[cancer research 28, 979-986,may 1968] nucleic acids of ... · both purines and pyrimidines were...

[CANCER RESEARCH 28, 979-986,May 1968]

Nucleic Acids of Bovine Lymphosarcoma and Normal Thymus1

Alfred Marshak2 and Celia Marshak

Department oj Pathology, Tulane University School of Medicine, New Orleans, Louisiana 7011S

SUMMARY

Prior work has shown that mild alkaline hydrolysis of nucleipreviously extracted with cold dilute acid and with lipid solvents yielded not only the expected RNA nucleotides butalso free adenine and guanine in amounts which varied inversely with the extent of preliminary lipid extraction. Thenucleic acid of the nuclei was identified as the source of thefree purines.

The experiments described here demonstrated that the DNAof the nuclei was the source of the free adenine and probablyalso of the guanine, and that the conditions to which thenuclei had been subjected were such that significant amountsof purines could not have been removed from isolated DNA.The combined results support the hypothesis that the DNAcontains purine deoxyribotides with direct or indirect linkagesto lipid which render the deoxyribosidic bonds labile to hydrolysis following extraction with lipid solvents.

The lymphosarcoma was found to have a high nuclear ribo-nucleic acid content when compared with thymus and myocardium. The cytoplasmic and nuclear ribonucleic acids of thelymphosarcoma were unusual in that each had a high cytidylicacid content.

In the analyses of the base content of the DNA, statisticallysignificant differences between lymphosarcoma and thymuswere found with respect to adenine and thymine.

INTRODUCTION

In the search for significant biochemical differences betweennormal tissue cells and also in the comparison of normal withneoplastic cells, nucleic acids have been extracted from suchcells or from their particulate components and analyzed fordifferences in their composition. This procedure leaves muchto be desired, for it often has left undetermined the question?of what portion of the total in any one category was represented by the fraction isolated for analysis, what alterationswere induced in the isolation process, and whether in the isolation, relationships that had existed between the nucleic acidsand other cell constituents might have been obscured orobliterated by the extraction process. If one could devise aprocedure for obtaining analytic data on the total nucleic acid

content of the cell or any cell particulate compartment, then,by comparison of the total yield with that obtained by summation of the fractions, agreement or disagreement might reveal the presence of components or relationships not predictedby preexisting information or theory. To this end a procedurewas developed for obtaining all of the bases of the nucleic acidor acids in cells or cell particulate fractions without prior extraction of the nucleic acids from the cell or the particulatefraction (12). This involved digestion of the cell or cell fraction with strong perchloric acid. The results obtained by thismethod compared well with those obtained by other investigators using different procedures when applied to the ribonucleic acid of the cytoplasm, but there was no agreement whendata obtained on nuclear ribonucloic acid were compared (14).With a simple modification of the perchloric acid digestionprocedure, it became possible to obtain accurate analytic datafrom high concentrations of nuclei in which the results hadpreviously been obscured by relatively large amounts ofhumins (14). Comparison of analytic data obtained on thealiquots of the same nuclei by applying the perchloric acidprocedure to obtain the bases and also mild alkaline digestionto obtain the nRNA3 nucleotides did not give agreement unless

there was added to the nucleotide yield the quantities of freeadenine and guanine which were also found, in which case allof the base found in the perchloric acid digest could beaccounted for. It was demonstrated that the free purines couldnot have been contaminants from the cellular pool of acidsoluble substances (14). It was also shown that the free purinesarose from the nucleic acid of the nuclei, and it was foundthat the yield of the free purines diminished with the extentof the preliminary extraction of the lipids (14).

The present experiments demonstrate that the source of thefree purines is the DNA. They also show that they arise fromDNA purine nucleotides whose glycosidic bonds have beenrendered labile to hydrolysis and have led to the hypothesisthat this lability is a consequence of the linkage of lipid tothese nucleotides.

Differences observed in the composition of the nRNA,

1 This investigation was supported by USPHS Research GrantCA 08099-03 PC and American Cancer Society Research Grant#P434. A preliminary report on some phases of this work hasbeen presented (13).

2 American Cancer Society Professor of Experimental Pathology.Received July 11, 1967; accepted January 18, 1968.

3 The following abbreviations are used : cRNA, cytoplasmicribonucleic acid; nRNA, nuclear ribonucleic acid; RCF, relativecentrifugal force; GMP, guanylic acid; CMP, cytidylic acid;AMP, adenylic acid; UMP, uridylic acid; At, adenylic acidwhich, when placed on the positive side of the electrophoresispaper strip nearest the positive pole, moved as a cation; G!, cytidylic acid which moved as a cation under the conditions mentioned above; Gj, guanine which showed no electrophoretic mobility when placed on the side of the paper nearest the negativepole.

MAY 1968 979

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Alfred Marshak and Celia Marshak

cRNA, and DNA of the lymphosarcoma and thymus are described and discussed.

MATERIALS AND METHODS

Tumor Tissue. We are indebted to Dr. Robert Marshak ofthe School of Veterinary Medicine of the University of Pennsylvania for making the lymphosarcomatous tissue availableto us. Lymphosarcomas 231 and 444 were obtained from severalenlarged mesenterio nodes which were removed immediatelyafter the animals were sacrificed; all necrotic and hemorrhagicareas were removed.

Thymus. The calf thymus tissue analyzed was obtained ata slaughter house and processing of the tissue began within20 minutes after sacrifice.

Nuclei and Cytoplasm. Nuclei and cytoplasm were isolatedin cold, 5% citric acid as previously described (10, 11). Nucleiisolated by this procedure were used because they could beobtained free of cytoplasmic contamination and because experiments with 32P-labeled nuclei showed that, when storedin the cold, such nuclei did not lose 32P- or UV-absorbing ma

terial to the medium (10, 11). They were then extracted 3times at 0Â°Cfor 10 minutes with 5% trichloracetic acid, cold

70% ethanol twice, cold 95% ethanol twice, 100% ethanolonce, and left in ethanohether (2:1) at 1-2Â°C overnight.

Further lipid extraction was carried out by refluxing for 10minutes using the following solvents: ethanol:ether twice,methanol:chloroform twice, petroleum ether (30Â°-60Â°Cboil

ing point fraction) twice. The preparations were then storedin fresh petroleum ether at room temperature overnight. Thefluid was decanted and the final traces of petroleum ether removed by evaporation at room temperature. The extracted,dried preparations were taken to constant weight in a vacuumdesiccator over silica gel. This pretreatment for the removalof acid soluble materials and lipids was similar to that used fornormal thymus nuclei, preparation 15-2, the analysis of whichis given in Marshak and Mullock (14).

14C-labeled DNA. Rats of the Charles River strain (males170-230 gm) were subjected to partial hepatectomy. Twenty

to 22 hours later, each of the 12 rats was given intraperitonealinjections of 4 /Â¿cof deoxyadenosine-8-14C (46.5 mc/mmole)and 4 /Â¿cof deoxycytidine-2-14C (30.4 mc/mmole) (New Eng

land Nuclear Corp., Boston, Mass.) in physiologic saline solution. Thirty-five days later the animals were sacrificed, thelivers perfused with cold saline, the nuclei isolated in cold 5%citric acid, and treated as described above for removal of theacid soluble and lipid constituents.

Methods

Alkaline Digestion. The basic procedure, reported earlier(14), consisted of hydrolysis of the dry preparations in 3.35ml of 0.3 N KOH per 100 mg at 30Â°C for 18 hours, with

intermittent stirring during the first hour. At the end of theincubation period, the solution was adjusted to pH 2 and wasallowed to stand for 1 hour at 1-2 Â°C.The precipitate was re

moved by centrifugation in the cold and washed twice with0.01 N perchloric acid. The solution obtained after removal ofthe precipitate was designated the supernatant fraction.

To obtain rapid penetration of the solvent in the investigations of varying hydrolysis times and liquids, the following procedure was used. The weighed dry nuclear powder in a 12-mIconical centrifuge tube, which also contained a polyethylenerod with a tapered end, was clamped to a vibrating machine(Vibro-mixer, Model El, Chemapec Inc., Hoboken, N. J.) whilethe tube was immersed in a constant temperature bath. Vibration was started before the liquid was added. All lumps wererapidly dispersed and solution was completed within 10 minutes.The time in contact with the test liquid was 1 hour. When thehydrolysis liquid was distilled water, the pH of the dispersionwas in the range 4-4.5. Undissolved solids were removed bycentrifugation at 2,250 RCF for 15 minutes at 1-2Â°C. Thesupernatant fluid was then filtered with suction throughsintered glass filters (porosity, 2-2.5 /*).

Volume Reduction. As in the previous experiments, reduction of the volume of the supernatant solution at pH 4 wascarried out with a stream of dry nitrogen impinging upon thesolution held in a boiling ethanol vapor bath (78Â°C). During

the concentration process the pH was repeatedly adjusted to4 with KOH and the precipitated KC104 removed.

In Experiment 39, where water was the hydrolysis liquid,the supernatant fluid volume was reduced in a flash evaporatorat 3-4 mm Hg with the flask immersed in a 20Â°Cwater bath.

Concentration time was 15 minutes.Electrophoresis. The apparatus and procedure for separa

tion of constitutents of the supernatant fluid of the alkalinedigest were the same as that previously described, but the potential drop was 4.7 volts/cm. Electrophoresis time was 3-4hours. Elution and spectrophotometric assay were carried outas previously described (14).

Determination of Bases. Digestion was in 70-72% HC104with added sucrose as previously described (14). The diluted,clarified digest was subjected to descending chromatographyon S & S No. 597 paper in an isopropanol:HCl solvent system(20). In a typical experiment, the supernatant fluid reducedto 1 ml volume was divided into two 0.5 ml portions, onealiquot being used for electrophoresis. The other, having beenreduced almost to dryness, received 4 mg sucrose and 0.2 mlof 11.9 x HCI04 and was then heated in a steam bath for 1hour to release the bases. The washed precipitate containingthe DNA, was collected at pH 2 and digested with 1.0 ml of11.9 N HC104 and 30 mg of added sucrose. Solids in the digestwere removed by centrifugation. Digests obtained in this waygave chromatograms free of interfering fluorescent substances.

In the search for thymine in the presence of large amountsof uracil, as in digests of cytoplasm, descending chromatog-raphy was also carried out with the butanol:! N HC1 (7:1)solvent systems.

In the experiments with the 14C-labeled rat DNA, where

both purines and pyrimidines were labeled, it was found thatthere was significant tailing of radioactivity from cytosine toadenine in chromatograms developed with the isopropanol:HClsystem and from thymine into adenine in the butanol :HC1.Wide separation of all bases was obtained by two-dimensionalchromatography employing butanol:0.1 N ammonia (7:1) inthe first dimension for 16 hours, and isopropanol:HCl in thesecond dimension for 22 hours. All blank areas adjacent to

980 CANCER RESEARCH VOL. 28



Lymphosarcoma and Thymus Nucleic Acids

UV-absorbing areas, when eluted, were found to be free ofradioactivity and, therefore, could not be a source of radioactive contamination in the base to be assayed.

UV Absorption. UV-absorbing spots were located and outlined, and the compounds eluted by shaking in 0.1 N HC1 for1 hour as previously described (14). Using a Gary 14 spectro-photometer (Gary Instruments, Monrovia, Calif.), absorptionspectra in the region 200-300 m/Â¿were taken of all eluates.Where necessary for identification, spectra were also obtainedin 0.1 N NaOH.

Measurement of Radioactivity. All counts were determinedby liquid scintillation, using a Tricarb 314-Ex-2 (Packard Instrument Co., Downers Grove, 111.).Preliminary data on countsof DNA bases separated by one-dimensional chromatographywere obtained directly from the chromatogram by cutting out astrip 40 x 77 mm containing the base to be measured, insertingthis strip with forceps into the glass vial so that the paperstrip completely lined the wall without overlapping, and thenadding the phosphor. Determinations of the amounts of thebase being counted were made by eluting other identical stripsof the chromatograms. However, this procedure was not usedfor the data summarized in Table 8, because it was found thattailing contributed to the radioactivity counts. As just mentioned, these data were obtained by two-dimensional chromatography, which eliminated tailing contamination.

Preliminary results on 14C measurement in cationic adenine

were obtained by pooling 0.1 N HC104 eluates of measuredamounts of adenine from electrophoresis strips and reducingthe volume under N2 in an ethanol vapor bath. The concentrated eluate was then streaked on to a strip of blank S & SNo. 597 filter paper 30 x 77 mm previously inserted into avial. However, it was found that it was not possible to securerigid control of losses of the compound during the multiplemanipulations involved in the pooling, concentration, andtransfer of the eluates.

The following procedure was used to obtain the data reported in Table 8, which includes DNA bases separated bytwo-dimensional chromatography and the RNA nucleotidesand bases separated by electrophoresis. After obtaining the UVabsorption spectrum of the eluates in 0.1 N HC1, measuredamounts of each category (from several chrcmatograms orelectrophoresis strips) were pooled after filtration through asintered glass filter to remove traces of paper fiber. The volumes were reduced to approximately 1 ml in a stream of nitrogen and the concentrated solutions transferred to stripsof S & S No. 597 paper 40 x 77 mm previously inserted intovials. Transfers were made so that at no time was there freesolution in the vials. An infrared lamp above the vials maintained a temperature of 50Â°C during the period when the solu

tion was being evaporated. In control tests it was found that,after taking to dryness the contents of vials containing 1 equivalent of HC1 in 1 ml, 97% of the HC1 had been removed byevaporation. The residual acid was removed by adding andevaporating two successive portions of distilled water. Trialswith solutions of adenine showed uniform distribution of thecompound after drying when examined with the UV lamp, andit was found by titration that there was no measurable amountof acid.

Two scintillation fluids were used in counting. The first system was composed of 2,5-diphenyloxazole, 4.9 gm; l,4-bis-2-(5-phenoxazolyl)benzene, 0.1 gm; and toluene (MathesonSpectral Grade) to 1 liter. After completion of this series ofcounts, the fluid was decanted and vials rinsed twice withtoluene and then allowed to dry under an infrared lamp. Thevials then received the scintillation fluid designated as EDAMby Davidson (3). In the first system in which the radioactivecompounds were not soluble, it was found that quenching wasconsiderably higher than in the second. Results are, therefore,presented only for the counts done in EDAM. Quenching wasmeasured in all samples using an internal standard (9.88 /A ofa toluene-14C solution) (Packard) containing 4,238 dpm. By

counting twelve successive cycles over a period of several days,it could be established that the solutions were not phosphorescent, and also that the toluene-14C in the amount added re

mained uniformly distributed in the solution. Previous trialsshowed that larger volumes of toluene would separate withtime. The counting efficiency had a mean value of 74% anda range of 61-75%, the individual efficiencies being dependent

upon the nature and amount of the purine or pyrimidine inthe solution.

RESULTS

Cytoplasmic RNA

Nucleotides. The yields and molar proportions of the nucleotides of the tumor cRNA are shown in Table 1. For comparisonthe proportions of the bases found previously in cRNA of normal calf thymus (12) are also presented. In the tumor theproportion of GMP was lower by 37% than the GMP of normal thymus cRNA. The CMP was 17% higher than in normalthymus.

Substances Moving as Cations. Only two of the substancespreviously classified as cations (14) appear in the supernatantfraction of the alkaline digests of cytoplasm. These are desig-

Table 1

ElectrophoresisNucleotidesLIL2

T2CationsLIBases"L,AMP0.441.01.0AI0.45A0.49GMP0.52

121.9ChromiG0.46CMP0.74

1.71.4Ci0.78itographyC0.72UMP0.43

1.00.9U0.34Total2.132.01

Bovine lymphosarcoma cytoplasmic RNA products found in thealkaline hydrolysate. L, lymphosarcoma; T, normal thymus; 1,micromoles/10 ing acid-extracted, fat-free nuclei; 2, molar proportions with AMP equal to 1 ; A, adenine; G, guanine; C, cytosine;U, uracil ; AMP, adenylic acid ; GMP, guanylic acid ; CMP, cy ti-dylic acid; UMP, uridylic acid; Aj, adenylic acid as cation, Cj,cytidylic acid as cation.

0 From an aliquot of the KOH hydrolysate digested in 70%HC104 at 100Â°C.

MAY 1968 981




nated as Ax and Cj in Table I. Their absorption spectra in 0.1N HCl and 0.1 N NaOH were found to be identical with thatof AMP and CMP, respectively. The yield of Aj was approximately equal to AMP and the yield of Ct approximatelyequal to CMP. With reference to the yield of bases obtainedby digestion of the supernatant fraction with perchloric acid(Table 1), it was clear that the sums AMP + Aj and CMP+ GÃŒwould constitute amounts far in excess of the adenineand cytosine, respectively, that were recovered by chromatog-raphy. In each category, either the nucleotide or the cationwould account for all the base found in the perchloric aciddigest. From these quantitative relationships and from thespectral data it appeared that cations Ax and Cl were identicalwith the nucleotides AMP and CMP, respectively. When thematerials to be separated electrophoretically were placed onthe positive side of the paper strip, the AMP and CMP movedtoward the negative pole and, when placed on the negativeside, they moved toward the positive pole. As in the case ofthe cRNA of normal thymus or myocardium (14), there wereno nonnucleotide cationic substances in the supernatant fraction from the cRNA.

Cytoplasmic DNA

The portion of the alkaline digest which was precipitatedat pH 2 was washed and digested in 11.9 N HC104. No thymine

Table 2

NucleotideLymphosarcoma

231Lymphosarcoma444aThymus32*Thymus

15-2Â«AMP1.001.001.001.00GMP1.671.601.401.58proportionsCMP1.451.481.171.21UMP1.050.981.030.91YieldÂ«1.031.390.270.60

Bovine thymus and lymphosarcoma, nuclear RNA. See Table 1for abbreviations.

" Micromoles per 10 mg acid-extracted, fat-free dry nuclei.6The animal from which this gland was taken was unusually

small. In the isolation of its nuclei, much finely divided materialwas found in the suspension which gave a more viscous characterto the suspension and required more than the usual number ofwashings to obtain the clean nuclei. The data are presented without bias as to whether this was a normal gland from a normalanimal.

cFrom A. Marshak and B. Mullock (14).

could be found after chromatography. An amount of thymineequal to 5% of the uracil found would have been detectedreadily by the methods used.

Nuclear RNA

Nucleotides. The analytic data obtained for the nRNA oftwo lymphosarcomas are given in Table 2. Included also forcomparison are the molar proportions of two preparations ofnuclei from normal bovine thymus. Although in the cRNA itwas the GMP proportion which deviated most from the valuesobtained with normal thymus, in the nRNA the CMP wassignificantly higher while the difference in GMP was of onlymarginal significance. Thus the lymphosarcoma differed fromthe thymus in having a high cytidylic acid content in both thecRNA and nRNA.

Cations. The bases adenine and guanine, as well as thesubstances A1( C,, and Gl previously found in bovine thymusand myocardium nRNA (14), were also obtained in the tumornRNA. Table 3 gives the data for lymphosarcoma 231. Thecationic substances Aj and C1 were found in amounts equalto the AMP and CMP. The At and Cj of the nRNA also hadthe spectral characteristics of AMP and CMP.

G! was equal to the cationic guanine (G) in quantity and inits spectral characteristics in both acid and alkaline solutions.G! therefore appears to be identical with guanine which, whenplaced on the side of the paper strip nearest the negative pole,does not move appreciably toward that pole but, when placedon the positive side of the paper, moves as a cation. Theguanine recovered after perchloric acid digestion shows goodagreement with the sum of guanine from guanylic acid pluscationic guanine.

The purines adenine and guanine were obtained as cationsin the supernatant fraction of the alkaline digests of both thymus and lymphosarcoma nuclei. The yields are given in Table4. In the data listed, thymus nuclear preparations 15-1 and 17differed in the extent of lipid extraction in the pretreatment ofthe nuclei (14). Although the yields of the purines varied forthe individual preparations, the yield of guanine relative toadenine was about 0.5 in all but one of the tissues analyzed.

Table 5 gives the ratio of the individual nRNA nucleotidesto the free adenine found in nuclei of the normal tissues and inlymphosarcomatous lymph nodes. The values of the ratios foreach of the nucleotides from the normal tissues were in the

Table 3

ElectrophoresisNucleotidesCationsAMP0.200

Â±0.004A

AIÂ»0.106

Â±0.003 0.206 Â±0.004GMP0.334

Â±0.008G0.051

Â±0.003CMP0.2ÃœO

Â±Oi"0.054

Â±0.0050.003UMP0.210

Â±0.007CiÂ»0.304

Â±0.004Total1.0340.721ChromatographyBases0A0.362

Â±0.006G0.373 Â±0.007C0.341 Â±0.008U0.208 Â±0.011.284

Bovine lymphosarcoma nuclear RNA products" found in the alkaline hydrolysate. See Table 1 for abbreviations.a Micromoles per 10 mg acid-extracted, fat-free dry nuclei.0Aj and Cj as in Table 1 ; Gj, electrophoretically immobile guanine.Â»From an aliquot of the KOH hydrolysate digested in 70% HC1O4 at 100Â°C.





Table 4 Table 6

Lymphosarcoma18*Lymphosarcoma

231Lymphosarcoma444Thymus15-1"Thymus15-2"ThymusIT"Thymus

32^Adenine0.0340.1060.0950.3270.2400.1270.040YieldÂ«Guanine0.0260.0510.0550.1510.1200.0580.021Guanine/

adenine0.770.480.580.460.500.460.52

Bovine thymus and lymphosarcoma purines in alkaline digestsof nuclei.

" Micromoles per 10 mg acid-extracted, fat-free dry nuclei.6 From A. Marshak and B. Mullock (14)."See Table 2, footnote b.

range 0.7 to 1.0, while those for the lymphosarcoma were inthe range 2.8 to 3.9. Thus the difference in the results for thetwo categories of nuclei was not limited to one or two nucleotides but was characteristic of all of the nucleotides in eachcategory.

Table 6 gives the yields of the purines and nucleotides obtained by alkaline digestion of nuclei from the two types ofnonneoplastic tissues and from two sets of enlarged mesentericlymph nodes from lymphosarcomatous cows. These nuclei hadbeen subjected to similar schedules of preliminary extractionwith cold dilute acid and lipid solvents. The amount of purinesrecovered from the lymphosarcoma was only half of that obtained from the thymus. However, the nucleotide yield fromthe lymphosarcoma was two times greater than that for thethymus. The ratio of purines to nucleotides was 0.5 in the normal tissues compared with 0.1 in the lymphosarcoma.

Release of Adenine and Guanine from Bovine Lymphosarcoma Nuclei. Table 7 gives the results obtained when therelease of the adenine and guanine from the fat-free, acid-extracted bovine lymphosarcoma nuclei was obtained undervarious conditions using the vibrator as described in the sectionon Methods. The purine yield after 1 and 18 hours of treatment with 0.3 N KOH was essentially the same; in the firstcase no individual nucleotides could be separated by electro-phoresis of the supernatant fraction, while in the latter casethe individual nucleotides were well separated by electropho-resis. Substitution of distilled water for KOH did not affectthe yield of the purines appreciably, and avoidance of contactof the nuclear powder with solution at pH below 4 loweredthe yield of purines only to a slight extent. The guanine toadenine ratio, under all experimental conditions, was 0.5 to 0.6.

Table 5

AMP GMP CMP UMP

Normal tissues" 0.74 1.07 0.83 0.82Lymphosarcoma" 2.76 3.56 3.92 3.56

Nuclei of bovine tissues and lymphosarcoma; nucleotides relative to free adenine. See Table 1 for abbreviations.

" Means of 4 preparations of nuclei from thymus and 1 from

myocardium.6 Means of 3 preparations obtained from nuclei of lymphosar

comatous lymph nodes of 3 animals.

Thvmus15-2&MyocardiumIS"MeanLymphosarcoma

231Lymphosarcoma444aMeanLymphosarcomaPurines

(adenineand guanine)0.360.170560.1570.1500.154050Nuclear

RNAnucleotides0.600.410.501.031.391.21240Purinesnucleotides0.520.13

Thymus and myocardium

Yields" of purines and nucleotides in the alkaline hydrolysate of

nuclei.o Micromoles per 10 mg acid-extracted, fat-free nuclei.6 From Marshak and Mullock (14)

Molecular Source of the Adenine and Guanine of Rat LiverNuclei. As shown in Table 8, there was radioactivity in bothDNA adenine and cationic adenine, and the specific activities ofboth were the same. On the other hand, no detectable radioactivity was found in the nucleotide adenine from the nRNA.These results demonstrated that the DNA was the molecularsource of the cationic adenine. As shown in the section onMethods, none of the radioactivity in DNA adenine isolated bytwo-dimensional chromatography could be attributed to contamination. Similarly none of the radioactivity in the cationicadenine could be assigned to contaminating compounds. Thefirst sample of cationic adenine listed in the table (97.6 fig)included most of the UV-absorbing spot identified as cationicadenine. Preceding this spot was a horizontal band which alsoabsorbed UV and had a slight blue fluorescence. The compounds in this band could not be identified. However, if theassumption were made that the radioactive substance in itwere adenine, it could be calculated that it would have a specific activity of 7,200-7,700 cpm/^mole. This was about 30times greater than the specific activity of the adenine in theDNA adenine spot itself. Therefore, it appeared probable thatthe band contained one or more other compounds labeled with14C. Also, the possibility of contamination of the adenine spot

with these compounds had to be examined. For this purposethe spot was eluted in two sections, one portion (9.59 ftg)furthest removed from the band and the other (3.80 /Â¿g)adjacent to the band. The specific activity of the former was 247dpm/^mole and the latter 223 dpm//Â¿mole. No radioactivitywas found in eluates of the paper on either side of the adeninespot nor in the paper between the guanine and adenine spots.We concluded, therefore, that the radioactivity found in theeluate from the adenine spot was due to adenine-14C only.Consequently, the molecular source of the cationic adeninewas clearly the DNA adenine.

Since deoxyadenosine may also be a precursor of DNAguanine but not directly, it was to be expected that the DNAguanine would be labeled but at a specific activity lower thanthe DNA adenine. This was found. The cationic guanine wasalso labeled, but its specific activity was less than half that ofthe DNA guanine. However, the specific activity of the guanineis in question because of difficulty in measuring the quantityof guanine. The UV-absorbing material was present as two

MAY 1968 983




Table 7

ExperimentNo.35Â«36373839Reagent0.3

NKOH0.3NKOHH20H20H20Hours118111LowestpH2.02.02.04-4.54-4.5iimoles/10Adenine0.0910.0950.0880.0710.063mgGuanine0.0490.0550.0500.0370.029Guanine/

adenine0.540.580.560.520.47

Release of purine bases from nuclei of bovine lymphosarcoma 444."Volume reduction in Experiments 35-38 was attained by the use of a N0 stream with bath

at 78Â°C; in Experiment 39, volume reduction was attained by flash evaporation with bathat 20Â°C.

spots, one much larger than the other, separated by a regioncontaining a substance with light red fluorescence which alsoextended beyond either spot. The fluorescent material had anabsorption maximum at 272 rmt and a minimum at 240 m/Â¿.The eluate of the combined TJV-absorbing guanine spots hadan absorption maximum at 272 m/t, but any peak that mighthave been present at 248 m/x was obscured. Therefore, theamount of guanine was estimated from the peak at 272 imiand found to be 93.3 fig, which is too high by an unknownamount because of the absorption due to the fluorescent material. The fluorescent material and the eluate from the paperblanks on either side of the guanine spots were found to contain no radioactivity.4 However, the other categories of nucleic

acid derivatives had specific activities either greater by about2 orders of magnitude (DNA cytosine and thymine) or had no

4 The bovine cationic guanine appeared as single spots with no

obvious associated fluorescent material and had absorption spectraidentical with that of pure guanine.

Table 8

AdenineDNACation6AMP"AI'GuanineDNACation6CytosineDNACMP


Table 10

BaseAdenineGuanineCytosineThyminen18181818DNAThymusmi28.1421.6822.3227.86Lymphnaarmma

mlm2ami0.030.060.030.07n15151515mg27.6121.7422.1528.50um

20.090.190.150.13TOIâ€”77120.530.060.170.76ai0.090.200.150.15"d60.315

Bovine thymus and lymphosarcoma. n, number of chromatograms; m, mean as % of totalbase; a, standard error; aa, standard error of difference.

The present experiments with radioactively labeled DNAshow that the free adenine had the same specific activity asthe adenine of the DNA and, therefore, must have been incorporated into DNA at the same time and in the same way.Hence the source of the free adenine was certainly the DNA,and the data also show that the free guanine most likely alsohad the same source.

The free purine yield from thymus and myocardium wasroughly equivalent to about 5 percent of the DNA, an amountwhich could be readily detected and measured. However, experiments by Thomas and Doty (19) indicated that degradation of purified calf thymus DNA in dilute acid (pH 2.6)proceeded at a very much lower rate than that which wouldyield amounts of purines comparable to those found with theisolated nuclei in these experiments.

The conditions for the formation of apurinic acid in theexperiments of Tamm et al. (18) were sufficiently differentfrom ours to preclude quantitative comparisons. Qualitatively,there were striking differences in the release of adenine andguanine from nuclei in our experiments when compared withthe release of these bases from isolated calf thymus DNA subjected to acid at elevated temperatures. Greer and Zamenhof(7) found that, under most conditions of heating, slightly moreguanine than adenine was released while, in our experiments,the guanine yield was about half that of the adenine (see Table4). Tamm et al. (18) found a large increase in the amount ofpurine released at pH 1.75 as compared with pH 2.3. At pH 4they found no adenine or guanine released after treatment for24 hours. In our experiments there was only a 19% increasein purines released at pH 2 compared with pH 4. In our experiments lowering the temperature from 78Â°to 20Â°C reduced

the yield only 7%. If unprecipitated DNA in the supernatantfraction were the source of the purines, a much larger effectof temperature would be expected from the data reported inthe literature. Other experimental evidence that unprecipitatedDNA is not the source of the purines is given in Marshak andMullock (14) where it was found that huge increases in theamount of DNA remaining in the supernatant solution didnot alter the purine yield. They also found, as we did in thepresent experiments, that, when there was no free thymidylicacid in the supernatant fluid, no thymine was found when theresidue of the dried supernatant fluid was digested in perchloric acid.

In our experiments when water was substituted for KOHsolution, approximately the same yield of purines was obtained.Also, there was little difference in yields on exposure to KOHfor 1 hour and for 18 hours. The release of purines was, there

fore, not dependent upon the action of alkali. The absence ofhydroxyl ion effects is consistent with the results of Helleinerand Butler (8) who found little DNA hydrolysis with hotsodium hydroxide unless barium hydroxide was also present.

We conclude that the release of purines from the nuclei wasdetermined by factors different from those controlling the rateof release of purines from isolated DNA. In the former case,the molecules released must have come from sites on the DNAwhere the attachment was by bonds much more labile to hydrolysis, the lability appearing after extraction with lipid solvents.

In the previous experiments it was shown that the amountof purines obtained from nuclei of the same thymus glandvaried inversely with the extent of prior extraction with lipidsolvents (14). This observation and the results of the presentexperiments together suggest that the purine deoxyribonucleo-tides in the DNA, which produced the free purines, differedfrom the usual DNA nucleotides (as observed in isolated calfthymus DNA) in having their glycosidic bonds rendered labileto hydrolysis by linkage of the nucleotides to lipids, directlyor indirectly, for example by lipoproteins. Accepting this, itfollows that the quantity of purines obtained from nuclei whichare subjected to similar extraction with lipid solvents might betaken as a measure of lipid-associated purine deoxyribotidesin the DNA of these nuclei.

Table 6 shows that the yield of purines for the lymphosarcoma was 43% of that found for the thymus. To compare thenumber of labile DNA deoxyribosidic bonds, these yields mustbe related in terms of equal numbers of nuclei or equal amountsof DNA since, in the different classes of nuclei, the DNA mayrepresent different proportions of the weight. Table 9 givesthe amounts of DNA found in each of these categories ofnuclei. It is apparent that a given weight of thymus nuclei willcontain 20% more DNA than an equal weight of lymphosarcoma nuclei. After correction to comparable amounts of DNA,the yield of purines for the lymphosarcoma is 0.185 AmÃ³lesascompared with the 0.36 AmÃ³lesof purines of the thymus nuclei,and the lipid-associated DNA deoxyribonucleotides in thelymphosarcoma was 51% of the amount in the thymus. Theresults also show that the low purine yield of the lymphosarcoma was not a peculiarity of the method of presentation ofthe data.

The yield of nRNA nucleotides from the lymphosarcomanuclei was twice that of the thymus nuclei. The nRNA of boththymus and myocardium were low, although myocardium hada low purine yield comparable to that of the lymphosarcoma.There is no correlation, therefore, between low purine and high

MAY 1968 985




nRNA yields. The high nRNA thus appears to be characteristicof the lymphosarcoma. Smith and Kaplan (17) found anelevated RNA content in the thymus of newborn mice and inneoplastic adult thymus which they related to the mitoticactivity of both these tissues. Since their data do not distinguish between nRNA and cRNA, their observations are notdirectly comparable to ours. The correlation of high nRNAcontent and mitotic activity does exist in our data, butobviously both conditions may be precipitated by another common factor, for example, by virus infection. Virus-like bodieswhich resemble the Type C of Bernhard (1) have been foundin the cytoplasm of spleen and lymph node cells and in themilk of lymphosarcomatous cows. However, there exists nodefinitive evidence that a virus is the causative agent of thisdisease (4, 5, 15).

On separation of bovine thymus cRNA nucleotides by columnchromatography, Osawa et cd. (16) found nucleotide proportions which were similar to those of the bases obtained fromrat liver cRNA by perchloric acid digestion by Marshak andMullock (14). Similar data for rat and rabbit liver cRNA wereobtained by Crosbie et al. (2) and by Elson and Chargaff (6).The high cytidylic acid content which we found in the cRNAand in the nRNA of the lymphosarcoma is in contrast withthese results. The data reported for the composition of thenRNA of normal tissues by Crosbie et al. (2), Osawa et al.(16) and Marshak and Mullock (14) showed no elevation ofthe cytidylic acid content in the nRNA of these tissues.

Kit (9) compared the base composition of microsomal RNAand nRNA of mouse spleen, lymphoma, and leukemia cells. Hefound no difference in the results from the normal and neoplastic cells. The guanine and cytosine proportions were higherin all the tissues he examined compared with those mentionedabove. However, he recognized that his data are not comparableto others because they were obtained by analysis of nucleicacids obtained by extraction with aqueous phenol, a procedurewhich fractionates the RNA. In the comparison of the results,he found no marked differences in the normal and neoplastictissues with respect to the proportions of the bases.

The diversity of the normal mammalian tissues, which haveshown no elevated cytidylic acid in the RNA such as we foundin the bovine lymphosarcoma, suggests a correlation with acondition existing in the lymphosarcoma and not in the normaltissues examined. Whether this is a reflection of rapid cell proliferation, of a possible virus proliferation, or of the neoplasticcondition per se remains to be determined.

Smith and Kaplan (17) found no differences in the DNA oftheir normal and neoplastic tissues. In Tables 9 and 10 wehave presented our data for the composition of the DNA ofthymus and lymphosarcoma. There are statistically significantdifferences with respect to the adenine and thymine but notthe other two bases.

We plan to continue these experiments applying more sensitive methods to the comparison of the nucleic acids of thelymphosarcoma with those of normal tissue cells.

ACKNOWLEDGMENTS

We are indebted to Mildred Eby for technical assistance withthe work here described.

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1968;28:979-986. Cancer Res Alfred Marshak and Celia Marshak Nucleic Acids of Bovine Lymphosarcoma and Normal Thymus

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