[CANCER RESEARCH 29, 391—398, February 19691

the use of the tritium-labeled compound, glucose-6-3H, forincorporation in mouse tumor and reticuloendothelial tissues.

SUMMARY

The need for an in vitro cytotoxic drug sensitivity test covering a broad metabolic field has led to the development of amethod for simultaneous assessment of RNA, DNA, and protein synthesis in vitro by mouse tumor and reticuloendothelialtissue slices using glucose-6-3H as a common precursor.3H/1 4C ratios are given following incubation with both glucose-6-3H and glucose-6-'4C, and the time course of 3H incorporation into protein and the nucleic acids by slices of mousespleen, thymus, lymph nodes, and tumor is presented. The useof actinomycin D and puromycin shows that incorporationinto protein, RNA, and DNA can be separately measured on

the same sample. This is further supported by detailed examination of the hydrolysis products of protein and the nucleicacids. Results are discussed in relation to other methods ofmeasuring nucleic acid and protein biosynthesis and to theknown pathways of anabolic conversion of glucose to proteinand nucleic acids.

INTRODUCTION

The investigations described in this paper were undertakenpreliminary to the design of a sensitivity test aimed at

ing in vitro the relative effects of cytotoxic drugs on reticuloendothelial and tumor tissues in mice and humans. Since thesite or mechanism of action of many such drugs, particularlythe alkylating agents, is not fully understood (33), it appearedimportant to utilize a common precursor, the metabolism ofwhich would cover a wide range of the reactions, particularlythose involved in the biosynthesis of protein, RNA, and DNA.The technic of using a common precursor, in addition tocovering a large area of metabolism, could also enable precursor incorporation to be measured simultaneously into thesecompounds on the same tissue sample, an important factorwhen dealing with small quantities of tissue such as mouselymph nodes or human biopsy samples.

Glucose labeled at the carbon-6 position was selected as thecommon metabolic precursor, and the present study examines

1Supported by a generous grant from the Board of Governors of St.

Bartholomew's Hospital.Received April 17, 1968; accepted October 7, 1968.

MATERIALS AND METhODS

Materials

Glucose oxidase and peroxidase were obtained in a “Biochemica Test Combination― tor blood sugar from the Boehringer Corporation Ltd., (London, W.5.). The DNA (type 1),RNA (type Xl), deoxyribonuclease 1 (DN-C), and the deoxyribonucleoside standards were all obtained from Sigma Chemical Co. (London, S.W.6.). Mylase P (obtained from A. oryzae)was obtained from Koch-Light Laboratories Ltd. (Colnbrook,Bucks., England.)

Glucose-6-14C, glucose-6-3H, and lcucine-U-'4 C were obtamed from The Radiochemical Centre (Amersham, Bucks.).

Actinomycin D was obtained from Merck, Sharp & DohmeInc. (Rahway, N. J.) and puromycin dihydrochloride fromNutritional Biochemicals Corp. (Cleveland, Ohio).

All other chemicals used in the investigation were obtainedfrom British Drug Houses Ltd. (Poole, Dorset).

Animals and Tumor

Young adult female mice from inbred colonies of NIH strain(Scientific Animal Services, Elstree, Herts.) or DBA.2J strain(Jackson Laboratory,Maine) were used. The tumor was anisologous implanted mammary adenocarcinoma (CaD2) main

tamed in the strain of origin (DBA.2J) by serial solid transfer.Experiments were carried out approximately 11 days afterimplantation when the tumor weighed 300—500 mg.

Tissue Preparations and Incubation

Mice were killed by cervical fracture; the tissues were rapidlydissected and dropped into ice-cold Krebs-Ringer phosphate

medium, pH 7.3 (32) containing 0.01% unlabeled glucose.When lymph glands were studied, the axilary pair were takenfrom each side of the animal. When necessary (lymph glandsand thymus) the tissues were freed of contaminating fat andconnective tissue with the aid of a dissecting microscope. Tis

sues were blotted to remove excess moisture, rapidly weighed,

and then sliced mechanically in a tissue chopper based on that

FEBRUARY 1969 391

Simultaneous Measurement of RNA, DNA, and Protein Synthesis in

Mouse Tumor and Reticuloendothelial Tissue Slices Using

Glucose-6- H as a Common Precursor

G. F. RowlandSurgical Professorial Unit, St. Bartholomew's Hospital, London, E. C. 1., England

on June 15, 2021. © 1969 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

G.F.Rowland

of Mcllwain and Buddle (22) set to slice at 0.5-mm intervals.The sliced tissues were transferred to 25-mi conical flasks contaming 1 ml of ice-cold Krebs-Ringer medium. Except whereotherwise stated, not more than 40 mg wet weight tissue weretaken per incubation flask. In the case of lymph glands, thequantity used could be as little as 8 mg per flask. A further1-mi sample of the medium, containing 5 jic (0.01 @.tmoles)ofglucose-6-3H, was then added unless otherwise indicated in thetables of the Results section. The flasks were incubated in ashaking water-bath at 37°C for the required times. At the endof incubation, flasks were cooled in crushed ice, and 1 ml ofan ice-cold 10% aqueous solution of unlabeled glucose wasadded.

Separation of RNA, DNA, and Protein

The standard procedure used for the experiments (exceptwhere otherwise stated) was partly based on the method of Ogurand Rosen (24). The details are as follows. The tissue andmedium were transferred to a small hand-operated homogenizer with a nylon sphere pestle (30) of 10 ml capacity. Afterbrief homogenization the mixture was transferred to a 15-miconical centrifuge tube, and 1.5 ml of ice-cold 10% (w/v) perchloric acid was added. The precipitate containing protein,lipids, and nucleic acids was centrifuged and washed by foursuccessive resuspensions and centrifugations in 3 ml of ice-cold2% (w/v) perchloric acid. Lipids were extracted by successivewashes in 1 ml ethanol, 2 ml chloroform:ethanol (1: 1) twice,and finally 1 ml ethanol. After carefully draining the tubes ofethanol, the precipitate was resuspended in 1 ml ice-cold Nperchloric acid and left for 18 hours at 4°Cto extract RNA.The tube was then centrifuged, the extract set aside, and theprecipitate resuspended in 0.5 ml cold N perchloric acid andrecentrifuged. The supernatant was combined with the firstextract. For extraction of DNA, the precipitate was resuspended in 1 ml 0.5 N perchloric acid and heated in a waterbath at 70°C for 20 mm. The extract obtained by centrifugation was set aside, and the precipitate was reextracted with 0.5ml 0.5 N perchloric acid at 70°Cfor 20 mm. The extracts werecombined. The residual protein precipitate was washed twicewith 2 ml distilled water and then dissolved in 1 ml N KOH bywarming at 50°Cfor 1 hour.

Determination of Specific Radioactivity of RNA,DNA, and Protein

The RNA and DNA extracts were neutralized with a fewdrops of 10 N KOH, and, after cooling, the precipitated potassium perchlorate was removed by centrifugation. The absorption spectra were measured in a Unicam SP 800 spectrophotometer (Unicam Instruments Ltd., Cambridge, England)and were found to correspond closely to the spectra of thepurified commercially obtained DNA and RNA standardsmeasured under similar conditions. In practice it was foundconvenient to measure the extinction at 260 mp ( 1 cm pathlength) of 0.3 ml aliquots of the RNA extracts diluted with3.0 ml distilled water and to compare this with a knownamount of RNA standard. DNA was determined similarly at270 m@.z.Another aliquot (0.6 ml) of neutralized RNA or DNA

extract was transferred to a counting vial for determination ofradioactivity as described below. Protein content was determined by the following modification of the method of Lowryet al. (21). An 0.1-mi sample of the protein dissolved in NKOH was taken, and 6 ml of 2% (w/v) Na2 CO3 containing0.01% CuSO4 @5H2O and 0.02% potassium tartrate wereadded. After 10 mm, 0.1 ml of diluted (1: 1) Folin and Ciocalteu's reagent (21) was added, and the extinction at 750 mjzwas measured after exactly 30 mm. A standard curve was constructed using crystallized bovine albumin (BDH, Poole,Dorset) under these experimental conditions. A further aliquot(0.6 ml) of the dissolved protein was transferred to a countingvial. Radioactivity was determined using a Beckman liquidscintillation spectrometer (Type LS.200) in which quench correction is obtained by ratio of an external standard counted in

two channels. Samples were mixed with 16 ml of the followingscintillation mixture : 4 gm of 2,5-bis-5'-tert-butylbenzoxazolyl-2'-thiophene (BBOT, CIBA Ltd., Duxford, Camb.).,400 ml 2-methoxyethanol, 80 gm naphthalene (molecular weight determination grade), and 600 ml toluene (A.R.).The samples were then counted twice for 20 mm or until20,000 counts were collected. Correction was made for background, decay, and quenching (using a series of standards prepared at different quenching levels in the same scintillationmixture). This enabled results to be obtained in terms of abso

lute radioactivity (pj.zc). The radioactivity was divided by theweight of RNA, DNA, or protein measured as above to givespecific radioactivity (p@ic/100 pg).

Experiments to Examine the Specific Activities of theComponents of RNA, DNA, and Protein

In order to obtain sufficient material of high activity forexamination of hydrolysis products, the pooled spleens of sixmice were used to yield 500 mg of sliced tissue, and this wasincubated in 20 ml of incubation medium containing 500 pcof glucose-6-3H (1.0 pmoles) for 60 mm. After incubation, 5ml of 20% (w/v) glucose was added and the sample was cooledand homogenized. Two 1-mi samples were taken for separationof RNA, DNA, and protein by the standard procedure asdescribed above. The remainder was divided into two equalportions. One portion was centrifuged and extracted with MNaCl as described by Wyatt (34) for preparation of purifiedDNA for enzymic hydrolysis as described below. The otherportion was used for extraction and hydrolysis of RNA andalso of protein as described below.

Separation of Deoxyribonucleosides

The dried purified DNA was dissolved in 2 ml 0.01 M Trismaleate buffer, pH 6.7, containing 100 @zgdeoxyribonuclease,5 mg mylase-P, 15 jimoles MgSO4 , and ethylmercurithiosalicylate to a final concentration of 0.01%. This mixture wasincubated at 30°C for 20 hours to liberate deoxyribonucleosides from the DNA (5). After incubation, the mixture wasdeproteinized by emulsification three times with chloroform:octanol (8: 1 v/v) (5) followed by centrifugation. The deproteinized hydrolysate was taken to dryness in a vacuum desiccator at room temperature over P2O5 and then redissolved in

392 CANCER RESEARCH VOL.29

on June 15, 2021. © 1969 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

Simultaneous RNA, DNA, and Protein Synthesis

about 0.1 ml water for loading on to a thin-layer chromatogram. Separation of the nucleosides was carried out on a 20 x20 cm plate coated with cellulose (MN-cellulose powder 300,Macherey, Nagel & Co., Duren, Germany) at a thickness of0.25 mm. The plate was developed in two dimensions; the firstsolvent was distilled water and the second was isopropanol: 5.7N HC1 (65:35 v/v). The deoxyribonucleosides were located by

examining the dried plates under short-wave length UV lightand identified by comparing positions with those of standardstreated in a similar manner. The spots were then eluted withwater and UV absorption spectra measured at pH 2.0. Thesewere compared with the spectra of the standard nucleosidesand with published data (10, 29) to verify identification andto give a quantitative estimation of each nucleoside. It wasfound that deoxyguanosine was decomposed to the free baseby the second solvent, but, fortunately, this nucleoside couldbe separated from the other three in the first solvent (water).The RF values in water were 0.49 for deoxyguanosine, 0.64for deoxyadenosine, 0.60 for deoxycytidine, and 0.69 forthymidine. After quantitating each nucleoside, the solutionwas evaporated to dryness in a vacuum desiccator over P2O5to remove both water and HC1. The dry residue was redissolved in 0.5 ml water and mixed with scintillation fluid fordetermination of radioactivity.

Extraction and Hydrolysis of RNA and Separation ofRibonucleotides

The homogenized sample was precipitated with perchloricacid, washed, and extracted with lipid solvents as in the standard procedure described above. After the final ethanol extraction, however, the portion used for separation of ribonucleotides was washed twice with ether and the precipitatedried. RNA was then extracted with 10% (w/v) NaC1, purified,and hydrolyzed as described by Graymore (13). The neutralized hydrolysate was subjected to high-voltage paper electrophoresis to separate the ribonucleotides (25). The nucleotidespots were eluted with 2 ml water, and 0.6-mi aliquots were

taken for determination of radioactivity as described above.The remainder of the eluate was scanned in a Unicam S.P. 800spectrophotometer (Unicam Instruments Ltd., Cambridge) toverify and measure the nucleotides by means of their specificabsorption characteristics in the UV region (3).

Hydrolysis of Protein and Separation of Amino Acids

The protein, after extraction of nucleic acids and lipids, waswashed in acetone and ether and air-dried; 2.5 mg dry weightprotein were mixed with 10 ml of 6 N HC1 and heated in a

sealed glass bomb at 1100C for 18 hours. On cooling, thebomb was opened and the contents taken to dryness in arotary evaporator. The sample was redissolved in 0. 1 N HC1 foramino acid analysis and fractionation. This was carried outusing a Technicon Analyser with the buffer gradient as described by Crawhall et a!. (7). In order to obtain amino acidsfor radioactivity determinations, the column effluent wasdivided at the bottom of the column so that approximately80% was collected in a “RadiRac―fraction collector (LKBInstruments Ltd., London) in 2-mi fractions. The remaining

20% was pumped into the optical system of the analyzer via aproportionating pump manifold modified as follows: sampletubing diameter reduced from 0.035 cm (internal diameter) to0.015 cm, nitrogen tubing reduced from 0.045 cm (internaldiameter) to 0.035 cm, ninhydrin tubing diameter unchanged.On completion of the 20-hour analysis, the quantity of eachamino acid was calculated from the recording chart and the

position of the amino acids in the fractionated effluent ascer

tamed. Samples containing the same amino acid were pooledand 0.8-mi aliquots taken for determination of radioactivity asdescribed above. Aliquots of the effluent not containing aminoacid were also taken for radioactivity determination.

RESULTS

Preliminary to the use of labeled glucose as common precursor for protein and nucleic acid synthesis in tissue slices, it wasnecessary to determine whether the overall concentration ofglucose in the incubation medium, namely 0.01%, was suffi

cient for tissue requirements during the incubation period.Several experiments were thus carried out using the glucoseoxidase, peroxidase method to determine the rate of disap

pearance of glucose from the incubation medium in the presence of mouse spleen or tumor slices. It was found that the

mean glucose disappearance rate from five experiments inwhich the tissue quantity per 2-mi incubation medium rangedfrom 55 to 160 mg (wet weight) was 1.03 ±0.10 (S.D.)

@molesglucose disappeared/100 mg wet weight spleen/hour.Similar experiments with tumor slices gave values of 0.80 ±0.03 (S.D.). Therefore, with the glucose concentration in themedium at 0.01% (1.11 j.imoles in 2 ml) by limiting the maximum tissue quantity to 40 mg in 2 ml, more than half thegIucose remains in the medium after one hour's incubation.

In order to demonstrate further that the glucose concentration in medium did not limit tissue requirements in macromolecular biosynthesis, experiments were carried out in whichimmediate precursor incorporation into protein was deter

mined at 0.01% glucose and compared with that using amedium containing glucose at 0.1@Y7o.L-Leucine-U-'4 C (1 j.ic,19.7 jig per 2 ml medium) was used with 40 mg wet weight ofspleen slices. The mean specific activities (j.zpc/100 jig) of protein from six determinations were 1167 ±36 (S.E.) with thelow glucose medium and 1112 ±50 (S.E.) with the high glucose medium. Thus a tenfold increase in glucose concentration

in the incubation medium did not increase the rate of incorpo

ration of amino acid into protein. Experiments were then carried out to compare the specific activities of RNA, DNA, andprotein with respect to 3H and ‘4Cfrom tissue incubated withboth glucose-6-3H and glucose-6-'4C in order to consider thepossibility of increasing lability of the labeled hydrogen atoms

during the incubation period. Table 1 shows the results ofthese experiments. It can be seen that the 3H/'4C ratios inRNA and DNA do not alter significantly during the last 40

mm of the incubation but that the ratio falls slightly when

measured in the protein of spleen slices.

The time course of incorporation into RNA, DNA, and protein was then studied in slices of three tissues of the reticuloendothelial system. The results using thymus, spleen, andaxillary lymph nodes are shown in Table 2. It can be seen that

FEBRUARY 1969 393

on June 15, 2021. © 1969 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

Table 1Ratio

3H/'4C―Incubation

time(mm)RNADNAProteinSpleenTumorSpleenTumorSpleenTumor204.87

±0.264.79 ±0.664.22 ±0.394.58 ±0.243.57 ±0.143.63 ±0.05404.94±0.264.78 ±0.103.97 ±0.104.05 ±0.083.26 ±0.113.41 ±0.11604.61±0.204.43 ±0.173.98 ±0.204.10 ±0.103.16 ±0.143.34 ±0.03

Table2.

IncubationtimeSpecific

activity (i.@c/100jig)―RNADNAProtein(mm)ThymusSpleenLymph

nodesThymusSpleenLymph nodesThymusSpleenLymphnodes1539±917±254±1641±224±227±418±520±549±630117±2794±16207±

872± 869± 9156±2058± 759± 586±645130±13130±13269±12090±1491±9212±13121±1071±3213±1960197

±15168 ±15231 ± 18162 ±10162 ±10225 ±43145 ± 7132 ±12167 ±475195±17227±21223±42167±17209±13255±24133±20145±14223± 9

Table4TreatmentSpecific

activity (i.q,tc/100 @g)aProteinRNADNANone

Actinomycin D2ig/ml1011g/ml

Puromycin5@.LgIml5O1zglml139±9

162±6135±4

96±336±4163±11

112±1444± 6

175±18156±22159±13

179±10181±21

173±21156±11

Table3Incubation

(mm)Specificactivity

(MMc/1001Lg)0RNADNAProtein15

30456075122±

5296 ±23588±40765±36827±42216±17

470 ±36774±24995±371090±4882±10

179 ±11408 ±32505±28593 ±20

G.F. Rowland

3H1 ‘4Cratios in RNA, DNA and protein of mouse spleen and tumor slices incubated with a mixture of glucose-6-3H and

glucose-6-14C for varying times. The incubation medium was basically as described in the text but for these experimentscontained 5 @.tcglucose-6-3H(0.01 @imole),1 j.zcglucose-6-'4C (0.3 zmole), and 1.1 pmoles unlabeled glucose.

aResults are the means ±S.D. of four tissue samples at each time.

Time course of 3H incorporation into RNA, DNA, and protein by mouse thymus, spleen, and lymph node slices incubated with glucose-6-31-1.aMean ±SE. of 4 determinations for each result.

incorporation by thymus and spleen is very similar, althoughincorporation by thymus reaches a plateau at about 60 min inthe case of RNA and DNA while in spleen it continues for the75 mm of the experiment. Incorporation by lymph glands intoRNA, DNA, and protein proceeds initially at a significantlyhigher rate than with thymus and spleen. A plateau, however,is reached sooner, by about 45 mm. Similar experiments were

carried out using slices of the mammary tumor (Table 3). Itcan be seen that considerably higher specific activities wereobtained with tumor than with reticuloendothelial tissues andthat incorporation into RNA and DNA was appreciably higherthan into protein. Incorporation appeared to be leveling off by60—75 mm in RNA and DNA but still increasing in protein.

Table 4 shows the results of six experiments in which spleenslices were incubated in the presence of varying concentrationsof either actinomycin D or puromycin. Actinomycin D hasbeen shown to act by specific block of DNA-dependent RNAsynthesis (11). Following the incubation of spleen slices withglucose-6-3H, incorporation into DNA or protein was not

significantly altered by concentrations of actinomycin Dwhich produced over 72% inhibition of incorporation intoRNA. Similarly, puromycin produced inhibition in incorporation into protein without affecting RNA or DNA. Table 5shows similar results using tumor slices.

In order to examine in more detail the nature of the incorporation into protein and nucleic acids, experiments were carriedout to compare the specific activities of protein, RNA, andDNA, as obtained by the standard procedure, with the specificactivities of their components.

In these experiments approximately 500 mg of spleen sliceswere incubated in 20 ml of medium in which the final specificactivity of the labeled glucose was increased tenfold compared

Effects of actinomycin D and of puromycin on incorporation of 3H intoprotein, RNA, and DNA during incubation of mouse spleen slices withglucose-6-3H. Incubation time was 60 mm. The pooled spleens of 12 micewere used.

aMean ±S.E. of 6 determinations for each result.

Time course of 3H incorporation into RNA, DNA, and protein by mousetumor slices incubated with glucose-6-3H.

aMean ±SE. of 5 determinations for each result.

394 CANCER RESEARCH VOL.29

on June 15, 2021. © 1969 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

Table5TreatmentSpecifIc

Activity (@.ti.tc/IO0pg)aProteinRNADNANone

Actinomycin D2pglml10@.zg/ml

Puromycin

5@LgIml593

±20

647±29788±119

238 ±10683

±37

517±24275±16

700 ±97995

±37

1116±681183±134

887 ±59 Table7FractionSpecific

activity (MMc/I 00pg)―Unhydrolysed

protein(prepared as in the standardprocedure)1485Aspartic

acidSerine33819120Threonine217Glutamic

acid2365Proline3742Glycine

Alanine683705Valine461Isoleucine90Leucine213Tyrosine

PhenylalanineLysine172

8464Histidine93Arginine83Activity

of protein as calculated from theactivities of the constituent amino acidsb1 521

Table6FractionSpecific

Activity (@&c/1OOag)―RNADNAUnhydrolyzed(extracted

as in the standard procedure)3150 ±1671213(mean of2)Cytidylic

acid1809 ±183Adenylicacid9041 ±247Guanylicacid3790 ±56Uridylic

acid1300 ±90Deoxycytidine1015

±148Deoxyadenosine540±94Deoxyguanine1156±95Thynsidine1578±179Activity

of RNA and DNA asca1culated@'from

the activities oftheir components35221210

Specific activities of ribonucleotides from RNA and deoxyribonucleosidesfrom DNA of spleen after incubating slices with glucose-6-3H.

“Mean±S.E. of 4 determinations.bSum of (sp. activity of each component x quantity of that component

present in hydrolysate)

Simultaneous RNA, DNA, and Protein Synthesis

experiment. It may be seen that only four amino acids arehighly labeled, serine, glutam.ic acid, proline, and alanine; ofthese serine is by far the most active. A calculated value ofspecific activity for the protein as obtained from the aminoacid values is also given and agrees well with that of the unhydrolyzed starting material.

Effects of actinomycin D and of puromycin on incorporation of 3H intoprotein, RNA, and DNA during incubation of mouse tumor slices withglucose-6-3H. Incubation time was 60 mm. The pooled tumor tissue from10 mice was used.

“Mean±S.E. of 6 determinations for each result.

with that of the standard procedure. The specific activities ofribonucleotides from alkaline hydrolysis of RNA, and deoxyribonucleosides from enzymic hydrolysis of DNA, are shownin Table 6. The results show that the purine-based ribonucleotides are more highly labeled than the pyrimidine nucleotides and that adenylic acid in particular is highly labeled.Following fractionation of DNA, the deoxyribonucleosidewith the highest specific activity is thymidine while deoxyadenosine labeling is low. Also from the data of radioactivitiesof the ribonucleotides and quantity of each present in theRNA hydrolysate, it is possible to calculate a value for specificactivity of RNA to which only the four nucleotide components have contributed. This value is given in Table 6 and

agrees reasonably with the value for RNA as obtained by thestandard procedure. A similar value is also calculated for DNAand again shows good agreement with the original DNA. Acidhydrolysis of protein from such experiments was also carriedout followed by fractionation into the constituent aminoacids. Table 7 shows the specific activities from a typical

Specific activities of amino acids from an hydrolysate of spleen proteinafter incubating spleen slices with glucose-6-3H.

a Results are from an experiment representative of a group of three similar

experiments.bSum (Sp activities of each amino acid x quantity of each amino acid)

Sum of quantity of each amino acid

DISCUSSION

The present study has arisen from the need for an in vitrotest system with two main requirements. First, a measure ofincorporation into RNA, DNA, and protein was required usingsmall quantities of tissue. Assessment on a single sample oftwo of these parameters could be achieved by the use ofdouble-labeling technics, but not of all three. The secondrequirement was for a system covering a broader field of metabolic pathways for nucleic acid biosynthesis than provided forby the use of labeled nucleosides. It has been shown thatcertain fluoropyrimidine antimetabolites, while inhibitingDNA synthesis and mitosis in mammalian cells (2), do notinhibit incorporation of thymidine (6), the most widely usedprecursor for DNA synthesis studies. These findings suggesteda block in the de novo synthesis of DNA which could not bedetected by the use of labeled thymidine. Since studies areplanned using such antimetabolites and also cytotoxic agentswhose mechanism of action is unknown, it seemed unwise tolimit the scope and accuracy of such studies by the use oflabeled nucleosides. Labeled glucose appeared to offer a morephysiologic approach to the de novo synthesis of nucleic acidprecursors while at the same time providing a measure ofincorporation of some amino acids into protein.Sum of quantities of each component present in hydrolysate

FEBRUARY 1969 395

on June 15, 2021. © 1969 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

G.F. Rowland

In studies on the utilization of glucose-6-'4 C for synthesis ofribose and deoxyribose of rat tissue nucleic acids in vitro (16,17), it was shown that incorporation in liver slices was muchlower than in thymus or spleen. This was ascribed to extensiveglycogenolysis during incubation, with a resultant dilution oflabeled glucose. The technic described in the present study isthus not suitable for a tissue such as liver with large stores ofglycogen but is ideally suited to reticuloendothelial tissues andto tumor tissues, known to have a very low glycogen content(23). Care must be taken, however, not to limit the availabilityof glucose in the medium, and this has been achieved by allowing an ample margin of glucose in the medium over that disappearing during the incubation period.

The use of glucose-6-3H as opposed to glucose-6-'4C wasprompted by the much lower cost of the 3H derivative. Thiscould become an important factor when considering the use oflarge numbers of samples necessary to produce statisticallymeaningful results in the testing of cytotoxic drugs. Using thisisotope could, however, present some problems that must beconsidered. A loss of tritium relative to carbon has been observed in the intact rat after administration of both types oflabeled glucose (9) and a possible metabolic cleavage of theC-H bond at C-6 suggested. In rat liver and diaphragm slices invitro, however, Bloom and Foster (2) found that no tritium waslost from C-6 of glucose formed intracellularly from doublelabeled glycerol. Examination in the present study of carbon/tritium ratios in protein and the nucleic acids after incubatingspleen slices showed the expected loss of tritium in protein as

a result of dehydrogenation steps in the citric acid cycle. Inaddition, however, the protein 3H/'4 C ratio fell by about 10%during the last two-thirds of the incubation period, indicatinga small loss of tritium. The ratio was more constant in thenucleic acids, suggesting that the relative loss of tritium didnot occur at the level of glucose or glucose-6-phosphate but atsome later stage in the metabolic pathway not common to allthree end-products. The higher ratios in RNA compared withDNA observed throughout the incubation are hard to explain,and the possibility of an isotope discrimination effect cannotbe completely ruled out. Such effects have been observed inthe metabolism of tritiated glucose (19), although the maineffects appeared to be directed at the C-i position and not atC-6. The results of the present study, however, would suggestthat if any isotope discrimination effect is present it is directedat some later metabolite, possibly a precursor of RNA.

The somewhat similar rates of progress of incorporation intoRNA and DNA of the three tissues of the reticuloendothelialsystem examined accord in general with the in vivo studies of

Itzhaki and Whittle (18), who showed that labeling rates ofribose and deoxyribose in rat spleen and thymus were similarafter injection of glucose-6-'4C. In studies in vitro, however,(17) they found that, after three hours incubation under oxygen, the relative specific activity of thymus RNA ribose washigher than that of spleen. In the present studies the incorporation into spleen continued to increase after thymus hadreached a plateau. Since, however, the incubation was ccrriedout under air and not oxygen, survival of metabolic functionsmay be prolonged by the presence of erythrocytes in thespleen slices. The relatively higher statistical variation in resultswith lymph node nucleic acids reflects some of the problems

inherent in the use of very small quantities of tissue consistingof a heterogeneous mixture of cells of widely differing metabolic activities. Incorporation by tumor slices was considerablyhigher than by slices of reticuloendothelial tissue; specificactivity of protein was approximately three times and nucleicacids up to five times as high. This result may be expectedsince the implanted tumor is a rapidly dividing and growingtissue. Measurement of tumor growth rate at the time the micewere killed showed tumor weight to be increasing by 27% ofthe wet weight each day.

Reich et a!. (26) showed that actinomycin D inhibits RNAbut not DNA synthesis of mammalian tissue cell cultures.They further showed that protein biosynthesis is not suppressed. The block in RNA synthesis has been ascribed to

specific inhibition of DNA-dependent RNA polymerase (12)

and may prove to be related to the formation of a complexbetween actinomycin D and DNA (27). In the present study,actinomycin D only inhibited incorporation into RNA, evidence that no gross contamination with RNA occurred in theDNA and protein fractions. The concentrations of the drugrequired to produce inhibition were, however, somewhathigher than is sometimes observed with tissue slices. Thus, Ilanet a!. (15), studying the effects on beetle pupa slices, obtained80% inhibition with actinomycin D at 1.6 jig/rnl, and Dickmanand Yang (8) used 0.5 j.zg/ml for inhibition in dog pancreasslices. However, both the mouse spleen slices and the tumorstudied here have a high DNA content, and, since DNA hasbeen demonstrated to protect against the inhibitory action ofactinomycin D (11), more actinomycin may be required toproduce the same inhibition in lymphoid and tumor tissueslices than in tissues with relatively less DNA.

Puromycin is now widely accepted as a specific inhibitor ofprotein synthesis, acting by premature release from the ribosomes of incomplete polypeptide chains (1). The 74% inhibition of incorporation into protein by concentrations which didnot affect incorporation into RNA and DNA is further proofof a satisfactory separation in the present study.

The most direct evidence that incorporation into protein,RNA, and DNA can be separately assessed using glucose-6-3Has a common precursor comes from an examination of thehydrolysis products. In the case of the amino acids, labelingwas very low in all the essential amino acids. Any labeling ofthese amino acids could be explained by the inaccuracies ofcounting the very low activity samples obtained from theamino acid fractionation. Of the nonessential amino acidsmeasured, aspartic acid and glycine had very little activity,whereas serine was very highly labeled. This can be explainedby consideration of the metabolic pathways leading to thesynthesis of these amino acids. Ichihar? and Greenberg (14)

reported formation of serine from glyceric acid and 3-phosphoglyceric acid in mammalian tissues. Present results suggesta high rate of synthesis by these routes in spleen slices. Thesubsequent conversion of serine to glycine involves themoval of the 13-carbon and its hydrogen atoms (31), thusexplaining the lack of labeling of glycine in the present study.

Aspartic acid, synthesized by transamination from oxaloacetic acid, could be expected to be unlabeled due to removal

of labeled hydrogen in the succinic and malic dehydrogenasesteps of the citric acid cycle, whereas glutamate is expected to

396 CANCER RESEARCH VOL.29

on June 15, 2021. © 1969 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

Simultaneous RNA, DNA, and Protein Synthesis

be labeled, arising as it does from a-ketoglutarate, in which thetritium-carbon bond is still intact. Both these predictions areverified by present experiments.

On examination of the RNA hydrolysis products, it wasnoted that the pyrimidine nucleotides were labeled less than

the purine nucleotides. Itzhaki and Whittle (17), using glucose6)4C as a precursor for RNA ribose in vitro, showed verysimilar results. In the present studies, however, considerationmust be given to any possible contribution due to labeling ofthe bases. The pyrimidine skeleton is synthesized from carbamylphosphate and aspartic acid (4). Since aspartic acid labeling is very low, it is unlikely that the cytidylic and uridylicacids of RNA are labeled with anything other than ribose. Inthe case of purine synthesis, however, C-2 and C-8 and theirassociated H-atoms are derived from the @3-carbon of serine(28), which is very highly labeled in the present experiments.This could, therefore, contribute to the overall labeling ofadenylic and guanylic acids. The relatively lower specific activity of guanylic acid could also be explained on this basis sincein guanine the labeled hydrogen on C-2 of the purine skeletonwould be replaced by an amino group.

Similar considerations may be applied to the DNA hydrolysates. Thus the relatively high labeling of thymidine could be

explained by the contribution made to the pyrimidine moietyof the methyl group on C-5, a major source of which is againthe 13-carbon of serine (4). The low activity of deoxyadenosinein the DNA hydrolysates is difficult to explain and may indicate partial destruction of this nucleoside in the chromatographic procedure, as was observed with deoxyguanosine.

The values of specific activity calculated for RNA, DNA, andprotein from the relative contributions of their componentsagree with the specific activity as measured by the standardprocedure adopted in this investigation. This further supports

the view that labeling is free from artefact and represents thein vitro anabolic conversion of glucose to protein and nucleic

acids.It is believed that the method described can form the basis

of an in vitro test of broad application to the selection ofcytotoxic drugs for cancer chemotherapy and also act as astarting point for the elucidation of mechanisms of action ofsuch drugs.

ACKNOWLEDGMENTS

I wish to thank Mr. C. G. Francis for skillful technical assistance andProfessor G. W. Taylor for the facilities of his department. I am alsoindebted to Miss M. Sumner for the transplantation of tumors.

REFERENCES

1. Allen, D. W., and Zamecnik, P. C. The Effect of Puromycin onRabbit Reticulocyte Ribosomes. Biochim. Biophys. Acta, 55:865—874,1962.

2. Beaver, G. H., Holiday, E. R., and Johnson, E. A. Optical Properties of Nucleic Acids and their Components. In: E. Chargaff and J.N. Davidson (eds.), The Nucleic Acids, Vol. 1, pp. 493—553. NewYork: Academic Press, 1955.

3. Bloom, B., and Foster, D. W. Hexose Synthesis in Liver and MuscleStudies with Glycerol-3-3H, 1,3-14C. J. Biol. Chem., 239:967—970, 1964.

4. Carter, C. E. Metabolism of Purines and Pyrimidines. Ann. Rev.Biochem., 25: 123—146, 1956.

5. Chargaff, E., Vischer, E., Doniger, R., and Green, C. The Composition of the Desoxypentose Nucleic Acids ofThymus and Spleen. J.Biol. Chem., 177: 405—416, 1949.

6. Cheong, L., Rich, M. A., and Eidinoff, M. L. Mechanism of GrowthInhibition of H.Ep. 1 Cells by 5-Fluorodeoxycytidine and 5-Fluorodeoxyuridine. Cancer Res., 20: 1602—1607, 1960.

7. Crawhall, J. C., Thompson, C. J., and Bradley, K. H. Separation ofCystine, Penicillamine Disulfide and Cysteine-Penicillamine MixedDisulfide by Automatic Amino Acid Analysis. Anal. Biochem., 14:405—413, 1966.

8. Dickman, R. , and Yang, J. Effects of Pancreozymin, Urecholineand Actinomycin D on the Metabolism of Ribonucleic Acid byCanine Pancreas. Biochem. J., 99: 56P—57P, 1966.

9. Dunn, A., and Strahs, S. A Comparison of 3H and ‘4C-GlucoseMetabolism in the Intact Rat. Nature, 205: 705—706, 1965.

10. Fox, J. J., and Shugar, D. Spectrophotometric Studies of NucleicAcid Derivatives and Related Compounds as a Function of pH. II.Natural and Synthetic Pyrimidine Nucleosides. Biochim. Biophys.Acta, 9: 369—384, 1952.

11. Goldberg, I. H., and Rabinowitz, M. Actinomycin D Inhibition ofDeoxyribonucleic Acid-Dependent Synthesis of Ribonucleic Acid.Science, 136: 315—316, 1962.

12. Goldberg, I. H., Rabinowitz, M., and Reich, E. Basis of Actinomycm Action, 1. DNA Binding and Inhibition of RNA PolymeraseSynthetic Reactions by Actinomycin. Proc. Nail Acad. Sci. U. S.,48: 2094—2101, 1962.

13. Graymore, C. N. Studies on the Effect of Pituitary Growth Hormone on Fat Metabolism and on the Turnover ofNucleic Acids inthe Liver. Ph.D. Thesis: University of London, 1956.

14. Ichihara, A., and Greenberg, D. M. Further Studies on the Pathwayof Serine Formation from Carbohydrate. J. Biol. Chem., 224:331—340, 1957.

15. Ilan, J., Ilan, J., and Quastel, J. H. Effects of Actinomycin D onNucleic Acid Metabolism and Protein Biosynthesis during Metamorphosis of Tenebrio molitor L. Biochem. J., 100: 441—447,1966.

16. Itzhaki, S. Metabolic Studies on the Sugars of'Nucleic Acids. I. TheIsolation of Pyrimidine-Bound Sugars and Utilization of Glucose6-14C for Ribose Formation in Mammalian Tissues in vitro. Biochim. Biophys. Acta, 87: 541—553, 1964.

17. Itzhaki, S., and Whittle, E. D. Metabolic Studies on the Sugars ofNucleic Acids. II. 14C-Labeling of Ribose and Deoxyribose and itsComparison with 32P Incorporation into Ribonucleic Acid andDeoxyribonucleic Acid of Rat-Thymus Cells. Biochim. Biophys.Acta, 87: 554—563, 1964.

18. Itzhaki, S., and Whittle, E. D. Metabolic Studies on the Sugars ofNucleic Acids III 14C-labeling of Ribose and Deoxyribose in RatTissues in vivo. Biochim. Biophys. Acta, 91: 190—198, 1964.

19. Katz, J., Rognstad, R., and Kemp, R. G. Isotope DiscriminationEffects in the Metabolism of Tritiated Glucose. J. Biol. Chem.,240: 1484—1486, 1965.

20. Lindner, A. Cytochemical Effects of 5-Fluorouracil on Sensitiveand Resistant Ehrlich Ascites Tumor Cells. Cancer Res., 19:189—194, 1959.

21. Lowry, D. H., Rosebrough, N. J., Fare, A. L., and Randall, R. J.Protein Measurement with the Folin Phenol Reagent. J. Biol.Chem., 193: 265—275, 1951.

22. Mcllwain, H., and Buddle, H. L. Techniques in Tissue Metabolism.I. A Mechanical Chopper. Biochem. J., 53: 412—420, 1953.

23. Nirenberg, M. W. A. Biochemical Characteristic of Ascites TumorCells. J. Biol. Chem., 234: 3088—3093, 1959.

24. Ogur, M., and Rosen, G. The Nucleic Acids of Plant Tissues. I. TheExtraction and Estimation of Desoxypentose Nucleic Acid and

397FEBRUARY 1969

on June 15, 2021. © 1969 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

G.F. Rowland

Pentose Nudeic Acid. Arch. Biochem. Biophys., 25: 262—276,1950.

25. Rees, K. R., and Rowland, G. F. The Metabolism of Isolated RatLiver Nuclei. Biochem. J., 78: 89—95,1961.

26. Reich, E., Franklin, R. M., Shatkin, A. J., and Tatum, E. L. Effectof Actinomycin D on Cellular Nucleic Acid Synthesis and VirusProduction.Science,134: 556—557,1961.

27. Reich, E., and Goldberg, I. H. Actinomycin and Nucleic AcidFunction. In: J. N. Davidson and W. E. Cohn (eds.), Progress inNudeic Acid Research and Molecular Biology, Vol. 3, pp.184—234.New York: Academic Press, 1964.

28. Reichard, P. Biosynthesis of Purines and Pyrimidines. In: E.Chargaff and J. N. Davidson (eds.), The Nucleic Acids, Vol. 2, pp.277—308.New York: Academic Press, 1955.

29. Reichard, P., and Estborn, B. Preparation of Desoxyribonucleo

tides from Thymonucleic Acid. Acta. Chem. Scand., 4: 1047—1053, 1950.

30. Rowland, G. F. Metabolic Studies on Nuclei Isolated from Normaland Precancerous Rat Liver Cells. Ph.D. Thesis: University of London, 1961.

31. Shemin, D. The Biological Conversion of 1-Serine to Glycine. J.Biol. Chem., 162: 297—307, 1946.

32. Umbreit, W. M., Burns, R. H., and Stauffer, J. F. Methods forPreparation and Study of Tissues. In: Manometric Techniques, 3rd.ed., pp. 135—169.Minneapolis: Burgess Publishing Co., 1959.

33. Wheeler, G. P. Some Biochemical Effects of Alkylating Agents.Federation Proc., 26: 885—892,1967

34. Wyatt, G. R. The Purine and Pyrimidine Composition of Deoxypentose Nudeic Acids. Biochem. J., 48: 584—590, 1951.

398 CANCER RESEARCH VOL.29

on June 15, 2021. © 1969 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

1969;29:391-398. Cancer Res G. F. Rowland

H as a Common Precursor3Slices Using Glucose-6-Synthesis in Mouse Tumor and Reticuloendothelial Tissue Simultaneous Measurement of RNA, DNA, and Protein

Updated version

http://cancerres.aacrjournals.org/content/29/2/391

Access the most recent version of this article at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

.pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/29/2/391To request permission to re-use all or part of this article, use this link

on June 15, 2021. © 1969 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/content/29/2/391http://cancerres.aacrjournals.org/cgi/alertsmailto:pubs@aacr.orghttp://cancerres.aacrjournals.org/content/29/2/391http://cancerres.aacrjournals.org/