380 BIOCHIMICA ET BIOPHYSICA ACTA

BBA 95050

R I B O N U C L E I C ACID S Y N T H E S I S AND T U R N O V E R IN RA T L I V E R

AND IN A R A P I D L Y G R O W I N G T R A N S P L A N T A B L E H E P A T O M A

J. M. WHITCUTT A~D JAY S. ROTH

Institute o] Cellular Biology, University o/ Connecticut, Storrs, Conn. (U.S.A.)

(Received January 3ISt, 1964)

SUMMARY

Rats bearing the McCoy MDAB hepatoma and normal animals were killed at differ- ent times after injection with [a2P]phosphate, and liver and hepatoma post-mito- chondrial supernatants were separated into fractions from which the RNA was isolated. Rapidly labeled RNA was found in a membrane and high-speed supernatant fraction, while liver ribosomal RNA separated in a different fraction from that in which the hepatoma ribosomal RNA was found. Starch-gel electrophoresis of RNA extracts gave a number of components, and the base compositions of these were determined. The same types of RNA appear to be present in both liver and hepatoma, though the amounts and turnover are different. A possible mechanism through which ribosomal RNA controls the rate of protein synthesis is suggested.

INTRODUCTION

Cancer cells differ in composition and metabolism from the normal tissue of origin and these changes are presumably preceded by corresponding changes in the struc- ture or functioning of their nucleic acids. Evidence of abnormalities in the distri- bution, turnover and base composition of tumor RNA has been presented 1-3, but the significance of these changes is uncertain. The present investigation is an at tempt to define some of the differences between normal rat liver and a rapidly growing transplantable hepatoma by comparing the RNA isolated at different times from corresponding tissue fractions.

EXPERIMENTAL AND RESULTS Materials

Male albino rats of the Holtzman Wistar strain weighing 2oo-25o g were used (unstarved). The transplantable tumor was the McCoy MDAB hepatoma.

An approx. 20 % suspension of non-necrotic tumor was prepared in 0. 9 % NaC1 solution with a loose-fitting Ten Broeck glass homogenizer and I-ml aliquots were immediately injected intraperitoneally into stock Holtzman strain animals. The tumor developed as an amorphous mass attached to the membranes of the upper abdomen and at various other sites, but the latter material was not used. Animals died of internal bleeding within a week, at which time up to io g of tumor could be recovered. However, experiments described herein were carried out at an earlier

Biochim. Biophys. Acta, 87 (1964) 38o-387

RNA TURNOVER IN RAT LIVER AND HEPATOMA 381

stage (4 days) when the tumor weighed about 5 g- Animals were given intraperitoneal injections of a~PO4a- (I mC in I ml supplied by Iso-Serve Inc., Cambridge, Mass.) at various intervals before being killed by breaking the neck and 5-1o g of liver or tumor was removed and immediately homogenized in a Teflon homogenizer at high speed.

Tissue /ractionation

This was done at o-5 °. Nuclei were prepared from separate tissue samples by homogenizing in 30 ml of 0.25 M sucrose+o.oo2 M CaC1, solution. The suspension was also given a single stroke in a close-fitting Ten Broeck homogenizer before being layered over 40 ml of 0.88 M sucrose solution and centrifuged for 15 min at 2000 ×g. The upper layer and the top IO ml of the lower layer were removed by suction and the remainder of the lower layer was immediately used in the extraction procedure described below.

Other tissue fractions were prepared from samples homogenized in 30 ml of 0.25 M sucrose solution (no CaC12) and centrifuged for IO rain at 15 ooo×g in a Spinco model L ultracentrifuge. The supernatant (except for the bottom 3 ml) was immediately recentrifuged for I h at 80 ooo ×g. This supernatant was decanted and stored at --20 ° while the pellet was allowed to drain for a few minutes and then suspended in 20 ml of 1 % sodium deoxycholate solution (pH 7.6) with several strokes of a ground-glass homogenizer. The suspension was centrifuged for IO rain at 15 ooo×g after which it was layered over 20 ml of 0.3 M sucrose, 0.6 M KC1, o.oi M MgC12 (ref. 4) and centrifuged 2 h at 40 ooo × g for the first hour and at 80 ooo × g thereafter. The upper 30 ml were decanted, and the tubes were inverted and allowed to drain for a few minutes. With liver preparations, a brown upper layer becomes detached from the colorless lower pellet and can be removed by washing with a small volume of deoxycholate solution (light ribosome fraction). Analytical ultracentri- fugation showed that this fraction consisted almost entirely of 6I-S particles, while the lower pellet was somewhat heterogeneous at higher sedimentation coefficients. A flow sheet of the preparations is illustrated in Scheme i.

Extraction o/ RN A

This was done at room temperature. Neutralized 5 % sodium lauryl sulfate solution (pH 7.2) was added to diluted tissue fractions to give the following volumes of i % lauryl sulfate solutions: nuclei, 50 ml; supernatant, IOO ml; membrane, 50 ml; light ribosomes, 15 ml; heavy ribosomes, 15 ml. These solutions were shaken for 2 min after which an equal volume of 9 ° % aqueous phenol containing o.I % 8-hydroxy- quinoline was added and intermittent shaking was continued for I h. The emulsion was then centrifuged at IOOO ×g until a clear lower phase was obtained (20-60 rain). The upper layer and interface were removed by suction and mixed with two vol- umes of 3 % potassium acetate in 95 % ethanol. The precipitate was allowed to co- agulate and then packed by centrifuging for 5 min at IOOO ×g. The pellet was re- suspended in IO ml of 1 % lauryl sulfate solution, centrifuged IO min at 2000 ×g, and this supernatant was decanted, mixed with two volumes of potassium acetate- ethanol and the precipitate packed by centrifuging 5 min at IOOO ×g. The pellet was dried in air for a few minutes (to remove ethanol) and was suspended in 2 ml of

Biochim. Biophys. Acta, 87 (z964) 380-387

382 J. M. WHITCUTT, J. S. ROTH

SCHEME I

Liver homogenate

15 ooo×g / / ~ IO rain

Microsomes and supernatant Nuclei, mitochondria etc.

80 ooo × g

Supernatant (S) Microsomes I layered over o. 3 M sucrose, 0.6 M

KC1, o.oi M MgCI~ / ~ I h at 4 ° ooo × g, I h at 80 ooo × g

/ \ \ Supernatant Colorless pellet covered by loosely membrane fraction adhering brown upper layer

(M) wash with deoxycholate

Brown loose layer Colorless pellet light ribosomes heavy ribosomes

(LR) (HR)

Scheme i. Scheme for separation of fractions from liver and McCoy MDAB hepatoma; details in text. With hepatoma, only a trace or no light ribosomal fraction was obtained.

water and frozen before addition of 2 ml dimethylsulfoxide 5. Finally, 2 ml of 2.5 M phosphate solution (prepared as described by KIRBYe), was added with thorough shaking and the mixture was centrifuged 5 min at IOOO ×g. The upper layer was removed by suction, leaving behind the interracial precipitate and this was treated with potassium acetate-ethanol as above. The precipitate, this t ime part ly liquid, was centrifuged, taken up in 2 ml of water and dialyzed 4 h against water, and was then reprecipitated with potassium acetate-ethanol, washed with ethanol and lyophilized. The dry flakes were taken up in a measured volume of 25 mM sodium borate, 5 mM chloride buffer (pH 8.5) and stored at --20 °.

This procedure gives good purification and satisfactory recovery with small amounts of RNA. For the nuclear fractions, maximum nucleotide recovery was required and the following procedure was used. After hot phenol extraction 7 the aqueous layer and the interface were precipitated separately with potassium aceta te- ethanol and the precipitates were taken up in 2 ml of 0.5 N KOH and incubated 16 h at 37 °. Hydrolysates were cooled and neutralized with 5 N HC104. Precipitates were centrifuged and washed with water and the combined supernatant and washings for each was lyophilized. The residues were made up to I ml with water and adjusted to pH 3-4.

Determination o] RNA

This was done by the orcinol method s using phenol-extracted yeast RNA as standard. 2o-~1 aliquots were assayed in a final volume of 2 ml.

Biochim. Biophys. Acta, 87 (1964) 38o-387

RNA TURNOVER IN RAT LIVER AND HEPATOMA 383

Base analysis

For highly labeled samples (12 and 24 h experiments) 2o-#1 aliquots in 2-ml centrifuge tubes were diluted to o . I2ml with yeast RNA solution (Iomg/ml); otherwise o.I-ml aliquots were taken and the RNA was precipitated with 0.3 ml of potassium acetate-ethanol (see above). The precipitate was centrifuged and drained of ethanol by briefly sucking air through the inverted tube, KOH solution (o.I ml of 0.5 N) was added, the tube was sealed with parafilm and incubated for 16 h at 37 °. The contents were mixed by shaking after I h and were frozen at the end of the in- cubation period. A trace of bromocresol green was added to the frozen sample and then 5 N HCIO 4 until acid. The precipitate was removed by centrifugation and washed with 50 ul of water, and the combined supernatant and washings were applied 2o#1 at a time on a line 18 cm from one end of a 2.5 ×45 cm strip. This was cut from Whatman AE-8I ion exchange paper which had been pretreated by soaking for 24 h in 0.5 M ammonium formate (pH 5) then 24 h in several changes of water, before being dried.

The chromatographic procedure is based on that of JACOBSON 9. Strips were de- veloped in the long direction with 0.2 N formic acid for 9 ° rain, were dried in air, and then cut under ultraviolet light between U, G and A. The U, G part was chro- matographed backwards for 60 rain with 4 N formic acid, dried, and the two parts were taped together again. Base ratios can be assayed by the optical method 1°, but in most cases the radioactivities of the C, A, U and G regions were determined and ratios were calculated from these.

Measurement o] radioactivity 2o-#1 aliquots were counted directly or (12 and 24 h experiments) were diluted

to IO ml from which I-ml aliquots were taken and counted at infinite thinness on aluminum planchettes using a thin-window gas-flow counter (Nuclear-Chicago D-47 detector and Model 165 scaling unit). Samples were counted for 5 min (5oo--lO ooo counts) and were corrected for background averaged over 20 min.

Paper chromatograms for base analysis were passed twice through a Model RSC I6o gas-flow scanner (Atomic Accessories, N.Y.) connected to a Model 162o B ana- lytical count ratemeter (Nuclear-Chicago) and the speed was adjusted to give a stable base line on the recorder. Areas under the peaks were cut out and weighed, and their ratios calculated.

Starch-gel electrophoresis

Gels were prepared from Starch-Hydrolyzed (Connaught Medical Research Laboratories, Toronto) and electrophoresis was done in duplicate as described by WHITCUTT et al. n. After electrophoresis, gels were sliced in half horizontally and fixed in a solution of o.I % (w/v) acridine orange in ethanol-water-acetic acid (50 : 50 : IO, v/v) with occasional stirring for 30 rain. They were then transferred to tubes containing 4 ° ml of ethanol-water-acetic acid, which eluted enough acridine to give maximum contrast between the dark red RNA bands and the yellow fluo- rescent background when viewed under ultraviolet light.

Recovery o[ RNA [rom the gels Individual bands were cut from the gels, the outer edges were trimmed, and

Biockim. Biophys. Acta, 87 (1964) 380-387

384 j . M . WHITCUTT, J. S. ROTH

the bands were suspended in a convenient volume (2o-4o ml) of ethanol-water- acetic acid with a ground-glass homogenizer. The suspension was centrifuged for 5 rain at 2ooo×g and the pellet was resuspended in potassium acetate-ethanol (see above) and resedimented. The pellet was extracted twice with a total of four times its volume of 4 M urea solution (centrifuged 5 rain at 15 ooo ×g), and o.2 ml of phenol-extracted yeast RNA solution (IO mg/ml) was added to the extract, which was then precipitated with potassium acetate-ethanol. Dimethylsulfoxide extraction and dialysis were carried out as previously described* and the final precipitate was used directly in the base analysis procedure.

DISCUSSION

The tissue fractionation procedure described above separated the post-mitochondrial supernatant into four separate fractions in liver and into three hepatoma fractions. Most of the hepatoma RNA was recovered from the colorless ribosomal pellet, and though liver preparations gave a similar pellet, it contained only a small amount of RNA and much larger amounts were found in the loose brown upper layer. I t ap- pears that liver ribosomes, like the nuclear ribosomes 12, are dissociated by excess deoxycholate in media of high ionic strength, and the stability of the hepatoma ribo- somes may be attr ibuted to the fact that they are more actively engaged in protein synthesis 1.-15. Over short periods hepatoma preparations took up label several times faster than the corresponding liver fractions and for both tissues the specific activities of the supernatant and membrane fractions were considerably higher than for ribo- somal fractions. Table I lists the percentage of RNA, its specific activity and over- all base composition for the different fractions obtained from liver or tumor at various time intervals after injection of 32p.

Methods for the isolation of RNA by phenol extraction have been modified several timesT, 16. Dimethylsulfoxide extraction 5 was included to remove protein and polysaccharide still present in the preparations. Considerable amounts of radio- activity remained in some residues and could be released by mild alkaline hydrolysis, mostly as inorganic phosphate.

Starch-gel electrophoresis proved a convenient technique for separating low- molecular-weight RNA into different components, but the acridine staining is prob- ably only suitable for double-stranded structures*. Recovery of RNA from the gels was not quantitative, though there were indications that the bulk of the supernatant and membrane RNA moved with the front bands while the most of the ribosomal RNA was in the slow component.

Starch-gel electrophoresis of liver supernatant RNA (Fig. I) gave a broad fast- moving band which almost certainly corresponds to the mixture of amino acid transfer RNA's; the rapid uptake of label by cytidylate would then be due to turnover of the terminal --pCpCpA group 1~.

Taken together with the high rate of labeling, the base composition and starch- gel pattern of the hepatoma supernatant RNA suggest that it contains appreciable

* In later experiments it was found tha t if the interracial pellet was removed and extracted wi th o.5-1 ml water, much bet ter recoveries were obtained.

* Under conditions favorable to breaking hydrogen bonds; low and high pH, urea, heating; staining of RNA was poor.

Biochim. Biophys. Acta, 87 (1964) 380-387

RNA TURNOVER IN RAT LIVER AND HEPATOMA 385

T A B L E I

DISTRIBUTION, SPECIFIC ACTIVITY AND BASE COMPOSITION OF FRACTIONS PREPARED FROM NORMAL LIVER AND

McCoY MDAB HEPATOMA

Specific Sample % R N A * activity * *

Base compog*t*on

C A U G

L ive r S u p e r n a t a n t 35 14oo 2 h Membrane 13 141o

L i g h t 52 2 i o r ibosome

L ive r S u p e r n a t a n t 28 43 4 °0 24 h Membrane 17 41 ooo

L i g h t 49 34 2oo r ibosome

H e a v y 7 4 ° 60o r ibosome

McCoy S u p e r n a t a n t 25 685o M D A B Membrane 12 669o h e p a t o m a H e a v y 63 2250 2 h r ibosome

McCoy S u p e r n a t a n t 21 177 ooo M D A B Membrane io 181 ooo h e p a t o m a H e a v y 69 153 ooo 12 h r ibosome

L ive r Nucle i 80 t 1280 3 ° min

McCoy Nucle i 9o t 32 90o M D A B h e p a t o m a

3 ° m i n

53 i i 18 18 23 44 i i 22 28 24 19 28

28 18 27 26 21 22 27 18 21

25 19 22

27 18 26 19 25 16

29 19 28 2o 28 17

17 21

23 16

29 25 25

22 22 21

34

27 31 35

34

26

3 ° 34

3 o 31 34

27

* Of t o t a l s u p e r n a t a n t fract ion. ** Counts / ra in / rag RNA.

*** Moles / Ioo moles, based on a2p ac t i v i t y . t Recovery from to t a l R N A in nuc lear fract ion.

C A U G

I 27 17:>828

C A U G

?_5 i8 27 30

26 23 23 28 I

J M

C A U G ~ C A U G

27 19 23 31

22 18 24 37 I~

] 29 30 22 30

LR HR

Fig. I. R e p r e s e n t a t i o n of s t a rch gel e lec t rophores is p a t t e r n s of four subf rac t ions ob t a ined from r a t l iver 24 h a f te r in jec t ion of 32p. The d i rec t ion of m i g r a t i o n is f rom the base to the top of the f igure and the r e l a t i ve a m o u n t s of R N A are shown by the w i d t h and shad ing of the bands . The va lues to the r i g h t of each p a t t e r n are base ra t ios of those bands t h a t were assayed b y me t hods descr ibed in the tex t . S = s upe r na t a n t , M = membrane , L R = l igh t r ibosome and H R = h e a v y

r ibosome fract ion.

B i o c h i m . B i o p h y s . A c t a , 87 (1964) 380-387

386 j . M . WHITCUTT, J. S. ROTH

amounts of components usually associated with the membrane fraction. In spite of small differences in base composition the front component of the membrane fraction is probably transfer RNA, and it is believed that the second component is a pre- cursor of ribosomal RNA.

In view of the high initial specific activity and the unusual base composition of the liver membrane preparation, this fraction probably contains all the messenger RNA associated with the microsomes. There was no evidence, however, for a high A: U ratio (indicative of messenger RNA) in any of the hepatoma fractions.

I t is not clear whether the two fast components of the light ribosome fraction of liver (Fig. I) correspond to any other bands; the front one does not resemble transfer RNA either in appearance or base composition, unlike the front band of the heavy ribosome fraction of hepatoma (Fig. 2) which does correspond to transfer RNA.

t C A U G I 25 18 26 31

• 26 18 24 32

I

m e | S M HR

C A U G

Z8 17 27 28

25 19 26 30

C A U G

• 26,6 ~ 30

m 27 17 23 33

Fig. 2. R e p r e s e n t a t i o n of s ta rch-gel e lectrophoresis p a t t e r n s of th ree subf rac t ions ob ta ined f rom McCoy M D A B h e p a t o m a I2 h a f te r in jec t ion of 32p. The d i rec t ion of mig ra t i on is f rom the base to t he top of t he f igure a n d t he re la t ive a m o u n t s of R N A are shown by t he w id th and shad ing of t he bands . The va lues to t he r igh t of each p a t t e r n are base ra t ios of those b a n d s t h a t were a s sayed by m e t h o d s descr ibed in t he tex t . S = s u p e r n a t a n t , M = m e m b r a n e and H R = h e a v y r ibosome

fract ion.

Experiments involving the isolation of RNA from liver and hepatoma nuclei after 30 min labeling emphasize the difference in the rate of uptake of labeled phos- phate. The base assays in the U, G region obtained from liver nuclear RNA are not given because there was some contamination with inorganic phosphate. The reversed C :A ratio in liver and the normal ratio in the hepatoma confirm the results of GEORGIEV AND ~¢~ANTIEVA 18.

The data presented in this paper, as well as that of othersT, 19-21, are consistent with the suggestion that messenger R N A of DNA-like base composition is incor- porated at a very early stage in the nucleus into a relatively stable, high-molecular- weight complex consisting of the message with attached transfer R N A arranged in a ribbon-like structure (see ZI;BAY~2). Protein and precursor ribosomal RNA may also be a part of this complex and it is possible that a major portion of the transfer RNA of the cells is normally bound in this way.

During protein synthesis it appears that the precursor ribosomal RNA is used up, and undergoes a slight modification of its base composition, characterized principally by an increase in G, and finally becomes incorporated into the ribosome. The complex can be used repeatedly provided sufficient precursor material is avail-

Biochim. Biophys. Acta, 87 (1964) 380-387

RNA TURNOVER IN RAT LIVER AND HEPATOMA 38 7

able. Ultimately the rate of protein synthesis is determined by the rate at which ribosomal RNA is produced 23 assuming that there are adequate supplies of messenger- transfer complex, amino acids and high energy compounds.

The results presented herein show no significant abnormality in the RNA of the hepatoma but do indicate a massive total synthesis of ribosomal RNA and to a lesser extent of transfer RNA. The possibility that the hepatoma also uses a different set of messenger templates cannot be excluded.

ACKNOWLEDGEMENTS

T h e w o r k was s u p p o r t e d b y g r a n t s f r o m t h e A m e r i c a n Can ce r Soc ie ty , P-3o3, a n d

t h e N a t i o n a l C a n c e r I n s t i t u t e , N a t i o n a l I n s t i t u t e s of H e a l t h , C 5264. K i n d l y s e n d

inqu i r i e s to J . S. RUTH.

T h e w o r k of J . S. R. was s u p p o r t e d b y r e sea r ch ca ree r g r a n t RC 31-63 f r o m t h e

N a t i o n a l Cance r I n s t i t u t e , N a t i o n a l I n s t i t u t e s of H e a l t h .

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Biochim. Biophys. Acta, 87 (1964) 380-387