Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

The incorporation of formate-C¹⁴ into the nucleic acids of rats with regenerating liver and Novikoff… Nixon, John Charles 1958

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1958_A6_7 N4 I6.pdf [ 3.6MB ]
Metadata
JSON: 831-1.0106096.json
JSON-LD: 831-1.0106096-ld.json
RDF/XML (Pretty): 831-1.0106096-rdf.xml
RDF/JSON: 831-1.0106096-rdf.json
Turtle: 831-1.0106096-turtle.txt
N-Triples: 831-1.0106096-rdf-ntriples.txt
Original Record: 831-1.0106096-source.json
Full Text
831-1.0106096-fulltext.txt
Citation
831-1.0106096.ris

Full Text

THE INCORPORATION OF FORMATE-C"^ INTO THE NUCLEIC ACIDS OF RATS WITH REGENERATING LIVER AND NOVIKOFF HEPATOMA. by John C h a r l e s Nixon  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 standard r e q u i r e d from candidates for the degree of MASTER OF SCIENCE.  Members of the Department Biochemistry  of  The U n i v e r s i t y of B r i t i s h Columbia April,  1958.  ABSTRACT  A comparison has t e e n made of the formate-C ^ 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 p u r i n e s and thymine of r e g e n e r a t i n g r a t The e f f e c t s host  l i v e r and Novikoff hepatoma  in vivo.  of these t i s s u e s on one a n o t h e r , and on the  t i s s u e s has been s t u d i e d .  mate by the n u c l e i c a c i d s generating r a t  The u t i l i z a t i o n of  of N o v i k o f f  for-  hepatoma and r e -  l i v e r was not s i g n i f i c a n t l y a l t e r e d  in  animals c o n t a i n i n g both of these r a p i d l y d i v i d i n g t i s sues.  The r e s u l t s  i n d i c a t e d that  the demand for  formate  by one of the r a p i d l y growing t i s s u e s d i d not lower  the  uptake of formate by the n u c l e i c a c i d s  tis-  sue. thesis  of the other  Furthermore i t was i n d i c a t e d that n u c l e i c a c i d synin regenerating  of n u c l e i c a c i d s and N o v i k o f f  l i v e r d i d not a l t e r  in other  tissues.  hepatoma had no e f f e c t  the  synthesis  Regenerating  liver  on the n u c l e i c a c i d  metabolism of the host  t i s s u e s of animals b e a r i n g one or  both of these t i s s u e s .  These r e s u l t s are not completely  i n agreement  w i t h those r e p o r t e d i n the  literature.  In a p r e l i m i n a r y experiment a r a d i o a c t i v e pension of Novikoff  hepatoma was t r a n s p l a n t e d  into r a t s .  Twenty percent of the i n j e c t e d r a d i o a c t i v i t y was i n the u r i n e d u r i n g the f i r s t The s p e c i f i c  2 4 hours of  recovered  tumor growth.  a c t i v i t j * * of the n u c l e i c a c i d bases of  tumor, obtained a f t e r  sus-  the  2 4 hours of growth, were n e g l i g i b l e .  These f i n d i n g s of  i n d i c a t e d that the n u c l e i c  acids  the donor tumor suspension were not u t i l i z e d  i n the s y n t h e s i s of the n u c l e i c a c i d s of growing tumor.  the  ACKNOWLEDGMENTS The personal Research C o u n c i l cal  a s s i s t a n c e of the N a t i o n a l  i n the form of two Graduate  Research F e l l o w s h i p s  is gratefully  The author a l s o wishes  Zbarsky d u r i n g the course of t h i s Mrs.  acknowledged.  to express h i s  c i a t i o n of the a s s i s t a n c e and counsel H.  Medi-  V. Creelman, Miss B. S.  appre-  g i v e n hy D r .  S.  research. F i n d l a y , Miss  L . Maddex, and Miss M. M. Wei of the Department  of  B i o c h e m i s t r y , U n i v e r s i t y of B r i t i s h Columbia, k i n d l y performed the tumor  transplantations.  The author acknowledges advice  the a s s i s t a n c e and  of D r . H. E . T a y l o r , Head of the Department  of  P a t h o l o g y , and D r . K. C o l e , Department of B i o l o g y and Botany, U n i v e r s i t y of B r i t i s h Columbia, i n the logical  preparation  mitotic  counts.  of the  histo-  tumor t i s s u e p r i o r to  the  T h i s r e s e a r c h was supported by grants from the N a t i o n a l Research C o u n c i l and the N a t i o n a l Cancer I n s t i t u t e of Canada.  TABLE OF CONTENTS Page INTRODUCTION I. II. III. IV. V.  1  S t u d i e s Comparing Tumor and Non-tumor Tissues The E f f e c t s of Tumor on Host T i s s u e s . S t u d i e s of Regenerating L i v e r M e t a b o l i c I n t e r r e l a t i o n s of Regenerat i n g L i v e r and Tumor T i s s u e s The Eresent I n v e s t i g a t i o n  EXPERIMENTAL  2 4 6 8 10 14  A. METHODS. I. II. III. IV. V. VI. VII. VIII. B.  Materials P a r t i a l Hepatectomies M i t o t i c Counts 1. "Standard" Technique 2. "Squash" Technique C o l l e c t i o n of E x p i r e d C 0 2 C o l l e c t i o n of Urine I s o l a t i o n of Purines and P y r i m i d i n e s . U l t r a v i o l e t Spectrophotometry R a d i o a c t i v e Counting Procedures  14 14 15 17 17 lj 18 l8 19 20  RESULTS. I. II.  III.  M i t o t i c Counts Formate I n c o r p o r a t i o n i n t o the Nucl e i c Acids of T i s s u e s of P a r t i a l l y Hepatectomized and Tumor-Bearing Rats i . P a r t i a l l y hepatectomized r a t s i i . Tumor-bearing r a t s i i i . P a r t i a l l y hepatectomized-tumorbearing r a t s Iv. E x c r e t i o n of Radio a c t i v i t y i n the E x p i r e d COo and U r i n e v . Tumor-bearing Raxs I n j e c t e d w i t h Forraate-C 1 ? f o l l o w e d by P a r t i a l Hepatectomy T r a n s p l a n t e d Radio a c t i v e N o v i k o f f hepatoma  DISCUSSION SUMMARY" BIBLIOGRAPHY  22  23 25 2o 27 29 33 35 39  ,..  52 54  Page 1.  Relative specific  activities  of n u c l e i c  a c i d p u r i n e s of 1 i v e r . . 2.  Relative specific  activities  .  of n u c l e i c  a c i d p u r i n e s of N o v i k o f f  hepa-  .. . . A . . f a c e  toma 3.  Relative specific  activities  Relative specific  activities  26  of n u c l e i c  a c i d purines of s p l e e n . 4.  25  S.\ . 0  .. . . A .. f a c e  of n u c l e i c  a c i d purines of i n t e s t i n a l  mucosa..  (to f o l l o w ) 5.  Relative specific  activities  27  of DNA  thymine of l i v e r , N o v i k o f f  hepatoma,  spleen and i n t e s t i n a l mucosa.(A 0 . / A 6.  Relative specific  activities  c e  )  28  of n u c l e i c  a c i d purines and thymine of tumorh e a r i n g d e l a y e d hepatectomized and tumor-bearing c o n t r o l animalsS.\°. ,f>ce) 3 4  27  TABLES Page  I II III  Mitotic  indices  of N o v i k o f f hepatoma  Time r e l a t i o n s h i p of p r o c e d u r e s . ^ . 0 .  23  R a d i o a c t i v i t y excreted  i n expired C 0 2 ^ P . £  IV  R a d i o a c t i v i t y excreted  i n ur i n e . . . ^ P . £ a P f ^ .  V  T o t a l r a d i o a c t i v i t y excreted  Specific  activities  a c e  )  3° 32  in expired  C 0 2 and ur ine VI  24  fpD-PV).  .(.*?. f&9?)... of n u c l e i c a c i d  32  purines  and thymine of growing N o v i k o f f hepatoma VII  R a d i o a c t i v i t y excreted  (to  37  i n urine during  growth of r a d i o a c t i v e toma  follow)  N o v i k o f f hepa(to f o l l o w )  37  - 1 INTRODUCTION In s t u d y i n g the n u c l e i c a c i d metabolism of tumor and non-tumor t i s s u e ,  i t was f e l t  that u s e f u l  information  c o u l d be obtained from a comparison of a n e o p l a s t i c and a r a p i d l y growing n o n - n e o p l a s t i c t i s s u e . of  such a study, Novikoff  were eminently s u i t a b l e  tissues,  s i n c e the N o v i k o f f  blished i n a transplantable  form by Novikoff  other hand r e g e n e r a t i n g r a t  liver  reasonable  tissue.  purposes  hepatoma and r e g e n e r a t i n g  i s a neoplasm which had been induced i n r a t  non-neoplastic  For  tissue  liver  liver hepatoma  and e s t a -  (l).  On the  p r o v i d e d a r a p i d l y growing  A comparison of these t i s s u e s  i n view of t h e i r common c e l l  origin.  seemed  Investigators  i n the past who have s t u d i e d the n u c l e i c a c i d metabolism of tumor and normal t i s s u e s have for plastic  the most part  compared neo-  and normal t i s s u e s which were u n r e l a t e d i n c e l l  Regenerating l i v e r  has been e x t e n s i v e l y used as a source of  r a p i d l y growing n o n - n e o p l a s t i c t i s s u e and compared to but a g a i n most of the data  which d i d not o r i g i n a t e  from h e p a t i c  Therefore the present  to tumor t i s s u e  tissue.  study was undertaken i n order  to compare the n u c l e i c a c i d metabolism of N o v i k o f f and r e g e n e r a t i n g r a t tissues  of N o v i k o f f  liver.  tumors,  obtained from s t u d i e s of the n u c l e i c  a c i d metabolism of t h i s t i s s u e have been r e l a t e d  two  origin.  hepatoma  Furthermore the presence  of  i n v i v o p r o v i d e d an o p p o r t u n i t y to study the hepatoma and r e g e n e r a t i n g  these effect  l i v e r a n the n u c l e i c a c i d  metabolism of other t i s s u e s of animals c o n t a i n i n g one or both  - 2 of the above r a p i d l y growing t i s s u e s , as w e l l as effect  of the hepatoma and r e g e n e r a t i n g  liver  the  on each  other. I.  S t u d i e s Comparing Tumor and Non-tumor  Tissues.  Studies on the u t i l i z a t i o n of s e v e r a l a c i d p r e c u r s o r s by malignant pared  and benign tumors as com-  to non-tumor t i s s u e s have been c a r r i e d out by  several  investigators.  Brues,  Tracy and Cohn ( 2 ) demon-  s t r a t e d that the r a t e of uptake of of a hepatoma was intermediate regenerating berger small  nucleic  liver.  by the n u c l e i c  acids  between that of r e s t i n g  and  On the other hand LePage and H e i d e l -  ( 3 ) found the i n c o r p o r a t i o n of g l y c i n e - 2 - C ^ was i n the thymus of r a t s ,  and g r e a t e s t i n r e g e n e r a t i n g carcinoma.  greater  l i v e r and  Subsequent experiments  s t r a t e d a greater  i n the normal  Flexner-Jobling  ( 4 - 7 ) have a l s o demon-  i n c o r p o r a t i o n of g l y c i n e - 2 - C  i n t o tumor t i s s u e as Reddy and coworkers  a n  d P  compared to non-tumor t i s s u e . (8) have found a higher  i n v i t r o of P-^2 i n t o the r i b o n u c l e i c a c i d malignant  liver,  human o v a r i a n tumors as  Recently  incorporation  (RNA) of  several  compared to benign human  o v a r i a n tumors.  A higher  i n c o r p o r a t i o n of P-^2 i n t o  deoxyribonucleic  a c i d (DNA) of most of the malignant  was a l s o noted, but the d i f f e r e n c e s nitude.  Wells and Winzler  incorporation nucleotides  were of a smaller  the tumors mag-  ( 9 ) have a l s o observed a higher  i n v i t r o of f o r m a t e - C 1 ^  of human leukemic c e l l s  as  i n t o the  acid-soluble  compared to normal  -  leucocytes,  3 -  the descending  being acute leukemia,  order  of s p e c i f i c  chronic granulocytic  c h r o n i c lymphocytic leukemia and normal Similar  findings  had been r e p o r t e d  (10) who found that leukemic  activity leukemia,  leucocytes.  by T u t t l e and a s s o c i a t e s  i n f i l t r a t i o n i n t o the  liver,  s p l e e n and lymph nodes of mice, was accompanied by an i n c r e a s e i n the uptake and r e t e n t i o n of r a d i o a c t i v e phorus  by the n u e l e o p r o t e i n s  of these  and a c i d - s o l u b l e  fractions  tissues. I n v e s t i g a t i o n s of the i n c o r p o r a t i o n of  preformed  p r e c u r s o r s i n t o the n u c l e i c a c i d s of tumor t i s s u e s a l s o been c a r r i e d out. a higher  Weed and Wilson (11,12) have  slices.  of o r o t i c acid-6-C*^ compared to l i v e r .  was a t t r i b u t e d and u t i l i z e cursor,  tumor s l i c e s as  In c o n t r a s t H u r l b e r t and P o t t e r  obtained a r e l a t i v e l y  as  have found  i n c o r p o r a t i o n of o r o t i c a c i d - 2 - C * ^ i n t o t h y m i d y l i c ,  u r i d y l i c and c y t i d y l i c a c i d s of liver  phos-  compared (13)  i n t o subcutaneous tumor The d i f f e r e n c e  i n the  transplants incorporation  to the greater a b i l i t y of the l i v e r U s i n g u r a c i l - 2 - C 1 ^ as  Rutman and coworkers  (14) observed a higher  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 of r a t to those of normal l i v e r . et a l  to absorb a  pre-  incor-  hepatoma as  compared  Comparable r e s u l t s were  obtained  (15) who found that the  a c t i v i t y of the n u c l e i c a c i d s and a c i d - s o l u b l e was higher  have  small amount of i n c o r p o r a t i o n i n v i v o  the o r o t i c a c i d .  by Heidelberger  to  specific nucleotides  i n a subcutaneous tumor than i n normal r a t  liver  f o l l o w i n g the a d m i n i s t r a t i o n latter  authors note that the i n c r e a s e d  i s not s p e c i f i c  to tumor since  c o n v e r s i o n of u r a c i l of  of u r a c i l - 2 - C  intestinal  they found a  mucosa of normal r a t s .  comparable pyrimidines  Other authors  that there was no  ence i n the r a t e of i n c o r p o r a t i o n of adenine n u c l e i c a c i d s of normal and tumor The E f f e c t s  II.  The e f f e c t  for  differ-  into  the  tissues.  of Tumor on Host T i s s u e s . of  metabolism of s e v e r a l monstrated  The  incorporation  i n t o the n u c l e i c a c i d  (16) had p r e v i o u s l y r e p o r t e d  .  tumors on the n u c l e i c  t i s s u e s of  several  tumors.  acid  the host has been deCerecedo et a l  (17-21)  have shown an i n c r e a s e i n c o n c e n t r a t i o n of n u c l e i c i n the l i v e r ,  lungs,  sarcoma-bearing lungs  kidney,  thymus, and lymph nodes of  mice; and i n the l i v e r , k i d n e y s ,  of 12 to 20 day g e s t a t i o n a l mice ( 2 2 ) .  was not observed  acids  and  This  in r a t s containing regenerating  effect  liver  (23) ; and r a t s c o n t a i n i n g Walker carcinoma showed a drop i n n u c l e i c a c i d c o n c e n t r a t i o n of the thymus and the RNA of the lymph nodes, w i t h an i n c r e a s e i n the n u c l e i c c o n c e n t r a t i o n of  acid  the spleen and the RNA of bone marrow  (24) . K e l l y and a s s o c i a t e s  (25-27) have shown an  i n c r e a s e d r a t e of i n c o r p o r a t i o n of p 3  2  i n t o the DNA of  l i v e r s and spleens of mice and r a t s b e a r i n g planted  tumors and i n pregnant  several  mice and r a t s .  trans-  Similar  i n c r e a s e s i n the i n c o r p o r a t i o n of g l y e i n e - a - C f o r m a t e - C 1 ^ i n t o the DNA of host t i s s u e s of bearing mice have been r e p o r t e d and Heidelberger  and  tumor-  ( 2 8 ) , although  (3) obtained no s i g n i f i c a n t  LePage  difference  between the i n c o r p o r a t i o n of g l y c i n e - 2 - C 1 ^ i n t o the DNA and RNA of the l i v e r of r a t s b e a r i n g carcinoma as  compared to normal l i v e r s .  i t has been demonstrated bearing F l e x n e r - J o b l i n g  In c o n t r a s t ,  (5) that the l i v e r s  of r a t s  carcinoma have a higher  of P ^ 2 i n t o the DNA n u c l e o t i d e s livers.  Flexner-Jobling  as  uptake  compared to normal  Others (29»30) have observed a s i m i l a r  i n tumor-bearing mice,  i n which there was a  effect  stimulation  of the i n c o r p o r a t i o n of adenine-S-C^"^ i n t o the n u c l e i c a c i d s of the host t i s s u e s . using  in v i t r o techniques,  tumor t i s s u e ,  Preliminary studies  indicated a stimulation,  of the i n c o r p o r a t i o n of adenine  DNA of normal organs.  the  i n c o r p o r a t i o n of  guanine  i n t o the KNA of c e r t a i n host  tissues  of r a t s bearing t r a n s p l a n t e d  human tumor.  these observations on the e f f e c t  Annau and coworkers  Further  of tumor on the  p o r a t i o n of n u c l e i c a c i d p r e c u r s o r s  i n t o host  of  to  incor-  tissues,  (33) and Malmgren (34) have  an increased m i t o t i c r a t e i n the l i v e r s mice and r a t s .  into  by  In a d d i t i o n , B a l i s and coworkers  (31»32) r e p o r t e d an i n c r e a s e d and hypo~xanthine  (36)  reported  tumor-bearing  - 6 III.  Studies  of Regenerating  The e a r l y l i t e r a t u r e liver  p e r t a i n i n g to  has been reviewed by Fishback ( 3 5 ) .  Anderson (36) made a c a r e f u l of  Liver*  the l i v e r  study of the  regenerating  H i g g i n s and restoration  i n the white r a t and observed that a f t e r  the  removal of approximately 70% of the l i v e r , the remnant more than doubles 72 h o u r s .  in size  i n the course of the f o l l o w i n g  These s t u d i e s were extended by Brues et a l  who found that  the remnant of l i v e r  tween 50 and 60% d u r i n g the f i r s t but observed no i n c r e a s e during this period. increase  in size  increased  24 hours  (37)  i n s i z e toe-  postoperatively,  i n the number of h e p a t i c  cells  T h i s phenomenon was a t t r i b u t e d to an  of the h e p a t i c c e l l s and was f o l l o w e d on  the second day by a marked i n c r e a s e  in c e l l  d i v i s i o n which  d i m i n i s h e d from then on (37). By u s i n g the e a r l y stages of l i v e r i n r a t s and other animals s u b j e c t e d i t was p o s s i b l e  to i n v e s t i g a t e  to p a r t i a l  a c i d metabolism.  Since the i n i t i a l  i n c o r p o r a t i o n of p r e c u r s o r s  In p a r t i c u l a r ,  (39-53).  l i v e r d u r i n g the f i r s t  toy a subsequent  re-  study toy Brues et a l  have been r e p o r t e d on the  i n t o the n u c l e i c a c i d s Johnson and A l b e r t  a r a p i d uptake of P - ^ i n t o the RNA and DNA of rat  that  has been w i d e l y used i n s t u d y i n g n u c l e i c  ( 3 8 ) , numerous i n v e s t i g a t i o n s  generating l i v e r  hepatectomy  c e r t a i n processes  o c c u r r e d i n a r a p i d l y growing t i s s u e . generating l i v e r  regeneration  24 hours a f t e r  of r e -  (41) observed regenerating  operation,  followed  d e c l i n e which was accompanied by a marked  increase  in mitosis.  w i t h the r e s u l t s  T h i s was i n general  of P r i c e and L a i r d  that on a per c e l l  basis,  agreement  ( 5 4 ) who found  the amounts of n u c l e i c a c i d ,  w i t h the e x c e p t i o n of nuclear RNA, reached t h e i r maximum  value before an i n c r e a s e  cells  c o u l d be d e t e c t e d .  r a t e s of DNA s y n t h e s i s  i n the number of  Others have r e p o r t e d maximum  in regenerating rat  and 2 4 to 30 ( 4 7 ) hours a f t e r  (44) after  the onset  t h e s i s tends servations  of a c t i v e m i t o s i s  to f a l l  dividing  l i v e r at 2 4  hepatectomy,  the r a t e of DNA syn-  to a lower l e v e l  (44)•  S i m i l a r ob-  have been obtained u s i n g p a r t i a l l y  tomized mice  (45»52)»  but  hepatec-  and mice on which l i v e r  regener-  a t i o n was induced by the a d m i n i s t r a t i o n of carbon  tetra-  chloride ( 5 0 ) .  1^ Using g l y c i n e - N E l i a s s o n et a l  J  as  precursor,  ( 4 0 ) found maximum i n c o r p o r a t i o n i n t o the  p u r i n e s of the n u c l e i c a c i d s at about 30 hours a f t e r trast  the i s o t o p i c  of r e g e n e r a t i n g r a t  partial  hepatectomy,  to the aforementioned r e s u l t s ,  but i n con-  E l i a s s o n et a l  served that the maximum r a t e of p o l y n u c l e o t i d e c o i n c i d e d i n time with the maximum of m i t o s i s In  more recent papers  found an i n i t i a l RNA  purines at  initial of  (55-57)  liver  the l a t t e r  ob-  synthesis frequency.  investigators  peak of g l y c i n e i n c o r p o r a t i o n i n t o the  14 hours a f t e r  operation.  However t h i s  h i g h peak d i d not c o i n c i d e w i t h the maximum r a t e  synthesis  of RNA, and a p p a r e n t l y r e p r e s e n t e d  turnover process d i s t i n c t  from net s y n t h e s i s  some  of new mole-  - 8 cules.  A smaller broader  peak of g l y c i n e uptake  RNA o c c u r r e d around 26 to 56 hours  into  postoperatively,  and was found to c o r r e l a t e w i t h the maximum r a t e of RNA s y n t h e s i s .  The major peak of i n c o r p o r a t i o n i n t o  DNA occurred at 26 - 32 hours and c o i n c i d e d with the peak of m i t o s i s , f r e q u e n c y and to a l a r g e extent  with  the time of maximum DNA s y n t h e s i s . IV.  Metabolic I n t e r r e l a t i o n s L i v e r and Tumor  of Regenerating  Tissues.  A number of workers have r e p o r t e d r e s u l t s experiments  of  i n which the n u c l e i c a c i d metabolism of  tumor was s t u d i e d i n animals c o n t a i n i n g r e g e n e r a t i n g liver.  At the same time the e f f e c t  of tumor on regenera-  t i n g l i v e r has been n o t e d , as w e l l as  the e f f e c t  combination of these two t i s s u e s on other host  of  the  tissues.  Experiments of t h i s type have been r e p o r t e d by LePage and Heidelberger  ( 3 ) u s i n g p a r t i a l l y hepatectomized  r a t s b e a r i n g subcutaneous  implants of F l e x n e r - J o b l i n g  carcinoma.  i n d i c a t e d that the presence  Their r e s u l t s  tumor and r e g e n e r a t i n g l i v e r  i n one animal had no  of  effect  on the i n c o r p o r a t i o n of g l y c i n e - 2 - C ^ i n t o r e g e n e r a t i n g liver  or tumor as compared to animals c o n t a i n i n g e i t h e r  regenerating l i v e r  or tumor.  other t i s s u e s of the a n i m a l . ved that  They p r o v i d e d no d a t a Paschkis et a l  for  ( 5 8 ) obser-  i n the presence of r e g e n e r a t i n g l i v e r , the growth  of two tumors of e p i t h e l i a l o r i g i n was a c c e l e r a t e d , a c c e l e r a t i o n was noted i n two sarcomatous  but no  tumors under  similar  conditions. Liver  the presence  r e g e n e r a t i o n was enhanced i n  of a growing tumor r e g a r d l e s s of whether  the growth of the l a t t e r regeneration.  was i n f l u e n c e d by the  The l a t t e r  authors  postulated  liver  the r e l e a s e  of a "growth promoting agent" from the r e g e n e r a t i n g i n t o the c i r c u l a t i o n which was s e l e c t i v e "target"  tumors.  in its  of one member of a p a r a b i o t i c and m i t o t i c a c t i v i t y of  pair  the l i v e r  hepatectomy  causes i n c r e a s e d weight of the other  intact  The nature of the "growth promoting agent"  known as  is  para-  i s un-  the mechanism u n d e r l y i n g the enhancement of  regeneration  i n the presence  In a d d i t i o n B a l i s and a s s o c i a t e s greatly  on  ( 5 9 ) and Wennecker and  vSussman ( 6 0 ) who have r e p o r t e d that p a r t i a l  liver  effect  In agreement w i t h t h i s h y p o t h e s i s was  the work of Bucher and coworkers  biont.  liver  of a growing tumor ( 5 8 ) .  ( 6 l ) have observed a  i n c r e a s e d uptake of adenine, 2 , 6 d i a m i n o p u r i n e ,  hypoxanthine, and g l y c i n e i n t o the purines of t i s s u e s of hamsters trols.  containing regenerating  the presence  as  compared to con-  The same authors have a l s o r e p o r t e d  human sarcoma t r a n s p l a n t e d  animals.  l i v e r , as  ( 6 l ) that  i n t o hamsters was a f f e c t e d  of r e g e n e r a t i n g  liver  i n the  a by  sarcoma-bearing  Using 2 , 6 d i a m i n o p u r i n e , guanine or hypoxanthine  precursor,  there was no a p p r e c i a b l e  change i n the  uptake  by the tumor of the a d m i n i s t e r e d p u r i n e , but when adenine was g i v e n , there was a three f o l d of  t h i s precursor  by the tumor.  increase  i n the uptake  The u t i l i z a t i o n of  by the tumor, on the other hand, was r e d u c e d .  glycine  There were  - 10 no s t r i k i n g changes noted i n the  i n c o r p o r a t i o n of  p r e c u r s o r s i n t o the h o s t ' s t i s s u e s of the hepatectomized-tumor-bearing V..  The Present The  partially  hamsters.  Investigation.  experiments  to compare  the  to be d e s c r i b e d were  in  order  in  N o v i k o f f hepatoma and r e g e n e r a t i n g  undertaken  the metabolism of the n u c l e i c  determine any p o s s i b l e e f f e c t s  rat  acids  l i v e r and to  of one or both of  these  t i s s u e s on the n u c l e i c a c i d metabolism of the t i s s u e s of the h o s t . The  maximum m i t o t i c a c t i v i t y was used as an i n -  dex of the maximum r a t e of growth of the hepatoma and r e generating  liver.  Therefore r a t s c o n t a i n i n g about 4 0 hour  postoperative regenerating approximated  rat  l i v e r were used s i n c e  this  the p e r i o d of maximum m i t o t i c frequency ( 3 6 ,  37,40,41,54,55).  The m i t o t i c  index of the N o v i k o f f hepa-  toma was determined on s u c c e s s i v e days a f t e r  transplan-  t a t i o n and found to be maximal d u r i n g the f o u r t h day of intraperitoneal  growth.  hour r e g e n e r a t i n g compare  In most cases r a t s c o n t a i n i n g 4 0  l i v e r and f o u r t h day tumor were used to  the i n c o r p o r a t i o n of sodium formate-C*^ i n t o  n u c l e i c a c i d purines and p y r i m i d i n e s of these Formate was used as a measure of the de novo of  n u c l e i c a c i d since  it  is  incorporated  tissues. biosynthesis  into positions  and 8 of the purine r i n g v i a the formate donor tetrahydro of  folic acid  thymine.  and i n order  (62,63),  a n  In v i v o experiments  d  2  N^-formyl  i n t o the methyl  were performed  to i n c r e a s e the s i g n i f i c a n c e  the  of the  group  throughout findings,  -  11 -  groups of 2 to 4 r a t s were used i n each experiment and the t i s s u e s p o o l e d . The r e s u l t s  to toe d e s c r i b e d i n t h i s  demonstrated a greater  i n c o r p o r a t i o n of f o r m a t e - C * ^ i n t o  the n u c l e i c a c i d of r e g e n e r a t i n g normal l i v e r .  thesis  As w e l l ,  liver  as  compared to  a higher 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 of the hepatoma was observed, compared to r e g e n e r a t i n g s u l t s of o t h e r s ,  liver.  i n which p a r t i a l l y  b e a r i n g animals were s t u d i e d , i n d i c a t e d that the presence Novikoff  In c o n t r a s t  as  to the r e -  hepatectomized-tumor-  the r e s u l t s  described herein  of r e g e n e r a t i n g  l i v e r and  hepatoma i n the same r a t had no s i g n i f i c a n t  on the i n c o r p o r a t i o n of formate of  i n t e s t i n a l mucosa,  as  compared to the a p p r o p r i a t e  effect  i n t o the n u c l e i c a c i d bases  spleen, regenerating l i v e r control.  or tumor,  These r e s u l t s  were  i n t e r p r e t e d as an i n d i c a t i o n of the autonomy of tumor and regenerating  l i v e r , since  growing t i s s u e s  the presence  of these two r a p i d l y  i n one a n i m a l , d i d not a f f e c t  to i n c o r p o r a t e formate  their a b i l i t y  into their nucleic acids.  f i n d i n g s were even more s t r i k i n g i n view of the which showed that  the a v a i l a b l e  r a p i d l y d e p l e t e d soon a f t e r  radioactive  These results  formate was  a d m i n i s t r a t i o n as a r e s u l t  of  the r a p i d and ej&tensive e x c r e t i o n of r a d i o a c t i v i t y i n the e x p i r e d C 0 9 and u r i n e .  - 12  Therefore  i t was of i n t e r e s t  mine the i n c o r p o r a t i o n of formate generating  to  deter-  i n t o the r e -  l i v e r of tumor-bearing animals which  had been i n j e c t e d w i t h formate-C 1 ^" p r i o r to partial  hepatectomy.  ment i n d i c a t e d that the formate of  The r e s u l t s of t h i s  there was l i t t l e d i f f e r e n c e  than  of the l i v e r s of the tumor-  bearing control animals. formate uptake  in  acids  l i v e r s of these animals  i n t o the n u c l e i c a c i d s  similar  experi-  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  the r e g e n e r a t i n g  the  Furthermore the extent  of  i n t o the tumor and host t i s s u e s was  i n the two s e t s of  animals.  A p r e l i m i n a r y experiment was  performed  to determine the d i s t r i b u t i o n of r a d i o a c t i v i t y i n animals which had been i n j e c t e d w i t h a suspension  of N o v i k o f f  hepatoma.  radioactive  It was hoped that  such an experiment would a l s o provide an o p p o r t u n i t y to study the n u c l e i c a c i d metabolism of the growing Novikoff  hepatoma.  Although many of the r e s u l t s  t h i s experiment were not s i g n i f i c a n t , dent that approximately 20 per c e n t ,  i t was  evi-  of the r a d i o -  a c t i v i t y a d m i n i s t e r e d i n the tumor suspension, excreted  of  i n the u r i n e d u r i n g the f i r s t  was  24 hours of  tumor growth. The r e s u l t s  of the experiments which have  -  13 -  been d e s c r i b e d are not completely i n a c c o r d w i t h the f i n d i n g s of o t h e r s . apparent d i f f e r e n c e s  The s i g n i f i c a n c e  has been  discussed*  of these  - 14 EXPERIMENTAL A.  METHODS I.  Materials. Male Sprague-Dawley  the colony at  rats  (180-200 grams) from  the U n i v e r s i t y of B r i t i s h Columbia, were  used i n a l l experiments.  The animals were fed  Buckerfield*s  1 Vita-Gras Pellets  ad l i b i t u m .  The N o v i k o f f hepatoma has been m a i n t a i n e d ,  in  the Department of B i o c h e m i s t r y , U n i v e r s i t y of B r i t i s h Columbia, ml.  by weekly i n t r a p e r i t o n e a l  t r a n s p l a n t i o n of 0.3 or 0 . 4  of a 1 i n 5 d i l u t i o n of a minced tumor c e l l  in physiological herein,  saline.  For the experiments  0.5 ml* of the tumor c e l l  p l a n t e d i n t o each r a t  suspension  described  suspension was  trans-  i n order to o b t a i n a l a r g e r  mass of  tumor. Sodium formate-C*4  w  a  s  obtained from Atomic Energy  of Canada and from Merck and Company L i m i t e d .  For  i t was d i s s o l v e d  water.  II.  Partial  i n 5 m l . of s l i g h t l y a l k a l i n e Hepatectomies.  The p a r t i a l  hepatectomies  anesthesia, using e s s e n t i a l l y Higgins and Anderson ( 3 6 ) .  were performed under  ether  the same method as d e s c r i b e d by  No p r e c a u t i o n s  Manufactured by B u c k e r f i e l d 1 s Columbia.  injection  were taken to  L i m i t e d , Vancouver, B r i t i s h  obtain a s t e r i l e  t e c h n i q u e , although c l e a n l i n e s s  observed d u r i n g the procedure. was l i g a t e d at and e x c i s e d .  The l e f t  lateral  the p e d i c l e u s i n g 00 b l a c k s i l k  lobe suture  The middle lobe was then e x c i s e d  s i m i l a r manner.  was  in a  The abdomen was c l o s e d i n one l a y e r  u s i n g 000 or 00 b l a c k s i l k continuous s u t u r e s .  The  animals were maintained on t h e i r usual d i e t before and after  operation.  A low p o s t o p e r a t i v e  m o r t a l i t y was ob-  tained. Removal of the l e f t of  the r a t  liver  lateral  and median lobes  r e p r e s e n t s a l o s s of approximately 70%  (36) of the i n t a c t l i v e r .  The normal l i v e r s ,  of  the  animals used, had a wet weight of 6 . 5 to 8 . 5 grams. T h e r e f o r e the remnant remaining a f t e r  p a r t i a l hepatectomy was  c a l c u l a t e d to weigh 2.0 to 2.5 grams. r e g e n e r a t i o n the l i v e r  After  40 hours of  remnant weighed 3.0 to 4 . 5 grams,  i n d i c a t i n g an increment i n l i v e r weight of 1 to 2 grams d u r i n g t h i s p e r i o d of r e s t o r a t i o n .  The wet weight of f o u r t h  day tumor was approximately 1.5 to 2.5 grams. significant difference, of r e g e n e r a t i n g  liver  from these v a l u e s ,  There was no  i n the weight  or tumor i n the p a r t i a l l y  hepatecto-  mized-tumor b e a r i n g a n i m a l s . III.  M i t o t i c Counts. In comparing the i n c o r p o r a t i o n of  i n t o the n u c l e i c a c i d s  of N o v i k o f f  rat  that  l i v e r , i t was f e l t  formate-C^  hepatoma; and r e g e n e r a t i n g  the d e t e r m i n a t i o n of formate i n -  c o r p o r a t i o n i n t o each t i s s u e should be made at  the time of  -  16 -  maximal r a t e of growth of each t i s s u e . n u c l e i c a c i d metabolism under comparable  In t h i s way the  of each t i s s u e c o u l d be  conditions  of growth.  studied  The p e r i o d of  maximum m i t o t i c frequency was used as an index of maximum r a t e of growth of each  tissue.  As p r e v i o u s l y s t a t e d , the l i t e r a t u r e frequency  i n f o r m a t i o n obtained  from  i n d i c a t e d that the p e r i o d of maximum m i t o t i c  of r e g e n e r a t i n g  hours a f t e r  the  the p a r t i a l  l i v e r o c c u r r e d approximately  hepatectomy.  In order  40  to d e t e r -  mine the p e r i o d of maximum m i t o s i s of the N o v i k o f f hepatoma, m i t o t i c  indices  of  intervals  after  paration,  the n u c l e i of  the microscope,  transplantation.  individual fields  injecting colchicine prior  were  in identifying  T h i s problem was overcome by  to removing the tumor t i s s u e ,  d e s c r i b e d by Leblond and tfalker  (64).  r a t was  w i t h c o l c h i c i n e (0.1  injected  subcutaneously  gram per 100 grams body weight) animals.  In t h i s way, c e l l  stopped at readily  and made for  d e t e r m i n a t i o n of the m i t o t i c Portions of  as  Each tumor-bear ing  6 hours p r i o r  milli-  to k i l l i n g  n u c l e i undergoing m i t o s i s  the metaphase s t a g e .  identified,  pre-  undergoing  because of d i f f i c u l t y  stages of m i t o s i s .  daily  were counted under  P r e l i m i n a r y m i t o t i c counts  and i n a c c u r a t e ,  the v a r i o u s  After h i s t o l o g i c a l  and the percentage of n u c l e i  m i t o s i s was determined. difficult  the tumor were determined at  the  were  T h i s phase of m i t o s i s was a faster  and more a c c u r a t e  activity.  of the s o l i d hepatoma,  the tumor mass, were obtained at d a i l y  from the intervals  periphery after  transplantation  17 -  from separate tumor-bearing r a t s .  The  t i s s u e s were prepared for m i t o t i c counts u s i n g two techniques. 1. fixed  "Standard"  Technique.  The f r e s h  tumor t i s s u e was  i n f o r m a l i n , s t a i n e d with hematoxylin and e o s i n ,  s e c t i o n e d u s i n g a microtome.  and  S t a i n i n g and s e c t i o n i n g of  the t i s s u e was performed by members of the Department of Pathology,  U n i v e r s i t y of B r i t i s h Columbia.  were counted under o i l had been counted for 2.  "Squash"  immersion u n t i l  Technique.  into a layer  of $ 0 0 n u c l e i  A small p o r t i o n of the  one c e l l  slide  Aceto-carmine i s a dye which  s t a i n s chromosomes,  1,000  were counted under o i l  and t h e r e f o r e  immersion u n t i l  n u c l e i had been counted f o r  the  The "squash" was  tageous i n the d e t e r m i n a t i o n of m i t o t i c c o u n t s . fields  fresh  to d i s p e r s e  in thickness.  then s t a i n e d w i t h a c e t o - c a r m i n e . preferentially  a total  fields  each tumor.  tumor was squashed on a microscope tissue  Different  is  advan-  Different  a total  of  each tumor.  IV. C o l l e c t i o n of E x p i r e d C O p . In those experiments  i n which the r a d i o  of the e x p i r e d COg was determined, by Zbarsky and Wright  the procedure as  (65) was u s e d .  was passed.  The e x p i r e d COg was  described  The animals were  i n a s e a l e d metabolism cage through which carbon air  activity  'bubbled'  placed  dioxide-free  through c a r «  bonate-free 5, 10 or 15$ NaOH, depending on the p e r i o d of time d u r i n g which the C 0 2 was c o l l e c t e d .  The a l k a l i n e  c o n t a i n i n g the e x p i r e d C 0 p was q u a n t i t a t i v e l y  solution  collected,  and  - 18 made to volume with C 0 2 - f r e e water.  Appropriate a l i q u o t s  were removed ( i f necessary n o n - i s o t o p i c Na 2 C0^ was added as c a r r i e r ) , and the carbonate V . C o l l e c t i o n of  p r e c i p i t a t e d as  BaCO^.  Urine.  Urine specimens were c o l l e c t e d i n a l l i n which f o r m a t e - C 1 ^ was a d m i n i s t e r e d .  In those  experiments experiments  i n which the e x p i r e d CO^ was c o l l e c t e d , the u r i n e was c o l l e c ted at  3, 6, 12, 24, 3&» 48 e t c .  the r a d i o a c t i v e was c o l l e c t e d at  formate.  the i n j e c t i o n of  other experiments  the u r i n e  12 hour i n t e r v a l s . Toluene or mercuric  c h l o r i d e was added as VI.  In a l l  hours a f t e r  preservative.  I s o l a t i o n of Purines and P r y i m i d i n e s . The animals were k i l l e d by e x s a n g u i n a t i o n under  the a n e s t h e s i a and the t i s s u e s q u i c k l y removed and f r o z e n . The v a r i o u s  t i s s u e s from the group of animals were p o o l e d ,  and the l i p i d s e x t r a c t e d ethanol-ether  by homogenizing i n 95$ e t h a n o l ,  (3:1) and e t h e r .  d r i e d and the sodium n u c l e a t e s i n 10% NaCl hours.  The f a t - f r e e  t i s s u e s were  e x t r a c t e d by c o n t i n u a l s t i r r i n g  (20 m l . per 0.6 gram d r y weight) at 85°C for 6  The sodium n u c l e a t e s  were p r e c i p i t a t e d by the a d d i t i o n  of 3 volumes of 95% ethanol and c h i l l e d o v e r n i g h t .  The pre-  c i p i t a t e d sodium n u c l e a t e s were e x t r a c t e d w i t h 10 volumes of 5% NaCl by s t i r r i n g at 85°C f o r 15 minutes, and r e p r e c i p i t a t e d w i t h 3 volumes of 95% ethanol and c h i l l e d f o r 2 to 3 h o u r s . The sodium n u c l e a t e s were c o l l e c t e d by c e n t r i f u g a t i o n , w i t h e t h a n o l , and e t h e r ,  and d r i e d .  washed  For t i s s u e s w i t h a wet  - 19 weight of l e s s  than 1 gram, the n u c l e i c a c i d s were  ex-  t r a c t e d a c c o r d i n g to the method of Hecht and P o t t e r  (47 )•  RNA and DNA were separated by the procedure of T y n e r , Heidelberger and LePage  (5).  The DNA and r i b o n u c l e o t i d e s  were then h y d r o l y s e d w i t h p e r c h l o r i c a c i d as d e s c r i b e d by Marshak and Vogel  (66).  The h y d r o l y s a t e was n e u t r a l i z e d  toy the a d d i t i o n of $0% potassium h y d r o x i d e , c h i l l e d ,  and  the p r e c i p i t a t e d potassium p e r c h l o r a t e removed by c e n t r i f u g a tion.  The purines and p y r i m i d i n e s , c o n t a i n e d i n the  natant,  super-  were separated by ion exchange chromatography on  columns of Dowex-50 ( 2 0 0 - 4 0 0 mesh ), H  form.  In most i n -  stances columns measuring 1 x 15 cm. were used, however f o r smaller q u a n t i t i e s ,  the columns were reduced to 1 x 8 cm.  D i s t i l l e d water was used as f o l l o w e d by 1 . 5 N H C 1 .  e l u e n t for  the f i r s t  20 to 30 m l . ,  The e l u a t e was c o l l e c t e d i n  fractions  of 2 to 3 ml- u s i n g a Technicon Timeflow F r a c t i o n C o l l e c t o r . VII.  U l t r a v i o l e t Spectrophotometry. The o p t i c a l d e n s i t i e s ,  fractions  the  from the i o n exchange columns were determined u s i n g  a Beckman DU spectrophotometer. individual dryness,  at 260 m i l l i m i c r o n s , of  The f r a c t i o n s  c o n t a i n i n g the  p y r i m i d i n e s and p u r i n e s were p o o l e d , evaporated  d i s s o l v e d i n 0 . 1 N H C l and made up to volume.  to  The  a b s o r p t i o n spectrum of each base was determined u s i n g a Beckman DK-2 r a t i o r e c o r d i n g spectrophotometer,  and the concen-  t r a t i o n s determined from the f o l l o w i n g molar e x t i n c t i o n coefficients2:  Determined for  this  instrument by A . H o r i .  - 20 uracil  -  7.56 x 10  thymine -  7.91  3  at 257.5 m i l l i m i c r o n s ,  x 10-^ at 263.5 m i l l i m i c r o n s ,  c y t o s i n e - 10.36 x 10^ at 274*5 m i l l i m i c r o n s , guanine - 11.42 x 10^ at 248  millimicrons,  adenine - I3.5O x 10-^ at 261.5 m i l l i m i c r o n s . V I I I . R a d i o a c t i v e Counting Procedures. All l e s s gas  r a d i o a c t i v e c o u n t i n g was done i n a window-  flow counter.  Suitable aliquots  of the p u r i n e  and p y r i m i d i n e s o l u t i o n s were counted on aluminum planchets covered w i t h lens paper.  Specific a c t i v i t i e s  of the purines  and p y r i m i d i n e s were expressed as counts per minute per micromole ( c . p . m . /uM) and the r e s u l t s for each experiment compared i n terms of r e l a t i v e s p e c i f i c a c t i v i t y equal  (R.S.A.)  to: s p e c i f i c a c t i v i t y of purine or p y r i m i d i n e ( c . p . m . / u M ) x 10-* s p e c i f i c a c t i v i t y of i n j e c t e d formate (c.p.m./jiM) Barium carbonate  t h i c k " samples.  samples were counted as  The minimum weight of barium carbonate  q u i r e d to produce an " i n f i n i t e l y be 120.0  mg. for  "infinitely re-  t h i c k " sample was found to  the p a r t i c u l a r assembly used (67).  In order  to c o r r e c t to t h i n samples the counts were m u l t i p l i e d by a factor  of 4«33*  T h i s f a c t o r was determined by o x i d i z i n g a  s u i t a b l e a l i q u o t of f o r m a t e - C l 4 to C 1 ^ 0 2 , which was as BaC  The l a t t e r  was counted and the amount of  a b s o r p t i o n determined by reference the f o r m a t e - C l 4 (67).  precipitated self  to a t h i n sample count of  - 21 In order  to prevent the l o s s of v o l a t i l e  active acidic material, alkaline.  each u r i n e c o l l e c t i o n was made  The sample was made up to volume with a l k a l i n e  water and the r a d i o a c t i v i t y determined by d r y i n g on aluminum p l a n c h e t s . absorption.  radio-  aliquots  No c o r r e c t i o n was made for  self  -  22  Bu RESULTS. I.  M i t o t i c Counts. On examining the s l i d e s prepared by the  technique i t was found that  the tumor n u c l e i c o u l d be  e a s i l y d e t e c t e d and counted, s i n c e d i s p e r s i o n of the tumor c e l l s . not c o n t a i n tumor c e l l s  "squash"  there was adequate  Furthermore the f i e l d s  i n which the n u c l e i were a b s e n t .  the other hand, s l i d e s prepared by the " s t a n d a r d " were d i f f i c u l t one c e l l  many c e l l s nuclei.  to examine because most f i e l d s  in thickness, were  the c e l l s  i t was decided that  gave more a c c u r a t e m i t o t i c counts  On  technique,  were more than  were crowded t o g e t h e r ,  cut by the microtome so as  Therefore  did  and  to exclude  their  the "squash" method  than the " s t a n d a r d "  tech-  nique. An e s t i m a t i o n  of the m i t o t i c a c t i v i t y of tumor  growing d u r i n g the 1st and 2nd days a f t e r  transplantation  was not p o s s i b l e because i n s u f f i c i e n t  tumor t i s s u e was ob-  tainable  index of 7th day tumor  at  these times.  The m i t o t i c  was a l s o not determined because by t h i s animals have d i e d as a r e s u l t  time many of  of the tumor; and moreover  tumor obtained from those animals which do s u r v i v e n e c r o t i c and hemorrhagic mitotic  is  is  the  too  to permit the d e t e r m i n a t i o n of  the  index. From the r e s u l t s of the m i t o t i c counts  it  the  evident  that N o v i k o f f  hepatoma  (Table  i s most a c t i v e  c a l l y d u r i n g the 4th day of i n t r a p e r i t o n e a l  growth.  I)  mitotiDuring  the f o l l o w i n g two days of growth, the m i t o t i c frequency  de-  - 23 c r e a s e s , and on the 6 t h day only about are  \% of  the n u c l e i  in mitosis.  TABLE I of  M i t o t i c i n d i c e s of N o v i k o f f Hepatoma, as t o t a l n u c l e i i n metaphase.  percent  Percent of t o t a l Nucl e i i n Metaphase "Standard" Technique -'Squash' technique (1000 n u c l e i counted) (500 n u c l e i counted)  Day a f t e r transplantation  3rd  10  16  4th  10  23  5th  6  8  6th  1  •*  Although the m i t o t i c similar  to that  index of 3 r d day tumor was  obtained for 4 t h day tumor u s i n g  "squash" t e c h n i q u e , 4 t h day tumor was p r e f e r r e d of  the l a r g e r  the because  mass of tumor on that day and a l s o  of the p o s s i b i l i t y  that  the m i t o t i c  because  index might be higher  than 3 r d day tumor i n view of the r e s u l t s o b t a i n e d from the " s t a n d a r d "  technique counts.  Therefore animals w i t h  4 t h day tumor were used i n subsequent experiments pare the i n c o r p o r a t i o n of formate-C*^ i n t o rat  to com-  regenerating  l i v e r and N o v i k o f f hepatoma. II.  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 s of T i s s u e s of P a r t i a l l y Hepatectomized and Tumor-bearing E a t s . In a l l  experiments  the s o l u t i o n of sodium  formate-C  - 24 was i n j e c t e d subcataneously rat.  The q u a n t i t i e s  animal,  of f o r m a t e - C 1 ^ i n j e c t e d i n t o each  i n the experiments  Table I I .  i n t o the dorsum of each  to be d e s c r i b e d , are  T h i s t a b l e a l s o summarizes  shown i n  the time r e l a t i o n -  14 s h i p of l i v e r injection, as  regeneration,  as w e l l as  tumor growth, and formate-C  showing the number of animals  a group for each experiment.  Data for  the 6 t h day  tumor-bearing r a t s were obtained by Ztoarsky et a l The data shown for of r e s u l t s  these animals represent  used  (68).  the average  obtained from two or three s i n g l e animal  ex-  per iments. The s p e c i f i c were determined i n a l l found to be n e g l i g i b l e .  activities  experiments,  and c y t o s i n e  and i n most cases were  In some instances  t i o n of f o r m a t e - C 1 ^ i n t o u r a c i l cant,  for u r a c i l  the i n c o r p o r a -  and c y t o s i n e was  signifi-  but was always c o n s i d e r a b l y l e s s than the i n c o r p o r a -  t i o n i n t o adenine and guanine, and g e n e r a l l y l e s s than t h y mine.  Therefore only the data  guanine are p r e s e n t e d .  for  thymine, adenine and  The s p e c i f i c  a c t i v i t y of DNA ade-  nine and guanine was higher than thymine i n a l l However the d i f f e r e n c e  in specific  tissues.  a c t i v i t y between the DNA  p u r i n e s and thymine was not constant and v a r i e d from one t i s s u e to another. ments,  that  It was a l s o noted i n d u p l i c a t e  t h i s d i f f e r e n c e was not constant  for a  experisingle  t issue. Figures 1-4 activities  illustrate  the r e l a t i v e  specific  of adenine and guanine obtained from the DNA  and ENA of l i v e r , tumor, spleen and i n t e s t i n a l mucosa of  TABLE II Time r e l a t i o n s h i p o f l i v e r r e g e n e r a t i o n , tumor g r o w t h , a n d f o r m a t e - C A 4 i n c o r p o r a t i o n a t time r a t s were s a c r i f i c e d . Number o f r a t s i n e a c h g r o u p . Q u a n t i t i e s of formate and C i n ,jec ted • C. p.m. of Mg o f Number Day o f Description of Hours formate i 4 tumor of of C injecinjected r at s gr ow t h 1 iver ted i n t o into in i n Secanimals each e a ch r a t gr oup tion rat 1.  Partiaily hepa t e c toinized  2.  Tumorbear i n g  Partially hepatectoroizedtumor bear i n g 4. Tumorbear i n g delayed hepatectomy 5 . Tumorbear i n g con t i o l for 4. b . Normal -  40  3.  -  3  3.72  x  10  7  4th 6th  3 2-3  3.33 7.5O  x  10 10  7  3.33 4.02  x  7  x  10 10  (single)  ma t e - C 4 1  incorporation i n hours  O.85  24  1.02 1.03  24 24  1.02 1.02  24  40  4 th  40  4th  3 2  40  6 th  4  3.96  x  10  7  1.02  72  6th  4  3.96  x  10  7  1.02  72  2-3  3.96 7.50  x I07 x 1077  1.02 1.03  72  6 th —  (single) *  x  7  Per i o d of f o r -  D a t a o f Z'barsky ex a l  (68).  7  24  24  To  race rage  R N A APE  NINE  G UAN  I N E  o«  D N A A D E N I N E GU AN I N E  >-  >  300-  O  <l  o o  200-  UJ  CL CO LU > — I <  100-  _J  LU  or  Partially F o u r t h day Partialiy  hepatectomized tumor-bearing. hepatectomized-tumor-bearing.  Normal  F i g u r e 1.  R e l a t i v e s p e c i f i c a c t i v i t i e s of n u c l e i c a c i d p u r i n e s of 1 i v e r from p a r t i a l l y hepatectomized, tumor-bearing, p a r t i a l l y hepatectomized-tumorb e a r i n g , a n d n o r m a l r a t s ; 24 h o u r s a f t e r t h e i n j e c t i o n of formate-  p a r t i a l l y hepatectomized,  tumor-bearing,  hepatectomized-tumor-bearing, relative  specific  these t i s s u e s are (i)  activities illustrated  partially  and normal r a t s .  The  of the DNA thymine from i n F i g u r e 5»  P a r t i a l l y hepatectomized r a t s .  In order  to  determine the i n c o r p o r a t i o n of formate-C^^ i n t o the nucleic acids  of r e g e n e r a t i n g r a t  r a t s was p a r t i a l l y hepatectomized w i t h formate-C*4 16 hours a f t e r four hours a f t e r sacrificed.  the formate  l i v e r , each of  three  ( 3 6 K and i n j e c t e d  the o p e r a t i o n .  Twenty-  i n j e c t i o n the animals were  The n u c l e i c a c i d purines and p y r i m i d i n e s  from the r e g e n e r a t i n g  l i v e r , s p l e e n , and i n t e s t i n a l  mucosa  were analysed for r a d i o a c t i v i t y as d e s c r i b e d .  The r e s u l t s  obtained for the purines and thymine of 40 hour  regenera-  t i n g l i v e r alte shown i n F i g u r e s 1 and 5 r e s p e c t i v e l y . comparing the r e l a t i v e  specific  activities  On  of the n u c l e i c  a c i d adenine, guanine and thymine of the r e g e n e r a t i n g with the corresponding n u c l e i c a c i d f r a c t i o n s liver,  i t i s evident that the n u c l e i c a c i d s  liver  of normal  of the former  t i s s u e i n c o r p o r a t e formate-C*^ to a much greater degree than those of normal l i v e r .  The d i f f e r e n c e  i s most  apparent  on comparing the purine d a t a . Examination of the d a t a obtained for s p l e e n and i n t e s t i n a l mucosa ( F i g u r e s presence  3-5)  indicates  of 40 hour r e g e n e r a t i n g r a t  that  the  l i v e r has no e f f e c t  the i n c o r p o r a t i o n of f o r m a t e - C ^ i n t o the n u c l e i c a c i d  on  To f a c e Page 2b,  R A D E N I N E  N  A  D  G UAN  INE  N  A D E N I N E  A  GUANINE  >^ 3 0 0  o < CJ ^200LLI CL CO LU  >  IOOLU  or  F o u r t h day t u m o r - b e a r i n g . Partially  h e p a t e c t o m i z e d - t u m o r - b e a r i ng.  S i x t h day tumor-bearing.  F i g u r e 2.  Relative specific a c t i v i t i e s of nucleic acid p u r i n e s o f N o v i k o f f hepatoma from p a r t i a l l y h e p a t e c t o m i z e d , t u m o r - b e a r i n g , p a r t i a l l y hepat e c t o m i z e d - t u m o r - b e a r i n g , a n d n o r m a l r a t s ; 24 hours a f t e r the i n j e c t i o n of formate-cH.  - 26 purines and thymine of e i t h e r spleen or i n t e s t i n a l mucosa. ( i i ) Tumor-bearing r a t s .  As p r e v i o u s l y s t a t e d ,  animals b e a r i n g 4 t h day Novikoff hepatoma were for  selected  these experiments because of the h i g h m i t o t i c a c t i -  v i t y observed d u r i n g the 4 t h day of i n t r a p e r i t o n e a l growth.  Three r a t s were i n j e c t e d i n t r a p e r i t o n e a l l y w i t h  0.5 m l . of a tumor suspension. p e r i o d 48 to 72 hours a f t e r  At a time d u r i n g the  transplantation,  each animal  was i n j e c t e d subcutaneously with formate-C*4, and s a c r i f i c e d 24 hours a f t e r  the formate  injection.  each r a t contained 4th day tumor at  Therefore  the time i t  wassacri-  f i c ed. The r e s u l t s activities  obtained for  the r e l a t i v e  of the n u c l e i c a c i d bases of l i v e r , tumor,  spleen and i n t e s t i n a l mucosa are represented 1 to 5.  specific  The data shown for  in Figures  the 4 t h day tumor ( F i g u r e s 2  and 5) i n d i c a t e d a h i g h degree of i n c o r p o r a t i o n of  for-  mate-C*4 i n t o the n u c l e i c a c i d purines and thymine. extent  of i n c o r p o r a t i o n i n t o the tumor i s greater  The  than  that obtained for r e g e n e r a t i n g l i v e r and normal s p l e e n , but approximately of the same order as testinal  that shown for i n -  mucosa. It may be noted a l s o that the presence of 4 t h  day Novikoff hepatoma has l i t t l e or no i n f l u e n c e on the de novo s y n t h e s i s  of the n u c l e i c a c i d s  of the host  liver,  R  N  A D E N I N E  A  D  G UAN  I N E  N  A  A D E N I N E  GU A N I N E  ^300  o  Partially  hepatectomized.  F o u r t h day tumor-bearing. Partially  hepatectomized-tumor-bearing.  Normal  F i g u r e 3.  Relative s p e c i f i c a c t i v i t i e s of nucleic acid p u r i n e s . o f spleen from p a r t i a l l y hepatectomized, tumor-bearing, p a r t i a l l y hepatectomized-tumorb e a r i n g , and n o r m a l r a t s ; 24 h o u r s a f t e r t h e i n j e c t i o n of formate-C . i 4  -  27  -  s p l e e n and i n t e s t i n a l mucosa.  In most i n s t a n c e s i t i s  seen that the degree of i n c o r p o r a t i o n of formate the n u c l e i c a c i d p u r i n e s and thymine  into  of the host t i s s u e s  of the tumor-hearing animals i s s i m i l a r  to the formate  i n c o r p o r a t i o n i n t o the c o r r e s p o n d i n g f r a c t i o n of the t i s s u e s of normal r a t s . Figure 3 ,  The one e x c e p t i o n , as shown i n  i s the t w o - t h i r d s decrease i n r e l a t i v e  a c t i v i t y of ENA  specific  adenine obtained from the spleens of the  4 t h day tumor-bearing animals, compared to the ENA of normal  adenine  spleen. The d a t a obtained by Zbarsky et a l (68)  for  6 t h day N o v i k o f f hepatoma are presented i n F i g u r e s 2 and 5.  In c o n t r a s t  to the r e s u l t s o b t a i n e d from m i t o t i c  counts of 4 t h and 6 t h day tumor, which i n d i c a t e d a n e g l i g i b l e m i t o t i c a c t i v i t y d u r i n g the 6 t h day of toneal growth;  the d a t a shown i n d i c a t e no  intraperi-  significant  d i f f e r e n c e i n the degree of i n c o r p o r a t i o n of formate the n u c l e i c a c i d s of 4 t h  into  and 6 t h day tumor.  ( i i i ) P a r t i a l l y hepatectomized-tumor-bearing Since n e i t h e r r e g e n e r a t i n g l i v e r  rats.  or 4 t h day N o v i k o f f hepa-  toma had an e f f e c t on the i n c o r p o r a t i o n of formate  into  the n u c l e i c a c i d s of the host t i s s u e s of animals c o n t a i n i n g e i t h e r one of the above r a p i d l y growing tissues, i t was interest  of  to determine whether the two t i s s u e s present i n  one animal would a f f e c t host t i s s u e s .  the formate i n c o r p o r a t i o n i n t o the  At the same time an o p p o r t u n i t y would  be  provided to study the e f f e c t of Novikoff'hepatoma and r e -  To f o l l o w Page 2 7 .  R  N  A D E N I N E  A  D  G UAN  INE  N  A D E N I N E  A G U A N I N E  >3^300  o < o o  2 0 0 -  LU  CL CO UJ  > 100-  UJ  or Partially  hepatectomized.  Fourth day tumor-bearing. Partially  hepatectomized-tumor-bearing.  Normal  F i g u r e 4.  R e l a t i v e s p e c i f i c a c t i v i t i e s of n u c l e i c a c i d p u r i n e s o f i n t e s t i n a l mucosa f r o m p a r t i a l l y h e p a t e c t o m i z e d , t u m o r - b e a r i n g , p a r t i a l l y hepat e c t o m i z e d - tumor - b e a r i n g , a n d n o r m a l r a t s ; 24 hours a f t e r the i n j e c t i o n of f o r m a t e - C . l 4  To f a c e  Partially F o u r t h day  P a t e 2b.  hepatectomized. tumor-hearing.  Partially  hepatectomized-tumor-bearing  S i x t h day  tumor-bearing.  ( 4 t h day),  Normal.  Figure  5.  R e l a t i v e s p e c i f i c a c t i v i t i e s o f DNA t h y m i n e o f l i v e r , N o v i k o f f h e p a t o m a , s p l e e n and i n t e s t i n a l m u c o s a , from p a r t i a l l y h e p a t e c t o m i z e d , t u m o r bearing, p a r t i a l l y hepatectomized-tumor-bearing, and n o r m a l r a t s ; 24 h o u r s a f t e r the i n j e c t i o n o f t ormate-C A '+.  - 28 generating  liver  on  each  With these regenerating Partial  liver  aims  and  i n mind, r a t s  Novikoff  The  which time  they  as w e l l a s 4th  day  animals  containing  hepatoma were  h e p a t e c t o m i e s were p e r f o r m e d on  bearing rats. at  other.  40  contained Novikoff  hour  prepared.  three  were s a c r i f i c e d  tumor-  40  hours  regenerating  hepatoma.  both  Similar  later,  liver  procedures  14 were c a r r i e d was  on a s e c o n d g r o u p o f 2 r a t s .  out  administered  sacrificed.  g r o u p s 24  to both  The  relative  nucleic  a c i d bases of  spleens  and  intestinal  Formate-C t h e y Were  hours before  specific  activities  the r e g e n e r a t i n g  of  livers,  the  tumors,  mucosa o f e a c h g r o u p were  deter-  mined. The  from  partially apparent  the  two  that  the  these  experiments  an a v e r a g e o f t h e  groups.  animal,  has  1  intestinal  simultaneous  mucosa, a s  any  hepatoma, or difference  no  regenerating  liver  the  and  shown  the  the r e s u l t s  Furthermore  since  formate uptake  fox  it is two  Novikoff  a c i d s of the host to  ob-  rapidly  hepatoma),  i n c o r p o r a t i o n of  of r e g e n e r a t i n g  converse,  i n the  on  compared  mucosa.  effect  the  effect  the n u c l e i c  s p l e e n and  to s u g g e s t  presence of  in  results  From the r e s u l t s  (regenerating l i v e r  formate-C ^ into  normal  for  hepatectomized-tumor-bearing animals  growing t i s s u e s i n one  presented  1 to 5 r e p r e s e n t  Figures tained  data  there into  there liver i s no  spleen  and  shown f o r i s no  on  evidence  Novikoff  significant  the n u c l e i c  or N o v i k o f f hepatoma i n a n i m a l s  acids con-  of  - 29 taining that  these  there  tissues  is  a  incorporation regenerating  remarkable  of  formate  liver  taining  one  ponding  functions  tissues. of  the  data  of  or  these  These  data  depleted  as  a  activity  in  the  into  the  even more expired  available  of  the  and  urine,  mediately  following  the  injection  partially  hepatectomized  COp  and  animals tumor,  UEx-cretion  Urine.  The  containing as  well  as  of  of  and  in  40  regenerating  both  groups  24  the  collected  of  formate  and  at  rapidly radio im-  into  4  liver  the  from  the  those 4th  or  partially  three  the  This  Expired  f i r s t  of  view  of  1  hourly  administration  in  hours  C o l l e c t i o n s were  the  both  was  rats.  for  corres-  urine.  tectomized- tumor-bear ing intervals  con-  containing  2  of  animals.  C0  from  the  formate-C  expired hour  to  the  Radio=activity was  bases  excretion  tumor-bearing  of  animals  formate-C^^  during  2  acid  in  seen  degree  striking  C0  extensive  CO2  (iv)  nucleic  animals  is  the  compared  from  the  It  in  hepatoma  as  were  for  result  the  Novikoff  results  together.  similarity  obtained  that  or  tissues  obtained  indicated  singly  hours  day hepa-  made  at  following  longer  the  intervals  thereafter. As each the  expired radio  stated, C0  2  collection  activity  were  corrected  (see  Methods)  for in  C0  the  of  the  self  order  2  contained was  in  an  aliquot  converted  to  BaCO-j,  latter  determined.  absorption to  convert  Ihe  and m u l t i p l i e d to  thiri  sample  of and  counts by  4*33  counts.  To f a c e  Page  30.  TABLE  III  B a d i o a c t i v i t y e x c r e t e d i n e x p i r e d CO^ d u r i n g 24 h o u r s i m m e d i a t e l y following subcutaneous i n j e c t i o n of f o r m a t e - C ^ .  Descr i p t i o n of Animals  Percent  of  total  Hours a f t e r  0-1  1-2  Badio a c t i v i t y  injected  Formate-C^^inlection  2-3  3-6  6-12  12-24  24 h o u r total  Partially hepatectomized  29.4  10.1 3.3  3-3  2.0  1.9  50.0  lumorbear i n g  29.4  8.9 2.0  1.9  1.5  1.5  45.3  16.8  12.0 2.8  3.1  2.1  1.8  38.6  Partially hepatectomized tumorbear i n g  -  30 -  In  t h i s way the t o t a l r a d i o a c t i v i t y  i n each c o l l e c t i o n  of  e x p i r e d COg was r e l a t e d to the t o t a l r a d i o  activity  a d m i n i s t e r e d as f o r m a t e - C ^ * The r a d i o a c t i v i t y d e t e r m i n a t i o n s f o r the exp i r e d COg a r e shown i n Table I I I .  The d a t a r e p r e s e n t the  r a d i o a c t i v i t y c o n t a i n e d i n the e x p i r e d CO2 expressed as the  percent of t o t a l r a d i o a c t i v i t y  injected.  Figures  g i v e n f o r the p a r t i a l l y hepatectomized tumor-bearing a n i mals r e p r e s e n t an average of the r e s u l t s o b t a i n e d from the two groups of animals. It  i s evident that a c o n s i d e r a b l e f r a c t i o n of the  a d m i n i s t e r e d formate i s o x i d i z e d to CjOg d u r i n g  the f i r s t  hour, a f t e r which the r a d i o a c t i v i t y of the e x p i r e d CO2 d e c r e a s e s . During the 3  r d  hour  the r a d i o a c t i v i t y  i n the  e x p i r e d COg has decreased to about one-tenth o f the i n i t i a l l e v e l , and slowly decreases t h e r e a f t e r . On t o t a l l i n g  i t i s seen that about 40 to 50% of the i n j e c t e d  experiment formate-C tween the  the r e s u l t s f o r the 24 hours of each  14 w  a  s  e x c r e t e d or e x p i r e d COg.  The d i f f e r e n c e be-  e x c r e t e d by the p a r t i a l l y hepatectomized a n i -  mals, and the 38.6% e x c r e t e d by the p a r t i a l l y hepatectomizedtumor-bearing animals, i s probably not s i g n i f i c a n t of  the small number of experiments performed.  i n view  Furthermore  these f i g u r e s are w i t h i n the range of 40 to 60% r e p o r t e d by others f o r normal animals (68). Urine samples were c o l l e c t e d from each group of animals a t p e r i o d i c  i n t e r v a l s f o l l o w i n g the i n j e c t i o n of the  formate-C ^. 1  31 -  Initially  the u r i n e was made to volume  w i t h d i s t i l l e d water and s u i t a b l e a l i q u o t s p l a t e d and d r i e d on aluminum p l a n c h e t s .  On counting these samples  i t was found that there was not s a t i s f a c t o r y agreement between the count of d u p l i c a t e  samples.  S i n c e the a l i -  quots of u r i n e had been d r i e d under an i n f r a - r e d lamp, i t was suggested that be  the d i s c r e p a n c y i n the counts might  the r e s u l t of the e v o l u t i o n of v o l a t i l e r a d i o  material.  Therefore a sample of u r i n e was p l a t e d and  counted. The same sample was then heated a t 100° about 1/2  of the sample was c o n s i d e r a b l y The pH of the u r i n e  found to be about 5 to 6 .  sample was determined and T h e r e f o r e i t was thought  that  of the u r i n e  due to the e v o l u t i o n o f v o l a t i l e r a d i o a c t i v e a c i d i c  material.  T h i s was a p o s s i b l e e x p l a n a t i o n  of agreement i n counts of d u p l i c a t e u r i n e this  heating  lower than the o r i g i n a l  the drop i n r a d i o a c t i v i t y a f t e r h e a t i n g was  C for  hour, and the r a d i o a c t i v i t y a g a i n determined.  It was found that the counts obtained f o l l o w i n g  count.  active  f o r the l a c k samples.  i n mind, each u r i n e sample was a d j u s t e d  With  to about pH  9 by the a d d i t i o n of sodium carbonate. A l i q u o t s of the a l k a l i n e u r i n e were p l a t e d , and where necessary, a l k a l i n e water was used to evenly d i s t r i b u t e the sample on the planchet.  In t h i s way s a t i s f a c t o r y agreement was o b t a i n e d  on counting d u p l i c a t e the u r i n e  Na2C03  samples.  In a l l f u t u r e  experiments  samples were made a l k a l i n e by the a d d i t i o n of  and/then d i l u t e d to volume u s i n g a l k a l i n e water.  32.  To f a c e Page TABLE IV  Badio a c t i v i t y excreted i n u r i n e d u r i n g 24 h o u r s i m m e d i a t e l y f o l l o w i n g subcutaneous i n j e c t i o n ? of formate- C H . Description of a n i m a l s  Partially hepatectomized Tumorbearing Partially hepatectomizedtumor*  Percent o f t o t a l Hours a f t e r  radio activity  injected 24 h o u r total  formate-C 4injection i  0-3  3-6  6-12  12 - 2 4  3-7  3.3  1.1  1.6  9.7  0.9  0.8  1.0  1.1  3.8  2.8  1.2  1.5  1,2  6.7  bearing  TABLE V T o t a l r a d i o a c t i v i t y e x c r e t e d i n e x p i r e d CO a n d u r i n e i n 24 h o u r s i m m e d i a t e l y f o l l o w i n g subcutaneous i n j e c t i o n o f formate-C ^-. 1  Descr i p t i o n of a n i m a l s  Percent of total  0 - 24 h o u r s a f t e r  co Partially hepatectomized. Tumorbearing Partially hepatectomizedtumorbearing  radio activity  2  Urine  injected  forinate-C*4 i n j e c t i o n C0  2  +  Urine  50.0  9.7  59.7  45-3  3.8  49.1  38.6  6.7  45.3  - 32 The  nature of the v o l a t i l e r a d i o a c t i v e a c i d i c  m a t e r i a l was  not  determined.  The u r i n e counts were not c o r r e c t e d for a b s o r p t i o n and  self  t h e r e f o r e the r e s u l t s shown i n Table  IV  are approximations of the r a d i o a c t i v i t y of each u r i n e sample.  The data  v i t y excreted the t o t a l  i n Table  IV r e p r e s e n t  the r a d i o  acti-  i n the u r i n e , expressed as percentages of  injected radio a c i t i v i t y .  of the i n j e c t e d r a d i o a c t i v i t y as compared to that e x c r e t e d i s a suggestion  Considerably  i s excreted  less  i n the  urine  i n the e x p i r e d C 0 2 .  There  that the e x c r e t i o n of r a d i o a c t i v i t y  the u r i n e i s g r e a t e s t d u r i n g  the i n i t i a l  in  few hours a f t e r  the formate i n j e c t i o n , however t h i s i s not as evident the data a l r e a d y presented  f o r the e x p i r e d CO2.  as  There  i s a l s o some v a r i a t i o n , between the s e v e r a l types of a n i mals, i n the percent urine during  of r a d i o a c t i v i t y excreted  the t o t a l 24 hour p e r i o d .  i n the  Again these  v a r i a t i o n s are not s i g n i f i c a n t because of the l i m i t e d number  of experiments performed.  c i d e with  As w e l l  they do not  the data given f o r the e x p i r e d C 0 2 ,  the e x c r e t i o n of r a d i o a c t i v i t y was  coin-  i n which  lowest i n the  partially  hepatec tomi zed-tumor-bear ing animals. On and u r i n e  totalling  (Table V),  the r e s u l t s f o r the e x p i r e d  i t will  be noted that about 4 5  C02 to  60%  of the i n j e c t e d r a d i o a c t i v e formate can be accounted f o r i n the C0 2  and u r i n e excreted d u r i n g the 24 hours immedi-  a t e l y f o l l o w i n g the i n j e c t i o n of formate-C* . 4  (v) Tumor-bearing  fiats  I n j e c t e d w i t h Formate-C  f o l l o w e d by P a r t i a l Hepatectomy,  ^  The uptake of formate  i n t o the n u c l e i c a c i d s of r e g e n e r a t i n g l i v e r and N o v i k o f f hepatoma i n p a r t i a l l y hepatectomized-tumor-bearing animals i n d i c a t e d an autonomy i n the n u c l e i c a c i d metab o l i s m of both r e g e n e r a t i n g r a t l i v e r and N o v i k o f f toma.  hepa-  Each of these t i s s u e s together i n one animal  was  a b l e to i n c o r p o r a t e formate i n t o t h e i r n u c l e i c a c i d s to the same degree as r e g e n e r a t i n g l i v e r separate animals.  or hepatoma i n  Furthermore the formate  incorporation  o c c u r r e d i n s p i t e of the r a p i d e x c r e t i o n of p r e c u r s o r i n the e x p i r e d CO2 and u r i n e . an experiment was  With these r e s u l t s i n mind,  planned to determine the degree of i n -  corporation of radio a c t i v i t y  into regenerating l i v e r i n  tumor-bearing animals which had been i n j e c t e d w i t h formateC*^ p r i o r  to the p a r t i a l  hepatectomy.  Each of 4» 3*d day tumor-bearing r a t s was i n j e c t e d subcutaneously w i t h formate-C^^.  The r a t s were  p l a c e d i n open metabolism cages and the f o l l o w i n g day p a r t i a l hepatectomies were performed on the a n i m a l s . r a t s were k i l l e d 40 hours l a t e r and the l i v e r , s p l e e n and i n t e s t i n a l mucosa a n a l y s e d . time the animals were s a c r i f i c e d  tumor,  T h e r e f o r e a t the  they c o n t a i n e d 6th  hepatoma, 40 hour r e g e n e r a t i n g l i v e r and had been with f o r m a t e - C  14  72 hours beforehand.  3 r d day tumor were i n j e c t e d w i t h f o r m a t e - C * later.  day injected  As c o n t r o l animals  f o r t h i s experiment, 2 groups o f 3 and 4 r a t s ,  f i c e d 72 hours  The  4  containing  and  sacri-  Figure 6. R e l a t i v e s p e c i f i c a c t i v i t i e s of n u c l e i c a c i d purines and DNA thymine of l i v e r , Novikoff hepatoma, spleen, and i n t e s t i n a l mucosa from p a r t i a l l y hepatectomized-tumor-bearing, and tumor-bearing c o n t r o l r a t s ; 72 hours a f t e r i n j e c t i o n o f forraate-C ^. 1  L I V E R Hepatectomized tumorbearing RNA  DNA  HEPATOMA  Tumorbearing control RNA  DNA  fepatectomizedtumorbearing RNA  Tumorbearing control  S P L E E N Hepatectomized tumorbearing  DNA  Hepatectomized tumorbearing  Tumorbearing control  RNA  100-  i  -p •H  -P  Tumorbearing control  MUCOSA  75-  O  o  •H <H •H  c_>  &  5 0  0) >  •H -P  i—I  s  25-  i II  Guanine  i  1  •^Adenine l o s t  Thymine  + One group o f 4 r a t s  Adenine  -  34  -  The r e s u l t s of these experiments t r a t e d i n F i g u r e 6.  The r e l a t i v e s p e c i f i c  are i l l u s activities  shown f o r the tumor-bearing c o n t r o l animals r e p r e s e n t an average of the v a l u e s o b t a i n e d from the two groups. It w i l l be noted that there a r e no s i g n i f i c a n t ces i n the i n c o r p o r a t i o n of formate  differen-  i n t o the n u c l e i c  a c i d p u r i n e s and thymine of the r e g e n e r a t i n g l i v e r s as compared to the c o r r e s p o n d i n g f r a c t i o n s of the l i v e r s from the tumor-bearing animals. for  As w e l l d a t a o b t a i n e d  the N o v i k o f f hepatoma and i n t e s t i n a l mucosa i n d i c a t e  no d i f f e r e n c e  i n the formate  i n c o r p o r a t i o n i n t o the  c o r r e s p o n d i n g n u c l e i c a c i d bases of these t i s s u e s i n the two types of animals.  However a d i f f e r e n c e i s noted i n  the ENA guanine of s p l e e n .  I t i s observed that the r e -  l a t i v e s p e c i f i c a c t i v i t y of the RNA guanine  of the spleens  of the tumor-bearing animals i s s l i g h t l y g r e a t e r than that found f o r the corresponding f r a c t i o n of the tumor-bearing delayed-hepatectomized  animals.  I t i s not p o s s i b l e to  compare the RNA adenine f r a c t i o n s of s p l e e n , s i n c e i n one of the experiments  t h i s f r a c t i o n was l o s t , although i t i s  seen that the RSA of RNA adenine spleen i s r e l a t i v e l y h i g h .  of the tumor-bearing  However there a r e no  cant d i f f e r e n c e s i n the formate  signifi-  incorporation into  corres-  ponding DNA l>ases from the spleens of the two s e t s of a n i mals. It w i l l  be noted that the r e l a t i v e  specific  a c t i v i t i e s obtained f o r the p u r i n e s and thymine i n these  - 35 experiments, 72  hours  before  siderably acid  lower  nal  is  obtained  formate  is  generally  as  the  already  incorporation bearing  of  of  other  activity  to  is  into  formate. the  incorporation of  intesticorres-  studied.  The  the r e l a t i v e l y  the RNA p u r i n e s  i n animals  active  opportunity Novikoff of  stated,  study  high of  suspension  to  study  hepatoma as the  tumor-  hepa-  experiment  of  the N o v i k o f f this  nature  grew w i t h i n  per-  radiowith  a  hepatoma.  Fur-  would p r o v i d e  an  the n u c l e i c a c i d metabolism it  was  the  of  the  peritoneal  rat.  Each of  c.p.m.  was  carried  out  i n the  following  two 6 t h d a y - t u m o r - b e a r i n g r a t s were  on two o c c a s i o n s ,  5 . 3 x 10?  this  the d i s t r i b u t i o n o f  of  The e x p e r i m e n t manner:  Novikoff  w h i c h had been t r a n s p l a n t e d  thermore an experiment  jected  the  were  than i n t o the  Transplanted Radio a c t i v e toma.  i n order  cavity  the  tissues  noted,  As p r e v i o u s l y  radio  nucleic  spleen.  III.  formed  con-  c a n be made r e g a r d i n g  higher  formate  are  which  i n j e c t i o n of  that  injected  corresponding  the n u c l e i c a c i d p u r i n e s  ponding purines exception,  of  w i t h one e x c e p t i o n ,  into  mucosa  were k i l l e d ,  from a n i m a l s  the  observation  that,  f o r m a t e - C ^ was  than those  24 h o u r s a f t e r  A further data  the  the a n i m a l s  fractions,  killed  of  i n which  at  an  interval  of  of C 1 ^ i n I . 3 6 m i l l i g r a m s  one h o u r , of  inwith  formate.  Ninety  minutes  after  36  the  were s a c r i f i c e d , and a transplantation rats the  were  second i n j e c t i o n ,  tumor  prepared  injected  -  cell  suspension for  4 groups of  into  was c o l l e c t e d .  In  of  the  some c a s e s t h e  donor  s e r v e d and a n a l y s e d .  was c a l c u l a t e d  2  rats  0.1  Duplicate  plated,  amount  the  the  of  ml.  of  ml.  portions  of  C ^.  it  of  these  suspeneach  110,100  No d e c r e a s e the  that a  detected  the  the  c.p.m.  same d o n o r s u s p e n s i o n  was i n d i c a t e d  in  in  alkaline negligible  the d o n o r  sus-  The n u c l e i c a c i d s were  f r o m the r e m a i n i n g d o n o r  specific activities  tumor  dose of  as f o r m a t e .  of  From  Therefore  1  the  re-  p l a t e d and c o u n t e d .  and c o u n t e d .  radio a c t i v i t y ,  was p r e s e n t  extracted  4  CO2  expired  was n o t e d a s compared t o  Therefore  pension,  of  received a total  samples. of  following  absorption.  0.5  that  ml ••. - p o r t i o n s  were made a c i d , activity  self  55.O5O c . p . m .  sion contained group of  each.  s u s p e n s i o n was  0.1  Duplicate  No c o r r e c t i o n was made f o r  radio  tumor  s u s p e n s i o n were made a l k a l i n e ,  it  two  of  The u r i n e was c o l l e c t e d f r o m e a c h g r o u p .  Part  counts  the  ml •  n u c l e i c a c i d p u r i n e s and p y r i m i d i n e s  tumors a n a l y s e d .  donor  Eight  w i t h 0.5  intraperitoneally  One g r o u p was s a c r i f i c e d e a c h d a y f o r  the  rats  i n the u s u a l manner.  s u s p e n s i o n and d i v i d e d  d a y s a n d the  the  tumor  s u s p e n s i o n and  purines and  pyrimidines  determined. One g r o u p o f days a f t e r  the  rats  was s a c r i f i c e d on s u c c e e d i n g  transplantation,  a n d the  tumor  tissue  - 37 p o o l e d and a n a l y s e d .  Therefore  specimens of  were o b t a i n e d w h i c h h a d b e e n g r o w i n g toneal^ of  this  for  24,  48,  experiment  mine  for  of  the d o n o r  hours of vities  each of  the  the  most c a s e s  the  count  midines of  the  growing  it  is  at  these  therefore  present the  times.  It  tumor  the 4 days of for  the  t h e amount  of  acti-  the  some o f  these  t i s s u e s were d e t e c t e d  the  present  growing  the  c o u l d be d e t e c t e d ,  the  24  tumor  tumor  latter  cases,  In  and  mass  was a t t a c h e d  on the  In pyri-  is negligible,  transplantation Therefore  amounts,  significant.  tumor  to d e t e c t  24  growth.  to  tissue  one a n d two d a y t u m o r - b e a r i n g  In  thy-  p u r i n e s and  t a m e r s were n o t  T h e s e were removed a n d a n a l y s e d . no tumor  tumor  ob-  after  specific  was o b s e r v e d t h a t  omentum.  and examined.  However  the  transplantation  3 days a f t e r  greater  growth,  difficult  e x c i s e d from the  of  suspension.  obtained  stated,  a n d 48 h o u r s a f t e r  was  had d e c r e a s e d to n e g l i g i b l e  a n d r e m a i n e d so d u r i n g  As p r e v i o u s l y  VI.  n u c l e i c a c i d p u r i n e s and  tumor  tumor  results  high specific a c t i v i t y  intraperitoneal  of  The  shown i n T a b l e  A relatively tained  intraperi-  72 a n d 96 h o u r s .  are  tumor  was  rats,  i s o l a t e d masses greater  omentum.  those cases  entire  greater  in  which  omentum  was a n a l y s e d . In  order  radio activity  to d e t e r m i n e  was e x c r e t e d ,  whether  the u r i n e  the  administered  and e x p i r e d  C02  To f o l l o w Page 3 7 . TABLE V I S p e c i f i c A c t i v i t i e s (c.p.m. /fill", of n u c l e i c a c i d thymine, guanine and. adenine i s o l a t e d from N o v i k o f f hepatoma, a t d a i l y i n t e r v a l s of growth, 110,100 c.p.m. of C A 4 i n j e c t e d i n donor tumor suspension. Hours of Tumor Growth  Specific DNA thymine  0  (Donor suspension)  24 48 72 96  Activity  guanine  c.p.m./uM  adenine  RNA  guanine  adenine  12,020  $,280  12,400  7.080  15,660  NS* . NS NS NS  NS NS NS NS  249  175 306  NS NS NS  NS NS  * NS - counts not s i g n i f i c a n t  TABLE V I I Radio a c t i v i t y e x c r e t e d i n u r i n e d u r i n g i n t r a p e r i toneal growth of r a d i o a c t i v e N o v i k o f f hepatoma, 110,100 c.p.m. of C * 4 i n j e c t e d i n donor tumor suspens ion. P e r i o d of Tumor Growth (hours)  0 12 24 36 48 72  -12 - 24 - 36 - 48 - 72 - 96  Number of Groups Averaged  J  3 2 1  C.p.m. excreted in urine  %  Total c.p.m. injected  22.2 2.2 1.4  I'542 Trace Trace Trace  M  Total'  25.8  NS NS NS  221  - 38 "  were analysed.  The  some of the groups.  e x p i r e d CO2 was In a l l cases  c o l l e c t e d from insignificant  amounts of r a d i o a c t i v i t y were found i n the e x p i r e d d u r i n g the 4 days of tumor growth.  COg This i s  i n agreement w i t h the aforementioned r e s u l t s which i n d i c a t e d that the donor suspension or no f o r m a t e - C . l4  v i t y was ticularly  little  However c o n s i d e r a b l e r a d i o a c t i -  found i n the u r i n e samples (Table V I I ) , 12  i n those e x c r e t e d d u r i n g the f i r s t  of tumor growth. 25$  contained  It w i l l be noted that  of the administered  0*^  parhours  approximately  has been e x c r e t e d  during  the 36 hours immediately f o l l o w i n g the i n j e c t i o n of the r a d i o a c t i v e tumor  suspension.  In view of the l a c k of s i g n i f i c a n t obtained  counts  f o r the n u c l e i c a c i d f r a c t i o n s of the growing obtained  it  t h i s experiment u s i n g a donor  i s planned to repeat  tumor suspension activity.  from the e x p i r e d  CO2,  tumor, as w e l l as those  w i t h a c o n s i d e r a b l y higher  radio  - 39  -  DISCUSSION The  experiments described  were u n d e r t a k e n i n o r d e r  in this  to s t u d y , i n v i v o ,  n u c l e i c a c i d metabolism of n e o p l a s t i c neoplastic  tissues.  maintained  in a transplantable  studied.  The  the  and  the  non-  h e p a t o m a , w h i c h can  of  f o r m , was  the  formate-C*  into  4  of formate~C*^ i n t o the  of r e g e n e r a t i n g  rat  liver,  normal  C o n s i d e r i n g the of  liver  nucleic  tissues,  growth of N o v i k o f f  liver,  i t was  or b o t h o f t h e s e  common c e l l  of  o r i g i n and  h e p a t o m a and  the  with  c e r t a i n h o s t t i s s u e s of r a t b e a r i n g N o v i k o f f toma, r e g e n e r a t i n g  and  hepa-  tissues.  rapid  rates  regenerating  rat  i n t e r e s t t o compare t h e n u c l e i c  metabolism i n these Several  acid  tissues. (2  investigations  have demonstrated a h i g h e r  -  12,  incorporation  f o r m a t e , g l y c i n e , o i r o t i c a c i d and  uracil  14, of  15) phosphorous,  into  the  n u c l e i c a c i d f r a c t i o n s o f tumor t i s s u e t h a n i n t o mal  tissue.  part  The  r e s u l t s reported  i n agreement w i t h  tigations, for  the  it  i n 4th  and  6th  i s i n n o r m a l s p l e e n and  herein  findings  i t i s evident  f o r m a t e i s more e x t e n s i v e l y biosynthesis  be  neoplasm  of t h i s n e o p l a s m were compared  incorporation  acids  Novikoff  incorporation  nucleic acids  thesis  are  of the  for the  above  from these r e s u l t s utilized day  most  investhat  for nucleic  Novikoff  liver.  nor-  hepatoma  acid than  However t h e h i g h  for-  -  40  -  mate i n c o r p o r a t i o n i n t o the hepatoma i s not  speci-  fic  ex-  to tumor s i n c e normal i n t e s t i n a l mucosa  h i b i t s a s i m i l a r degree of formate i n t a k e .  This  i s i n accord with the f i n d i n g s of Heidelberger al  (15)  who  of u r a c i l  observed that the degree of  et  conversion  i n t o the n u c l e i c a c i d p y r i m i d i n e s  of i n -  t e s t i n a l mucosa i n v i v o was  s i m i l a r to that  Flexner-Jobling  Therefore  a high  rate  of n u c l e i c a c i d s y n t h e s i s or turnover  i s not  unique  carcinoma.  of  to n e o p l a s t i c t i s s u e .  It i s b e l i e v e d that the  ferences  of formate i n c o r p o r a t i o n i n t o  i n the extent  the n u c l e i c a c i d s of the  dif-  t i s s u e s s t u d i e d are r e l a t e d  to d i f f e r e n t degrees of growth or c e l l  renewal i n  these t i s s u e s .  intestinal  Since  the c e l l s of the  mucosa are c o n t i n u o u s l y p l a c e d by new  cells  being  (69),  a r e l a t i v e l y high l e v e l in this tissue. corporation liver  re-  one would a n t i c i p a t e f i n d i n g of n u c l e i c a c i d  Furthermore the higher  i n t o the n u c l e i c a c i d s of  than i n t o normal r e s t i n g l i v e r  i n view of the greater mer  sloughed o f f , and  biosynthesis formate i n -  regenerating i s understandable  c e l l m u l t i p l i c a t i o n i n the  for-  tissue. It would a l s o appear from these r e s u l t s that  4th  and 6th  day  Novikoff  v i d i n g t i s s u e s than 40 However Hecht and  hepatoma are more r a p i d l y d i -  hour r e g e n e r a t i n g  Potter  (47)  rat  liver.  found that the  incor-  - 41 poration of orrotic acid-6-C  24-30  into  4  t h e DNA  hour r e g e n e r a t i n g x a t 1 i v e x was  t w i c e t h a t o f 40 h o u r r e g e n e r a t i n g  of  approximately  liver.  Similar (44)  r e s u l t s w e r e o b t a i n e d by C a t e r , Holmes a n d Mee on m e a s u r i n g t h e u p t a k e o f P ^ generating r a t l i v e r .  2  i n t o t h e DNA  Others have r e p o r t e d  of r e (40,  41»)  55^51) m a x i m a l r a t e s o f n u c l e i c a c i d s y n t h e s i s i n r e generating  rat liver  at earlier  generation  t h a n 40 h o u r s .  stages  Therefore  i n the r e -  i t i s possible  that the 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 s of 24 -  30 h o u r r e g e n e r a t i n g r a t l i v e r  could  approxi-  mate t h e u p t a k e o f f o r m a t e by t h e n u c l e i c a c i d s o f 4th  and 6th  d a y N o v i k o f f hepatoma.  In c o n t r a s t t o  the aforementioned r e s u l t s , B r u e s , Tracy found a higher  i n c o r p o r a t i o n of P^  2  i n t o the n u c l e i c  a c i d s o f 96 h o u r r e g e n e r a t i n g r a t l i v e r a t r a n s p l a n t e d hepatoma. Heidelberger 2-C ^ 1  (3)  a s compared t o  F u r t h e r m o r e LePage and  observed a higher  uptake of g l y c i n e -  by 63 h o u r r e g e n e r a t i n g r a t l i v e r  8 day-old  (2)  a n d Cohn  a s compared t o  F l e x n e r - J o b l i n g carcinoma t r a n s p l a n t s .  In  v i e w o f the low r a t e s o f n u c l e i c a c i d s y n t h e s i s obt a i n e d by o t h e r s a t a p p r o x i m a t e l y stage  of r a t l i v e r  t h e 63 or 96 h o u r  r e g e n e r a t i o n , the r e s u l t s  h e r e i n i n d i c a t e t h a t 4"th a n d 6 t h  day N o v i k o f f  a r e more r a p i d l y d i v i d i n g t i s s u e s t h a n  reported hepatoma  the t r a n s p l a n t e d  - 42 hepatoma of Brues and Flexner-Jobling  coworkers, and  8  day-old  carcinoma-transplants.  Although the m i t o t i c counts of hepatoma i n d i c a t e d that 4 t h  day  tumor i s a more  r a p i d l y d i v i d i n g t i s s u e than 6 t h  day  formate i n c o r p o r a t i o n s t u d i e s do not this finding.  I t i s evident  hepatoma, the corroborate  that there  d i f f e r e n c e i n the uptake of f orniate-C* a c i d s of 4 t h  day  T h i s apparent d i s c r e p a n c y  p l a i n e d on  the b a s i s of a turnover  hepatoma.  4  is l i t t l e by  the n u c l e i c  hepatoma, as compared to 6 t h  hepatoma.  day  c o u l d be  In such a process formate  day  Novikoff  incorporation  i n t o the n u c l e i c a c i d s would occur u n a s s o c i a t e d cell division. at  i s not  experiments performed.  Anderson (57)  the RNA  of 9 -  process  14 hour regenera-  t i n g l i v e r , at which time they found no net  synthesis  RNA. It i s e v i d e n t ,  from 4 t h  day  hepatoma has  from the r e s u l t s  tumor-bearing animals, that no  i n f l u e n c e on the  obtained Novikoff  incorporation  formate i n t o the n u c l e i c a c i d s of l i v e r and nal  justified  However A q v i s t  observed such a turnover  between g l y c i n e and  of  with  t h i s time i n view of the small number of m i t o t i c  counts and and  Such an e x p l a n a t i o n  ex-  process between  the n u c l e i c a c i d s of 6 t h  formate and  Novikoff  mucosa of the h o s t .  No  of  intesti-  e f f e c t of tumor on  the  - 43  -  i n c o r p o r a t i o n of formate i n t o the n u c l e i c a c i d guanine, DNA  adenine, and  thymine of spleen  observed. However the r e l a t i v e s p e c i f i c of RNA  adenine of spleen was  bearing animals.  The  i s not understood.  was  activity  decreased i n the tumor-  s i g n i f i c a n c e of t h i s f i n d i n g  Pertinent  to t h i s r e s u l t i t i s  of i n t e r e s t to note that others  (24)  have observed  a drop i n n u c l e i c a c i d c o n c e n t r a t i o n of r a t s bearing Walker carcinoma. that the presence of N o v i k o f f  of host  tissues  However i t appears  hepatoma has  f l u e n c e on the de novo b i o s y n t h e s i s  of the  no i n nucleic  a c i d s of the host t i s s u e s of the r a t .  In agreement  w i t h t h i s i s the f i n d i n g of LePage and  Heidelberger  (3)  incorporation  who  found no d i f f e r e n c e i n g l y c i n e  i n t o the n u c l e i c a c i d s of normal l i v e r as compared to l i v e r s of r a t s b e a r i n g the other  Flexner-Jobling  carcinoma.  On  coworkers (5)  found that  the  hand Tyner and  presence of F l e x n e r - J o b l i n g the uptake of P ^ K e l l y and creased  mice.  i n t o the DNA (25  associates  uptake of P-^ , 2  i n t o the DNA  - 28)  stimulate  nucleotides  of r a t  liver.  have observed an i n -  glycine-2-C ^ 1  and  formate-C ^ 1  of host t i s s u e s of tumor«bearing r a t s  Others have r e p o r t e d  formed purine  carcinoma d i d  precursors  (29  -  32)  that s e v e r a l  were u t i l i z e d to a greater  gree by the n u c l e i c a c i d s of host t i s s u e s of  tumor-  and prede-  - 44 -  bearing animals of  normal  than  animals.  the In  obtained with several Novikoff  n u c l e i c a c i d s of  view  tumors,  hepatoma h a s l i t t l e  nucleic  a c i d metabolism of  However  until  precursors, sider  of  further  are  Novikoff  the it  results  of  others  is significant  or no e f f e c t  other  tissues  on  t i s s u e s of  the the  studies, using a variety  performed,  it  i s not  hepatoma a s a u n i q u e  that  host. of  p o s s i b l e to tumor  in  con-  this  re-  gard. No e v i d e n c e was o b t a i n e d cept  of  the r e l e a s e  f r o m the others  of a "growth  regenerating  liver  of  to  support  promoting"  rats  mucosa was n o t  presence  liver  regenerating  more n e i t h e r  Novikoff  had a n e f f e c t of  one a n o t h e r  of  results  Flexner-Jobling carcinoma. a g r e e w i t h the or w i t h the The l a t t e r  growth  investigators  several  into  the  Further-  the  liver nucleic  hepatectomized-tumor-  (3)  using  with  partially  subcutaneous t r a n s p l a n t s However  experiments  observations of  animal.  acids  by  had. b e e n o b t a i n e d  by LePage a n d H e i d e l b e r g e r bearing  altered  regenerating  formate  in p a r t i a l l y  Similar  hepatectomized r a t s  take of  i n the  hepatoma n o r  on t h e u p t a k e  bearing r a t s . glycine  agent  a s p r o p o s e d by  by s p l e e n a n d i n t e s t i n a l  acids  con-  The de novo b i o s y n t h e s i s o f n u c l e i c  (58)'  of  the  these of  f i n d i n g do n o t  P a s c h k i s et  B a l i s and a s s o c i a t e s  found a s t i m u l a t i o n  preformed p r e c u r s o r s i n t o  of  the  in  al  (58)  (61). the  up-  purines  of  45  -  tissues of p a r t i a l l y hepatectomized  Furthermore  hamsters.  they observed an i n c r e a s e d i n c o r -  poration of adenine  i n t o t h e tumor o f p a r t i a l l y  hepatectomized hamsters  b e a r i n g a human t u m o r .  On t h e o t h e r h a n d t h e u t i l i z a t i o n o f g l y c i n e by t h e human tumor i n t h e p a r t i a l l y h a m s t e r was r e d u c e d . that the presence the preformed  hepatectomized  These r e s u l t s w o u l d  of regenerating 1iver,stimulates  pathway o f n u c l e i c a c i d  i n tumor, b u t d e p r e s s e s  hepatectomized r a t s  toma i n d i c a t e no e f f e c t de n o v o b i o s y n t h e s i s  biosynthesis  t h e de novo pathway.  ever the r e s u l t s o b t a i n e d from t h i s partially  suggest  How-  study using  b e a r i n g N o v i k o f f hepa-  o f r e g e n e r a t i n g l i v e r on t h e  of the n u c l e i c a c i d s of the  hepatoma. Although there i s a slight relative  specific  activity  a c i d p u r i n e s and thymine tially  of the t i s s u e s  f i c a n c e of these f i n d i n g s  in,the  rats,  par-  the s i g n i -  i s d o u b t f u l i n view o f the  l i m i t e d number o f e x p e r i m e n t s  performed.  I t would  the combination of r e g e n e r a t i n g l i v e r and  N o v i k o f f hepatoma  i n one r a t d o e s n o t a l t e r t h e  n u c l e i c a c i d metabolism testinal  i nthe  o f most o f t h e n u c l e i c  hepatectomized-tumor-bearing  seem t h a t  decrease  mucosa.  o f t h e h o s t ' s s p l e e n or i n -  Similar  B a l i s and coworkers  r e s u l t s were o b t a i n e d b y  (6l) i n partially  tumor-bearing hamsters.  hepatectomized-  The r e s u l t s o f t h e l a t t e r  - 46 investigators also e f f e c t on  i n d i c a t e d t h a t tumor has  the n u c l e i c a c i d metabolism of  ting liver,  i n the  no  regenera-  combined hepatectomized-tumor-  bear i n g hamsters.  The  r e s u l t s of t h i s study  are  i n agreement, f o r i t i s observed that N o v i k o f f toma d o e s n o t  alter  the  i n c o r p o r a t i o n of formate i n -  t o the n u c l e i c a c i d s of r e g e n e r a t i n g t i a l l y hepatectomized-tumor-bearing As rat  s t a t e d , the  l i v e r does not  bolism  liver  in  par-  rats.  f i n d i n g that  regenerating  a f f e c t the n u c l e i c a c i d meta-  of N o v i k o f f  r e s u l t s obtained  hepa-  hepatoma, i s i n c o n t r a s t  by B a l i s a n d  d i f f e r e n c e c o u l d be  coworkers  to  the  (61).  This  a t t r i b u t e d to d i f f e r e n c e s i n  m e t a b o l i s m . o f t h e human tumor u s e d by B a l i s a n d c i a t e s and  the N o v i k o f f  hepatoma used i n t h e s e  Furthermore the d i f f e r e n t r e s u l t s could of the It  occur  t h e r e f o r e , to note the (6l),  o f B a l i s and  associates  from s i m i l a r  experiments w i t h other  because  tumor w e r e n o t On  activity  always  that p r e l iminary. d a t a t u m o r s and  c o n s i d e r i n g the r a p i d e x c r e t i o n of  liver  aniliver  found.  i n the e x p i r e d C0  regenerating  rats.  observation  mals i n d i c a t e d t h a t the e f f e c t s of r e g e n e r a t i n g on  assostudies.  s p e c i e s d i f f e r e n c e between h a m s t e r s and  i s of i n t e r e s t ,  the  2  of  or N o v i k o f f  these t i s s u e s , i t i s evident  radio  the r a t s b e a r i n g  either  h e p a t o m a or  of  that  both  the a v a i l a b l e f o r -  - 47 mate-C*4 i n the t i s s u e s of t h e s e a n i m a l s i s r a p i d l y depleted  the 24  during  hours immediately  the  formate-C ^ injection.  60%  o f the a d m i n i s t e r e d  S i m i l a r l o s s e s o f 40  1  t a i n e d by o t h e r s  (68,  following  r a d i o a c t i v i t y have been 70)  Therefore i t appears that  u s i n g normal  the e x c r e t i o n o f  formate  s i g n i f i c a n t l y a l t e r e d by  or two  a d d i t i o n a l r a p i d l y growing t i s s u e s , which a f u r t h e r demand on  formate.  The  the p r e s e n c e of  one  "pool" of a v a i l a b l e  s i g n i f i c a n c e of these f i n d i n g s i s not  understood, although "pool"  the  ob-  animals.  i s not  place  -  i t is possible  that  the  formate  of the r a t s i s l a r g e enough t h a t a d d i t i o n a l  mands on t h e s u p p l y  of formate would not  be  de-  manifested  i n s i g n i f i c a n t a l t e r a t i o n s i n t h e e x c r e t i o n o f C02* Although u r i n a r y formate determinations f o r m e d the creted  r e s u l t s i n d i c a t e that  Nevertheless  i n accord  with  many m e t a b o l i c but  a few  o f a l k a l i n e and  recovered  ex-  observed  i n the u r i n e .  administered This  finding  the e s s e n t i a l p o s i t i o n of formate i n  pathways.  U r e a , a l l a n t o i n and c i r e a t i n i n e  of the u r i n a r y c o n s t i t u e n t s w h i c h  account for r a d i o a c t i v i t y wing the a d m i n i s t r a t i o n of The  per-  acidic urine.  a s i g n i f i c a n t amount o f t h e  r a d i o a c t i v i t y was  are  f o r m a t e - C ^ ^ " was  i n the u r i n e because of the d i f f e r e n c e  i n the r a d i o a c t i v i t y  is  were not  observation  i n the u r i n e - o f  could  rats,  follo-  C^. that  formate i n t o the n u c l e i c a c i d s  the  incorporation  of r e g e n e r a t i n g  of  liver  and  • 48 N o v i k o f f hepatoma was  not altered, i n p a r t i a l l y  hepatectomized-tumor-bearing  r a t s was c o n s i d e r e d  as an i n d i c a t i o n of autonomy i n the n u c l e i c metabolism  of these t i s s u e s .  The autonomous growth  of m a l i g n a n c i e s i s f r e q u e n t l y observed. i t was  of i n t e r e s t  of r a d i o a c t i v i t y  acid  to determine the  Therefore  incorporation  into regenerating l i v e r  i n tumor-  b e a r i n g animals which had been i n j e c t e d w i t h formateC ^ 1  p r i o r to the p a r t i a l hepatectomy.  It i s evident  that the n u c l e i c a c i d s of the r e g e n e r a t i n g l i v e r s i n these animals i n c o r p o r a t e d approximately the same amount of r a d i o a c t i v i t y as the n o n - r e g e n e r a t i n g l i v e r s of the tumor-bearing c o n t r o l s .  However the  l i v e r s of the c o n t r o l animals were a b l e to i n c o r p o r a t e r a d i o a c t i v i t y d u r i n g the p e r i o d immediately  following  the f o r m a t e - C ^ i n j e c t i o n , at which time the  concen-  1  t r a t i o n of a v a i l a b l e f o r m a t e - C ^ was 1  r e l a t i v e l y high.  On the other hand the r e g e n e r a t i n g l i v e r was  f i r s t able  to i n c o r p o r a t e r a d i o a c t i v i t y 24 hours a f t e r  the formate-  C 4 A  was  i n j e c t e d a t which time approximately 50$ of the  r a d i o a c t i v i t y had been e x c r e t e d . T h e r e f o r e the r a d i o a c t i v i t y d e t e c t e d i n the n u c l e i c a c i d s of the regenerating liver  c o u l d be d e r i v e d from the formate a l r e a d y i n -  c o r p o r a t e d i n t o the remnant at the time of the hepatectomy, as w e l l as the formate i n c o r p o r a t e d by the regenerating liver.  In view of the low r e l a t i v e s p e c i f i c  acti-  -  vities  49  -  o b t a i n e d f o r t h e n u c l e i c a c i d p u r i n e s and  thymine  of the r e g e n e r a t i n g l i v e r ,  that there i s l i t t l e  available  i n t h e r a t d u r i n g t h e p e r i o d 24 the subcutaneous also  i t would  appear  formate-C ^  present  1  t o 72 h o u r s  after  i n j e c t i o n of f o r m a t e - C .  The  1 4  data  i n d i c a t e that the n u c l e i c a c i d s of r e g e n e r a t i n g  l i v e r do n o t  incorporate radio a c t i v i t y  sources of r a d i o a c t i v i t y mals.  from  indirect  i n f ormate-C' -in jected  ani-  1 4  T h i s i s i n d i c a t e d by t h e r e s u l t s o b t a i n e d f o r  tumor, s p l e e n and  intestinal  that regenerating l i v e r  mucosa w h i c h  has n o t w i t h d r a w n  from the n u c l e i c a c i d s of these t i s s u e s . difference  i s noted  i n the r e l a t i v e  of the n u c l e i c a c i d p u r i n e s and  demonstrate radio Little  specific  thymine  activity or  no  activities  o f tumor  and  i n t e s t i n a l mucosa, o f t h e t u m o r - b e a r i n g a n i m a l s w i t h d e l a y e d h e p a t e c t o m i e s , as compared t o the c o r r e s p o n d i n g f r a c t i o n s of the tumor-bearing c o n t r o l a n i m a l s . is also  n o  difference  o f c o r r e s p o n d i n g DNA  i n the r e l a t i v e bases  specific  of s p l e e n of the  There activities  hepatec-  t o m i z e d - tumor-bear i n g a n i m a l s as compared t o the animals.  However t h e r e i s a d e c r e a s e  specific activity hepatectomized It  o f t h e RNA  guanine  i n the  control  relative  of s p l e e n of the  group as compared t o the c o n t r o l  group.  i s n o t p o s s i b l e t o compare t h e c o r r e s p o n d i n g a d e n i n e  fractions,  b u t i t i s n o t e d t h a t t h e RNA  adenine  of  the  -  of  50-  the c o n t r o l group i s r e l a t i v e l y " h i g h .  f i c a n c e of these f i n d i n g s i s not on  the  The  signi-  understood.  However  o a s i s of these r e s u l t s i t would appear t h a t  generating  l i v e r does not  grow a t  the  expense of  reother  tissues. The  r e s u l t s of the  e x p e r i m e n t c a r r i e d out  to  d e t e r m i n e the d i s t r i b u t i o n of r a d i o a c t i v i t y  following  the  hepatoma  t r a n s p l a n t a t i o n of radio a c t i v e Novikoff  are d i f f i c u l t  to assess,  t i o n s d i d not y i e l d  s i g n i f i c a n t data.  t h a t a p p r o x i m a t e l y 20%  evident activity  i s excreted  t h e r e was  little  suspension.  The  p o u n d s w h i c h had  the a d m i n i s t e r e d  results also  the  i s the  the  indicate  of formate e x c r e t i o n .  12 that  tumor  radio a c t i v i t y  r e s u l t of the  incorporated  radio  first  f r e e f o r m a t e - C * ^ i n the donor  I t i s assumed t h a t  sent i n the u r i n e  tuents  of  determina-  However i t i s  i n the u r i n e d u r i n g  h o u r s o f tumor g r o w t h .  sult  b e c a u s e many o f t h e  catabolism  f o r m a t e , and  not  preo f com-  the  re-  Therefore urinary c o n s t i -  such as a l l a n t o i n , B u r e i d o i s o b u t y r a t e ,  B amino-  i s o b u t y r a t e , a r e a t q m i n e , a r e a t i n e , m e t h i o n i n e and  serine  c o u l d become r a d i o a c t i v e f o l l o w i n g t h e 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 a tumor s u s p e n s i o n l a b e l l e d w i t h In agreement w i t h radio a c t i v i t y or  the r a p i d appearance  i n the u r i n e ,  i s the  insignificant specific activities  of the  tumor o b t a i n e d  C*^.  a f t e r 24  of  f i n d i n g of n e g l i g i b l e i n the n u c l e i c  acids  hours of i n t r a p e r i t o n e a l  -  51  -  growth.  This would i n d i c a t e a r a p i d turnover  b o t h RNA  and  DNA  d u r i n g the f i r s t  of the 24  i n j e c t e d N o v i k o f f hepatoma  hours of growth.  However  n u c l e i c a c i d _ o f t h e d o n o r tumor s u s p e n s i o n t a i n e d o n l y a b o u t 2% the  suspension.  of the t o t a l  Therefore  the degree of t u r n o v e r  from these  the  con-  radio activity  i t is difficult  the l a c k of s i g n i f i c a n t d a t a . difficult  of  to  in  assess  r e s u l t s because  Furthermore  of  i t is  t o o b t a i n a d e q u a t e amounts of tumor,  separate  f r o m g r e a t e r omentum, 24 a n d 48  hours a f t e r t r a n s p l a n -  t a t i o n , and  shown f o r tumor  t h e r e f o r e the d a t a  t a i n e d at these "pure"  t i m e s , may  tumor t i s s u e .  not  be r e p r e s e n t a t i v e o f  Nevertheless  i t is felt  i t w o u l d be a d v a n t a g e o u s t o r e p e a t  this  u s i n g a more r a d i o a c t i v e d o n o r tumor The  results  a c i d metabolism  of these  host  both of these  t i s s u e s , are not  obtain s t a t i s t i c a l l y  s h o u l d be r e p e a t e d . perform  suspension.  s t u d i e s of the n u c l e i c  similar  Novikoff  t i s s u e s o f r a t s c o n t a i n i n g one  w i t h t h e r e s u l t s o b t a i n e d by to  that  experiment  of r e g e n e r a t i n g r a t l i v e r ,  hepatoma, and  completely  others.  However, i n o r d e r  s i g n i f i c a n t data  the  s t u d i e s u s i n g a v a r i e t y of Similar  or  in accord  experiments  I t w o u l d a l s o be o f i n t e r e s t  nucleic acid precursors.  ob-  to  labelled  s t u d i e s performed  l a r g e g r o u p s o f m i c e s h o u l d a l s o be  considered.  on  SUMMARY A study has been made of the i n c o r p o r a t i o n i n v i v o of f o r m a t e - C ^ 1  i n t o the n u c l e i c a c i d s of regenera-  t i n g r a t l i v e r and N o v i k o f f hepatoma. l i v e r was tomy.  Regenerating  obtained 40 hours a f t e r p a r t i a l  N o v i k o f f hepatoma was  hepatec-  o b t a i n e d on the 4 t h  6 t h day a f t e r t r a n s p l a n t a t i o n .  The  or  i n f l u e n c e of one  or both of these t i s s u e s on the host t i s s u e s has  also  been examined. The  i n c o r p o r a t i o n of formate  purines and thymine of 4 t h  i n t o the n u c l e i c  acid  day N o v i k o f f hepatoma and  r e g e n e r a t i n g r a t l i v e r d i d not appear  to be  signifi-  c a n t l y i n f l u e n c e d by the simultaneous presence of both of these r a p i d l y d i v i d i n g t i s s u e s i n one animal. These f i n d i n g s have been d i s c u s s e d i n the l i g h t  of  evidence which i n d i c a t e d that approximately 50  P  cent of the a d m i n i s t e r e d r a d i o - a c t i v i t y was  e r  excreted  i n the u r i n e and C02« Regenerating r a t l i v e r and 4 t h had no s i g n i f i c a n t e f f e c t  day N o v i k o f f hepatoma  on the 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 s of the s p l e e n and  intes-  t i n a l mucosa of the host animal b e a r i n g one or both of these  tissues.  The r e l a t i v e s p e c i f i c a c t i v i t i e s of the n u c l e i c  acid  bases of 40 hour r e g e n e r a t i n g r a t l i v e r were g r e a t e r than the r e l a t i v e s p e c i f i c a c t i v i t i e s of c o r r e s p o n -  -  53  -  d i n g f r a c t i o n s of normal  liver.  t h e u p t a k e o f f o r m a t e by 4 t h h e p a t o m a was  greater  On 6th  and  t h a n 40  the  other  day  hand  Novikoff  hour r e g e n e r a t i n g  rat  1iver. The  results  mal  m i t o t i c frequency  ever  of m i t o t i c counts i n d i c a t e d a maxii n 4th  day  the n u c l e i c a c i d s of 4th  hepatoma. 6th  and  day  HowNovikoff  hepatoma i n c o r p o r a t e f o r m a t e to a p p r o x i m a t e l y  the  same e x t e n t . In a p r e l i m i n a r y experiment a r a d i o a c t i v e suspension  o f N o v i k o f f h e p a t o m a was  a group of r a t s .  transplanted  N e g l i g i b l e or  insignificant  s p e c i f i c a c t i v i t i e s were o b t a i n e d a c i d bases of the 20  Approximately a c t i v i t y was period.  24  tumor a f t e r per  cent  excreted  Although  of  into  f o r the n u c l e i c hours of  growth.  the a d m i n i s t e r e d  i n the u r i n e d u r i n g  formate analyses  radio-  this  were not  per-  formed, p r e l i m i n a r y experiments i n d i c a t e d that "free"  formate-C  pension.  The  l 4  was  present  i n the donor  s i g n i f i c a n c e of these  no  sus-  findings  has  been d i s c u s s e d . The  results  obtained  been d i s c u s s e d others.  from these  in relation  e x p e r i m e n t s have  to the  f i n d i n g s of  54 **  BIBLIOGRAPHY 1.  N o v i k o f f , A. B., C a n c e r R e s e a r c h , J_2, 1010  (1957).  2.  B r u e s , A. M., T r a c y , M. M., a n d C o h n , W. E . , J . B i o l . Chem,, 135, 619, (1944).  3.  LePage,  G.A. , a n d H e i d e l t o e r g e r , C , J . B i o l .  1S8,  Chem.,  593» (195D-  4.  LePage,  5.  T y n e r , E.P., H e i d e l b e r g e r , C , a n d L e P a g e , C a n c e r R e s e a r c h , 1_3,*186, (1953).  6.  L e P a g e , G.A., P o t t e r , V.R., B u s c h , H., H e i d e l t o e r g e r , C , a n d E u r l t o e r t , R.B. , C a n c e r R e s e a r c h , 12,  G.A., C a n c e r  R e s e a r c h , 13, 178,  (1953)* G.A. ,  153» (1952).  7.  T y n e r , E.P. , H e i d e l t o e r g e r , C , a n d L e P a g e , G.A., C a n c e r R e s e a r c h , 12, 158, (1952).  8.  R e d d y , D.V.N., B r e i g e r , H., a n d O r c h e n , M., C a n c e r  R e s e a r c h , 1J, 677, (1957)9.  W e l l s , W. , a n d W i n z l e r , R . J . , F e d e r a t i o n  382, 10.  T u t t l e , L.W., E r f , L.A., a n d L a w r e n c e ,  J.A., J . C l i n .  57, (1941)•  Weed, L . L . , a n d W i l s o n ,  435.  D.W., J . B i o l .  Chem., 1 8 9 .  U95D-  12.  Weed, L . L . , C a n c e r  13.  P o t t e r - , V.R., a n d H u r l t o e r t , R.B. , J . B i o l .  257,  15,  (1956).  Invest.,20, 11.  Proc,  R e s e a r c h , 11., 470, (1951). Chem., 1 9 5 ,  (1952).  14»  Rutman, R . J . , C a n t a r o w , A., a n d P a s c h k i s , K. E . , C a n c e r R e s e a r c h , L 4 , 119, (1954).  15.  H e i d e l t o e r g e r , C , Lietoman, K.C., H a r t o e r s , E . , a n d B h a r g a v a , P.M., C a n c e r R e s e a r c h , 1_Z» 399, (1957).  16.  G r a f f , S., E n g e l m a n , M., G i l l e s p i e , H.B., a n d G r a f f , A.M., C a n c e r R e s e a r c h , 11, 388, (1951).  17.  C e r e c e d o , L. R., R e d d y , D.V.N., P i r c i o , A., Lomtoardo, M.E., a n d T r a v e r s , J . J . , P r o c . S o c . E x p e r . B i o l .  & Med., 2 8 , 683, (1951).  -  55 -  18.  C e r e c e d o , L . B . , R e d d y , D.V.N., L o m b a r d o , M.E., M c C a r t h y , P.E., a n d T r a v e r s , J . J . , P r o c . S o c . E x p e r . B i o l . & Med., 8 0 , 7 2 3 , ( 1 9 5 2 ) .  19.  L o m b a r d o , M.E., R e d d y , D.V.N., M c C a r t h y , P., S i n g e r , E., a n d C e r e c e d o , L.R., F e d e r a t i o n P r o c , 11, 250, (1952).  20.  L o m b a r d o , M.E., T r a v e r s , J . J . , a n d C e r e c e d o , L.R., J . B i o l . Chem., 1^3, 4 3 , ( 1 9 5 2 ) .  21.  R e d d y , D.V.N., a n d C e r e c e d o , L.R. , F e d e r a t i o n 10, 236, ( 1 9 5 D -  22.  R e d d y , D.V.N., a n d C e r e c e d o , L.R., J . B i o l . 122 , 57.  Proc,  Chem.,  (195D-  23.  L o m b a r d o , M.E., C e r e c e d o , L.R., a n d R e d d y , D.V.N., J . B i o l . Chem., 2 0 2 , 9 7 , ( 1 9 5 3 ) .  24.  B r e s n i c k , E . , a n d C e r e c e d o , L.R., J . B i o l . 2 2 5 , 297, (1957).  25.  Kelly,  26.  K e l l y , L . S . , P a y n e , A.H., W h i t e , M.R., a n d J o n e s , H.B., C a n c e r R e s e a r c h , 11, 6 9 4 , ( 1 9 5 1 ) .  27.  P a y n e , A.H., K e l l y , L . S . , a n d W h i t e , M.R., C a n c e r R e s e a r c h , 12,65, (1952).  28.  P a y n e , A.H., K e l l y , L . S . , B e a c h , L . , a n d J o n e s , H.B., C a n c e r R e s e a r c h , 12, 4 2 6 , ( 1 9 5 2 ) .  29.  Way, J . L . , M a n d e l , H.G. a n d S m i t h , P.K., C a n c e r  L.S. S c i e n c e . I l l , 222,  R e s e a r c h , J J . , 812,  Chem.,  (1950).  (1954).  30.  G r i f f i n , A.C., T e x . Rep. B i o l .  31.  B a l i s , M.E. , V a n Praa<g, D. , a n d B r o w n , G. B., C a n c e r R e s e a r c h , 1 J , 673, (1955). B a l i s , M.E., V a n P r a a g , D., a n d A e z e n , F., C a n c e r R e s e a r c h , 16, 628, (1956).  32. 33.  & Med., 13,  l6l,  (1957).  Annati, E. , M a n z i n e l l i , A., a n d R o t h , A., C a n c e r R e s e a r c h , 11,  304, (1951).  34.  Malmgren,  35.  F i s h b a c k , F.C. , A r c h . P a t h . , 2» 955»  R.A., C a n c e r R e s e a r c h , 16,  232,  (1956).  (1929)..  - 56 -  36.  Higgins,  G . M . , and A n d e r s o n , R . M . ,  37*  E r u e s , A . M . , D r u r y , D.R.,and B r u e s , M . C . , A r c h . P a t h . , 2 2 , 658, (1936).  38.  Brues, A . M . , Tracy,  12, 186, (1931).  39.  Eliasson, N.A.,  Nygaard, Daoust,  Acta.  Cater,  0.,  46.  D.B.,  Jardetzky, Hecht,  P.,  and  5 , 4 3 1 , (1951)•  Arch.  Biochem.,  35,  Cancer R e s e a r c h ,  15,  (1955)-  R. , L e b l o n d , C P . ,  J.  S.,  and R u s c h , H . P . ,  Nadler,  N.J.,  and E n e s c o ,  221, 727, (1956).  B . E . , a n d Mee,  L.K.,  Acta  4 6 , 655, (1956).  C D . ,  Biol. L.I.,  Chem.,  Holmes,  Radiol., 45.  Reichard,  Chem. S c a n d . ,  and A l b e r t ,  M. , J . B i o l . 44.  E.,  (1952).  240, 43.  Hammarsten,  S.,  Johnson, R.M.,  340, 42.  Biol.  (1948).  Aqvist, 41.  J.  Bergstrand, A . , E l i a s s o n , N.A., Hammarsten,.E,, N o r b e r g , B . , R e i c h a r d , P . , and U b i s c h , A . V . , C o l d S p r i n g Harbor Symp., Quant. B i o l . , 13,  22,  40.  Cohn, W . E . ,  Path.,  619. (1944).  155,  Chem.,  M.M.,  Arch.  Barnum, C P . ,  Chem.,  and Vermund,  H. ,  222, 421, (1956).  and P o t t e r ,  V.R. , Federation  Proc,  15, 271, (1956). 47*  Hecht,  L.I.,  and P o t t e r ,  V.R.,  Cancer  Research,  16, 988, (1956). 48.  Takagi, y . , Hecht, R e s e a r c h , 16,  49*  Hecht,  50.  Jardetzky,  L.I.,  L.I.,  and P o t t e r ,  16, 999, (1956). C D . ,  Kelly,  L.S.,  W., 52.  Hirsch,  Tex.  Rep.  J.D.,  Jardetzky, Biol.  Cancer  V.R.,  Cancer  Research,  C P . , A r c h . Biochem.  350. (1957).  Cancer R e s e a r c h ,  Barnum, C P . ,  V.R.,  a n d Barnum,  & Biophys., 62, 51.  and P o t t e r ,  994, (1956).  Beach, G . , and  IJ, C D . ,  & Med.,  Palmer,  117, (1957). and H a l b e r g ,  F.,  1 5 , 134, (1957).  - 57'53'  S c h n e i d e r , J . H . , a n d P o t t e r , V.R. , C a n c e r  54.  P r i c e , J.M., a n d L a i r d , A.K., C a n c e r  12, 701, 650,  55.  (1957).  Research, 10,  Research,  (1950).  H a m m e r s t e n , E . , A q v i s t , S., A n d e r s o n , E.P. , E l i a s s o n , N.A. , T h o r e l l , B., A c t a . Chem. S c a n d . , 10, I568,  (1956).  56.  A n d e r s o n , E.P. a n d A q v i s t , S., A c t a . Chem.  10, 57.  (1956).  A q v i s t , S., a n d A n d e r s o n ,  10, 58.  1576,  E . P . , A c t a . Chem.  P a s c h k i s , K.E., C a n t a r o w ,  A., S t a s n e y , J . , a n d H o b b s ,  519,  (1955)»  B u c h e r , N.L.R. , S c o t t , J . F . , a n d Auto.• » J.C. , C a n c e r  R e s e a r c h , i i , 457, 60.  Scand.,  1583, (1956).  J.H., C a n c e r R e s e a r c h , 1$, 59.  Scand.,  (1951).  W e n n e c k e r , A.S., S u s s m a n , N. , P r o c . S o c . E x p e r .  Biol.  & Med., 26, 683, (1951). 61.  B a l i s , M.E., V a n P r a a g , 0 . ,  a n d B r o w n , G.B.,  Cancer  R e s e a r c h , 16,632, (1956). 62.  J a e n i c k e , L., B i o c h i m . e t B i o p h y s .  A c t a . 12,  588,  (1955)63.  G r e e n b e r g , G.R., J a e n i c k e , L . , a n d S i l v e r m a n , M., B i o c h i m . e t B i o p h y s . A c t a . 1J, 589, (1955).  64.  L e b l o n d , C P . , a n d W a l k e r , B.E. , P h y s i o l . R e v . , 36,  255, 65. 66.  (1956).  Z b a r s k y , S.H., a n d W r i g h t , W.D. , C a n . J . Med. S c i . ,  Jl,  151» (1953).  M a r s h a k , A., a n d V o g e l , H . J . , J . B i o l .  597,  Chem.,  189,  (1951).  67.  P e r s o n a l communication  68.  Z b a r s k y , S.H., F i n d l a y , S., S c r i m g e o u r , K.G., a n d N i x o n , J.C., P r o g r e s s Report t o the N a t i o n a l C a n c e r I n s t i t u t e o f C a n a d a , December 15, 1956.  f r o m D r . S. H. Z b a r s k y .  -  58 -  S t e v e n s , C E . , D a o u s t , fi. , a n d L e b l o n d , J . B i o l . C h e m . , 202, 177, (1953)-  C P .  Friedmann, B . , Nakada, H . I . , and Weinhouse, J . B i o l . Chem. 210, 413, (1954).  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0106096/manifest

Comment

Related Items