Incorporation of Radioactive Phosphate into NucleicAcids of Regenerating Rat Liver*

ODDVAR NYGAARD AND HAROLD P. RUSCH

(AfcArdle Memorial Laboratory, Medical School, University of Wisconsin, Madison 6, Wis.)

It has been repeatedly demonstrated in tracerexperiments that deoxypentosenucleic acid (DNA)is biochemically inert in tissues where little or nocell division occurs (1, 10, 192, 17). However, inproliferating tissues, such as tumors (92,928) andregenerating liver (5, 6, 8, 14), labeled precursorsare rapidly incorporated into DNA. These observations have led to the concept that DNA issynthesized only during cell division, and, hence,that the incorporation of labeled precursors intoDNA can be used to measure the mitotic activityof a tissue.

Nucleic acid synthesis in regenerating rat liverwas first studied with tracer technics by Brueset al. (6). These workers found that radioactive

phosphate was incorporated extensively into theDNA fraction. This was confirmed by Bergstrandand co-workers (5) using glycine-N'5 as the precursor. The data of Brues et a!. further showed

that, in contrast to the ribosenucleic acids (RNA),the DNA retained its labeling for a considerabletime.

In subsequent studies on regenerating rat liverquantitative changes in cell constituents and datafrom tracer experiments have been correlatedwith mitotic activity. These studies have demonstrated that the concentration of RNA increasedat the time of most rapid growth (16, 926). However, Price and Laird (18) have found that, on aper cell basis, the amounts of the nucleic acids

with the exception of the nuclear RNA—reachedtheir maximum values before an increase in thenumber of dividing cells could be detected. In particular, the amount of DNA per cell increasedgreatly just prior to mitosis.

Eliasson and co-workers (8) and Johnson andAlbert (14) studied the incorporation of glycineN'5 and of p32, respectively, into rat liver constituents during constant periods at various times

5This work was supported by grants from the Alexanderand Margaret Stewart Trust Fund, from the American CancerSociety Institutional Grant No. 71A, and from the Jonathan

Bowman Memorial Fund.

Received for publication August 16, 1954.

after partial hepatectomy. In general agreementwith the results of Price and Laird, they foundthat the most rapid synthesis of both DNA andPNA, as judged from the isotope concentration inthe isolated nucleic acids, occurred at or prior tothe time of maximum mitotic activity.

The following report is a time study of the incorporation of @32into the nucleic •acids of thevarious cell fractions of regenerating liver. Thisstudy was carried out to gain further informationon the interrelationships Of @32incorporation,nucleic acid synthesis, and liver regeneration andalso to determine suitable conditions for the use ofregenerating liver in studying mitotic inhibitors.

MATERIALS AND METHODS

Male albino rats, weighing 185—92925gm., wereused. Partial hepatectomies were performed according to the method of Higgins and Anderson(11). At selected intervals after the operation eachrat received by intraperitoneal injection 0.92 pc ofP32/gm body weight as carrier-free inorganicphosphate.2 J@ the first two series the animals werefasted for 6 hours prior to operation and untilsacrificed; in the other series they were fed adlibitum throughout the experimental period. Allanimals had access to water.

At the time of sacrifice, the rats were anesthetized with ether and the livers perfused in situwith ice-cold O.92@5M sucrose. The liver lobes wereexcised and weighed, forced through a plastictissue press, and a 10 per cent homogenate wasprepared in 0.88 M sucrose.

Preparation of cellular fractions.—The homogenates werefractionated by differential centrifugation, essentially according to the procedure of Price et al. (19), into a nuclear, a largegranular, a microsomal, and a supernatant fraction.

The nuclear fraction was washed twice with either hypertonic or isotonic sucrose solution. The washed fraction contamed 10—15 per cent whole cells. The nuclei were furtherpurified by repeated washing and resedimentation in weak

I Obtained from the Holtzman Rat Co., Madison, Wis.

2 The PE was supplied to us by Dr. E. C. Albright of theDepartment of Medicine, University of Wisconsin, on allocation from the U.S. Atomic Energy Commission.

9240

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NYGAARD AND RUSCH—Phosphate into Regenerating Rat Liver 9241

acid, twice with a 2 per cent citric acid solution (3), and subsequently with a 2 per cent acetic acid solution. This lattermodification was used, since citric acid was found more effective than acetic acid in breaking up whole cells, whereasacetic acid caused less clumping of the nuclei during the subsequent washings.5 The nuclei were resuspended each time bymeans of a motor-driven plastic pestle fitting closely againstthe plastic tubes used for the centrifugation. The nuclei, whilein citric acid, were sedimented by centrifuging for 10 minutesat 600 X g. During the subsequent acetic acid washings theforce was reduced to 500 X g and the time shortened graduallyto 5, 2, and, finally, 1 minute. After about ten washings thesupernatant was water-clear, and microscopic examination ofthe sediment revealed little, if any, cytoplasmic contamination.

Preparation of RNA from cytoplasmicfractiona.—This wascarried out, as described by Barnum and Huseby (1), by extraction with 10 per cent NaCl at 100°C., and by precipitation of the sodium salt of RNA on addition of 2 volumes ofethyl alcohol. The precipitate was re-extracted and reprecipitated in the same way and then washed with cold 5 per centtrichloroacetic acid (TCA) to remove co-precipitated glycogen.The RNA was hydrolyzed with hot TCA according to Schneider (23).

Eztrac&m and separation of RNA and DNA from acidtseshed nuclci.—The add-washed iiuclei were twice suspendedin 95 per cent ethyl alcohol and sedimented by centrifugationto remove any remaining acetic acid. The nuclei were thensuspended in alcohol, methyl red and phenolphthalein addedas indicators, and an aqueous, molar solution of sodium carbonate was added dropwise until the suspension became brightyellow. The nuclei were sedimented and washed once more with95 per cent ethyl alcohol. The combined nucleic acids wereextracted from the washed and neutralized nuclei with NaCland precipitated with alcohol as described for the cytoplasmic RNA fractions. After re-extraction and reprecipitationthe nucleic acids were separated by a modified SchmidtThannhauser (22) procedure. The nucleic acids were dissolvedin 0.1 N NaOH and heated for 20 minutes at 80°C. Twovolumes of alcohol were added to the cooled solutions, andthe samples were left overnight in the cold. The sodium saltof DNA was sedimented by centrifugation. The supernatantliquid, containing the hydrolyzed RNA, was acidified toremove any traces of unprecipitated DNA. The Na-DNA wasredissolved in water; the DNA was precipitated with cold 5per cent TCA, washed with cold 5 per cent TCA, and finallyhydrolyzed with hot 5 per cent TCA (23).

Analytical methods.—Theisolated DNA sampleswere analyzed for the presence of RNA by acombination of the orcinol and diphenylamine reactions (3). Similarly, the nuclear RNA sampleswere checked for the presence of DNA. In bothcases contamination was below the limits ofresolution of the color tests employed.4

Aliquots of the purified nucleic acid sampleswere oxidized and phosphorus determined according to the method of Fiske and Subbarow (9). Theoxidized samples were also used for the determination of radioactivity by an annular GeigerMuller counter for liquid samples. Specific activities were calculated as counts/min4sg P in the oxi

3 This modification was worked out in cooperation with Dr.Walter Wiest, formerly of this laboratory.

4 The methods employed would permit the detection of contaminating nucleic acids present in excess of 1 per cent.

dized samples.5 The specific activity of the totalacid-soluble phosphorus (ASP) of each wholehomogenate was determined to serve as a reference value for the other radioactivity determinations.

The total nucleic acids in aliquots of the original liver homogenates were determined accordingto the method of Schneider (923). The number ofnuclei in diluted aliquots of unfractionated homogenate of the regenerating liver lobes werecounted essentially as described by Price et al.(920).@

RESULTS AND DISCUSSION

PRELIMINARY SURVEY OF P@ INCORPORATION INTO DNA

Eight animals were injected with @32immediately after the operation and sacrificed in pairsafter 192, 18, 924, and 30 hours. This group ofanimals was starved. Only the specific activities ofthe acid-soluble phosphorus and those of theDNA-phorphorus were determined.

Table 1 shows that, for the first 192hours after

TABLE 1

INCORPORATION OF P'@ INTO TIlE AcID-soLUBLE FRACTION (ASP) AND INTO DNAAT VARIOUS TIMES AFFER PARTIAL HuPA

TECTOMY

@32was injected immediately following

the operation.

SrzcivicACTIVITYHouseATT@U(cpm/pgP)OPERATIONASPDNA12101.70.461871.32.312453.34.903047.87.21

hepatectomy, only slight incorporation of @32intoDNA occurred, indicating very little DNA synthesis during this period. After 192 hours the incorporation of @32increased rapidly. This agreeswith the finding of Price and Laird (18) that therewas a steady increase in the amount of DNA pernucleus from 192hours after partial hepatectomyto the time when there was a detectable increasein the frequency of mitosis.

For a time study on @32incorporation intoDNA, the tracer preferably should be administered after the lag period in DNA synthesis.

a The specific activities, as presented here, can be convertedto Biological Concentration Coefficient (24),

BCC — (cpm/mae) X 100—cpm admin/gm body wt'

by multiplying by the factor 41.

6 Crystal violet was used instead of methyl green, following

a suggestion by Dr. A. K. Laird.

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92492 Cancer Research

PERIOD OP Mt,xwtm& INCORPORATIONOF P@ INTO DNA

In the studies of Eliasson and co-workers (8)and of Johnson and Albert (14), the highestvalues for the incorporation of labeled precursorsinto DNA were found about 9.4 hours after partialhepatectomy. However, further data were neededto determine the time of the maximum rate of incorporation more accurately.

Three groups of rats, each composed of threeanimals, were injected intraperitoneally with @3216@920, and 924hours after partial hepatectomy.The animals were all sacrificed 4 hours after theinjection.

As shown in Table 92the specific activities of theASP fraction were relatively uniform in the threegroups. Of special interest here are the specfficactivities of the DNA fractions. The data indicatethat DNA was most actively synthesized duringthe 920-924-hour period.

The specific activities of the nucleic acids reported in Table 92are in general higher than those

TABLE 2

INCORPORATION OF PE INTO NucLEIc ACIDS ATVARIous TIMES AF'r@RPARTIALHEPATECTOMY

.@ All animals were killed 4 hours

after injection of P@.Spzcii.ic ACY1YITY

(cpm/@.gP)Nuclear

ASP@ DNA RNA209 18.6 118204 34.2 117197 25.3 96.5

a Total cytoplasinic RNA. Cytoplasm was not fractionated in theseerperiments.

obtained during the corresponding 4-hour periodin subseqtient experiments (cf. Table 3). This difference may be related to the nutritional status ofthe animals, since the animals reported on inTable 92were fasted, while those in Table S werefed. Similar observations have been made byBarnum and Huseby (1) on fed and fasted normalmice.

TIME S@rimv ON THE INCORPORATION OF P@' INTONuci.zic Acms

In this series the incorporation of @32intonucleic acids was studied for periods of varyingduration. In all cases the radioactive phosphatewas injected 920hours after partial hepatectomy,since in this way the specific activity of the ASPwould be high at the time when@ the rate ofphosphorus incorporation was at its maximum,thus insuring rapid incorporation of @32during theearliest intervals studied.

Eight groups of animals, each consisting of threerats, were used in this series; one group served ascontrols; the remaining animals were partially

hepatectomized. All operations were carried outat approximately the same hour of the day. Sincesome of the groups were to be kept alive for arelatively long time after the operation, all animals in this series were fed.

Quantitative changes of liver constituents.—Thetotal number of nuclei, total DNA, and total RNAin the livers all increased during the regenerationperiod (Table 3). These increases correspondedfairly well to the increases in liver weight. Thecalculated values for DNA per nucleus are all

buss &rvsaOPZRATIC@

Injectionof F―

162024

CytoplasmicRNA*35.631.024.8

Sacrifice

. 20

2428

TABLE .3

QUANTITATiVE CHANGES IN CELL CoNsrnimNTs AND INCORPORATION OF P― iNTO NUCLEIC

AciDs OFVARIoUsCELLFRACTIONSAYI@ERPARTIALHEPATECTOMYHona. @rmOPZRATI@

Uxorzaarsn2554g8364467116ANIMALS*207

3.35445

5.9025.613.3201

3.55576

6.9034.111.9214

4.30670

7.4548.611.1198

208 1924.23 4.63 4.75

640 731 9509.03 9.30 11.3044.3 49.1@ 53.814.2 12.5 11.9201

6.301147

14.1071.412.3215

(7—9)(1400—1900)

(14—19)(60—80)

10.Otbuss

@jiza P@'ADMINISTRATIOS4a4811*4 479114.6

34.525.830.633.332.834.3

0.6614.711.49.68.7

Av. initial body wt. (gm.)Av. final liver wt. (gui.)No. nuclei per liver (XII)')DNA/liver (mg.RNA@liver (mg.DNA/nucleus (mg.X10')

Specific activities(cpm/@sg P):ASPDNANuclear RNASupernatant RNAMicrosomal RNALarge granular RNA

270197100.552.536.924.414.020.022.440.239.428.898.788.675.749.5

.40.734.9§18.1

12.611.130.4

25.523.836.8

36.537.337.4

36.836.638.6

40.139.413.8.

16.736.236.532.4 48.5Total P―in DNA, per liver 8.2(cpmX10@)

C The figures in parentheses are ranges found in normal animals of aiinilsrweiglits.

t Fromthe reportby PriceandLaird(18).:Theisotopewasinjected*0hour.aftertheoperation.I Cytoplasm not analysed.

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NYGAARD AND RUSCH—Phosphate into Regenerating Rat Liver

tamed for resting liver, as seen from Table 3 andas reported previously by other authors (1, 13).In all the cytoplasmic RNA fractions from re.generating liver the specific activity at 924hourswas considerably higher than that of the corresponding fractions from normal livers.

In the RNA of normal liver there is a constantturnover of phosphorus, as well as of other constituents, owing to simultaneous synthesis andbreakdown. The increased incorporation intoRNA in regenerating liver would seem to indicatethat the net increase in the amount of RNA duringregeneration is mainly owing to an increase in therate of synthetic reactions rather than to a decrease in the rate of breakdown.

/09a

@soU

@3O

20

2 4 8 16 24 47 %HOURS AFTER pU INJECTION

CHART 1.—Specific activity-time curves for total ASP, nu

clearRNA, and DNA of regenerating ratliver. P―was injected20 hours after partial hepatectomy. Both axes of the graph arelogarithmic.

The situation with respect to the incorporationof @32into DNA is simpler, inasmuch as labeledprecursors are normally incorporated into liverDNA to a very limited extent. Therefore, in thecase of DNA the increased incorporation is due to

an increase in the rate of synthesis.Relation between net DNA sijnthesie and @Z2

incorporation.—Table 3 also gives the cumulativeincorporation of @32into DNA. Except for thevalue at 47 hours, there was a steady increase inthe amount of @$2incorporated.

Approximate calculations demonstrate the rcationship between the incorporation of @32intoDNA and the amount of DNA synthesized. Forthe purpose of these calculations it was assumedthat DNA is synthesized from P-containingprecursors in the ASP fraction and, furthermore,that the synthesis of DNA is irreversible and proceeds at a relatively constant rate throughout theobservation period.

higher than those reported for normal livers (7,18, 927); however, they show no consistent changewith time. This agrees with the data reported byThomson et al. (927)but not with the observationsof Price and Laird (18), who found that in regenerating liver the amount of DNA per nucleusrises to a maximum about 924hours after partialhepatectomy and then decreases gradually.

Specific activity-time relationship of nucleic acidfractions.—Table S also contains the specificactivities for ASP and the nucleic acids isolatedfrom the various cell components. To demonstratemore clearly the relationship between the specificactivities of the various compounds, the specificactivity-time curves for ASP and the two nucleicacids from liver nuclei have been plotted in Chart1. The curves for the cytoplasmic RNA fractionsfollowed that for DNA very closely and havetherefore not been included.

The specific activity of DNA rose rapidly atfirst, indicating rapid synthesis of DNA. Twentyfour hours after injection of the isotope, thespecific activity of DNA exceeded that of the ASP,and then decreased slowly. Table S shows that at924hours the specific activity of DNA in normal(resting) liver was only@ as great as that ofDNA in regenerating liver.

The nuclear RNA is metabolically the mostactive among the nucleic acids in regeneratingliver, as has been previously found to be the casein resting liver (1, 192, 18, 15). The present datashow tl@at in regenerating liver the turnover of @32in the nuclear RNA is faster than is that of normalliver. Thus, in the present study the maximumspecific activity of the nuclear RNA must havebeen reached earlier than 92hours after the administration of pa2, while in resting rat liver thismaximum occurs after about 5 hours.7 Furthermore, the specific activity of the nuclear RNA inregenerating liver equals that of ASP less than924 hours after injection of P@, while in normalliver the specific activity of nuclear RNA at 924hours is still far below that of ASP (cf. Table 3).8

All the cytoplasmic RNA fractions showedspecific activity-time relationships similar to thatfound for DNA. The analyses at the earliest timesstudied showed that the supernatant RNA incorporated @82faster than the RNA of the particulate fractions. This agrees with the results ob

I Unpublished data.

8 An increase in the uptake of inorganic phosphate-P―

as well as of orotic acid-N― into the nuclear RNA of regenerating rat liver has also been observed by E. P. Anderson andS. E. G. Aqvist (J. Biol. Chem., 202:513—20, 1953). Theirreport came to our attention after the submission of thismanuscript.

- @@--—- I •

0—@ _

NUCLEAR@PN4

;@‘@@.—•—._

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9244 Cancer Research

The specific activity-time relationships of theDNA precursors are not known. However, Sacks(921)has shownthat in normalrats the variouscomponents of the liver ASP have acquired approximately equal labeling about 4 hours after theinjection of p32•If there is any difference betweennormal and regenerating liver in this respect, equallabeling would presumably occur earlier in thelatter tissue. It has therefore been assumed thatat times later than 4 hours after administration ofp32 the specific activity of the ASP will representan approximate value for the specific activity ofthe DNA precursors.

Table 4 gives calculations of the amounts of

TABLE 4

Av. specific activity of ASP(cpm/@@gP)t

Increase in DNA-P―(cpmXlO')

Calculated DNA synthesized(mg.)

Measured DNA increase(mg.)

Ratio (DNA cab/DNAmeas)

S Based on the data for the corresponding time intervals in Table S.t Averagespecificactivitywascalculatedbydividingtheareaunderthe

specific activity-time curve by the number of hours for each time period considered.

DNA synthesized in various periods starting 4hours after the injection of p32•The total numberof counts incorporated into DNA have beendivided by the average specific activity of the ASPin the same period, giving the amount of P incorporated and, hence, the amount of DNAsynthesized in this period. There is reasonableagreement between the calculated and measuredamounts of DNA laid down in the livers duringthe various time periods, indicating that thesynthesis of DNA is indeed an irreversible process.

With one exception the calculated amounts ofDNA synthesized are higher than the measuredones. This difference tends to increase with thelength of the time periods. This tendency is probably owing to the fact that the rate of the DNAsynthesis does not remain constant throughout theexperimental period, as was assumed, but decreases as regeneration approaches completion.This decrease in the rate of synthesis has beendemonstrated in previous experiments with labeledprecursors (8, 14), and it is also evident from thedata in the present report. As a result, the DNAprecursors are incorporated more rapidly when

their specific activity is higher than the estimatedaverage for the entire period.

Recently, Stevens et al. (92ô)and Barnum andco-workers (92) have presented data which theyinterpret as indicating that, during cell division,both daughter cells receive newly formed DNA,while the DNA originally present is broken downand enters the metabolic pool. However, Barton(4), in a preliminarynote, has reportedresultsincompatible with this hypothesis. He found that

@32and C'4, incorporated into DNA of regenerat

ing liver, were not lost when new DNA wassynthesized following a second partial hepatectomy. These findings provide strong evidencethat DNA, once formed in a cell, is biochemicallystable during subsequent cell division. The resultsreported here support this latter view.

The present data, together with those ofprevious authors, demonstrate the close correlation among incorporation of precursors into DNA,DNA synthesis, and cell division in regeneratingrat liver. In this tissue, therefore, DNA synthesiscan serve as an indicator of cell division, and, consequently, measurements of @32incorporation intoDNA may be used in the study of factors influencing the rate of mitosis.

It follows from the results that, if the effect ofmitotic inhibitors were to be studied in thissystem, the animals should preferably be injectedwith @32about 920hours after the operation andsacrificed approximately 920 hours later. Theseconditions would presumably be favorable, sincethey provide DNA of high specific activity in theabsence of an inhibitor.

SUMMARY1. In rats subjected to partial hepatectomy

there was very little incorporation of @32into thedeoxyribonucleic acid (DNA) of the liver duringthe first 192hours after the operation.

92. The most active incorporation of @32intoDNA of regenerating liver was found 920—924hoursafter partial hepatectomy.

3. When @32was injected 920hours after the op.eration, the DNA of the liver reached its maximum specific activity about @0hours later.

4. @32was more actively incorporated into theribonucleic acid (RNA) of the nucleus and of thevarious cytoplasmic fractions in regenerating liverthan in resting liver. However, the relative orderof the specific activities of the various RNA fractions was the same in both tissues.

5. Changes in the absolute amounts of liver

constituents during the regenerative period havebeen recorded. The observed increases in totalRNA, DNA, and number of nuclei per liver were

CoMPARIsoN BETWEENCALCULATEDAND MEASUREDAMOUNTS OF DNA SYNTHESIZED PER LIvER

DURING VARIous TIME PERIODS@

TIMEINTERVAL(hours after P'S mi.)

4—16 4—14 4—47

100.6 78.3 52.84—ge

35.0

22.4 22.7 18.6 34.7

2.23 2.90 8.52 9.90

2.13 2.40 4.40 7.20

1.05 1.21 0.80 1.87

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NYGAARD AND Ruscu—Phosphate into Regenerating Rat Liver 9245

roughly proportional to the increase in liverweight. The amount of DNA per nucleus showedno consistent change with time.

6. The synthesis of DNA, as calculated fromthe @32incorporation, has been compared with thenet increase in DNA of regenerating livers. Thegood agreement between the calculated andmeasured values indicates that the synthesis ofDNA is irreversible.

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1955;15:240-245. Cancer Res   Oddvar Nygaard and Harold P. Rusch  Regenerating Rat LiverIncorporation of Radioactive Phosphate into Nucleic Acids of

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