BIOCHIMICA ET BIOPHYSICA ACTA 663

BBA 8163

INTRACELLULAR DISTRIBUTION OF DEOXYRIBOSIDIC

COMPOUNDS IN NORMAL AND R E G E N E R A T I N G LIVER

AND IN NOVIKOFF HEPATOMA

RAM M. BEHKI* AND WALTER C. SCHNEIDER

Laboratory of Biochemistry, National Cancer Institute, National Institutes o[ Health, Department o[ Health, Education and Wel]are,

Bethesda, Md. (U.S.A.)

(Received May 2ist, 1962 )

SUMMARY

The intracellular distribution of deoxyribonucleosides and digestible deoxyribo- nucleotides was found to depend upon the medium employed for the isolation of various fractions. In an aqueous medium, these compounds were localized almost exclusively in the supernatant and the digestible forms were hydrolyzed during the process of homogenization and fractionation. The latter was prevented by the prior lyophilization of the tissue samples. However, nuclei prepared from lyophilized tissues in organic solvents contained a considerable amount of deoxyribonucleosides and almost all of the digestible deoxyribonucleotide compounds. The results indicate that the acid-soluble deoxyribosyl compounds are normally localized in the nucleus but are lost from the nucleus when tissues are fractionated in aqueous media.

INTRODUCTION

The presence of deoxyribosyl compounds in normal and malignant tissues has been reported previouslyl-L ROTHERHAM AND SCHNEIDER 7 reported an increased con- centration of deoxyribonucleoside monophosphates and of digestible deoxyribo- nucleotides in acid-soluble extracts of the Novikoff hepatoma and of regenerating livers 24 h post-operatively as compared to normal liver. On the assumption that these deoxyribosidic compounds serve as precursors of nuclear DNA their intra- cellular localization was of interest. Such a study might help in understanding the significance of their increased levels in growing tissues.

The results reported here indicate that in growing tissues the distribution of deoxyribosyl compounds and their increased levels after digestion are dependent upon the medium employed for cell fractionation, since a major portion of these compounds and their digestible forms was found to be present in nuclei when they were prepared in organic solvents as compared to their almost complete absence in nuclei prepared in aqueous medium.

* Fellow of tile Jane Coffin Childs Memorial Fund for Medical Research; present address: Dept. of Pathology, Massachusetts General Hospital, Boston, Mass.

Biochim. Biophys. Acta, 61 (1962) 663-667

664 R. M. BEHKI, W. C. SCHNEIDER

METHODS

Livers from rats were quickly removed, chilled and homogenized in ice-cold medium to give a IO °/o homogenate. Regenerating liver was obtained by partial hepatec- tomy according to the method of HIGGINS AND ANDERSON 8 and the liver was removed 24 h later and homogenized as above. The nuclei were prepared from fresh homo- genates in sucrose-CaCl~ medium by the method of HOGEBOOM et al. 9. The use of CaCI~ was later omitted since the supernatants obtained in its presence gave con- sistently higher values. Nuclei were isolated and washed four times by sedimenting from a 0.25 M sucrose upper layer to a 0.34 M sucrose lower layer. The other frac- tions were prepared as usuaF °.

In other cases the livers from normal and partially hepatectomized rats or transplants of the Novikoff hepatoma were quickly excised and dropped into liquid nitrogen. The pooled samples from a number of rats were broken into small pieces in a liquid-nitrogen chilled apparatus and lyophilized. In the initial stages of lyo- philization, the flasks were kept frozen by surrounding them with an alcohol-dry ice bath. The lyophilization was continued for 4 8 h and then the samples were removed, powdered and lyophilized once again for 4-6 h. A portion, generally 4-5 g, was used immediately for the preparation of nuclei and the remainder was stored in the freezer at --2o °.

For the preparation of non-aqueous nuclei, 4-5 g of lyophilized powder was homogenized with ice-cold light petroleum in a Pot ter-Elvehjem-type homogenizer which was cooled after I-mill of homogenization. About 1.5 h were required to ho- mogenize a 4-g sample. The suspension was filtered through four layers of muslin and the residue was homogenized and filtered again. The nuclei were prepared in carbon tetrachloride-benzene mixtures essentially as described by DOUNCE et al. n . Batches of the same tissue differed slightly in their sedimentation behavior in mixtures of different specific gravities. Variations in the specific gravity of the solvent mixtures were made to compensate for these differences. The preparations were examined under the microscope at each step. All of the centrifugations were carried out at o °. The sedimentation, homogenization and flotation procedures were repeated twice in most cases. The preparations of normal liver nuclei were iudged to be of good quality by their appearance under the phase-contrast microscope as well as after staining with aqueous crystal violet (I mg/ml) lz. Very little adhering cytoplasmic material could be observed. In the case of regenerating liver nuclei, however, the preparations were slightly contaminated with cytoplasmic material. Further purification of these nuclei only resulted in the loss of nuclei and in their breaking into smaller fragments. The nuclei isolated from the Novikoff hepatoma were almost free of any cytoplasmic contamination. DNA and RNA contents of the samples were estimated according to SCHNEIDER 13.

The deoxyribosyl compounds were estimated microbiologically by a modification of the method of HOFF-JoRGENSEN 14 in the neutralized acid-soluble extracts of various fractions. The digestible deoxyribonucleotides were also assayed rnicrobiologically after treatment of the extracts with snake venom 15 and calculated as the difference between untreated and venom-treated samples.

Biochim. Biophys. Acta, 61 (1962) 663-667

I N T R A C E L L U L A R DISTRIBUTION OF DEOXYRIBOSIDIC COMPOUNDS 665

RESULTS AND DISCUSSION

Table I gives the distribution of deoxyribosyl compounds before and after digestion with snake venom in various cell fractions prepared in 0.25 M sucrose. It is evident that the major portion of the tissue deoxyribosyl compounds was present in the supernatant fraction. The addition of CaC12 to the sucrose medium, which is required for the best preparations of nuclei gave inexplicably high values for the supernatant fraction and the addition of o.oi M sodium fluoride, to inhibit phosphatase action,

T A B L E I

CONTENTS OF DEOXYRIBOSYL COMPOUNDS IN VARIOUS FRACTIONS OF NORMAL AND REGENERATING RAT LIVER ISOLATED IN 0 .25 M SUCROSE

T h e r e s u l t s a r e e x p r e s s e d a s / * g of d e o x y c y t i d i n e p e r g of l i v e r or a n e q u i v a l e n t a m o u n t of e a c h f r a c t i o n .

Cell ]raction

Normal rat liver Regenerating rat liver

Be~ore A Iter Be/ore A ]ter digestion* digestion* digestion digestion

U n f r a c t i o n a t e d l i v e r 9.1 (8.1 - lO.O5) lO.4 (9.2 - 11.5) 19.3 25.6 2o.0 19.8 18.2 ~" 23.3""

N u c l e i o.12 ( O . l - O . 1 9 ) 0.35 ( 0 . 3 4 - 0 . 3 7 ) 2.1 2.9 1.3 1.7

M i c r o s o m e s o o i . o i . 2 S e d i m e n t 0.42 (0.27 - 0 .64) 0 .94 (0.45 - 1.4) 0 .88 1.38

( m i t o c h o n d r i a + o .7 1.4 m i c r o s o m e s )

S u p e r n a t a n t 8.2 (7.0 - 9.6) 18.3 23.3 18.o 17. 7 21.o 2o.8

" A v e r a g e of f o u r or m o r e d i f f e r e n t d e t e r m i n a t i o n s . T h e n u m b e r s i n p a r e n t h e s e s i n d i c a t e r a n g e s .

*" A p o r t i o n of t h i s r e g e n e r a t i n g l i v e r w a s f r o z e n in l i q u i d n i t r o g e n a n d a n a c i d - s o l u b l e e x t r a c t w a s p r e p a r e d for a s s a y . T h e v a l u e s o b t a i n e d w e r e 13.6 a n d 25.1 b e f o r e a n d a f t e r d i g e s t i o n re- s p e c t i v e l y .

did not affect the results. In the case of regenerating liver, moreover, digestible deoxyribosyl compounds were apparently hydrolyzed during the homogenization since the amount of these compounds was much less than in a sample of regenerating liver which was frozen and assayed directly (footnote, Table I). The nuclei and other particulate fractions contained only a small percentage of the normal and regenerating-liver deoxyribosyl compounds.

When the tissue samples were frozen immediately and lyophilized, the hydrolysis of digestible deoxynucleosidic compounds was apparently arrested since these com- pounds were present when a homogenate was prepared subsequently in 0.25 M sucrose (Table II). However, the deoxyribosyl componds were again found to be localized mainly in the supernatant fraction, both before and after hydrolysis. The results with lyophilized samples agree well with the values reported previously for tissues assayed directly ~.

When nuclei prepared in non-aqueous medium were assayed for deoxyribosyl compounds, the DNA contents of the isolated nuclei and of the whole tissue was used

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6 6 6 R . M . BEHKI, W. C. SCHNEIDER

T A B L E I I

DEOXYRIBOSYL COMPOUNDS IN HOMOGENATES AND SUPERNATANTS OF LYOPHILIZED SAMPLES OF REGENERATING LIVER AND NOVIKOFF HEPATOMA IN 0.25 m SUCROSE

Tile va lues a r e / t g of d e o x y c y t i d i n e per i oo mg of lyophi l ized t i s sue or an equ iva l en t a m o u n t of s u p e r n a t a n t .

Tissue ]faction • Deoxyribosyl compounds

Belore hydrolysis Alter hydrolysis

Regene ra t ing - l ive r 5.4, 5-4 9.5, 8.8 h o m o g e n a t e

Regene ra t ing - l ive r 5 .6, 5.7 IO.I, lO. 7 s u p e r n a t a n t

N o v i k o f f - h e p a t o m a 12.o, i i .o 16.o, 15.8 h o m o g e n a t e

N o v i k o f f - h e p a t o m a 13.o 19.o s u p e r n a t a n t

The resu l t s were ob t a ined on pooled, lyophi l ized samples f rom a n u m b e r of ra ts .

to calculate the recovery of nuclei. The results are presented in Table III. The yield of nuclei from regenerating liver was the lowest, probably because of their fragile nature. The yield of such nuclei was only about 2-4 % of the lyophilized sample weight but they contained from lO-25 % of the DNA in the lyophilized material from which they were isolated. In the case of the Novikoff hepatoma, 55-62 % of the

T A B L E I I I

DEOXYRIBOSYL COMPOUNDS IN LYOPHILIZED TISSUES AND IN NUCLEI ISOLATED IN ORGANIC SOLVENTS

Material Nuclei yield RNA DNA DNA Deoxyribosyl compounds*

Expt. dry weight ree°very*~ Be/ore A[ter Increase (%) (%) (°/°) (%) digestion digestion

Lyoph i l i z ed i ( ioo) 2. 5 1.o 4 (ioo) 3.85 4.57 o.72 n o r m a l l iver 2 (ioo) 2. 7 0.808 (ioo) 4-4 5 . ° ° 1.4

N o r m a l - l i v e r nucle i I 7 .8 3 .8 5.34 4o 1.8 2.45 0.65 2 5.7 4 .0 " 4-7 33 .2 2.7 3.1 0.4o

Lyoph i l i z ed i ( ioo) 2.7 1.14 (lOO) 3.46 7.1 3.64 r egene ra t i ng 2 (IOO) 0.865 (Ioo) 6.5 8.2 i .7 l iver 3 (IOO) 2.3 0.884 (IOO) 6.0 9.4 3.4

Regene ra t ed - l i ve r I 2. 5 4.1 6.08 13. 3 1. 5 3.64 2.14 nucle i 2 2.08 4.3 4.51 Ii .O 2.72 4.4 1.68

3 4.36 1.6 5 .ol 25.0 3.43 5.84 2-41 Lyoph i l i zed Novi- I (IOO) 2.92 2.62 (IOO) 2.8 4.55 1.75

koff h e p a t o m a 2 (ioo) 2.82 2.92 (IOO) 2. 4 4.6 2.2 H e p a t o m a nucle i I 9 3.6 18.1 62 1.8 3.4 1.6

2 7.05 4 .2 22.7 55 1.42 3.27 1.85

* # g of d e o x y c y t i d i n e per mg of DNA. "* (ioo) represen t s the a m o u n t of D N A in the in i t i a l lyophi l ized ma te r i a l .

DNA of the lyophilized tumor was recovered in the hepatoma nuclei, while in normal liver 32-40 ~o was present in the isolated nuclei. The results of the microbiological assays show that the nuclei prepared in non-aqueous medium contained a substantial amount of deoxyribosyl compounds. Furthermore, the amounts of digestible de-

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INTRACELLULAR DISTRIBUTION OF DEOXYRIBOSIDIC COMPOUNDS 66 7

oxyribosyl compounds recovered in the regenerating-liver nuclei and in the hepatoma nuclei was 59-94 % and 84-91% respectively, of that present in the original tissue indicating that these compounds may be almost exclusively localized in the nuclei of these tissues. Their presence in the supernatant fluid in the fractionation made in o.25 M sucrose may be due to release from the nuclei under these conditions. These deoxyribosyl compounds and their hydrolyzable forms may be of major significance in the metabolic activities of the nuclei since the increased levels of deoxyribosyl compounds in regenerating-liver nuclei and in hepatoma nuclei may be a charac- teristic feature of rapidly multiplying cells. The enzymic activity of these non- aqueous nuclei for the synthesis of deoxyribonucleic acid also points in this direction and will be considered in a subsequent paper.

R E F E R E N C E S

1 W. C. SCHNEIDER, J. Biol. Chem., 216 (1955) 287. 2 W. C. SCHNEIDER, jr. Natl. Cancer Inst., 18 (1957) 569- 3 ~V. C. SCHNEIDER AND L. V~'. BROWNELL, jr. Natl. Cancer Inst., 18 (1957) 579- 4 G. A. LE-PAGE, jr. Biol. Chem., 226 (1957) 135- 5 R. L. POTTER AND I{. L. SCHLESINGER, jr. Am. Chem. So6., 77 (1955) 6714.

R. L. POTTER, R. L. SCHLESINGER, V. BUETTNER-JANUSCH AND L. THOMPSON, .jr. Biol. Chem., 226 (1957) 381.

7 j . ROTHERHAM AND W. C. SCHNEIDER, .jr. Biol. Chem., 232 (1958) 853. 8 G. M. HIGGINS AND R. M. ANDERSON, Arch. Pathol., 12 (1931) 186. 9 G. }-[. HOGEBOOM, W. C. SCHNEIDER AND ]~. J. STRIEB1CH, J. Biol. Chem., 196 (1952) I I I .

10 G. H. I~OGEBOOM, in S. P. COLOWlCK AND N. O. I{APLAN, Methods in Enzymology, Vol. I, Academic Press, 1955, p- 16.

11 A. L. DOUNCE, G. t{, TISHKOFF, S. R. BARNETT AND R. M. FREER, jr. Gen. Physiol., 33 (195o) 629.

12 V. G. ALLFREY, I~. STERN, A. E. MIRSKY AND H. SAETERN, ./. Gen. Physiol., 35 (I952) 520 . 13 W. C. SCHNEIDER, in S. 13. COLOWICK AND N. O. ]~APLAN, ~lethods in Enzymology, Vol. 3,

Academic Press, 1957, p. 680. 14 E. ]:{OFF-JORGENSEN, Biochem. J., 5 ° (1952) 400. 15 \V. C. SCHNEIDER, J. Biol. Chem., 237 (1962) 14o 5.

Biochim. Biophys. Acta, 61 (1962) 663-667