synthesis  effects  of  6-mercaptopurine  de novo c a n be a t t r i b u t e d  ribonucleotide  of phosphoribosylamine  to  (6-MP)  or  i n h i b i t i o n by  synthesis.  6-MP  is  - 12  known to be metabolized ism,  and  by pathways of hypoxanthine metabol-  to i n t e r f e r e with the s y n t h e s i s and (Brockman, 1963).  of p u r i n e n u c l e o t i d e s that n a t u r a l p u r i n e s t h e s i s de novo was Gollub  -  (1959) who  The  interconversion observations  a f f e c t e d an e a r l y step i n p u r i n e  syn-  extended to p u r i n e analogues by Gots  and  6-thioguan-  observed that mercaptopurine and  ine were p a r t i c u l a r l y e f f e c t i v e as i n h i b i t o r s of the accumul a t i o n of 5-ami io-4-imidazole carboxamide r i b o n u c l e o s i d e  by  I  Escherichia c o l i  B-96.  Some i n t e r e s t i n g r e s u l t s were obtained Le C l e r c (1962) who  by Cogniaux-  s t u d i e d the e f f e c t of 8-azaguanine on  s y n t h e s i s of v a c c i n i a v i r u s .  The  antimetabolite  the  inhibited  the m u l t i p l i c a t i o n of v a c c i n i a , a DNA-containing v i r u s , a l though the presence of the analogue c o u l d be demonstrated o n l y i n the RNA  of the i n f e c t e d c e l l .  on v a c c i n i a v i r u s s y n t h e s i s was  The  a c t i o n of 8-azaguanine  s t u d i e d on c h i c k embryo mono-  l a y e r s , under agar o v e r l a y or i n l i q u i d medium. of t h i s study support the view that RNA ant p a r t  The r e s u l t s  must p l a y an  i n the development of v i r u s e s whose g e n e t i c  i s c o n s t i t u t e d by DNA.(Tamm, i 9 6 0 ; Tamm et a l . , 1963)  importmaterial Cog-  niaux-Le C l e r c o f f e r s 3 hypotheses on the mechanism of i n h i b i t i o n of v a c c i n i a v i r u s s y n t h e s i s by the analogue.  First,  that a v e r y low amount of 8-azaguanine i n c o r p o r a t e s  i n t o the  DNA-genetic m a t e r i a l of the v i r u s , g i v i n g r i s e to ious u n i t s .  Although i t i s d o u b t f u l  that the  non-infect-  i n h i b i t i o n of  -  13  -  v a c c i n i a v i r u s s y n t h e s i s i s r e l e v a n t to t h i s mechanism, i t cannot e n t i r e l y be  excluded.  Another p o s s i b i l i t y i s that  a n t i m e t a b o l i t e might have some i n d i r e c t of the v i r u s DNA.  e f f e c t on  I n t e r f e r e n c e with normal RNA  d u r i n g the l a g phase p r e v e n t s the onset of DNA most c e l l s  ( H a r r i s , 1959)«  The  the  synthesis  synthesis synthesis i n  t h i r d hypothesis  i s that  the  analogue i n h i b i t s p r i m a r i l y the s y n t h e s i s of the v i r u s p r o t eins.  - 14 -  Methods and M a t e r i a l s Antimetabolites 5-Fluorouracil were obtained  (NSC 19893) and thioguanine  (NSC 752)  through Dr. J.E. Bismanis as a g i f t from the  Cancer Chemotherapy N a t i o n a l S e r v i c e Center,  N a t i o n a l Cancer  I n s t i t u t e , Bethesda 14, Maryland. The  r e q u i r e d amount of the a n t i m e t a b o l i t e s were  weighed out e x a c t l y on a C h r i s t i a n Becker balance and made up to volume i n a v o l u m e t r i c  flask.  The c e l l u l a r  antimetabolites  were made up at a c o n c e n t r a t i o n of 10,000 micrograms per m i l l i liter  (ug./ml. ) i n p h y s i o l o g i c a l s a l i n e b u f f e r e d at pH 7*2.  A few drops of 5 thioguanine. for  ° H were needed to e f f e c t  Heating  s o l u t i o n of the  i n a water bath at 40°C was  necessary  the complete s o l u t i o n of the compounds. The  filter,  s o l u t i o n s were then f i l t e r e d  through a M i l l i p o r e  of 0 . 4 5 micron (u) pore s i z e , f i t t e d to a Swinny ad-  apter and a 30 was  N K  c c  « syringe.  A drop of the f i l t e r e d s o l u t i o n  t e s t e d f o r s t e r i l i t y on a blood agar p l a t e which was i n -  cubated at 37°C f o r 48 hours. f r o z e n at - l 8 ° C u n t i l  The stock s o l u t i o n s were kept  r e q u i r e d f o r use.  r i a t e d i l u t i o n s were made i n s t e r i l e sired  From these,  approp-  s a l i n e to o b t a i n the de-  concentrations. Each a n t i m e t a b o l i t e was i n j e c t e d by deep p e n e t r a t i o n  i n t o the y o l k sac by means of a s y r i n g e f i t t e d with a 26 gauge  -  l t j f  i n c h needle.  15 -  The e n t i r e needle was i n s e r t e d i n t o the egg  through a h o l e d r i l l e d  i n the s h e l l over the a i r sac r e g i o n ,  and the inoculum was then e x p e l l e d (Waddington et a l . , 1 9 5 8 ) . S o l u t i o n s of a n t i m e t a b o l i t e s were i n j e c t e d e i t h e r after  immediately  i n t r o d u c t i o n of the v i r u s or 1-g- hours l a t e r , as i n d i c a t -  ed. Eggs Source,  Incubation, P r e p a r a t i o n  Eggs were o b t a i n e d at the p o u l t r y farm at the Univers i t y of B r i t i s h Columbia, Vancouver, Canada. ated i n an egg incubator at 37°C  a  n  They were incub-  d turned d a i l y .  On the  t e n t h or e l e v e n t h day of i n c u b a t i o n the eggs were candled i n a dark room, and i n f e r t i l e eggs and dead embryos were d i s c a r ded.  The top of the embryo f r e e of blood v e s s e l s and the area  of the a i r sac were marked.  The working area of each egg was  swabbed with t i n c t u r e of i o d i n e f o l l o w e d by 70% a l c o h o l before drilling,  and a l s o p r i o r to and a f t e r i n j e c t i o n of the v i r u s  or a n t i m e t a b o l i t e . Allantoic Cavity Inoculation With the use of an e l e c t r i c r o t o r d r i l l  a small h o l e  was d r i l l e d i n t h e s h e l l above the embryo, c a r e being  taken  not to p i e r c e the s h e l l membrane.;, A s i m i l a r opening was made i n the middle of the a i r sac r e g i o n i n order to prevent  back  - 16 -  flow of the inoculum which was  c o n t a i n e d i n a t u b e r c u l i n sy-  r i n g e f i t t e d w i t h a 26 gauge, 3/4 was to  the a l l a n t o i c c a v i t y , and the v i r u s inoculum was After  paraffin,  needle leading injected  i n o c u l a t i o n both openings were s e a l e d w i t h meland the eggs r e i n c u b a t e d at 3 7 °  C h o r i o - a l l a n t o i c Membrane I n o c u l a t i o n A small h o l e was d r i l l e d sac  The  i n t r o d u c e d at an o b l i q u e angle i n t o the opening  there. ted  i n c h needle.  c  f o r 33 - 36 hours.  (CAM-inoculation)  i n the center of the a i r  and a t r i a n g u l a r a r e a marked o f f on the top of the  bryo.  After d r i l l i n g ,  em-  the t r i a n g u l a r p i e c e of s h e l l was  lift-  ed o f f w i t h a p a i r of f o r c e p s , and w i t h a s t e r i l e needle a small s l i t  was made i n the s h e l l membrane without p i e r c i n g  the c h o r i o - a l l a n t o i c membrane (CAM)  l y i n g d i r e c t l y beneath i t .  By means of a l a r g e p i p e t t e bulb, g e n t l e s u c t i o n was at  the h o l e i n the a i r sac u n t i l  s h e l l membrane. The  slit  the CAM  away from the  T h i s c o u l d be d e t e c t e d upon c a n d l i n g the egg.  i n the s h e l l membrane was  through i t the v i r u s inoculum was The window was  fell  applied  lengthened s l i g h t l y , d e p o s i t e d onto the  and  CAM.  subsequently covered over w i t h t r a n s p a r e n t tape,  and the eggs were then t i l t e d  s l i g h t l y to d i s t r i b u t e the i n -  oculum over the s u r f a c e of the CAM.  After  i n c u b a t i o n at  37°  C f o r 48 hours, the membranes were c o l l e c t e d and evaluated for  virus concentration.  - 17 -  Red Blood  Cells  Guinea p i g e r y t h r o c y t e s were f i r s t  employed i n hem-  a g g l u t i n a t i o n t i t r a t i o n s of Newcastle Disease V i r u s (NDV). Blood was o b t a i n e d by c a r d i a c p u n c t u r e from guinea p i g s r e a r e d f o r the Department of B a c t e r i o l o g y and Immunology at the U n i v e r s i t y of B r i t i s h Columbia.  A s t e r i l e 20 ml. s y r i n g e  f i t t e d w i t h a 20 gauge needle was used f o r withdrawal of b l o o d from the animal.  Approximately 10 ml. of blood c o u l d be ob-  t a i n e d from a s u c c e s s f u l heart b l e e d on one guinea p i g . The  b l o o d was c o l l e c t e d i n t o an Erlenmeyer f l a s k con-  t a i n i n g as a n t i c o a g u l a n t  a s o l u t i o n of 3.8%  sodium c i t r a t e .  A f t e r c e n t r i f u g a t i o n a t 2000 r.p.m. at 4°C f o r 15-20 minutes, the plasma was removed and the e r y t h r o c y t e s washed t h r e e times with  s t e r i l e saline.  suspension  i n saline.  The packed c e l l s were made up to a 10$ For hemagglutination  c e l l s were used as a 0.5% suspension  (HA) t e s t s the  i n saline.  E v e n t u a l l y , c h i c k e n e r y t h r o c y t e s were used i n a l l the HA t e s t s .  Blood was c o l l e c t e d at the p o u l t r y farm at the U n i -  v e r s i t y of B r i t i s h Columbia, and washed i n the same manner as f o r guinea p i g e r y t h r o c y t e s . more pronounced when these  With NDV, hemagglutination  e r y t h r o c y t e s were used than when  guinea p i g r e d c e l l s were employed i n HA t e s t s . hemagglutinin  was  Also, the  of v a c c i n i a v i r u s o f t e n a g g l u t i n a t e s the ery-  t h r o c y t e s of o n l y c e r t a i n s p e c i e s of fowls.  T h i s appears  - 18 -  to be a g e n e t i c c h a r a c t e r i s t i c , u n r e l a t e d to age, sex or breed ( C l a r k and Nagler, 1943)*  The hemagglutinin i s not an i n t e g -  r a l p a r t of the v i r u s and may be separated from the v i r u s particle. P r e p a r a t i o n of V i r u s Suspensions Newcastle  Disease V i r u s (NDV)  A suspension of NDV, 1962),  grown on HeLa c e l l s  (9 J u l y ,  and w i t h a t i t e r of l 6 0 h e m a g g l u t i n a t i n g u n i t s (HA un-  i t s ) , was o b t a i n e d from Dr. J.E. Bismanis approximately two months a f t e r storage at - l 8 ° C .  The v i r u s suspension was  thawed j u s t b e f o r e use and kept on i c e .  V i r u s d i l u t i o n s were  made i n s t e r i l e s a l i n e b u f f e r e d at pH 7» 2. 0.1 ml. of v i r u s suspension was i n o c u l a t e d into the a l l a n t o i c c a v i t y of 10-11  day o l d c h i c k embryos.  The openings  were s e a l e d and i n c u b a t i o n c a r r i e d out at 37°C f o r 33-36 hours. H a r v e s t i n g of A l l a n t o i c  Fluid  At the end of the i n c u b a t i o n p e r i o d the embryos were candled and dead embryos were d i s c a r d e d .  S u r v i v i n g embryos  were c h i l l e d at 4°C f o r about f o u r hours b e f o r e h a r v e s t i n g the a l l a n t o i c f l u i d .  The eggs were p l a c e d on end, the s h e l l  over the a i r sac was removed w i t h f o r c e p s , and the s h e l l membrane beneath t o r n away with s t e r i l e  instruments.  Allantoic  f l u i d was then withdrawn by means of a s t e r i l e Pasteur p i p e t t e  - 19 -  and c o l l e c t e d i n t o s t e r i l e t e s t tubes. s i g n of t u r b i d i t y was d i s c a r d e d .  F l u i d showing  any  A drop of the p o o l e d f l u i d  c o n t a i n i n g the v i r u s was p l a c e d on a b l o o d agar p l a t e which was  i n c u b a t e d at 37°C f o r 48 hours to t e s t the s t e r i l i t y of  the f l u i d .  The p o o l e d f l u i d was c e n t r i f u g e d at 2000 r.p.m.  at 4°C f o r 15 minutes f o r c l a r i f i c a t i o n .  An h e m a g g l u t i n a t i o n  t e s t was c a r r i e d out on the suspension which was subsequently d i s p e n s e d i n t o the screw-capped tubes i n 1 ml. q u a n t i t i e s f o r storage at -l8°C. Vaccinia Virus V i r u s was o b t a i n e d from g l a s s c a p i l l a r y tubes cont a i n i n g smallpox v a c c i n e as manufactured by Connaught Labora t o r i e s at Toronto, Canada.  Each c a p i l l a r y c o n t a i n e d approx-  i m a t e l y 0 . 0 5 ml. of the v a c c i n e . were d i l u t e d 1:10  The contents of c a p i l l a r i e s  i n s t e r i l e b u f f e r e d s a l i n e , and 0.1  ml.  i n o c u l a t e d onto the c h o r i o - a l l a n t o i c membrane of 10-11 o l d c h i c k embryos.  A f t e r three passages on the CAM,  was  day the  ti-  t e r of the v i r u s used i n the t e s t s was ^12 HA u n i t s per ml. H a r v e s t i n g of C h o r i o - a l l a n t o i c Membranes After  (CAM)  i n c u b a t i o n f o r 48 hours at 37°C, the eggs were  r e f r i g e r a t e d f o r approximately 4 hours b e f o r e h a r v e s t i n g . The s h e l l was removed to the l e v e l of the f a l l e n CAM,  and the  membranes removed a s e p t i c a l l y w i t h f o r c e p s and s c i s s o r s s t e r i l e p e t r i dishes containing saline.  into  The membranes were  - 20 -  e i t h e r homogenized immediately or s t o r e d overnight to f a c i l i t a t e the g r i n d i n g . i l e buffered s a l i n e (0.5 a P o t t e r homogenizer.  at  -l8°C  Membranes were suspended i n s t e r -  ml. per membrane) and ground up  The homogenate was  at 2000 r.p.m. f o r 15-20  minutes at 4°C,  f l u i d c o n t a i n i n g the v i r u s was  then c e n t r i f u g e d and the  dispensed  in  i n 1.0  supernatant ml.  quantit-  i e s i n t o screw-capped tubes which were s t o r e d at - l 8 ° C . was  This  subsequently used as v a c c i n i a v i r u s suspension.  T e s t s with A n t i m e t a b o l i t e s  and  Virus  In each experiment, s i x embryos were used f o r t e s t and f o r a p p r o p r i a t e  the  c o n t r o l s as f o l l o w s :  Test 0.1  ml.  of v i r u s d i l u t i o n was  l a n t o i c c a v i t y or the CAM  i n o c u l a t e d v i a the a l -  of s i x 10-12. day  by the a n t i m e t a b o l i t e v i a the y o l k sac. o u r a c i l employed was  0.5  ml.,  embryos,  The  followed  dose of 5 - f l u o r -  w h i l e that of thioguanine  was  0. 25 ml. Controls 0.1  ml.  of v i r u s d i l u t i o n was  l a n t o i c c a v i t y or the CAM 0.1  ml.  suspension was  of s i x 10-12  i n o c u l a t e d v i a the a l day  of normal a l l a n t o i c f l u i d or ground up  CAM  i n o c u l a t e d i n t o embryos, the former v i a the  a l l a n t o i c c a v i t y , and the l a t t e r v i a the 0.5  embryos.  ml.  of 5-FU  each embryo or O.25  ml.  was  CAM.  i n j e c t e d i n t o the y o l k sac of  of thioguanine  by the same route.  - 21  0.1  -  ml. of s t e r i l e b u f f e r e d s a l i n e was i n o c u l a t e d  v i a the a l l a n t o i c c a v i t y or the  CAM.  U n t r e a t e d eggs incubated f o r the same p e r i o d of  time  as those under t e s t . Methods of A s s a y i n g the P r o d u c t i o n of V i r u s - s p e c i f i c MateriaT Determination of the Hemagglutinating V i r w s - c o n t a i n i n g Fluid" Hemagglutination  T i t e r of the  t e s t s were c a r r i e d out i n d u p l i c a t e  and set up i n a g g l u t i n a t i o n tubes w i t h round bottoms. Newcastle Disease V i r u s Hemagglutinating  a c t i v i t y of NDV  was  assayed by the  p a t t e r n t e s t d e s c r i b e d by S a l k ( 1 9 4 4 ) , u s i n g equal volumes t w o - f o l d d i l u t i o n s of v i r u s and 0 . 5  of s e r i a l blood c e l l ing 0 . 5  ml.  suspension.  A c o n t r o l tube was  s a l i n e and 0.5  percent r e d  included contain-  ml. red blood c e l l s .  The racks of  tubes were shaken and allowed to stand at 4°C f o r 45-60 minutes. Vaccinia Virus Smaller q u a n t i t i e s of v a c c i n i a v i r u s were used,  O.25  ml. of v i r u s suspension and an equal volume of c h i c k e n e r y t h r o c y t e s ( C l a r k and Nagler,  1943)'  v a c c i n i a v i r u s hemagglutination  The optimum temperature  for  i s 3 7 ° C ( R h o d e s and VanRooyen)  The r a c k s of tubes were shaken v i g o u r s l y to o b t a i n  - 22 -  even d i s p e r s a l of the reagents and then allowed to stand at 37°  c  until  the c e l l s i n the c o n t r o l had s e t t l e d  (approximate-  l y 45-60 minutes). The  endpoint of the t i t r a t i o n was  read hy  determin-  ing the h i g h e s t d i l u t i o n of v i r u s to g i v e a g g l u t i n a t i o n of r e d c e l l s as i n d i c a t e d by the presence of a f i l m of r e d c e l l s on the bottom of the t e s t tube.  In the absence of a g g l u t i n -  a t i o n , the e r y t h r o c y t e s form a s o l i d r i n g at the bottom of the t e s t tube.  The r e c i p r o c a l of the v i r u s d i l u t i o n i n the  l a s t tube to show complete h e m a g g l u t i n a t i o n was hemagglutinating t i t e r of the v i r u s  taken as the  suspension.  Determination of the I n f e c t i v i t y T i t e r of the V i r u s Containing F l u i d T h i s method was  employed i n determining whether vac-  c i n i a v i r u s , h a r v e s t e d from embryos t r e a t e d w i t h  antimetabo-  l i t e s r e t a i n e d any of i t s i n f e c t i v i t y f o r the c h i c k embryo. E s t i m a t i o n of v i r u s a c t i v i t y was CAM  c a r r i e d out on the  of the c h i c k embryo by Burnet's method (Burnet,  1942).  The v i r u s suspension was prepared i n s e r i a l t e n - f o l d  dilut-  i o n s i n s t e r i l e b u f f e r e d s a l i n e , and 0.1  dilut-  i o n was  d e p o s i t e d onto the CAM  incubated f o r 10-11  ml. of each  of hens' eggs which had been  days at 37°C.  A f t e r a f u r t h e r 4 8 - 7 2 hours  i n c u b a t i o n the membranes were e x c i s e d and examined f o r the presence of s p e c i f i c f o c i or f o r t h i c k e n i n g of the membrane  - 23 -  i n d i c a t i v e of i n f e c t i o n .  S i n c e d i s c r e t e f o c i were not always  obtained, no attempt c o u l d be made to c a l c u l a t e the f i f t y per cent  i n f e c t i o u s dose The  (IDCJQ)  of the v i r u s suspension.  i n f e c t i v i t y t i t e r of the v a c c i n i a v i r u s f o r the  c h o r i o - a l l a n t o i c membrane of the c h i c k the highest  d i l u t i o n of v i r u s suspension to produce s p e c i f i c  f o c i or t h i c k e n i n g Controls  embryo was taken as  of the membrane i n d i c a t i v e of i n f e c t i o n .  were set up to account f o r any n o n - s p e c i f i c  reaction.  - 24 -  Results Tolerance  of the Chick Embryo f o r A n t i m e t a b o l i t e s  5-Fluorouracil The  c h i c k embryo of 10-11  days c o u l d s u r v i v e a dose  of 5,000 ug. of 5-FU f o r 48 hours at 37°C.  The analogue was  i n j e c t e d i n t o the y o l k sac of the egg, four embryos being used f o r each d i l u t i o n of a n t i m e t a b o l i t e i n determining ance dose.  Uninoculated  s a l i n e were a l s o set up.  the t o l e r -  c o n t r o l s and c o n t r o l s r e c e i v i n g o n l y The dose of a n t i m e t a b o l i t e i n the  t e s t s was the maximum c o n c e n t r a t i o n t o l e r a t e d by at l e a s t 3 out of 4 embryos f o r 48 hours at 37°C. mal  T h i s g i v e s the maxi-  e f f e c t i v e c o n c e n t r a t i o n of the a n t i m e t a b o l i t e . Thioguanine Four embryos were used f o r each d i l u t i o n of antimet-  a b o l i t e i n determining  the t o l e r a n c e dose.  c o u l d s u r v i v e 2,000 ug of thioguanine  The c h i c k embryo  f o r 48 hours at 37°C.  S i m i l a r c o n t r o l s were set up as f o r 5-FU. T e s t s on NDV w i t h 5 - F l u o r o u r a c i l Analogue added immediately a f t e r c h i c k embryo w i t h  virus.  Analogue added !•§• hours a f t e r embryo with the v i r u s . The  i n f e c t i o n of the  (Shatkin,  i n f e c t i o n of the c h i c k  1963)  t i t e r of NDV used was 640 HA u n i t s per ml.  Six  - 25 -  embryos were employed i n each t e s t and each c o n t r o l  group.  A 10" d i l u t i o n of t h e stock v i r u s was used i n the t e s t s . 2  R e s u l t s of Hemagglutination T e s t s when 5-FP was added at Zero Time Chicken RBC DILUTION OF ALLANTOIC 5-FU v s . NDV FLUID  1; 10 1:20 1:40 1:80 1:160 1:320 1:640 1:1280 1:2560  2* 2+ 3+ 3+ 2*  Control  — _ -—  +  2+ 2+ 3+ 3+ 2+  — —  NDV CONTROL 5-FU  + +  2+ 3t 3+ 2t  ± _  + +  3+ 3+ 3t 2*  NORMAL CONTROL ALLANTOIC FLUID CONTROL  _ _  _  — — —  --  *  — _ — —  — —  _  -  HA T i t e r s : C o n t r o l NDV=640 HA u n i t s / m l . of o r i g i n a l suspension Test NDVr640 HA u n i t s / m l .  virus  There i s no s i g n i f i c a n t r e d u c t i o n i n t h e HA t i t e r between the t e s t and t h e c o n t r o l Key to Symbols  *,2-».,3 + = degree of h e m a g g l u t i n a t i n g ± - p a r t i a l hemagglutination = no h e m a g g l u t i n a t i o n  activity.  The f o l l o w i n g r e s u l t s were o b t a i n e d when guinea p i g r e d b l o o d c e l l s were employed i n the h e m a g g l u t i n a t i o n t e s t s . The h e m a g g l u t i n a t i o n was l e s s pronounced than w i t h t h e c h i c k en e r y t h r o c y t e s which were employed i n subsequent  HA t e s t s .  - 26 -  Guinea P i g RBC  DILUTION OF ALLANTOIC FLUID  1:10 1:20 1:40 1:80 l:l6o 1:320 1:640 1:1280 1:2560 Control  HA t i t e r s :  5-FU v s . NDV  + 4  2+ 2+ 3+  4 4  2+ 2+ 2+  NDV CONTROL  4 +  2+ 3+ 2*  +  +  •  —  —  —  — -  -  — -  5-FU CONTROL  + 4  -  2+ 2+ 3*  4  -—  .. —  —  -  — -  C o n t r o l NDV • 640 HA u n i t s / m l . Test NDV = 640 HA u n i t s / m l .  There i s no s i g n i f i c a n t  r e d u c t i o n i n t h e HA t i t e r between t h e  t e s t and t h e c o n t r o l , R e s u l t s of T e s t s when 5-FU was added lj Hours after the Virus  DILUTION OF ALLANTOIC FLUID  1:10 1:20 1:40 1:80 1:160 1:640 1:1280 1:2560 Control  5-FU v s . NDV  ±  NDV CONTROL  4 4  2 2+  4  4  24 24  4  2+  34  3+  34  34  34  34  34  34  +  3+  —  _  —-  mm mm mm  *  -  —  HA T i t e r s : C o n t r o l NDV = 640 HA u n i t s / m l . Test NDV = 640 HA u n i t s / m l .  5-FU CONTROL  --  -  — _  -  - 27 -  Chicken e r y t h r o c y t e s  were used i n the  estimation  of  the h e m a g g l u t i n a t i n g a c t i v i t y of the v i r u s s i n c e they were found to g i v e more pronounced hemagglutination. There i s no  s i g n i f i c a n t change i n the hemagglutinat-  i n g p t i t e r of the v i r u s when the that of the analogue by The at a t i t e r  NDV  i n t r o d u c t i o n of v i r u s precedes  1§ hours.  c o n t r o l v i r u s gave p a r t i a l  of 1280  HA  u n i t s per ml.  hemagglutination  However, t h i s was  not  c o n s i d e r e d s i g n i f i c a n t s i n c e the h e m a g g l u t i n a t i n g t i t e r  of  the v i r u s suspension was  last  determined on the b a s i s of the  tube to show complete h e m a g g l u t i n a t i o n . T e s t s on V a c c i n i a V i r u s w i t h Antimetabolite with v a c c i n i a v i r u s . t e s t s was  512  each t e s t and v i r u s was  HA  was  The  u n i t s per  5-Fluorouracil  added 1^ hours a f t e r i n f e c t i o n  t i t e r of v a c c i n i a v i r u s used i n the ml.  c o n t r o l group.  used i n the t e s t s .  S i x embryos were employed i n A 10-1  d i l u t i o n of the  stock  - 28 -  Hemagglutination with Chicken E r y t h r o c y t e s  DILUTION OF CAM VIRUS  1.-2 1 4 1. 8 1. 16 1. 32 1' 64 1 128 1 256 1 512  Control  5-FU v s . VACCINIA  4"  VACCINIA CONTROL  44  +  2+ 2*  4  3+ 3+ 3-* 3+  2+ 2+  4-  4-  — _ —  —  -  2+  4  -  + 4  34 3+ 3-f  2+  -  4  -  HA T i t e r s : V a c c i n i a C o n t r o l - 5 1 2 HA u n i t s / m l . V a c c i n i a Test =128 HA u n i t s / m l .  5-FU CONTROL  -  UNINFECTED CONTROL  -  There i s a f o u r - f o l d r e d u c t i o n i n the hemagglutinati n g a c t i v i t y of V a c c i n i a v i r u s h a r v e s t e d from embryos t r e a t e d w i t h 5-FU.  - 29 -  T e s t s on Newcastle Disease V i r u s w i t h Thioguanine The t i t e r  of NDV employed was 640 HA u n i t s per ml.  S i x embryos were used i n each t e s t and c o n t r o l group as before.  A 10~  2  d i l u t i o n of v i r u s suspension was used i n the  tests. R e s u l t s of Hemagglutination T e s t s when Thioguanine was added at Zero Time.  DILUTION OF ALLANTOIC FLUID  THIOGUANINE vs. NDV  1:10 1:20 1:40 1:80 1:160 1:320 1:640 1:1280 1:2560  +  +  3* 3+ 3+ 3+  2+ 3+ 3+ 3+  2+ 3+ 3* 3+  +  +  +  2*  -—  Control  +  NDV CONTROL  THIOGUANINE CONTROL  2+  -  — NORMAL ALLANTOIC CONTROL  -  2+  + 3+ 3t 3* 2+  — —  _ —  +  - 30 -  C o n t r o l NDV = 2560 HA u n i t s / m l . Test NDV = 2560 HA u n i t s / m l .  HA T i t e r s :  There i s no r e d u c t i o n i n t h e hemagglutinating i t y between v i r u s o b t a i n e d from t r e a t e d and from  untreated  embryos. R e s u l t s of Hemagglutination T e s t s when 6-TG was Administered 1^ Hours a f t e r the V i r u s  DILUTION of ALLANTOIC FLUID  1:10 1:20 1:40 l:80 l:l6o  1:3:2o  1:640 1:1280 1:2560  6-TG v s . NDV  +  NDV CONTROL  —  -  4  3+ 3* 3+ 3+  2+  +  2+  3+ 3+ 3+  3+ 34 3+  •*  +  3* 3+ 3+ 3+  2+  -—  Control  6-TG CONTROL  2+  +  -—  -— NORMAL ALLANTOIC FLUID CONTROL  -  2+  -—  activ-  - 31 -  HA T i t e r s : C o n t r o l NDV = 2560 HA Test NDV = 2560 HA  units/ml. units/ml.  V i r u s - c o n t a i n i n g a l l a n t o i c f l u i d from 6-TG t r e a t e d embryos showed s i m i l a r h e m a g g l u t i n a t i n g a c t i v i t y w i t h v i r u s from u n t r e a t e d  embryos.  Test on V a c c i n i a V i r u s w i t h Thioguanine U n t r e a t e d embryos were i n f e c t e d w i t h v a r y i n g ions of the v i r u s suspension  harvested  membranes of 6-TG t r e a t e d embryos. was i n o c u l a t e d onto each CAM, -  10~* ).  0.1  dilut-  from c h o r i o - a l l a n t o i c ml. of the suspension  u s i n g 4 eggs per d i l u t i o n  (10 *  I n f e c t i v i t y was determined by the number of pocks  2  on the membrane, or by g e n e r a l i z e d t h i c k e n i n g of the CAM. Untreated Embryos  VIRUS DILUTION  AVERAGE NO. of ROCKS  10-1  Much t h i c k e n i n g of CAM  10-  -  2  68  Pf u/ml.  —— 6.8  x io4  io-3  3.0  3.0  x 104  10-4  0.5  5.0  x lo4  10-5  —  Average Number of P f u - 4 . 9 x 104  Pfu/ml.  - 32 -  T&-Treated Embryos  VIRUS DILUTION  io-  1  Pf u/ml.  AVERAGE NO. o f POCKS Thickening  of CAM  10-2  64.0  6 . 4 x 10 "4  10-3  4.0  4 . 0 x 10 4  10-4  Generalized  thickening  10-5  Generalized  thickening  Average Number of P f u = 5 . 2 x 10"4  Pfu/ml.  There was no r e c o g n i z a b l e r e d u c t i o n i n the i n f e c t i o n of membranes from both groups. eralized  At higher  t h i c k e n i n g of the CAMs were evident.  d i l u t i o n s gen-  - 33  -  Discussion The  a n t i m e t a b o l i t e 5 - f l u o r o u r a c i l , (5-FU), i s a  s t r u c t u r a l analogue of thymine which i s an e s s e n t i a l component of DNA.  Since the c a r b o n - f l u o r i d e bond i s strong,  the 5  s u b s t i t u t i o n of a f l u o r i n e atom f o r the hydrogen i n the p o s i t i o n of u r a c i l makes 5-FU  an i n t e r e s t i n g a n t i m e t a b o l i t e .  A t h e o r e t i c a l reason advanced f o r the s y n t h e s i s of 5-FU that the van der Waal r a d i u s ,  (1.35^)» °f the f l u o r i n e atom  i s v e r y c l o s e to that of hydrogen,  (1.2A*), and might cause  the compound to b l o c k the enzymatic r e a c t i o n to DNA w i t h greater  e f f i c i e n c y than other  i t i o n . (Welch, 196l) (Sadler, 1963), i t y with and  synthesis  s u b s t i t u e n t s on the 5-  However, i t was  i n c r e a s i n g van der Waal's r a d i i of the  that the f l u o r i n e atom, which has  activ-  substituents  the s m a l l e s t r a d i u s  p a r t from hydrogen, decreases a n t i - v i r a l a c t i v i t y the  5-FU  pos-  r e a l i z e d more r e c e n t l y  that there i s a g r a d a t i o n of a n t i v i r a l  I o d o u r a c i l and bromouracil  was  a-  least.  undergo dehyd.rogenation although  does not. 5-FU  i s metabolized  to 5 - f l u o r o u r i d i n e (FUR)  and  to f l u o r o u r i d i n e monophosphate (FUMP), or f l u o r o u r i d y l i c (See Appendix, F i g . 2.).  FUMP may  the normal m e t a b o l i t e  i n t o RNA,  i n t o RNA chenal  UMP  to form a f r a u d u l e n t  and Oettgen, i 9 6 0 ;  then acid  b l o c k the i n c o r p o r a t i o n of or i t may  be  incorporated  f l u o r i n e - c o n t a i n i n g RNA.(Bur-  Salzman et a l . , 1962).  The  analogue  - 34 -  5-FU  may  a l s o r e p l a c e thymine i n DNA.  If i n c o r p o r a t e d ,  a  s t r u c t u r a l analogue of a m e t a b o l i t e might cause l e t h a l thesis,  syn-  i . e . the p r o d u c t i o n of i n a c t i v e v i r a l progeny. (Tamm  and Eggers, 1 9 6 3 ) .  (1963) a l s o shares the o p i n i o n  Sadler  that the i n c o r p o r a t i o n of unnatural  bases or n u c l e o t i d e s i n -  to v i r u s n u c l e i c a c i d should n e c e s s a r i l y impair  replication.  For the RNA-containing v i r u s e s , i t would seem important inhibit  s p e c i f i c a l l y the s y n t h e s i s of RNA  the metabolism of  without  to  affecting  DNA.  On the c o n t r a r y , other workers r e p o r t that 5-FU no  i n h i b i t o r y e f f e c t on RNA  (1962) r e p o r t that 5-FU p o l i o v i r u s RNA. specific was  The  Munyon and  (Simon, 1961),  5-FU  s u b s t i t u t e d v i r u s had  i n h i b i t o r y e f f e c t on NDV  The  analogue  and a c c o r d i n g to Kaufman, (1963)  activity.  i s s a i d to have no  the same r  grown i n HeLa  i s i n c o r p o r a t e d i n t o n u c l e i c a c i d s and has no anti-viral  Salzman  i s e f f e c t i v e l y incorporated into  i n f e c t i v i t y as the u n s u b s t i t u t e d v i r u s .  found to have no  cells  viruses.  has  The  c o n v e r s i o n product  i n h i b i t o r y e f f e c t on RSV  5-FU  therapeutic of 5-FU,  FUDR  (Rich et a l . ,  1962). The  f i n d i n g i n my  hemagglutinating  experiments that no r e d u c t i o n i n  a c t i v i t y was  bryos were t r e a t e d with 5-FU,  o b t a i n e d when NDV-infected may  probably  the b a s i s t h a t , i f the analogue was the r e s u l t i n g ;:  virus  hemagglutinating  activity.  was  be e x p l a i n e d  incorporated into  not a l t e r e d w i t h r e s p e c t  The v i r u s of NDV  emon  RNA, to  agglutinates  - 35 -  e r y t h r o c y t e s of guinea p i g s and fowls. i n d i c a t i v e of the presence  Hemagglutination i s  of v i r u s p a r t i c l e s .  I t i s probab-  l e that i f a f r a u d u l e n t RNA i s formed, i t i s n e v e r t h e l e s s repl i c a t e d and produces i n f e c t i o u s v i r u s capable of hemagglutina t i n g these e r y t h r o c y t e s .  The a n t i m e t a b o l i t e was o r i g i n a l l y  a d m i n i s t e r e d to t h e embryos immediately the v i r u s v i a t h e a l l a n t o i c c a v i t y .  after  i n f e c t i o n with  In s t u d i e s on the r e p l i c -  a t i o n of t h e DNA v i r u s , - v a c c i n i a , S h a t k i n (1963)3 and ( S h a t k i n et  a l . , 1 9 6 3 ) , made t h e o b s e r v a t i o n t h a t v i r a l  commences w i t h i n 1-g- hours a f t e r  infection.  DNA f o r m a t i o n  Although  t h i s ex-  p r e s s e d the s i t u a t i o n i n a DNA v i r u s , w h i c h , l i k e NDV, r e p l i c a t e s i n the cytoplasm be  of the c e l l ,  i t was f e l t  that i t would  i n t e r e s t i n g to observe whether 5-FU, added l | hours a f t e r  i n f e c t i o n , would have any e f f e c t on an RNA v i r u s . t h e r e was no d i f f e r e n c e i n t h e hemagglutinating  However,  a c t i v i t y of  NDV under these c o n d i t i o n s . In DNA s y n t h e s i s , (Appendix, F i g . 2.) t h e normal uence of events from u r a c i l  i s v i a deoxyuridine  u r i d i n e monophosphate (DUMP) and t h y m i d y l i c a c i d 5-FU  seq-  (UDR), deoxy(TMP).  When  i s used i t i s c o n v e r t e d to FUDR and e v e n t u a l l y to f l u o r o -  d e o x y u r i d i n e monophosphate (FDUMP) which b l o c k s the methylation  of DUMP to form TMP. (Cohen et a l . , 1958; Salzman et a l . ,  1963).  The FDUMP i s a p p a r e n t l y not i n c o r p o r a t e d i n t o DNA. Some i n h i b i t i o n was o b t a i n e d with 5-FU and v a c c i n i a  v i r u s as evidenced by the r e s u l t s of the hemagglutination  tests;  - 36 -  t o t a l or complete i n h i b i t i o n was not obtained. f o u r - f o l d reduction  There i s a  i n t h e h e m a g g l u t i n a t i n g a c t i v i t y of vac-  c i n i a v i r u s when t h e 5-FU was administered bryo la hours a f t e r the v i r u s .  to the c h i c k em-  I t i s p o s s i b l e that the s e r -  i e s of events mentioned above takes p l a c e .  The f a c t that com-  p l e t e i n h i b i t i o n was not o b t a i n e d may depend on s e v e r a l o r s , among  which are:  fact-  the s t r a i n of embryo t r e a t e d , the  stage of development of the embryo when the compound i s i n troduced, t h e a b i l i t y of the drug to p e n e t r a t e bryonic  cells,  i n t o the em-  and the presence of p r o t e c t i v e mechanisms.  Other p o s s i b i l i t i e s w i l l be d i s c u s s e d  i n a later  section.  Some of t h e knowledge of the r e p l i c a t i o n of v a c c i n i a v i r u s was d e r i v e d f r o m the use of the metabolic i n h i b i t o r FUDB (Salzman, i 9 6 0 ) which t o t a l l y b l o c k s to TMP and thus t h e f o r m a t i o n  the conversion  of DNA.  d e r i v a t i v e of, and can be h y d r o l y s e d  of UDR  FUDR i s a metabolic to, the f r e e base 5-FU.  F l u o r o u r a c i l analogues then, may have these e f f e c t s : 1)  The i n h i b i t i o n of t h y m i d y l i c  2)  The i n c o r p o r a t i o n of FUMP i n t o RNA to g i v e a f r a u d u l e n t  acid  synthesis.  RNA. 3)  They may b l o c k t h e i n c o r p o r a t i o n of UMP i n t o RNA. The  events i n the s e q u e n t i a l  s y n t h e s i s of v i r a l com-  ponents i n HeLa c e l l s i n f e c t e d w i t h v a c c i n i a v i r u s a r e summarized i n t h e f o l l o w i n g c h a r t by S h a t k i n  et a l . ( I 9 6 3 ) .  (1963) and Salzman  - 37 Formation of V a c c i n i a V i r u s Components (Chart and d e s c r i p t ion f o l l o w i n g a r e from Shatkin, 1963')  ^  INFECTIOUS VIRUS RATE-LIMITING PROTEIN DNA-PROTEIN VIRAL PROTEIN  •i 1  0  VIRAL DNA  2 3 4 5 6 7 8 9 ' . Viral  10  11  12  13  14 Hours  DNA f o r m a t i o n commences w i t h i n 1^ hours a f t e r  i n f e c t i o n and i s completed at 6§- - 7 hours. V i r a l p r o t e i n s d e t e c t a b l e by t h e i n d i r e c t i c a l method, a r e a l s o formed w i t h i n 1 ^ - 2 ion,  immunolog-  hours a f t e r  infect-  but are s y n t h e s i z e d throughout the i n f e c t i o u s c y c l e . " R a t e - l i m i t i n g p r o t e i n " necessary f o r i n f e c t i o u s  v i r u s f o r m a t i o n i s s y n t h e s i z e d l e s s than 1 hour b e f o r e maturat ion. The a s s o c i a t i o n of v i r a l e a r l y as 2 hours a f t e r Whereas v i r a l at  DNA and p r o t e i n begins as  infection. DNA  s y n t h e s i s appears to be complete  7 hours, the amount of DNA a s s o c i a t e d w i t h v i r a l  c o n t i n u e s to i n c r e a s e throughout  protein  infection.  I n f e c t i o u s v i r u s i s formed b e g i n n i n g at 5 - 6 hours and i s complete at 13 - 14 hours a f t e r  infection.  - 38 -  In s t u d i e s on v a c c i n i a v i r u s s y n t h e s i s FUDR at 10""6  M  w  a  s  a  i n HeLa c e l l s ,  d d e d to c u l t u r e s at v a r y i n g times a f t e r  i n f e c t i o n and a l l c u l t u r e s were sampled and t i t r a t e d at 25 hours.  Any v i r u s formed a f t e r the a d d i t i o n of the i n h i b i t o r  must c o n t a i n DNA s y n t h e s i z e d p r i o r to the time the i n h i b i t o r was  added, s i n c e t h e e f f e c t of FUDR i n b l o c k i n g DNA  synthesis  i s b e l i e v e d to be immediate (Salzman et a l . , 1963). According  to J o k l i k ( 1 9 6 2 ) , when FUDR i s added to  i n f e c t e d HeLa c e l l s at any time up to 2| hours a f t e r ion,  no v a c c i n i a v i r u s i s formed i n these c e l l s  a f t e r 24 hours; t h e r e f o r e , no v i r a l  infect-  subsequently  DNA was s y n t h e s i z e d  before  2§ hours a f t e r i n f e c t i o n , i . e . DNA s y n t h e s i s was i n h i b i t e d by the a n t i m e t a b o l i t e inhibit  The a n t i m e t a b o l i t e  FUDR does not  the i n c o r p o r a t i o n of preformed thymidine d e r i v a t i v e s  i n t o DNA, but t h e r e in  FUDR.  i s so l i t t l e of these compounds present  the i n t r a c e l l u l a r pool  of HeLa c e l l s ,  that  t h e s i s of DNA, and t h e r e f o r e of v i r u s , stops the a d d i t i o n o f FUDR to the c e l l s .  i n practice syn?  immediately on  When the analogue i s add-  ed to t h e c e l l s 6 hours a f t e r i n f e c t i o n w i t h v i r u s , the f u l l complement of v i r u s i s produced, showing that i t i s i n t h i s p e r i o d between 2§- and 6 hours a f t e r i n f e c t i a n t h a t s u f f i c i e n t viral  DNA i s formed f o r i n c o r p o r a t i o n i n t o mature v i r u s .  These r e s u l t s g e n e r a l l y agree w i t h that of Salzman et a l . , (1963) and S h a t k i n that v i r a l  (1963),  DNA formation  except that t h e l a t t e r groups f i n d  commences w i t h i n !•§• hours a f t e r i n -  - 39 -  fection. Since FUDR and the f r e e hase 5-FU to which i t can be hydrolyzed,  both b l o c k t h y m i d y l i c a c i d f o r m a t i o n  et a l . , 1961) then t h e ^ f a c t that o n l y p a r t i a l  (Hartmann  i n h i b i t i o n of  v i r u s r e p l i c a t i o n was achieved may mean t h a t : 1)  The e f f e c t of 5-FU was not immediate.  2)  Some v i r a l  3)  A l a r g e r dose of a n t i m e t a b o l i t e i s needed f o r complete  DNA i s p r o b a b l y  synthesized before  l-§- hours.  inhibition. 4)  5-FU i s not a v e r y e f f e c t i v e i n h i b i t o r  since i t only  p a r t i a l l y b l o c k s DNA s y n t h e s i s i n c e l l s of the CAM of t h e c h i c k embryo. 5)  There i s a c o n s i d e r a b l e amount of thymidine d e r i v a t i v e s present  i n the n u c l e o t i d e p o o l of the c h i c k embryo  chorio-allantoic The  cells.  q u e s t i o n whether v i r a l p r o t e i n formation  on t h e s y n t h e s i s of v i r a l  DNA was examined by S h a t k i n  He made use of an immunological method designed r a l p r o t e i n s i n t h e absence of v i r u s maturation. s i s of immunologically  depends (1963).  to d e t e c t v i The synthe-  d e t e c t a b l e v i r a l p r o t e i n s i n HeLa c e l l s  i n f e c t e d with v a c c i n i a v i r u s was observed w i t h i n 2 hours a f ter  infection.  When DNA s y n t h e s i s i n i n f e c t e d c e l l s was  b l o c k e d by 10-6 ^ FUDR, t h e f o r m a t i o n tinued.  of v i r a l p r o t e i n s con-  The r a t e of v i r a l p r o t e i n s y n t h e s i s i n t h e presence  of FUDR was maximal f o r the f i r s t 4 - 6 hours a f t e r  infection  - 40 -  and  c o n t i n u e d at a d i m i n i s h e d r a t e d u r i n g t h e l a t t e r 8 - 1 0  hours of t h e i n f e c t i o u s teins  cycle.  l a t e r i n the i n f e c t i o u s  The f o r m a t i o n of v a c c i n i a proc y c l e as w e l l  as v i r u s maturat-  i o n a r e u n a f f e c t e d when t h e analogue i s added a f t e r the synthesis  of v i r a l  DNA. (Salzman et a l . , 1963)  Adenovirus, l i k e v a c c i n i a v i r u s , virus.  However, adenovirus DNA i s b e l i e v e d  the nucleus of the i n f e c t e d virus  i s a DNA-containing  i n the cytoplasm.  cell,  to r e p l i c a t e i n  and t h e DNA of v a c c i n i a  FUDR was used i n the study of the  r o l e of DNA s y n t h e s i s i n the r e p l i c a t i o n o f type 2 adenovirus i n suspension c u l t u r e s  of KB c e l l s .  When t h e analogue at 10"  M was added to a d e n o v i r u s - i n f e c t e d c u l t u r e s hours a f t e r and  infection,  uninfected c e l l s ;  protein,  i t stopped DNA s y n t h e s i s i n i n f e c t e d i t p e r m i t t e d the s y n t h e s i s of n o n - v i r a l  RNA and a c i d - s o l u b l e  n u c l e o t i d e s i n these, and i t  prevented v i r u s ; r e p l i c a t i o n i n i n f e c t e d f e r r e d from t h i s that us m u l t i p l i c a t i o n , enovirus-specific s i s of v i r a l itor after It  at times up to 7  cells.  DNA s y n t h e s i s i s e s s e n t i a l  (Green, 1962 a ) and that protein  I t can be i n f o r adenovir  f o r m a t i o n o f ad-  i s d i r e c t l y dependent on the synthe  DNA (Flanagan and Ginsberg, 1961).  When  inhib-  (FUDR) was added at d i f f e r e n t times from 8 - 2 1 hours infection,  seems that  hours a f t e r  increasing  amounts of v i r u s were formed.  adenovirus DNA i s made from 1 - 8 to about 21  infection.  Intracellular virus  from 13 - 14 hours to about 28 hours a f t e r  i s synthesized infection.  (Green,  - 41 -  1962 b ) . ial  The time d i f f e r e n c e s i n the events i n the sequent-  s y n t h e s i s of v i r a l  ings.  components a r e r e f l e c t e d i n these f i n d -  The s y n t h e s i s of adenovirus components l a g s behind that  of v a c c i n i a v i r u s .  Such i n f o r m a t i o n  i s v a l u a b l e because i t  g i v e s some i n d i c a t i o n of the most a p p r o p r i a t e  time to add i n -  h i b i t o r , depending on the system under study. In 1962, the a n t i v i r a l agent, 5 - i o d o - 2 ' - d e o x y u r i d i n e (IUDR) was found to cure corneal  infection,  i n rabbits,  caused by herpes simplex viruses.(Kaufman, et a l . , 1962 a, b; Kaufman, 1963).  This antimetabolite  i n h i b i t s the phosphory-  l a t i o n o f thymidine and the p o l y m e r i z a t i o n i n t o DNA.(Delamore and P r u s o f f , corporated  IUDR may a l s o be i n -  i n t o an abnormal and presumably n o n - f u n c t i o n a l  which may be no longer an e a r l i e r  1962).  of t h y m i d y l i c a c i d  infectious.  The analogue FUDR a c t s at  s i t e and i n h i b i t s thymidylate  then the phosphorylase. the same o v e r a l l  DNA  synthetase  rather  These two a n t i m e t a b o l i t e s produce  e f f e c t though h a v i n g d i f f e r e n t  s i t e s of a c t -  ion. It might be expected that p u r i n e analogue would i n t e r f e r e w i t h the metabolism of n u c l e i c a c i d s , and thus might seem l i k e l y to have secondary consequences on p r o t e i n formation.  The c l o s e r e l a t i o n s h i p between n u c l e i c a c i d and p r o t e i n  b i o s y n t h e s i s has suggested that i n c o r p o r a t i o n of analogues into polynucleotides  could result  s y n t h e s i s or the formation  i n i n h i b i t i o n of p r o t e i n  of f r a u d u l e n t p r o t e i n s v i a f r a u d -  - 42 -  (Henderson and Mandel, 1963).  u l e n t RNA  C e r t a i n p u r i n e analogues  a r e known to mimic the nat-  u r a l p u r i n e s i n i n h i b i t i o n of the de novo pathway of p u r i n e s y n t h e s i s i n microorganisms 1963)  and i n mammalian cells.(Brockman,  The r e a c t i o n s of p u r i n e bases w i t h p h o s p h o r i b o s y l p y r o -  phosphate to form r i b o n u c l e o t i d e s a r e c a t a l y z e d by n u c l e o t i d e pyrophosphorylases ences.  among, which there may be s p e c i e s d i f f e r -  A f t e r t h e r i b o n u c l e o s i d e monophosphate i s formed ( i .  e. the r i b o n u c l e o t i d e ) ,  i t may or may not be f u r t h e r phosphor-  y l a t e d to the d i - and t r i p h o s p h a t e s .  In experiments  w i t h 6-  t h i o g u a n i n e (6-TG) the monophosphates predominate, but d i and t r i p h o s p h a t e s a r e a l s o made. (Moore and Le Page, 195§) Formation  of d e o x y r i b o n u c l e o t i d e s i s necessary f o r  the a c t i o n of some a n t i m e t a b o l i t e s , and f o r those which a r e i n c o r p o r a t e d i n t o DNA.  T h i s may occur by r e d u c t i o n of the  r i b o n u c l e o t i d e , by t x a n s d e o x y r i b o s i d a t i o n or by p h o s p h o r y l a t ion  of a d m i n i s t e r e d deoxyr i b o n u c l eo s i des  ( i . e . ITJDR).  The  d e o x y r i b o n u c l e o t i d e of TG i s i n c o r p o r a t e d i n t o DNA (Le Page and Jones,  1961);  even n a t u r a l p u r i n e d e o x y r i b o n u c l e o t i d e s  are p r e s e n t i n c e l l s  i n o n l y v e r y small amounts compared to  those of the p y r i m i d i n e s . In g e n e r a l , the p u r i n e and p y r i m i d i n e a n t i m e t a b o l i t e s undergo the same types of c a t a b o l i c r e a c t i o n s as do the natu r a l compounds.  P u r i n e s a r e o x i d i z e d at t h e 2, 6, and 8  - 43  -  p o s i t i o n s ; deamination-may a l s o occur.  I n c o r p o r a t i o n of c e r -  t a i n p u r i n e and p y r i m i d i n e analogues i n t o n u c l e i c a c i d s been c l e a r l y e s t a b l i s h e d , (Matthews, 1958)  a  n  has  appears to  d  take  p l a c e by routes c h a r a c t e r i s t i c of n u c l e i c a c i d s y n t h e s i s . A n t i m e t a b o l i t e s which are i n c o r p o r a t e d i n t o n u c l e i c a c i d s of mammalian c e l l s u s u a l l y are a l s o i n c o r p o r a t e d i n t o m i c r o b i o l o g i c a l systems and v i r u s e s .  There has been c o n s i d e r a b l e  var-  i a t i o n observed i n the t o t a l amount of bases r e p l a c e d by analogues.  The  extent  of the a n t i m e t a b o l i t e  v a r i e s w i t h the metabolic  incorporated  a c t i v i t y of the system.  In v i r a l  systems where c e l l p r o l i f e r a t i o n i s r a p i d , the analogue s u b s t i t u t e completely dee,  1956).  f o r a normal component (Litman  Antimetabolites  the  and  may Par-  incorporated into nucleic acids  are not n e c e s s a r i l y u n i f o r m l y d i s t r i b u t e d as the normal components. No p u r i n e n u c l e o t i d e s are known to i n h i b i t t i d e . b i o s y n t h e s i s de novo. may  nucleo-  However, i t i s b e l i e v e d that  act to i n h i b i t n u c l e o t i d e metabolism.  6-TG  by some i n v e s t i g a t o r s (Le Page and Jones, 1961)  they  i s believed to be  similar  to or i d e n t i c a l with 6-mercaptopurine, (6-MP) i n i t s metabolic e f f e c t s because of the s i m i l a r i t y of the two though t h e r e i s s t i l l  Al-  l i t t l e agreement as to the mechanism of  i t s i n h i b i t o r y e f f e c t s , at the present h e l d view i s that 6-MP  structures.  time the most  widely  i n the form of i t s r i b o n u c l e o t i d e i n -  h i b i t s the c o n v e r s i o n of i n o s i n a t e to e i t h e r or x a n t h y l a t e or both.(Appendix, F i g . 3 . ) .  adenylosuccinate  Whether or not  - 44 -  6-TG  e x e r t s t h e same a c t i o n i s s t i l l  ably incorporated  i n doubt.  6-TG i s prob-  i n t o DNA or RNA, a b i o c h e m i c a l event which  p r o b a b l y produces t o x i c i t y to the c e l l s , h i b i t i n g the s y n t h e s i s  or i t may act by i n -  or the f u n c t i o n of n u c l e o t i d e  es concerned i n g l y c o l y s i s and r e s p i r a t i o n . ( L a s z l o 1961).  coenzymet a l . ,  Marked d i f f e r e n c e s i n the t o x i c i t i e s of these two com-  pounds have l e d to the d i s c o v e r y between them, c o n t r a r y  of biochemical  differences  to p r e v i o u s b e l i e f .  In the experiment undertaken, 6-TG produced no i n h i b i t i o n i n c e l l s i n f e c t e d e i t h e r w i t h NDV or v a c c i n i a v i r u s . This observation,  although not i n accordance w i t h other r e -  p o r t s on the t u m o u r - i n h i b i t o r y and  p r o p e r t i e s o f 6-TG ( S a r t o r e l l i  Le Page, 1958? Le Page and Jones, 1961) and w i t h the r e -  s u l t a n t i c i p a t e d i n t h i s experiment, i s not s u r p r i s i n g s i n c e it  i s known that a l t e r n a t e pathways of p u r i n e and p y r i m i d i n e  metabolism e x i s t . (Henderson and Mandel, 1963). i n h i b i t i o n o f growth t h e r e f o r e , be  inhibited.  For complete  a l l a l t e r n a t i v e pathways must  P u r i n e n u c l e o t i d e metabolism i s more complex  than that of p y r i m i d i n e  nucleotides,  and although p u r i n e an-  alogues have been shown to i n h i b i t tumour growth, t h e s i t u a t i o n i n a tumour system i s not n e c e s s a r i l y i n p a r a l l e l that of v i r a l the  with  r e p l i c a t i o n . B i o l o g i c a l e f f e c t s r e s u l t i n g from  i n c o r p o 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  analogues i n t o  i c a c i d s have been i n c o n s i s t e n t . Although some c l e a r - c u t ionships  nuclerelat-  e x i s t between i n c o r p o r a t i o n and a l t e r e d biochemical  - 45 -  functions,  t h e r e a r e many examples where a l t e r e d n u c l e i c  seem to behave l i k e normal n u c l e i c a c i d s .  acids  If the s u b s t i t u t i o n  of the analogue takes p l a c e at a s i t e i n the n u c l e i c a c i d c h a i n which n o r m a l l y i s a template f o r the a c t i v e s i t e of a particular  enzyme, an important change i n t h e s t r u c t u r e of the  s i t e may be produced l e a d i n g to impairment of the corresponding b i o l o g i c a l f u n c t i o n . at a d i f f e r e n t s i t e ,  However, i f s u b s t i t u t i o n takes p l a c e  the f u n c t i o n of the enzyme may be unaf-  f e c t e d so that t h e a n t i m e t a b o l i t e  may a c t u a l l y support the  biochemical reaction.  w h i l e i n many cases base an-  Therefore,  alogue i n c o r p o r a t i o n reduced i n f e c t i v i t y , ivity still  has been  reported.  considerable  infect-  - 46 -  up a r t  The  II  E f f e c t s of a P y r i m i d i n e A n t i m e t a b o l i t e  on  V a c c i n i a V i r u s i n Chick F i b r o b l a s t T i s s u e C u l t u r e  - 47 -  Part  II of t h i s t h e s i s i s the f o l l o w i n g  based on r e s e a r c h undertaken  at the U n i v e r s i t y of C a l i f o r n i a ,  at Berkeley, and which i s r e l e v a n t pursued  report  to the experimental work  at the U n i v e r s i t y of B r i t i s h Columbia.  - 48 -  Introduction During the course of some experimental work on the e f f e c t s of a p u r i n e and a p y r i m i d i n e analogue on Disease v i r u s terested  (NDV)  and v a c c i n i a v i r u s , the author hecame i n -  i n the r e c e n t l y p u b l i s h e d  the t h e r a p e u t i c  Newcastle  reports  of that time on  e f f e c t of 5 - i o d o - 2 ' - d e o x y u r i d i n e  herpes simplex v i r u s i n f e c t i o n i n v i v o  (IUDR ) on  (Kaufman and Maloney,  1962  b) and on v a c c i n i a v i r u s i n f e c t i o n (Kaufman et a l . , 1962  a).  S i n c e i t was  not p o s s i b l e  to pursue  i t further  at that  time, the author waited f o r the o p p o r t u n i t y to t r y an  exper-  iment of t h i s nature. In t h i s experiment  an attempt  was made to study the  e f f e c t of IUDR on v a c c i n i a v i r u s i n c h i c k f i b r o b l a s t monolayer c u l t u r e s . produces  The v a c c i n i a v i r u s employed i n the t e s t 48-  cytopathogenic changes i n CF monolayers i n about  60 hours a f t e r i n f e c t i o n . may  (CF)  The cytopathogenic e f f e c t  be seen as the rounding up of c e l l s ,  c e l l s and d e g e n e r a t i o n of the c e l l  sheet.  (CPE)  c e l l fragments,  giant  - 49 -  Materials  and Metnods Chick f i b r o b l a s t monolayer c u l t u r e s were prepared i n  a n u t r i e n t medium (5% lamb serum and 95% n u t r i e n t composed of 0.5% l a c t a l b u m i n ied  E a r l e ' s balanced s a l t Ten-fold  enzymatic h y d r o l y s a t e  solution i n modif-  solution).  d i l u t i o n s of stock v a c c i n i a v i r u s were made  i n m o d i f i e d Hanks' maintenance medium (MH).  When c e l l mono-  l a y e r s were complete, 3 oz. p r e s c r i p t i o n b o t t l e s were i n f e c t ed w i t h 0.2 ml. of a p p r o p r i a t e  virus dilution.  V i r u s was a l -  lowed to adsorb f o r 1 hour at 37°C w i t h o c c a s i o n a l  t i l t i n g of  the b o t t l e s to f a c i l i t a t e the spread of the v i r u s inoculum over the c e l l  sheet.  At the end of t h i s time the c u l t u r e s  were o v e r l a i d w i t h 6 - 8 ml.  of MH.  Uninfected  ml. MH.  Tubes were i n f e c t e d with 0.1  c o n t r o l s were always  included.  For plaque assay, b o t t l e s were o v e r l a i d w i t h methylc e l l u l o s e and incubated f o r 4 days, at 37°C. violet  0.1% c r y s t a l  i n 20% a l c o h o l was used to s t a i n the monolayers to ex-  amine f o r the presence of plaques. When CPE was observed, the f l u i d s were removed and t e s t e d f o r presence of v i r u s by i n f e c t i n g monolayer c u l t u r e s with varying  d i l u t i o n s of the f l u i d  CPE a f t e r s u i t a b l e  and o b s e r v i n g these f o r  incubation.  IUDR was added to MH i n c o n c e n t r a t i o n s  of 100 mg./ml.  - 50 -  and 1 mg./ml.  The a n t i m e t a b o l i t e was added to the medium  j u s t b e f o r e use. C u l t u r e s e x h i b i t i n g CPE were t r e a t e d with mediurai c o n t a i n i n g IUDR.  The medium was changed d a i l y because the  breakdown p r o d u c t s of IUDR, such as i o d o u r a c i l , to some extent  inhibit  the a n t i v i r a l  a c t i v i t y of the parent compound.  C o n t r o l c u l t u r e s were (treated i n an i d e n t i c a l manner w i t h medium f r e e of IUDR. A f t e r treatment w i t h the a n t i m e t a b o l i t e f o r 24 hours and 48 hours, f l u i d s were removed and t e s t e d f o r CPE on other CF  monolayers. Some c u l t u r e s were t r e a t e d w i t h IUDR f o r 24 hours  before i n f e c t i o n with v i r u s .  - 51 -  Results The f i r s t  set of monolayers prepared, were i n f e c t e d  a f t e r about 90 hours of i n c u b a t i o n s i n c e t h e r e were no compl e t e monolayers u n t i l to  10-6 vvere used.  cell  t h i s time.  D i l u t i o n s of v i r u s of I O * -  A f t e r an a d d i t i o n a l 12 hours' i n c u b a t i o n  sheets were beginning to f a l l  some of the c o n t r o l  o f f the g l a s s s u r f a c e .  (uninfected)tubes, intact c e l l  In  sheets  c o u l d be seen, but i n o t h e r s the c e l l s were a l s o beginning to fall  o f f the glass surface.  this  time.  The c u l t u r e was 1 days o l d at  48-hour CF monolayers were i n f e c t e d with v a c c i n i a v i r u s d i l u t i o n s of 1 0  _ 1  to 10"^ f o r o b s e r v a t i o n of the CPE.  B o t t l e c u l t u r e s were i n f e c t e d f o r assay of the stock v i r u s . The r e s u l t s of the plaque assay a r e shown i n T a b l e Monolayers to  i n f e c t e d w i t h v i r u s as i n the above began  show CPE a f t e r 48 - 60 hours.  and CPE,  10""  2  1.  Cultures i n f e c t e d with  10"  1  v i r u s d i l u t i o n s e x h i b i t e d d e f i n i t e and pronounced  rounding up of c e l l s , degeneration of the c e l l  the edges, c e l l  d e b r i s , and fragments  sheets were no longer continuous. ion CPE was j u s t  starting.  of c e l l s .  sheet at  The c e l l  At t h 10"3 and 10-4  dilut-  No changes were evident at the  h i g h e r d i l u t i o n except that c e l l  sheets were beginning to de-  t e r i o r a t e i n some of these as w e l l as i n the c o n t r o l  tubes.  - 52 -  I n f e c t e d c u l t u r e s were t r e a t e d w i t h 100 mg./ml. of a r r e s t e d a f t e r 24 hours t r e a t -  IUDS.  The CPE was a p p a r e n t l y  ment.  A f t e r 48 hours t h e r e was no p r o g r e s s i o n  i c changes.  In u n t r e a t e d  c o m p l e t e l y destroyed.  c o n t r o l s the c e l l  The CPE was v e r y  of the cytopath  sheets were almost  extensive.  Another set of c u l t u r e s was i n f e c t e d w i t h d i l u t i o n s of v a c c i n i a stock v i r u s .  48 - 60 hours l a t e r  pathogenic changes were obvious i n c u l t u r e s . ures were t r e a t e d with MH c o n t a i n i n g  varying  Infected  1 mg./ml. IUDK.  cytocult-  The r e -  s u l t s are shown i n T a b l e I I . S l i g h t CPE was observed i n c u l t u r e s exposed to IUDB b e f o r e i n f e c t i o n with the v i r u s . A l i q u o t s of f l u i d s from i n f e c t e d and t r e a t e d u r e s were t e s t e d f o r CPE on other monolayers.  F l u i d from un-  t r e a t e d c u l t u r e s showed v e r y c l e a r cut and extensive t e s t e d on other monolayers. and  cult-  CPE when  Samples of f l u i d from 24 hour  48 hour t r e a t e d c u l t u r e s e x h i b i t e d v e r y s l i g h t CPE.  - 53 Table I Plaque Assay of Stock V a c c i n i a  Virus Dilution  No. of Plaques  Virus Pfu/ml.  Average  6.65  152,-110  131.0  35138-  36.5  i8.:3  x io3  8.5  42.5  x io  3  3,-o.  1.5  75  x io  3  io-5  0 ;2.  1. 0  50  x io3  IO"  0;0  0  IO"  1  IO"  2  •  io-3 10-  4  6  T i t e r of V i r u s = 4 x 1 0  x IO  3  0 Pfu/ml.  4  T a b l e II V a c c i n i a V i r u s on Chick F i b r o b l a s t s  Virus Dilution  Observation Before Treatment  U n d i l u t ed  Extensive  CPE  IUDH 1 mg./ml. 24 h r s . 1  CPE  Progressed slightly  IUDR 1 mg./ml. "48 h r s . No f u r t h e r CPE  IO'  1  Pronounced CPE  S l i g h t CPE  No CPE  10"  2  Some CPE evident  No CPE  No CPE  S l i g h t CPE  S l i g h t CPE  S l i g h t CPE  No f u r t h e r CPE  No CPE  10-5  No CPE  No CPE  No CPE  io-  No CPE  No CPE  No CPE  io-3 io"  4  6  Extensive  CPE  - 54 -  Discussion With the v a c c i n i a v i r u s used, cytopathogenic changes were not evident  u n t i l 48 - 7  2  hours a f t e r i n f e c t i o n .  n e c e s s i t a t e d a w a i t i n g p e r i o d longer treatment of v i r u s - i n f e c t e d c e l l a t e d a problem i n that c e l l old  and f r a g i l e ( 5 - 7  complete, c e l l off  than a n t i c i p a t e d  l a y e r s w i t h IUDR.  This before  This  cre-  sheets by t h i s time were q u i t e  days),  so that before  treatment was  sheets were beginning to d e t e r i o r a t e and to f a l l  the s u r f a c e of the g l a s s . At f i r s t ,  the use of a 1:200 d i l u t i o n i n n u t r i e n t  medium of CF c e l l s f o r s e t t i n g up c u l t u r e s d i d not p r o v i d e complete monolayers i n 48 hours.  In subsequent  preparations  a 1:100 d i l u t i o n of c e l l s was used i n s t e a d ; t h i s produced l u x u r i a n t monolayers w i t h i n 48 hours. In c e l l tinct  cytopathogenic changes c o u l d be seen at 60 hours a f t e r  incubation. cells,  cell  Changes were m a n i f e s t e d as the rounding up of fragments, giant c e l l  t i n u i t y of c e l l most u n t r e a t e d 4 - 5  c u l t u r e s i n f e c t e d with v a c c i n i a v i r u s , d i s -  sheets.  formation  and l o s s of con-  Rounded c e l l s were abundant.  cultures c e l l  sheets were completely  In  destroyed  days a f t e r i n f e c t i o n ; t r e a t e d c u l t u r e s s u r v i v e d f o r a-  bout 7 days. In the c u l t u r e s t r e a t e d w i t h IUDR, t h e CPE d i d not appear to p r o g r e s s any f u r t h e r a f t e r 24 - 48 hours of t r e a t -  - 55 -  ment. ed,  When f l u i d from c o n t r o l and t r e a t e d c u l t u r e s were t e s t -  l e s s CPE was caused by the l a t t e r . The  5-  aromatic  r i n g i n IUDR i s halogenated i n p o s i t i o n  I t s s t r u c t u r a l formula  i s g i v e n i n f i g u r e 1.  i d i n e a n t i m e t a b o l i t e a c t s to i n h i b i t  T h i s pyrim-  the p h o s p h o r y l a t i o n of  thymidine and the p o l y m e r i z a t i o n of t h y m i d y l i c a c i d i n t o DNA. It may a l s o be i n c o r p o r a t e d  i n t o an abnormal and presumably  n o n - f u n c t i o n a l v i r a l DNA which i s rendered i s probable  that IUDR not o n l y prevents  uninfectious.  It  the i n f e c t i o n of c e l l s  which have been p r e v i o u s l y f r e e of v i r u s , but perhaps i s capable of stopping the s y n t h e s i s of v i r u s i n c e l l s a l r e a d y i n fected.  -  56  -  Summary The p y r i m i d i n e  antimetabolite,  5 - f l u o r o u r a c i l was  found to i n h i b i t v a c c i n i a v i r u s r e p l i c a t i o n i n the c h i c k bryo but was  em-  i n e f f e c t i v e a g a i n s t Newcastle d i s e a s e v i r u s .  Thioguanine, a p u r i n e analogue, had no e f f e c t on e i t h e r v i r u s . hemagglutination  The  t e s t and  inhibitory  assay methods employed were the  the i n f e c t i v i t y of the v i r u s f o r  the c h i c k embryo. It was iododeoxyuridine  p o s s i b l e to demonstrate to some extent can a r r e s t the cytopathogenic  that  changes i n  chick fibroblast c e l l s i n tissue culture, following vaccinia virus infection. preventing  This pyrimidine  major CPE  sure to the v i r u s .  i n c e l l s treated, with The  c e n t r a t i o n s of 100 mg. drug-containing  analogue seems capable of  a n t i m e t a b o l i t e was per ml.,  medium was  and  1 mg.  IUDR before  expo-  e f f e c t i v e at conper ml.  when the  changed d a i l y .  Major d i f f e r e n c e s i n the metabolism of the  various  antimetabolites  suggest d i f f e r e n c e s i n t h e i r mechanisms of  action.  the p o s s i b l e areas of i n h i b i t i o n are  Among  of the i n c o r p o r a t i o n of the normal m e t a b o l i t e , of the analogue i n t o RNA  or DNA  blocking  incorporation  to form f r a u d u l e n t n u c l e i c  acids. A l s o , the a n t i m e t a b o l i t e may  inhibit  s y n t h e s i s of  - 57 -  the normal m e t a b o l i t e or may Pyrimidine  b l o c k n u c l e o t i d e metabolism.  and p u r i n e analogues may  i c r e a c t i o n s , f o r example,  inhibit  enzymat-  the enzymatic p h o s p h o r y l a t i o n  of  the normal bases. Finally,  inhibition  of p r o t e i n s y n t h e s i s may be  as a p o s s i b l e mechanism of a c t i o n because of the c l o s e i o n s h i p between n u c l e i c a c i d and p r o t e i n  cited  relat-  synthesis.  C o r r e l a t i o n s between systems must be i n t e r p r e t e d with caution;  d e s p i t e many s i m i l a r i t i e s  o l i s m of a n t i m e t a b o l i t e s ferences  i n the a c t i o n s and metab-  in biological  have been uncovered.  systems s t u d i e d ,  dif-  -  58 -  Appendix  - 59 -  OH  .ft H  Thioguanine. ( 6 . ^ >  Guanine  1 0  '1 *  i.l,  . F l u o r o u r a c i l ( JrFU)  UracIT  J  OH  ti  Thymine  5--iodo-2-deoxyuridine (IUDR)  FIGURE I.  - 60 -  UMP  u  —3> UR  FU  FUR  -> FUMP  u  —5* UDR  -> DUMP  FU  -> FUDR  -> FDUMP  H>  RNA  i  —RNA  1  THF  F i g u r e 2.  DNA  rl > TMP  - Ci  Formation of RNA and DNA  Key to Symbols  u UR IMP RNA FU FUR FUMP UDR DUMP TMP DNA FUDR  -  uracil uridine u r i d i n e monophosphate ribonucleic  acid  fluorouracil fluorour idine fluorouridine  monophosphate  deoxyuridine d e o x y u r i d i n e monophosphate thymidine monophosphate deoxyribonucleic  acid  — fluorodeoxyuridine  FDUMP - f l u o r o d e o x y u r i d i n e monophosphate  - 61 -  Key toSymbols PRPP + Glutamine  PRPP - 5 - p h o s p h o r i b o s y l - l pyrophosphate  Pur ine nucleotides & analogs  FGAR - formylglycinamide r ibonucleot ide  Pho-spho r i bo sy 1 am ine  FGAMR - f o r m y l g l y c i n a m i d i n e ribonucleotide AIC  Glycinamide  - 5- i o-4-imidazole carboxamide a m  n  SAMP - a d e n y l o s u c c i n i c a c i d  ribotide  XMP - x a n t h y l i c a c i d IMP - i n o s i n e monophosphate  FGAR  Azaserine  Glutamine  AMP - adenosine monophosphat e GMP - guanosine mono phosphate  FGAMR  AIC  Ribotide  IMP  Mercaptopur ine r ibonucleot ide -SAMP AMP F i g u r e 3*  XMP GMP  Known S i t e s of A c t i o n of P u r i n e (Brockman, 1963)  Analogs.  J  - 62 -  Fluorodeoxyuridine -5'-phosphate  .Deoxyuridine-5' -phosphate  Thymidylic  Synthetase Bromodeoxyur i d i n e -5'-phosphate  Iododeoxyuridine -5'-phosphate  Thymidine-5 -phosphate 1  i Thymidylic  DNA  Phosphorylase  polymerase  1  DNA  F i g u r e A>»  S i t e of A c t i o n of P y r i m i d i n e Analogues  - 63 -  Bibliography Barry, R.D.,  D.R. 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