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Nature of rat liver cell sap factors inhibiting the DNA synthesis in tumour cells
324
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 96698
N A T U R E OF R A T L I V E R CELL SAP FACTORS I N H I B I T I N G T H E DNA S Y N T H E S I S IN TUMOUR CELLS
M I Z U E MIYAMOTO AND H I R O S H I T E R A Y A M A
Zoological Institute, Faculty of Science, University of Tokyo, Tokyo (Japan) (Received J u l y 2nd, 197 o)
SUMMARY
I. In the previous paper, we reported that the adult rat liver cell extract (sap) inhibits the incorporation of [14C]thymidine into DNA of ascites hepatoma cells in culture 3 and that there are at least two inhibitor components in the adult rat liver cell sap, one of which is arginase e. 2. Arginase is not present in the new-born rat liver, while the thermolabile inhibitor component is present in both the adult and the new-born rat liver. Experimental evidence presented in this paper suggests that the thermolabile inhibitor present in the rat liver cell extracts is thymidine hydrolase and/or phosphorylase. Malignant hepatomas lack both arginase and thymidine-decomposing enzymes. 3. New-born rat liver contains a factor stimulating [14C~orotate incorporation into RNA and DNA of the ascites hepatoma cells in culture.
INTRODUCTION
Although it was already known that the rat (and other mammalian) liver cell extract inhibits the proliferation of cultured cells 1,2, the inhibition of cellular DNA synthesis by the rat liver cell extract was reported first b y OTSUKA AND TERAYAMA3. TERAYAMA AND SASADA4 compared the inhibitory activities of various tissue extracts of different animals. The ability to inhibit the [laC]thymidine incorporation into DNA of ascites hepatoma, AH-4I 4 cells, was shared with the adult rat liver, new-born rat liver, foetal rat liver, regenerating rat liver and some of the so-called minimal deviation hepatomas, but the activity was absent from rapidly growing malignant hepatomas. Recently SASADA AND TERAYAMA5 presented evidence indicating that there are at least two inhibitory factors in the adult rat liver extract. One component is rather thermostable at 55 ° and has been identified as arginase in accord with the suggestion made by HOLLEYe. The other component, which is thermolabile, occurs in both adult and new-born rat livers. Therefore the new-born rat liver seemed to be a suitable material for investigating the second or thermolabile inhibitor component. The present paper deals mainly with the nature of the inhibitory factor present in the newborn rat liver cell sap. Biochim. Biophys. Acta, 228 (1971) 324-33o
DNA
SYNTHESIS INHIBITING CELL SAP
325
MATERIALS AND METHODS
The livers of Wistar albino rats weighing 15o g and neonatal rats within 2 days after delivery were used for preparing the liver cell extracts according to the procedures described in the previous papers 3-5. The rat ascites hepatoma AH-4I 4 cells or Ehrlich mouse ascites carcinoma cells were harvested from Donryu strain rats or ddN strain mice, respectively, 5-7 days after inoculation of the tumour cells as also described previously. The I-ml tumour cell pellet was suspended in a mixture consisting of 60 ml of Hanks salt solution containing penicillin (50 units/ml) and streptomycin (50/*g/ml), 6 ml of bovine serum (The Serum Institute, Chiba, Japan) and 2.4 ml of IO % lactoalbumin hydrolysate (enzymic, Nutritional Biochem. Inc., Cleveland, U.S.A.). For the assay of nucleic acid synthesis in the tumour cells, I2-14C]thymidine (Radiochem. Centre, Amersham, England, 60. 5 mC/mmole, 0.02 #C) or E6-1~Clorotate (Radiochem. Centre, Amersham, England, 60.8 mC/mmole, 0.5/*C) in o. I ml was added to a mixture of 3 ml tumour cell suspension and 0.5 ml adequately diluted tissue extract. As the control, 0.5 ml of the Hoagland Medium A (the medium used for preparing tissue extracts) was added. The whole mixture was incubated at 37 ° under gentle shaking. After the incubation, the cells were collected by centrifuging at 3000 rev./min. The cell pellet was then subjected to the procedure of SCHMIDTAND THANNHAUSER7 to fractionate RNA and DNA components. The radioactivity incorporated was measured by a Hitachi windowless gas-flow counter. Assays of RNA and DNA were carried out by conventional methods using orcinols and diphenylamine 9, respectively. Assays of thymidine hydrolase and thymidine phosphorylase activities were carried out according to FRIEDKIN AND ROBERTSTM. Enzyme solution(o.2 ml of lO-2O ~/o liver cell sap) was mixed with the following substrate solutions: for phosphorylase, 16/*moles of thymidine in I ml of 0.2 M TrisHC1, o.I M arsenate buffer (pH 7-5) was used; and for hydrolase the same solution without arsenate. After reaction at 38 ° for certain periods of time, the reaction was stopped by addition of i ml of 3 ~/o HC104 and the increase of absorbance at 3o0 nm of the acid-soluble supernate was measured. The enzymic activity was expressed in terms of/,moles of thymine generated per h per g liver. To check how far E14C]thymidine added to the incubation mixture (0.02/,C in 3.6 ml) was actually decomposed by the liver extract, after 3 h incubation the reaction mixture was acidified by the addition of trichloroacetic acid, and the acid-soluble supernate was repeatedly washed with ether and concentrated under reduced pressure. An aliquot was applied to a thin-layer chromatographic plate of cellulose. The plates were developed with isopropanol-concentrated HCl-water (650:167 : 183, by vol.)11. The spots corresponding to thymidine and thymine (detected by ultraviolet) were scraped off, and the radioactivity was ehited into hot water for 3 h and counted.
RESULTS
The time-course of C14C]thymidine incorporation into DNA in AH-4I 4 cells in the presence and absence of 0.5 ml of 4 ° % new-born rat liver cell sap is illustrated in Fig. I. The incorporation seemed to proceed almost linearly within the incubation Biochim. Biophys. Acta, 228 (i971) 324-33°
326
M. MIYAMOTO, H. TERAYAMA
time of 5 h. The presence of the new-born rat liver cell sap inhibited the incorporation strongly. The inhibition of the new-born rat liver cell sap in [14C~thymidine incorporation into AH-4I 4 cell DNA was shown to be dose-dependent when assayed after 3 h incubation. A similar dose-response curve was also found in Ehrlich carcinoma cells. If we adopt the definition that I unit of cell sap inhibitor corresponds to the 3 ° % inhibition of DNA-synthesis 4, the new-born rat liver possesses about 4 ° units of inhibitor potency, which is nearly one-half that of adult rat liver.
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1
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1 2 3 4 Incubotion time (h)
5
Fig. I. Time-course of [l*C]thymidine incorporation into D N A in A H - 4 I 4 cells in the absence and in the presence of n e w - b o r n r a t liver cell sap. F r e s h l y prepared 4 ° ~o n e w - b o r n r a t liver cell sap (0. 5 m]) was added to the incubation m i x t u r e in a final volume of 3.6 ml. The incubation conditions are described in METHODS AND MATERIALS. 0 - - 0 , in the presence of n e w - b o r n rat liver cell sap; G - C ) , in the absence of n e w - b o r n r a t liver cell sap (control). Fig. 2. Time-course of [l*C]orotate incorporations into R N A and D N A in A H - 4 I 4 cells, and the effect of n e w - b o r n r a t liver cell sap u p o n it. 0. 3 ml of 4 ° % n e w - b o r n rat liver cell sap was added to the incubation mixture. The incorporation of [14C]orotate into R N A and D N A in A H - 4 I 4 cells was m e a s u r e d as described in METHODS AND MATERIALS. • - - • and A - / x , incorporation of [14C]orotate into D N A in the presence and in the absence of n e w - b o r n r a t liver cell sap, respectively. 0 - 0 and Q - C ) , incorporation of [14C!orotate into R N A in the presence and in the absence of n e w - b o r n r a t liver cell sap, respectively.
As reported in the preceding papei 5, the inhibition b y the new-born rat liver cell sap cannot be reversed or prevented by an additional supply of L-arginine in the incubation mixture, in contrast to the marked effect of arginine observed for the adult rat liver cell sap inhibition, suggesting that the new-born rat liver cell sap inhibition is not related with the arginase activity. We have found another difference between the two liver cell saps. As shown in Fig. 2, the E14C]orotate incorporation into nucleic acids (RNA and DNA) in AH-4I 4 ceils was not inhibited, but rather markedly stimulated, in the presence of new-born rat liver cell sap. This stimulatory effect of new-born rat liver cell sap seemed to be more prominent in the short period Biochim. Biophys. Acta, 228 (i97 I) 324-330
DNA
327
S Y N T H E S I S I N H I B I T I N G C E L L SAP
incubations. On the other hand, the adult rat liver cell sap did not show any effect, either stimulatory or inhibitory, upon the [14Clorotate incorporations into RNA and DNA. In the previous paper it was reported that the adult rat liver cell sap also inhibited [14Clorotate incorporation 3. Repeated experiments of kinetic and doseresponse analysis, however, showed that the adult rat liver cell sap had no effect upon the E14Qorotate incorporations while the [l*Clthymidine incorporation into DNA was inhibited strongly in accord with previous reports. The above-described results suggested that an inhibitory factor commonly present in both adult and new-born rat livers may be related to the thymidine metabolism but not to the orotate metabolism. Since the metabolic pathways starting from thymidine and orotate are intermingled at the stage of thymidylate, the specific inhibition of thymidine incorporation by the liver cell sap seems to be due either to the impairment of thymidine kinase activity or to the decomposition of thymidine itself. In order to check the latter possibility thymidine phosphorylase and thymidine hydrolase activities of adult and new-born rat liver cell saps were investigated. As evidenced in Table I (hydrolase activity) and Table II (phosphorylase activity), both thymidine hydrolase and phosphorylase activities were detected in the liver cell saps from the adult and new-born rats. The levels of these enzymes in the adult rat liver were a few times higher than those in the new-born rat liver. The kinetics of these enzymic reactions, as evidenced in Tables I and II, also pointed to the lability of these enzymes: thermal treatment of the liver cell saps at 55 ° for several TABLE
I
THYMIDINE HYDROLASE ACTIVITY OF RAT LIVI~R CELL SAP AND ITS THERMOSTABILITY A s s a y of t h e e n z y m i c a c t i v i t y w a s c a r r i e d o u t in t h e a b s e n c e of a r s e n a t e .
Enzyme solution (0.2 ml)
Reaction time (rain)
AAsoo nm
Thymidine hydrolase activity i~moles thymine/ g tissue per h
%
IO % a d u l t r a t l i v e r cell s a p
3° 60 90 12o
0.099 o.124 o.139 o.155
48.1 3o.1 22. 5
20 % a d u l t r a t l i v e r cell s a p
3° 60 90 12o
o.178 0.233 o.252 0.276
43.2 28.3 2o. 4
20 % n e w - b o r n r a t l i v e r cell s a p
3° 60 90 I2O
0.042 o.088 o. lO6 O.lO 7
lO.2 lO.2 8.6
20 % a d u l t r a t l i v e r cell s a p t r e a t e d 3 m i n a t 55 ° t r e a t e d 5 m i n a t 55 °
90 9°
0.092 0.059
7.46 4.76
15. 5 9.9
20 % n e w - b o r n r a t l i v e r cell s a p t r e a t e d 3 m i n a t 55 ° t r e a t e d 5 m i n a t 55 °
90 90
O.Ol 3 o.ooo
1.o 5 o.oo
lO. 3 o
Biochim. Biophys. Acta,
IOO
IOO
228 ( i 9 7 I ) 3 2 4 - 3 3 o
328
M. MIYAMOTO, H. TERAYAMA
TABLE II THYMIDINE
PHOSPHORYLASE
ACTIVITY
OF RAT LIVER
CELL
SAP AND
ITS THERMOSTABILITY
Assay of the enzymic activity was carried out in the presence of arsenate.
Enzyme solution (0.2 ml)
Reaction time (rain)
z]A300nm
Thymidine phosphorylase activity I, moles thymine/ g tissue per h
%
20 °/o adult rat liver cell sap
3° 60 90 12o
0.282 0.378 0.453 0.503
68. 5 45.9 36.7
IOO
20 % new-born rat liver cell sap
3° 60 90 12o
0.086 o. 166 o.17o o.174
2o.8 20.0 13. 7
IOO
2 0 % adult rat liver cell sap treated 3 m i n at 55 ° treated 5 m i n at 55 °
90 90
0.273 0.206
18.6 16. 7
27 24
2 o % n e w - b o r n rat liver cell sap treated 3 m i n at 55 ° treated 5 m i n at 55 °
90 90
0.066 0.057
5.3 4.6
25 22
minutes destroyed the enzymic activities. The relation between the thermal inactivat i o n of t h y m i d i n e h y d r o l a s e a n d t h a t of t h e cell s a p i n h i b i t o r a c t i v i t y of t h e n e w b o r n r a t l i v e r cell s a p is s h o w n i n Fig. 3. I n o r d e r t o c o n f i r m t h a t t h y m i d i n e h y d r o l a s e a n d / o r p h o s p h o r y l a s e in t h e n e w - b o r n r a t l i v e r cell s a p is a c t u a l l y i n v o l v e d i n t h e i n h i b i t i o n of [ 1 4 C l t h y m i d i n e i n c o r p o r a t i o n i n t o D N A of A H - 4 I 4 cells, t h e e f f e c t of u n l a b e l l e d t h y m i n e (a p r o d u c t i n h i b i t o r of t h e e n z y m e s ) u p o n t h e i n h i b i t o r p o t e n c y of t h e n e w - b o r n r a t l i v e r cell
IOO~
~_>
\ ~o'6_"~.
o I~riod of heat treatment
~ (rnin)
Fig. 3. Thermal inactivation of the thymidine hydrolase activity and the cell sap inhibitor activity of new-born rat liver cell sap. 20 % new-born rat liver cell sap was treated at 55 ° for different periods of time (o, i, 2, 3 and 6 min), followed by immediate cooling in ice-water and by centrifugation. Aliquots of the supernate were added to the reaction mixture containing [14Clthymidine to measure the cell sap inhibitor activity. Another aliquot was used for the assay of thymidine hydrolase. In the figure, relative activities of thymidine hydrolase (O) and cell sap inhibitor (O) are plotted against the period of thermal (55 °) treatment.
Biochim. Biophys. Acta, 228 (1971) 324-33 °
D N A SYNTHESIS INHIBITING CELL SAP
329
TABLE I I I EFFECT BORN
OF ADDED RAT LIVER
THYMINE
UPON
[14C]THYMIDINE
INCORPORATION
AND
ITS INHIBITION
20 % new-born rat liver cell sap (ml)
Thymine conch. (M)
[x4CJThymidine incorporated (counts]rain × zo -4 per mg DNA)
None
o 1"1o-5 1.1o -4
I. I9 1.25 1.39 1.31 1.22 o.612 0.744 o.818 0.926 1.22
1.io
0.2
BY NEW-
CELL SAP
-s
3.io -a o I • lO-5 i • lO-4 I • IO-s 3" Io-s
Inhibition (%)
o -- 5 --17 --IO -- 3 49 4° 41 29 o
s a p has been i n v e s t i g a t e d . The results s u m m a r i z e d in T a b l e I I I i n d i c a t e t h a t t h e i n h i b i t o r y p o t e n c y is r e d u c e d m a r k e d l y b y t h e a d d i t i o n of u n l a b e l l e d t h y m i n e over a c o n c e n t r a t i o n r a n g e of I . lO-5-3 • IO -s M. The a d d i t i o n of u n l a b e l l e d t h y n l i n e itself showed no effect u p o n the [14Clthymidine i n c o r p o r a t i o n in t h e absence of new-born r a t liver cell sap. I n a n o t h e r experim e n t , [14Clthymine (25.2 mC/mmole, 0.02/~C) a d d e d to the i n c u b a t i o n m i x t u r e was n o t i n c o r p o r a t e d to a n y a p p r e c i a b l e e x t e n t into D N A (less t h a n 0. 5 % of t h e incorpor a t i o n of t h y l n i d i n e ) . These results s u p p o r t t h e view t h a t t h e change of t h y m i d i n e into t h y m i n e b y t h e e n z y m e s present in t h e liver cell s a p is a real m e c h a n i s m of t h e cell s a p inhibition. I n fact, u n d e r routine a s s a y conditions [l*C~thymidine a d d e d in t h e i n c u b a t i o n m i x ture was d e c o m p o s e d to the e x t e n t of 19 a n d 35 % after 3 h i n c u b a t i o n in t h e presence of o.I a n d 0.2 ml of 20 % new-born r a t liver cell sap, respectively. These values of [14Clthymidine d e c o m p o s i t i o n in t h e a s s a y s y s t e m are well correlated w i t h the degrees of inhibition of [14C]thymidine i n c o r p o r a t i o n b y the new-born r a t liver cell sap.
DISCUSSION The p r e s e n t i n v e s t i g a t i o n has shown t h a t new-born r a t liver cell sap, which was d e v o i d of arginase, could still i n h i b i t t h e [14Clthymidine i n c o r p o r a t i o n i n t o t u m o u r cell D N A . The e x p e r i m e n t a l evidence i n d i c a t e s t h a t t h y m i d i n e h y d r o l a s e a n d / o r p h o s p h o r y l a s e in t h e new-born r a t liver cell s a p are i n v o l v e d in t h e i n h i b i t o r y m e c h a n i s m of t h e cell sap. These e n z y m i c activities were also f o u n d in t h e a d u l t r a t liver cell s a p even a t higher levels, a n d seemed to c o n t r i b u t e to t h e i n h i b i t o r y p o t e n c y of t h e a d u l t r a t liver cell s a p in a d d i t i o n to arginase. T h e cell s a p from A H - 4 I 4 cells was d e v o i d of b o t h arginase a n d t h y m i d i n e - d e c o m p o s i n g enzymes, in accord w i t h t h e o b s e r v a t i o n t h a t the t u m o u r cell s a p showed no effect u p o n [14C1thymidine incorporation into DNA. The evidence o b t a i n e d so far suggests t h a t t h e D N A synthesis in t h e a d u l t r a t liver is u n d e r a d u p l i c a t e control m e c h a n i s m t h r o u g h b o t h arginase a n d t h y m i d i n e d e c o m p o s i n g enzymes, whereas t h a t in t h e new-born r a t liver is u n d e r a single c o n t r o l Biochim. Biophys. Acta, 228 (1971) 324-33o
330
M. MIYAMOTO, H. TERAYAMA
m e c h a n i s m t h r o u g h t h y m i d i n e - d e c o m p o s i n g e n z y m e s , a n d t h a t i n t u m o u r cells is e n t i r e l y free f r o m t h e s e c o n t r o l l i n g m e c h a n i s m s . I n a d d i t i o n t o t h e c o n t r o l m e c h a n i s m i n t h e s a l v a g e p a t h w a y of D N A s y n t h e s i s , t h e n e w - b o r n r a t l i v e r cell s a p c o n t a i n s a s t i m u l a t o r for t h e o r o t a t e i n c o r p o r a t i o n (a p a t h w a y de novo). F u r t h e r s t u d i e s o n t h e n a t u r e of t h i s s t i m u l a t o r as w e l l a s o n t h e m e c h a n i s m of t h e a r g i n a s e i n h i b i t i o n in the DNA synthesis are now in progress in our laboratory.
ACKNOWLEDGEMENT A p a r t of t h e e x p e n d i t u r e s for t h e p r e s e n t s t u d y w a s c o v e r e d b y t h e G r a n t - i n A i d for S c i e n t i f i c R e s e a r c h f r o m t h e M i n i s t r y of E d u c a t i o n .
REFERENCES I 2 3 4 5
6 7 8 9 io ii
I. LIEBERMANN AND P. OVE, Biochim. Biophys. Acta, 38 (196o) 153. M. I-IoRI AND T. UKITA, J. Biochem. (Japan), 51 (1962) 322. H. OTSUKA AND H. TERAYAMA, Biochim. Biophys. Acta, 123 (1966) 274. H. TERAYAMA AND M. SASADA, GANN, 59 (1968) 51. ~{- SASADA AND I-l_. TER&YAMA, Biochim. Biophys. Acta, 19o (1969) 73. R. V. HOLLEY, Biochim. Biophys. Acta, 145 (1967) 525 . G. SCHMIDT AND S. J. THANNHAUSER, J. Biol. Chem., 161 (1945) 83. W. MEJBAUM, Z. Physiol. Chem., 258 (1939) 117. Z. DISCHE, Mikroehimie, 8 (193 o) 3. M. FRIEDKIN AND D. ROBERTS, J. Biol. Chem., 207 (1954) 245. P. GRIPPS, M. ]ACCARINO, M. RossI AND E. SEA.RNO, Biochim. Biop~,s. Acta, 95 (1965) I.
Biochim. Biophys. Acta, 228 (1971) 324-330
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 96698
N A T U R E OF R A T L I V E R CELL SAP FACTORS I N H I B I T I N G T H E DNA S Y N T H E S I S IN TUMOUR CELLS
M I Z U E MIYAMOTO AND H I R O S H I T E R A Y A M A
Zoological Institute, Faculty of Science, University of Tokyo, Tokyo (Japan) (Received J u l y 2nd, 197 o)
SUMMARY
I. In the previous paper, we reported that the adult rat liver cell extract (sap) inhibits the incorporation of [14C]thymidine into DNA of ascites hepatoma cells in culture 3 and that there are at least two inhibitor components in the adult rat liver cell sap, one of which is arginase e. 2. Arginase is not present in the new-born rat liver, while the thermolabile inhibitor component is present in both the adult and the new-born rat liver. Experimental evidence presented in this paper suggests that the thermolabile inhibitor present in the rat liver cell extracts is thymidine hydrolase and/or phosphorylase. Malignant hepatomas lack both arginase and thymidine-decomposing enzymes. 3. New-born rat liver contains a factor stimulating [14C~orotate incorporation into RNA and DNA of the ascites hepatoma cells in culture.
INTRODUCTION
Although it was already known that the rat (and other mammalian) liver cell extract inhibits the proliferation of cultured cells 1,2, the inhibition of cellular DNA synthesis by the rat liver cell extract was reported first b y OTSUKA AND TERAYAMA3. TERAYAMA AND SASADA4 compared the inhibitory activities of various tissue extracts of different animals. The ability to inhibit the [laC]thymidine incorporation into DNA of ascites hepatoma, AH-4I 4 cells, was shared with the adult rat liver, new-born rat liver, foetal rat liver, regenerating rat liver and some of the so-called minimal deviation hepatomas, but the activity was absent from rapidly growing malignant hepatomas. Recently SASADA AND TERAYAMA5 presented evidence indicating that there are at least two inhibitory factors in the adult rat liver extract. One component is rather thermostable at 55 ° and has been identified as arginase in accord with the suggestion made by HOLLEYe. The other component, which is thermolabile, occurs in both adult and new-born rat livers. Therefore the new-born rat liver seemed to be a suitable material for investigating the second or thermolabile inhibitor component. The present paper deals mainly with the nature of the inhibitory factor present in the newborn rat liver cell sap. Biochim. Biophys. Acta, 228 (1971) 324-33o
DNA
SYNTHESIS INHIBITING CELL SAP
325
MATERIALS AND METHODS
The livers of Wistar albino rats weighing 15o g and neonatal rats within 2 days after delivery were used for preparing the liver cell extracts according to the procedures described in the previous papers 3-5. The rat ascites hepatoma AH-4I 4 cells or Ehrlich mouse ascites carcinoma cells were harvested from Donryu strain rats or ddN strain mice, respectively, 5-7 days after inoculation of the tumour cells as also described previously. The I-ml tumour cell pellet was suspended in a mixture consisting of 60 ml of Hanks salt solution containing penicillin (50 units/ml) and streptomycin (50/*g/ml), 6 ml of bovine serum (The Serum Institute, Chiba, Japan) and 2.4 ml of IO % lactoalbumin hydrolysate (enzymic, Nutritional Biochem. Inc., Cleveland, U.S.A.). For the assay of nucleic acid synthesis in the tumour cells, I2-14C]thymidine (Radiochem. Centre, Amersham, England, 60. 5 mC/mmole, 0.02 #C) or E6-1~Clorotate (Radiochem. Centre, Amersham, England, 60.8 mC/mmole, 0.5/*C) in o. I ml was added to a mixture of 3 ml tumour cell suspension and 0.5 ml adequately diluted tissue extract. As the control, 0.5 ml of the Hoagland Medium A (the medium used for preparing tissue extracts) was added. The whole mixture was incubated at 37 ° under gentle shaking. After the incubation, the cells were collected by centrifuging at 3000 rev./min. The cell pellet was then subjected to the procedure of SCHMIDTAND THANNHAUSER7 to fractionate RNA and DNA components. The radioactivity incorporated was measured by a Hitachi windowless gas-flow counter. Assays of RNA and DNA were carried out by conventional methods using orcinols and diphenylamine 9, respectively. Assays of thymidine hydrolase and thymidine phosphorylase activities were carried out according to FRIEDKIN AND ROBERTSTM. Enzyme solution(o.2 ml of lO-2O ~/o liver cell sap) was mixed with the following substrate solutions: for phosphorylase, 16/*moles of thymidine in I ml of 0.2 M TrisHC1, o.I M arsenate buffer (pH 7-5) was used; and for hydrolase the same solution without arsenate. After reaction at 38 ° for certain periods of time, the reaction was stopped by addition of i ml of 3 ~/o HC104 and the increase of absorbance at 3o0 nm of the acid-soluble supernate was measured. The enzymic activity was expressed in terms of/,moles of thymine generated per h per g liver. To check how far E14C]thymidine added to the incubation mixture (0.02/,C in 3.6 ml) was actually decomposed by the liver extract, after 3 h incubation the reaction mixture was acidified by the addition of trichloroacetic acid, and the acid-soluble supernate was repeatedly washed with ether and concentrated under reduced pressure. An aliquot was applied to a thin-layer chromatographic plate of cellulose. The plates were developed with isopropanol-concentrated HCl-water (650:167 : 183, by vol.)11. The spots corresponding to thymidine and thymine (detected by ultraviolet) were scraped off, and the radioactivity was ehited into hot water for 3 h and counted.
RESULTS
The time-course of C14C]thymidine incorporation into DNA in AH-4I 4 cells in the presence and absence of 0.5 ml of 4 ° % new-born rat liver cell sap is illustrated in Fig. I. The incorporation seemed to proceed almost linearly within the incubation Biochim. Biophys. Acta, 228 (i971) 324-33°
326
M. MIYAMOTO, H. TERAYAMA
time of 5 h. The presence of the new-born rat liver cell sap inhibited the incorporation strongly. The inhibition of the new-born rat liver cell sap in [14C~thymidine incorporation into AH-4I 4 cell DNA was shown to be dose-dependent when assayed after 3 h incubation. A similar dose-response curve was also found in Ehrlich carcinoma cells. If we adopt the definition that I unit of cell sap inhibitor corresponds to the 3 ° % inhibition of DNA-synthesis 4, the new-born rat liver possesses about 4 ° units of inhibitor potency, which is nearly one-half that of adult rat liver.
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o 6.o ×
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-~ 4.0
~2
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3.0 oO
2£
._~'~ D..~.
1,C
1
2
3
4
5
Incubotion time (h)
2io
0
1 2 3 4 Incubotion time (h)
5
Fig. I. Time-course of [l*C]thymidine incorporation into D N A in A H - 4 I 4 cells in the absence and in the presence of n e w - b o r n r a t liver cell sap. F r e s h l y prepared 4 ° ~o n e w - b o r n r a t liver cell sap (0. 5 m]) was added to the incubation m i x t u r e in a final volume of 3.6 ml. The incubation conditions are described in METHODS AND MATERIALS. 0 - - 0 , in the presence of n e w - b o r n rat liver cell sap; G - C ) , in the absence of n e w - b o r n r a t liver cell sap (control). Fig. 2. Time-course of [l*C]orotate incorporations into R N A and D N A in A H - 4 I 4 cells, and the effect of n e w - b o r n r a t liver cell sap u p o n it. 0. 3 ml of 4 ° % n e w - b o r n rat liver cell sap was added to the incubation mixture. The incorporation of [14C]orotate into R N A and D N A in A H - 4 I 4 cells was m e a s u r e d as described in METHODS AND MATERIALS. • - - • and A - / x , incorporation of [14C]orotate into D N A in the presence and in the absence of n e w - b o r n r a t liver cell sap, respectively. 0 - 0 and Q - C ) , incorporation of [14C!orotate into R N A in the presence and in the absence of n e w - b o r n r a t liver cell sap, respectively.
As reported in the preceding papei 5, the inhibition b y the new-born rat liver cell sap cannot be reversed or prevented by an additional supply of L-arginine in the incubation mixture, in contrast to the marked effect of arginine observed for the adult rat liver cell sap inhibition, suggesting that the new-born rat liver cell sap inhibition is not related with the arginase activity. We have found another difference between the two liver cell saps. As shown in Fig. 2, the E14C]orotate incorporation into nucleic acids (RNA and DNA) in AH-4I 4 ceils was not inhibited, but rather markedly stimulated, in the presence of new-born rat liver cell sap. This stimulatory effect of new-born rat liver cell sap seemed to be more prominent in the short period Biochim. Biophys. Acta, 228 (i97 I) 324-330
DNA
327
S Y N T H E S I S I N H I B I T I N G C E L L SAP
incubations. On the other hand, the adult rat liver cell sap did not show any effect, either stimulatory or inhibitory, upon the [14Clorotate incorporations into RNA and DNA. In the previous paper it was reported that the adult rat liver cell sap also inhibited [14Clorotate incorporation 3. Repeated experiments of kinetic and doseresponse analysis, however, showed that the adult rat liver cell sap had no effect upon the E14Qorotate incorporations while the [l*Clthymidine incorporation into DNA was inhibited strongly in accord with previous reports. The above-described results suggested that an inhibitory factor commonly present in both adult and new-born rat livers may be related to the thymidine metabolism but not to the orotate metabolism. Since the metabolic pathways starting from thymidine and orotate are intermingled at the stage of thymidylate, the specific inhibition of thymidine incorporation by the liver cell sap seems to be due either to the impairment of thymidine kinase activity or to the decomposition of thymidine itself. In order to check the latter possibility thymidine phosphorylase and thymidine hydrolase activities of adult and new-born rat liver cell saps were investigated. As evidenced in Table I (hydrolase activity) and Table II (phosphorylase activity), both thymidine hydrolase and phosphorylase activities were detected in the liver cell saps from the adult and new-born rats. The levels of these enzymes in the adult rat liver were a few times higher than those in the new-born rat liver. The kinetics of these enzymic reactions, as evidenced in Tables I and II, also pointed to the lability of these enzymes: thermal treatment of the liver cell saps at 55 ° for several TABLE
I
THYMIDINE HYDROLASE ACTIVITY OF RAT LIVI~R CELL SAP AND ITS THERMOSTABILITY A s s a y of t h e e n z y m i c a c t i v i t y w a s c a r r i e d o u t in t h e a b s e n c e of a r s e n a t e .
Enzyme solution (0.2 ml)
Reaction time (rain)
AAsoo nm
Thymidine hydrolase activity i~moles thymine/ g tissue per h
%
IO % a d u l t r a t l i v e r cell s a p
3° 60 90 12o
0.099 o.124 o.139 o.155
48.1 3o.1 22. 5
20 % a d u l t r a t l i v e r cell s a p
3° 60 90 12o
o.178 0.233 o.252 0.276
43.2 28.3 2o. 4
20 % n e w - b o r n r a t l i v e r cell s a p
3° 60 90 I2O
0.042 o.088 o. lO6 O.lO 7
lO.2 lO.2 8.6
20 % a d u l t r a t l i v e r cell s a p t r e a t e d 3 m i n a t 55 ° t r e a t e d 5 m i n a t 55 °
90 9°
0.092 0.059
7.46 4.76
15. 5 9.9
20 % n e w - b o r n r a t l i v e r cell s a p t r e a t e d 3 m i n a t 55 ° t r e a t e d 5 m i n a t 55 °
90 90
O.Ol 3 o.ooo
1.o 5 o.oo
lO. 3 o
Biochim. Biophys. Acta,
IOO
IOO
228 ( i 9 7 I ) 3 2 4 - 3 3 o
328
M. MIYAMOTO, H. TERAYAMA
TABLE II THYMIDINE
PHOSPHORYLASE
ACTIVITY
OF RAT LIVER
CELL
SAP AND
ITS THERMOSTABILITY
Assay of the enzymic activity was carried out in the presence of arsenate.
Enzyme solution (0.2 ml)
Reaction time (rain)
z]A300nm
Thymidine phosphorylase activity I, moles thymine/ g tissue per h
%
20 °/o adult rat liver cell sap
3° 60 90 12o
0.282 0.378 0.453 0.503
68. 5 45.9 36.7
IOO
20 % new-born rat liver cell sap
3° 60 90 12o
0.086 o. 166 o.17o o.174
2o.8 20.0 13. 7
IOO
2 0 % adult rat liver cell sap treated 3 m i n at 55 ° treated 5 m i n at 55 °
90 90
0.273 0.206
18.6 16. 7
27 24
2 o % n e w - b o r n rat liver cell sap treated 3 m i n at 55 ° treated 5 m i n at 55 °
90 90
0.066 0.057
5.3 4.6
25 22
minutes destroyed the enzymic activities. The relation between the thermal inactivat i o n of t h y m i d i n e h y d r o l a s e a n d t h a t of t h e cell s a p i n h i b i t o r a c t i v i t y of t h e n e w b o r n r a t l i v e r cell s a p is s h o w n i n Fig. 3. I n o r d e r t o c o n f i r m t h a t t h y m i d i n e h y d r o l a s e a n d / o r p h o s p h o r y l a s e in t h e n e w - b o r n r a t l i v e r cell s a p is a c t u a l l y i n v o l v e d i n t h e i n h i b i t i o n of [ 1 4 C l t h y m i d i n e i n c o r p o r a t i o n i n t o D N A of A H - 4 I 4 cells, t h e e f f e c t of u n l a b e l l e d t h y m i n e (a p r o d u c t i n h i b i t o r of t h e e n z y m e s ) u p o n t h e i n h i b i t o r p o t e n c y of t h e n e w - b o r n r a t l i v e r cell
IOO~
~_>
\ ~o'6_"~.
o I~riod of heat treatment
~ (rnin)
Fig. 3. Thermal inactivation of the thymidine hydrolase activity and the cell sap inhibitor activity of new-born rat liver cell sap. 20 % new-born rat liver cell sap was treated at 55 ° for different periods of time (o, i, 2, 3 and 6 min), followed by immediate cooling in ice-water and by centrifugation. Aliquots of the supernate were added to the reaction mixture containing [14Clthymidine to measure the cell sap inhibitor activity. Another aliquot was used for the assay of thymidine hydrolase. In the figure, relative activities of thymidine hydrolase (O) and cell sap inhibitor (O) are plotted against the period of thermal (55 °) treatment.
Biochim. Biophys. Acta, 228 (1971) 324-33 °
D N A SYNTHESIS INHIBITING CELL SAP
329
TABLE I I I EFFECT BORN
OF ADDED RAT LIVER
THYMINE
UPON
[14C]THYMIDINE
INCORPORATION
AND
ITS INHIBITION
20 % new-born rat liver cell sap (ml)
Thymine conch. (M)
[x4CJThymidine incorporated (counts]rain × zo -4 per mg DNA)
None
o 1"1o-5 1.1o -4
I. I9 1.25 1.39 1.31 1.22 o.612 0.744 o.818 0.926 1.22
1.io
0.2
BY NEW-
CELL SAP
-s
3.io -a o I • lO-5 i • lO-4 I • IO-s 3" Io-s
Inhibition (%)
o -- 5 --17 --IO -- 3 49 4° 41 29 o
s a p has been i n v e s t i g a t e d . The results s u m m a r i z e d in T a b l e I I I i n d i c a t e t h a t t h e i n h i b i t o r y p o t e n c y is r e d u c e d m a r k e d l y b y t h e a d d i t i o n of u n l a b e l l e d t h y m i n e over a c o n c e n t r a t i o n r a n g e of I . lO-5-3 • IO -s M. The a d d i t i o n of u n l a b e l l e d t h y n l i n e itself showed no effect u p o n the [14Clthymidine i n c o r p o r a t i o n in t h e absence of new-born r a t liver cell sap. I n a n o t h e r experim e n t , [14Clthymine (25.2 mC/mmole, 0.02/~C) a d d e d to the i n c u b a t i o n m i x t u r e was n o t i n c o r p o r a t e d to a n y a p p r e c i a b l e e x t e n t into D N A (less t h a n 0. 5 % of t h e incorpor a t i o n of t h y l n i d i n e ) . These results s u p p o r t t h e view t h a t t h e change of t h y m i d i n e into t h y m i n e b y t h e e n z y m e s present in t h e liver cell s a p is a real m e c h a n i s m of t h e cell s a p inhibition. I n fact, u n d e r routine a s s a y conditions [l*C~thymidine a d d e d in t h e i n c u b a t i o n m i x ture was d e c o m p o s e d to the e x t e n t of 19 a n d 35 % after 3 h i n c u b a t i o n in t h e presence of o.I a n d 0.2 ml of 20 % new-born r a t liver cell sap, respectively. These values of [14Clthymidine d e c o m p o s i t i o n in t h e a s s a y s y s t e m are well correlated w i t h the degrees of inhibition of [14C]thymidine i n c o r p o r a t i o n b y the new-born r a t liver cell sap.
DISCUSSION The p r e s e n t i n v e s t i g a t i o n has shown t h a t new-born r a t liver cell sap, which was d e v o i d of arginase, could still i n h i b i t t h e [14Clthymidine i n c o r p o r a t i o n i n t o t u m o u r cell D N A . The e x p e r i m e n t a l evidence i n d i c a t e s t h a t t h y m i d i n e h y d r o l a s e a n d / o r p h o s p h o r y l a s e in t h e new-born r a t liver cell s a p are i n v o l v e d in t h e i n h i b i t o r y m e c h a n i s m of t h e cell sap. These e n z y m i c activities were also f o u n d in t h e a d u l t r a t liver cell s a p even a t higher levels, a n d seemed to c o n t r i b u t e to t h e i n h i b i t o r y p o t e n c y of t h e a d u l t r a t liver cell s a p in a d d i t i o n to arginase. T h e cell s a p from A H - 4 I 4 cells was d e v o i d of b o t h arginase a n d t h y m i d i n e - d e c o m p o s i n g enzymes, in accord w i t h t h e o b s e r v a t i o n t h a t the t u m o u r cell s a p showed no effect u p o n [14C1thymidine incorporation into DNA. The evidence o b t a i n e d so far suggests t h a t t h e D N A synthesis in t h e a d u l t r a t liver is u n d e r a d u p l i c a t e control m e c h a n i s m t h r o u g h b o t h arginase a n d t h y m i d i n e d e c o m p o s i n g enzymes, whereas t h a t in t h e new-born r a t liver is u n d e r a single c o n t r o l Biochim. Biophys. Acta, 228 (1971) 324-33o
330
M. MIYAMOTO, H. TERAYAMA
m e c h a n i s m t h r o u g h t h y m i d i n e - d e c o m p o s i n g e n z y m e s , a n d t h a t i n t u m o u r cells is e n t i r e l y free f r o m t h e s e c o n t r o l l i n g m e c h a n i s m s . I n a d d i t i o n t o t h e c o n t r o l m e c h a n i s m i n t h e s a l v a g e p a t h w a y of D N A s y n t h e s i s , t h e n e w - b o r n r a t l i v e r cell s a p c o n t a i n s a s t i m u l a t o r for t h e o r o t a t e i n c o r p o r a t i o n (a p a t h w a y de novo). F u r t h e r s t u d i e s o n t h e n a t u r e of t h i s s t i m u l a t o r as w e l l a s o n t h e m e c h a n i s m of t h e a r g i n a s e i n h i b i t i o n in the DNA synthesis are now in progress in our laboratory.
ACKNOWLEDGEMENT A p a r t of t h e e x p e n d i t u r e s for t h e p r e s e n t s t u d y w a s c o v e r e d b y t h e G r a n t - i n A i d for S c i e n t i f i c R e s e a r c h f r o m t h e M i n i s t r y of E d u c a t i o n .
REFERENCES I 2 3 4 5
6 7 8 9 io ii
I. LIEBERMANN AND P. OVE, Biochim. Biophys. Acta, 38 (196o) 153. M. I-IoRI AND T. UKITA, J. Biochem. (Japan), 51 (1962) 322. H. OTSUKA AND H. TERAYAMA, Biochim. Biophys. Acta, 123 (1966) 274. H. TERAYAMA AND M. SASADA, GANN, 59 (1968) 51. ~{- SASADA AND I-l_. TER&YAMA, Biochim. Biophys. Acta, 19o (1969) 73. R. V. HOLLEY, Biochim. Biophys. Acta, 145 (1967) 525 . G. SCHMIDT AND S. J. THANNHAUSER, J. Biol. Chem., 161 (1945) 83. W. MEJBAUM, Z. Physiol. Chem., 258 (1939) 117. Z. DISCHE, Mikroehimie, 8 (193 o) 3. M. FRIEDKIN AND D. ROBERTS, J. Biol. Chem., 207 (1954) 245. P. GRIPPS, M. ]ACCARINO, M. RossI AND E. SEA.RNO, Biochim. Biop~,s. Acta, 95 (1965) I.
Biochim. Biophys. Acta, 228 (1971) 324-330