spontaneous release of dna by human blood lymphocytes …suisse de ia recherche scientifique nos....

[CANCER RESEARCH 35, 2375-2382, September 1975]

SUMMARY

Human blood lymphocytes released DNA in vitro in theabsence of any stimulation. Once purified from the complexappearing in the supernatant, this DNA exhibited typicalcharacteristics as shown by its UV absorption curve, itsdeoxyribose coloration, and its sensitivity to DNase. Elution patterns on hydroxyapatite columns indicated that theexcreted DNA is double stranded. The released DNA wassmaller than the cellular DNA; its molecular weight rangedfrom 3.5 x l0@to 3.7 x 106 daltons.

The DNA appearing in the supernatant does not seem tobe due to dead or dying cells since: (a) the same amount ofDNA was found in the medium whether the incubationlasted 2 hr or as long as 16 hr; (b) cell death rate had noeffect on the amount of extracellular DNA; (c) when thelymphocytes were centrifuged and placed in a new mediumseveral times in a row, a similar amount of extracellularDNA was isolated from each of the successive supernatants,whereas if, after centrifugation, the lymphocytes were putback in their original medium, no increase in the amount ofextracellular DNA was observed, suggesting an activeregulatory mechanism independent of a mechanical effect;(d) it took more than 1hr for extracellularDNA to reachitsmaximum concentration, a fact that also argues against amechanical effect; (e) the specific activity of the releasedDNA was different from that of the cellular DNA, depending on the time of labeling; and (f) the cells that had cxcreted DNA kept their functional integrity, as shown bytheir fully maintained capacity to increase DNA synthesisafter stimulation.

The extracellular DNA hybridized specifically with cellular DNA. The hybridization curves indicate that the DNAexcreted is highly complex, and they suggest that it is cornposed of part of the newly synthesized DNA. The higherspecific activity of the released DNA, compared with that ofthe cellular DNA after a long labeling period, can be cxplained only by a preferential release of the newly synthesized DNA.

INTRODUCTION

Bacteria in culture spontaneously release DNA (3, 8, 13,22, 23, 30, 32, 33). In transformable bacteria, it has beenshown that this extracellular DNA is genetically active (8,13, 22, 23) and is released in a sequence-specific order (8).

We have previously reported that not only prokaryotesbut also whole organs of higher organisms, such as frogauricles, release DNA during in vitro incubation (3, 32).Moreover, extracellular DNA was found in vivo in bovinecerebrospinal fluid (3). It has also been found that rabbitspleen tissues cultured in vitro excreted DNA (21).

Human lymphocytes labeled with [3H]TdR2 release acidinsoluble radioactive molecules in their culture mediumafter PHA stimulation (27). In a preliminary communication (5), we reported that nonstimulated lymphocytes alsoshed DNA.

In this paper, we present further evidence that humanblood lymphocytes spontaneously release DNA in theirculture medium without any previous stimulation, and wecharacterize this extracellular DNA.

MATERIALS AND METHODS

Lymphocyte Culture and Separation of the Supernatant.Human peripheral blood lymphocytes were prepared understerile conditions by the Ficoll-Isopaque gradient technique(9), with heparinized whole-blood samples from healthydonors. After 2 washings with Hanks' TC medium, thelymphocytes were suspended at a concentration of 10' perml in TC 199 buffered with 0.04 M HEPES and, unlessotherwise specified, with the addition of 20% homologousserum of the same blood group as the donor. The sourcesof the chemicals were Pharmacia Fine Chemicals, Uppsala, Sweden, for Ficoll; Difco Laboratories, Detroit, Mich.,for Hanks' TC and TC 199; and Calbiochem, La Jolla,Calif., for HEPES. Sterility was secured by supplementingthe medium with 120 units of penicillin (Mycofarm, Delft,Holland) and 180 units of colimycin (Laboratoire Bellon,Neuilly, France) per ml. The WBC in such preparations

1 This work was supported by grants from the Ligue Genevoise contre Ic

Cancer, from the Ligue Suisse contre Ic Cancer, from the Fonds NationalSuisse de Ia Recherche Scientifique Nos. 3776.72 and 3832.72, from theScidl Hentsch Fund, and from the OJ. Isvet Fund Nos. 101 and 102.

Received March 7, 1974; accepted May 19, 1975.

2 The abbreviations used are: [3HJTdR, tritiated thymidine; PHA,

phytohemagglutinin; HEPES, N-2-hydroxyethylpipcrazine-N'-2-ethanesulfonic acid; Con A, concanavalin A; C,j, concentration at time 0 (mole xsec/liter).

SEPTEMBER 1975 2375

Spontaneous Release of DNA by Human Blood Lymphocytes asShown in an in Vitro System'

Philippe Anker, Maurice Stroun, and Pierre A. Maurice

Division of Onco-Hematology, Department of Medicine, Cantonal Hospital, 1205 Geneva [P. A ., P. A . M.] and Department of Plant Physiology,Faculty ofScience, University of Geneva, 1211 Genevd4, Switzerland tM S.]

Research. on February 5, 2021. © 1975 American Association for Cancercancerres.aacrjournals.org Downloaded from

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P. Anker et a!.

of culture, or Con A (Calbiochem), 50 sg/ml of culture.After 12 hr of labeling with [3H]TdR (1 @sCi/m1), theacid-insoluble radioactivity was determined (20). All experiments were performed on cultures in triplicate.

DNA Extraction. DNA was extracted from the cells orfrom the supernatants as follows. Sodium lauryl sulfate(Fisher Scientific Company, Fair Lawn, N. J.) was addedto a final concentration of 2% either to cells resuspended inTC 199 medium (2 x l0@ lymphocytes/mI) or to thecell-free supernatant. This solution was shaken for 15 mm inthe presence of an equal volume of phenol saturated withsterile water. After centrifugation for 15 mm (8000 rpm,Sorvall RC 2-B), the aqueous phase was collected andsubmitted to another phenol treatment. Phenol was removed by ether extraction. Residual ether was eliminatedby bubbling nitrogen through the solution, and KC I wasadded to a concentration of 3 M. The solution was thenloaded on a hydroxyapatite column (7) (DNA grade;Bio-Rad Laboratories, Munich, Germany), which waswashed successively with phosphate buffer solutions (equimolar amounts of Na2HPO4 and NaH2PO4, pH 6.8) asfollows: 150 ml of a 0.03 M solution, 150 ml of 0. 12 Msolution, and 50 ml of a 0.20 M solution. Finally, thedouble-stranded DNA was eluted with 0.48 M phosphatebuffer and collected in l-ml fractions. All phosphate bufferswere adjusted to 3 M KCI. To remove all the phosphate andKC1, the fractions containing the DNA, as indicated by UVabsorption at 258 nm, were centrifuged overnight at 45,000rpm in a Spinco L 65 centrifuge. The pellet of DNA wasresuspended in the suitable solvent, depending on thesubsequent characterization procedure.

As a control, TC 199 medium with 20% serum wassubjected to the same extraction procedure.

Characterization of DNA. The following studies wereperformed on the DNA extracted from the cells and fromthe supernatants. (a) Their amount was determined on aBeckman Model DB spectrophotometer by UV absorptionat 258 nm and by deoxyribose colorations (1 1, 14). Theabsence of protein contamination was controlled (19). (b)The double-stranded nature of the DNA was checked byheat denaturation followed by elution on a hydroxyapatitecolumn (7). (c) Enzyme sensitivity was tested by acid precipitation (28) after digestion for 2 hr at 37Â°with the following DNase's at a concentration of 50 zg/ml; commercialsplenic DNase II (Fluka, Buchs, Switzerland), splenicDNase II, highly purified by Professor G. Bernardi (Laboratoire de GÃ©nÃ©tiqueMolÃ©culaire, University of Paris), andhighly purified snail DNase (also kindly provided by Professor Bernardi). Other enzyme sensitivity tests were performed with pancreatic RNase (Sigma Chemical Co., St.Louis, Mo.), 100 @zg/ml, in which DNase was inactivatedby heating at 90Â°for 15 mm, or with Pronase (Grade B;Calbiochem) previously submitted to autodigestion for 8 hrat 37Â°.(d) The molecular weight was estimated by zonalcentrifugation on linear gradients of 5 to 20% sucrose in thepresence of sedimentation markers (23, 16, and 5 5 RNA;Miles Laboratory, Slough, England). (e) Cellular and extracellular DNA were submitted to CsCl density-gradientcentrifugation ( I7). (J) DNA specificity was studied by

were approximately 98% lymphocytes. Aliquots (35 ml) ofsuspension were put in capped plastic tubes (8 tubes/series)and incubated at 37Â° under air atmosphere for varyinglengths of time according to the protocol described below.After Incubation, the lymphocytes were centrifuged a 1sttime at 1000 rpm for 10 mm in a Christ Heraeus II4KScentrifuge (Martin Christ A. G., Osterhode am Harz,Germany) at 4Â°.Twenty-five ml @ofthe supernatant werecollected by pipetting, leaving 10 ml of medium with thesedimented lymphocytes in order to prevent cell loss. Thesupernatant was then cleared of any remaining cells and celldebris by a 2nd centrifugation at 20,000 rpm in a SorvallRC 2-B centrifuge (Ivan Sorvall Inc., Norwalk, Conn.). Asan ultimate precaution, a 3rd centrifugation was performedat 45,000 rpm for 2 hr at 4Â°in a Spinco Model L 65 centrifuge (Beckman Instruments, Inc. Fullerton, Calif.), and theresultant supernatant was collected and kept frozen untilDNA extraction. The lymphocytes put aside after the 1stcentrifugation were resuspended in the remaining 10 ml ofsupernatant. An aliquot of this suspension was taken for cellcounting and for viability tests, as described below. In someexperiments, when the lymphocytes were incubated successively several times in renewed medium, 25 ml of fresh medium were added each time to the 10 ml of cell suspensionfor the ensuing incubation pÃ§riod.At the end of the incubation, sterility of the supernatants was tested by plating aliquots on Agar Nutrient Broth (Difco).

Labeling and Radioactivity Measurement. [3H]TdR [10 or40 Ci/mmole, labeled in the methyl group, purity 98%(Radiochemical Center, Amersham, England)] was addedto the cell suspension for various periods of time at a finalconcentration of 10 to 20 zCi/ml. After DNA extraction,radioactivity was counted in the presence of the suitablephosphor (16) in a Beckman Tri-Carb liquid Scintillator. In order to exclude the possibility that, after extraction, the radioactivity carried by the DNA could be dueto a radioactive contaminant, perchloric acid-hydrolyzed[3HJDNA was chromatographed (29); chromatographyshowed that radioactivity migrated with the thymidine spotonly.

Cell Counting and Control of Cell Viability. Lymphocyteswere counted in a hemocytometer at Time 0 and after eachincubation. Four separate samples totaling about 1000 cellsfor each series were counted. At the same time, thelymphocytes were also examined for their ability to excludetrypan blue. Cells were resuspended at a concentration of 3x 106 cells/mI, and 2 drops of culture were mixed with 1drop of 0.2% trypan blue. A total of 400 cells/series werecounted in a hemocytometer chamber. The results areexpressed as percentage of cells taking up the dye.

In some instances, the lymphocytes were tested further atthe end of various incubation periods for their capacity tosynthesize DNA under a mitogen-induced stimulation. Forthis purpose, lymphocytes treated for The same time periodsas in the DNA release experiments were compared withfresh control lymphocytes from the same donor. After thesevarious treatments, they were incubated for 72 hr at 37Â°inTC 199 medium containing 20% homologous serum andPHA-M (Difco), 0.05 ml of the commercial solution per ml

2376 CANCER RESEARCH VOL. 35



Amount of DNA(jug/culture)Lymphocyte

counts(cells xlO'/ml)DeadExtra

ReleasedDNABeforeAfterlymphocytesTimeofincubationCellular cellular(% ofcellularDNA)incubationincubation(%)Experiment

1 (with 20%serum)A.8hr94121.72.21.451.430.9B.2hr(lstincubation)2 hr(2nd incubation)2hr(3rdincubation)2 hr (4th incubation)21

â€˜@19 @,8723 (

974 24 ,72.1

@1.9@ 8 Q2.3 ( â€˜2.4 ,Y1.441.43

1.461.40I .421.5

20.6

0.6Experiment

2 (withoutserum)A.8hr778 24.531.04-1.049B.2hr(Istincubation)2hr(2ndincubation)2hr(3rdincubation)2 hr (4th incubation)23.5

@20 @76II (

900 2 1.5 )2.6'@

2.2 t,841.2@ â€˜2.4 ,P1.011.02

0.820.820.74II

102345

Spontaneous Re/ease of DNA by Human Lymphocytes

DNA-DNA hybridization. [3H]DNA extracted either fromthe supernatant or from the cells was hybridized against anexcess of nonlabeled human splenic DNA. C0t curves wereperformed according to the method of Britten and Kohne(10). Both unlabeled and labeled DNA were sheared at 0Â°by sonic treatment at 20,000 cps, 100-watt output, for a totalsonic treatment of 15 mm, using a MSE sonic oscillator(M.S.E., Ltd., London, England). This reproducibly gave adouble-stranded DNA sedimenting at 6 to 8 S. The [3H]-DNA was adjusted to a very low concentration to avoid selfhybridization, and it was mixed with a high concentration ofunlabeled DNA at a mass ratio of 1:100 or 1:400. Themixture was boiled for 10 mm in an oil bath at 120Â°adjustedto 0. 12 M phosphate buffer and incubated at 60Â°. After thedesired incubation period, the samples were collectedand quickly cooled on ice. The mixture was then passedthrough a hydroxyapatite column equilibrated at a temperature of 60Â°in 0. 12 M phosphate buffer with 0.2% sodiumlauryl sulfate to separate the double-stranded hybrid DNAfrom single-stranded unhybridized DNA. The singlestranded DNA was washed from the column by 0. 12 Mphosphate buffer. The completion of the washing waschecked by counting the final eluant. The hybrid DNA wasthen eluted with 0.48 M phosphate buffer, and the completion of the elution was checked as described above. Therecovery of DNA after. these elutions was more than 95%.The controls consisted of incubation of labeled DNAwithout any cold DNA under identical conditions ofphosphate molarity and incubation temperature. The valuesobtained in these controls were subtracted from the hybridization experiments to account for possible self-annealing5

RESULTS

Amount of Released DNA. Table 1 shows that the sameamount of DNA was found in the supernatant whether theincubation lasted 2 or 8 hr. In the course of an 8-hrexperiment in which the medium was renewed every 2 hr,similar amounts of DNA were detected in each of the 4successive incubation solutions. The cumulative amount ofDNA recovered in these experiments corresponded to about8% of the total cellular DNA. This percentage was the samewhether the lymphocytes were incubated in the presence orabsence of serum, in spite of the much higher cell lossobserved in the latter condition. Thus, after a series of 4successive incubations, lymphocyte recovery was practicallycomplete in the presence of serum whereas, in its absence, a25% cell loss was observed. Similarly, significant differencesin cell mortality were detected with the trypan blue-exclusion test. The cell mortality remained negligible after anincubation in the presence of serum and reached 45% in itsabsence. However, most experiments performed withoutserum did not show the high degree of cell mortalityindicated in Table I, and this example has been chosen onpurpose as an extreme case.

Table 2 shows that the amount of DNA recovered in thesupernatant increased with each renewal ofthe medium, butwhen the lymphocytes were centrifuged in the same way andresuspended into the same medium, no increase was observed.

Table 3 illustrates the fact that some DNA appeared inthe medium within I mm and that its amount increased

TABLE IA mount of DNA extracted from lymphocytes and from their supernatants after various periods of incubation in culture medium

with or without serum

Lymphocyte cultures (200 ml) were started at a concentration of I .45 x 108 cells/mI in TC 199 and 20% serum (Experiment I)and 1.04 x 10' cells/mI in TC 199 only (Experiment 2). They were incubated for 8 hr at 37Â°either (A) continuously or (B) in 4successive periods of 2 hr with medium renewals. After the various periods of incubation indicated on the table, the DNA wasextracted from the cells and from the medium and its amount was determined. Cell counts were performed before and after eachincubation period, and cell viability was estimated by the trypan blue-exclusion test. The mortality rates indicated for eachexperiment under Part B concern the same population of lymphocytes and, therefore, cannot be added to each other.

SEPTEMBER 1975 2377



Amount of DNALymphocytecounts(jig/culture)â€”Extra

Released DNA(% of cellular(cells

xl0'/ml)Dead

lymphocytesBefore.AfterConditionsofincubationCellularcellularDNA)incubationincubation(%)Four

successive 2-hrincubations:A.Lymphocytesresuspended-each

time in the samemediumlstincubation1.321.451.52nd

incubationI.362.53rdincubation1.314th

incubation847 2631.43.5B.

Lymphocytesresuspendedeachtime in a newmediumIstincubation

2ndincubation3rd incubation4th incubation24

â€˜@19 !@@23 (

891 24 J2.7

â€˜@2.1 t.@10 12.6 ( â€˜2.7 J1.321.46

1.41.381.391.5

1.51I

LymphocyteAmountof DNAcounts(cells(pg/culture)Releasedx

10/ml)DNADeadTime

of(%Before Afterlymphoincuba Extra ofcellu incu- incucyteslionCellular

cellularlar DNA)bationbation(%)I

mm40.481.901.932.530mm6.80.821.971lhr16.521.9012hr824

18.22.2822

P. A nker el a!.

Table 2Amount of DNA extracted from lymphocytes and from their supernatants after successive incubations with or without medium

renewal

A culture oflymphocytes(l.32 x 10' cells/mI in TC 199medium containing 20% serum) was divided in 2 parts of200 ml. Thesecultures were submitted to 4 successive incubations of 2 hr: (A) the lymphocytes were centrifuged at the end of each incubationperiod, the supernatant was removed and the lymphocytes were put back in the same medium; (B) the lymphocytes were centrifugedat the end of each incubation period and put back in a new medium.

sharply during the 1st hr, reaching a plateau after approximately 2 hr.

Specific activity of the released DNA depended on whenthe releasing cells were labeled (Table 4). Values farexceeding cellular DNA specific activity were observed onextracellular DNA samples released by lymphocytes labeledduring a previous incubation and resuspended in newnonradioactive medium (Experiment 1), whereas the contrary was observed for DNA released at the time of celllabeling (Experiment 2).

Finally, the absence of any detrimental effect of a priorincubation on the ability of lymphocytes to synthesize DNAupon stimulation with PHA or Con A is demonstrated inTable 5. Whether the lymphocytes had been incubated for 8or 16 hr with or without medium renewals prior tostimulation, or were immediately stimulated, [3H]TdRuptake remained unaltered.

No DNA was found in the culture medium beforeincubation with lymphocytes.

Characterization of the Released DNA. After purification, the released DNA reacted as well as the cellular DNAwith diphenylamine or with indole colorations, in conformity with coloration curves of standard deoxyribose solutionsand with what could be predicted from UV absorption. InUV, the released DNA showed a typical absorption curvewith a maximum at 258 nm and a minimum at 230 nm.

The material extracted was sensitive to all DNase's used.After DNase treatment, over 95% (practically 100% withhighly purified splenic or snail DNases) of the acid-insolubleDNA became acid soluble. On the other hand, the releasedDNA remained completely insensitive to either RNase orPronase treatment. After heat denaturation, over 90% of theDNA was eluted from hydroxyapatite by 0. 12 Mphosphatebuffer.

Table 3Amount ofDNA extractedfrom lymphocytes andfrom their supernatants

after increasing time periods of incubation

Conditions were similar to those described in Table I, except that alllymphocyte suspensions were incubated for increasing periods of time inIC 199 medium containing 20% serum. Each incubation time correspondsto a separate culture.

Chart 1 shows that the molecular weight of the releasedDNA was not as homogeneous as that of the cellular DNA.Whereas the cellular DNA had 1 peak at 18 S [about 5 xl0 daltons, according to the formula of Eigner and Doty(12)], the extracellular DNA had 2 main peaks, 1 sedimenting at 16 S (about 3.7 x 10' daltons) and 1 sedimenting at 7S (about 3.5 x l0@daltons).

The DNA released by lymphocytes banded at the samedensity (1.700 g/cu cm) as the cellular DNA (Chart 2). Bothextracellular and cellular [3HIDNA present an importanthomology with nonlabeled cellular DNA (Chart 3). In bothcases, a portion representing about 20% hybridized veryrapidly. The 2nd pattern is one of a more homogeneouscurve in which the C,j Â½occurred at a value of approximately l0@-After that point, however, while the cellular




Incubations andlabelingAmount

of DNA(pig/culture)

ExtraCellular cellularSpecific

activity(cpm/@.cg)Lymphocyte

counts(cells x l0'/mI)Dead

lymphocytes(%)ExtraCellular cellularBeforeincubationAfterincubationExperiment

1lstincubation(l6hr)inthepresenceof[3H]TdR

followed by a 2nd incubation (4 hr) without label1007.321.33241,4001.381.31.6Experiment

21st incubation ( 16 hr) without label followed by a

2nd incubation (4 hr) in the presence of [â€˜H]TdRI100.4 24.2263 36I .44I .382.1

Incubation preceding DNA[â€˜HJTdR

uptake (cpm/culture) bylymphocytes afterLymphocyte

counts in ConA-stimulated cultures

(cells x l0ml)NoPHA

M Con AInitialFinalsynthesisstimulationstimulationstimulation stimulationcountcountControl

without mitogen1 ,98IControlwithno prior incubation130,455 142,1141.011.0l6hrofpreincubation170,3881.018

hr of preincubationI 39, 157160,6760.994successive 2-hr preincubationsI 34,386 181,7471.01with

medium renewal

Spontaneous Re!ease of DNA by Human Lymphocytes

Table 4Specific activity of the cellular and extracellular DNA in relation to the time oflabeling with [â€˜H]TdR

Lymphocyte cultures (200 ml, 1.4 x 10' cells/mI) were subjected to 2 successive incubations of 16 and 4 hr. respectively, with medium renewal (TC 199with 20% serum). [â€˜H]TdR (10 @tCi/ml) was added to the medium either during the 1st incubation (Experiment I) or during the 2nd incubation(Experiment 2). The cellular and extracellular DNA was extracted after the 2nd incubation and the amount and specific activity were determined.

Table 5DNA synthesis ability oflymphocyte cultures upon stimulation with PHA or Con A with or without previous

incubations for various periods of time

Lymphocyte suspensions (2 ml, 1.01 x 10' cells/mI) in TC 199 medium and 20% serum were treated as inDNA-release experiments (Table I, 2, and 3) and subsequently were stimulated with PHA-M (0.05 ml Difcostandard solution per ml of culture) or Con A (SO @zg/mlof culture) for 72 hr. Control cultures were directlystimulated without prior incubation. [â€˜H]TdR(I @&Ci/ml)was added 12 hr before the culture was terminated,and the acid-insoluble radioactivity was determined. Final lymphocyte counts were performed on ConA-treated suspensions only, because the PHA-treated cultures were too clumped for accurate counting.

[3H]DNA hybridization curve continued to go down, thereleased [3HJDNA curve reached a plateau. This plateauwas the same whether the mass ratio oflabeled extracellularDNA to unlabeled cellular DNA was 1:100 or 1:400. At C0:100, neither the released [3H]DNA nor the cellular[3H]DNA hybridized with nonlabeled Escherichia co/iDNA.

DISCUSSION

Is the DNA Released by Living Cells? These resultsdemonstrate that normal human lymphocytes release DNAwhen incubated in vitro. The possibility that a majorfraction of the DNA appearing in the supernatant couldhave originated from dying or dead cells seems to be ruledout by the following observations. (a) The same amount ofDNA was found in the medium whether the incubationlasted 2 or 8 hr (Table 1), or 4 hr, compared with 16 hr(Table 4). One would expect the extracellular DNA concentration to increase with time if the phenomenon were due todying cells. (b) Furthermore, Table 1 also shows that the

amount of DNA recovered from the supernatant wasunaffected by the cell death rate. It appears from the sameexperiments that similar quantities of released DNA arerecovered whether the percentage of cell recovery is almosttotal or whether one-fourth of the cells has been lost. Thisshows that dead cells or even fragments of lysed cells cannotbe held responsible for the DNA in the supernatant but arepelleted during the different centrifugations described. (c)In the course of an 8-hr experiment in which the culturemedium was renewed every 2 hr. similar amounts ofextracellular DNA were detected in each of the 4 successiveincubating solutions (Tables 1 and 2), whereas no increasewas observed if the lymphocytes were put back in theoriginal medium (Table 2). The constant renewal of DNAshed at each transfer to new solutions suggests an activemechanism. In this respect, the possible role of a mechanical stress (because of centrifugation, for instance) seems tobe ruled out by the relatively long time (1 hr) necessary forextracellular DNA to reach its maximal concentration(Table 3). However, the best argument against a stress effectdue to centrifugation is illustrated by the fact that no in

SEPTEMBER 1975 2379



renewal of extracellu1ar DNA has been observed with frogauricles (32), which can be gently transferred to a new medium without centrifugation. The active process involved isdifficult to understand. The constancy of the DNA concentration in the supernatant could be due to some inhibitingfactor such as a nuclease that starts working only when acertain concentration of extracellular DNA is reached. Thishypothesis is nevertheless unlikely since the released DNA(although once purified sensitive to DNase) is not destroyedin the medium by the DNase's we have tried, and thus seemsto be released as a complex. There is also the possibility thatthe released DNA enters other cells, as has been seen withDNA spontaneously released from bacteria, which can betaken up either by other bacteria (8, 13, 22, 23) or byeukaryotic cells (4, 30, 31, 33, 34). In this case, the uptake ofDNA could be the limiting factor of the extracellular DNAconcentration. Experiments made with purified DNA haveshown that a certain concentration of DNA is necessary forthe uptake process to take place (2). It should be noted thathuman lymphocytes have been reported to take up purifiedforeign DNA (2 1). (d) The differences in the specificactivity of the cellular and the extracellular DNA in relationto the time of labeling (Table 4) also argues against the lysedcell origin of the released DNA. The fact that after a longperiod of labeling (Experiment I) the extracellular DNA hasa higher specific activity than the cellular DNA can be cx

I plained if one assumes that the newly synthesized DNA is

@ preferentially excreted. However, the lower specific activity! of the extracellular DNA, compared with the cellular DNA

@ after a short time of labeling (Experiment 2) indicates that0 there is a certain delay between synthesis and release. (e)

@ Finally, the conservation of a functional cellular integrity af4 ter the time periods of our experimental conditions was con

firmed by the capacity of the previously incubated lymphocytes to increase DNA synthesis upon PHA or Con A slimulation just as well as nontreated lymphocytes (Table 5).

Characterization of the Released DNA. The typicalabsorption curve in UV, the deoxyribose colorations, thesensitivity to DNase, and the insensitivity to other enzymespoint out that the material extracted consists of DNA. It isdouble stranded, as shown by its elution characteristics onhydroxyapatite columns.

The molecular weight of the released DNA, although

P. Anker et a!.

crease in extracellular DNA was observed when the lymphocytes had been centrifuged 3 times and put back eachtime into the same medium (Table 2). Moreover, the same

2

CELLULAR DNA

.

I

E

I

I

F

fraction numblr

Chart I. Sucrose gradient centrifugations of [â€˜H]DNA from lymphocytes and from their supernatants. Five @&gof [â€˜HJDNA were purified fromlymphocytes or from the supernatant in which they were labeled for 8 hr.The [â€˜H]DNA was mixed with 40 @igof 23, 16, and S S unlabeled reference RNA. The samples were layered on S-mI linear gradients of 5 to20% sucrose in 0.015 MNaCI and 0.0015 Msodium citrate, and centrifugedin an SW 50.1 rotor at 45,000 rpm at 20Â°for 2 hr 30 mm.

fraction numb.r

EXTRACELLULAR DNA

EXTRACELLULAR DNA1.731g/cucm

1.lOOg/cucm

[email protected]â€˜\gi@cucm

CELLULAR DNA

Chart 2. Density gradient of [3H]DNA fromlymphocytes and from their supernatants. Diagram of CsCI ultracentrifugation (27,000 rpm for62 hr at 20Â°)of 5 @gof [3H]DNA extracted from

T lymphocytes (d = 1.700 g/cu cm) and from the! supernatant in which they were labeled with

@ [â€˜H]TdRfor 8 hr. Unlabeled Clostridium perfrin@ gens DNA (d = 1.691 g/cu cm) and Micrococcus

4 lysodeikticus DNA (d = 1.731 g/cu cm) were

added as reference.

a

a

IFU

EU

@onnum@ fractÃ©ctanumblr




Spontaneous Re/ease of DNA by Human Lymphocytes

sence of hybridization with unlabeled E. co/i DNA. Thepossibility that the released material could come from acontaminating microorganism is also ruled out by thecomplexity of the extracellular DNA without mentioningthe sterility of the supernatants.

It is difficult to know exactly the origin of the releasedDNA. It could be composed of extra copies of part of thegenome, for instance, the working genes involved in transcription or regulation (24â€”26). Once used, these extracopies could migrate to the membrane (15, 18) and beexcreted as a transfer ofgenetic information from cell to cell(2, 6, 35). In the particular case of lymphocytes, such amechanism has been postulated in order to explain thecooperation process observed between thymus- and bonemarrow-derived cells during immune reactions (15, 18, 21,26, 27). It should be noted that some characteristics of ourexcreted DNA (density and molecular weight) are similar tothose of the DNA bound to the lymphocyte membranes (15,18). In any case, the release of DNA seems to be a generalphenomenon occurring not only in prokaryotes (1, 8, 13, 22,23, 32) but also in eukaryotes (3, 5, 21, 27, 32).

ACKNOWLEDGMENTS

We are grateful to Professor G. Bcrnardi and Professor A. Bernardi(Laboratoire de GÃ©nÃªtiqueMolÃ©culaire, University of Paris VI) for theadvice and help they gave us. We also thank Dr. M. Odjha (Laboratoire deMicrobiologic, University ofGeneva) and Dr. S. Modak (Institut Suisse deRecherches ExpÃªrimentales sur Ic Cancer, Lausanne) for useful commentsand Dr. J. Hedgpeth (lnstitut de Biologic MolÃªculaire, University ofGeneva) for reading the manuscript. J. Henri, C. Lederrey, and A. Cattaneo should be thanked for their excellent technical assistance.

REFERENCES

I. Anderson, E. S. Possible Importance of Transfer Factors in BacterialEvolution. Nature, 209: 637-638, 1966.

2. Anker, P. Sort du DNA BactÃ©rienIntroduit chez Solanum Lvcopersicum esc., Thesis No. 1492, Edition MÃ©decineet Hygiene, pp. 76-78.University of Geneva, GenÃ¨ve, 1969.

3. Anker, P., and Stroun, M. Spontaneous Release of Nucleic Acids byLiving Bacteria and Cells of Higher Organisms. Abstracts. EighthInternational Congress of Cell Biology, Brighton, p. 3, 1972.

4. Anker, P., and Stroun, M. Bacterial Ribonucleic Acid in the FrogBrain after a Bacterial Peritoneal Infection. Science, 178: 62 I -623,1972.

S. Anker, P., Stroun, M., and Maurice, P. Spontaneous Extracellular

Synthesis of DNA Normally Released by Human Lymphocytes. Abstracts of the XIth International Cancer Congress, Florence, Vol. 2,p. 24, 1974.

6. Bendich, A., Wilczok, T., and Borenfreund, E. Circulating DNA asa Possible Factor in Oncogenesis. Science 148: 374-376, 1965.

7. Bernardi, G. Chromatography of Nucleic Acids on Hydroxypatite.Nature, 2tM: 779, 1965.

8. Borenstein, A., and Ephrati-Elizur, E. Spontaneous Release of DNAin Sequential Genetic Order by Bacillus Subtilis. J. Mol. Biol., 45.137â€”152,1969.

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10. Britten, R. J., and Kohne, D. E. Repeated Sequences in DNA. Science,161:529-540,1968.

z

0z

Chart 3. Comparison of C,t curves of cellular [â€˜H]DNAand released[â€˜H]DNA hybridized with an excess of nonlabeled cellular DNA.[â€˜H]DNAwas purified from the cell-free medium and from the lymphocytes labeled for 8 hr. The [â€˜HJDNA was sheared to 7 5 size. The unlabeledhuman spleen DNA was treated in the same manner. The [â€˜HIDNAwasadded to the nonlabeled DNA in a mass ratio of I: 100. This mixture wasseparated in different aliquots containing up to 10 A/mI in tightly closedPyrex tubes. They were denatured in a silicone oil bath at 120Â°for IS mmand cooled rapidly on ice. DNA solutions previously maintained in 0.03 Mphosphate buffer were adjusted to 0. 12 M of the same buffer and incubatedat 60Â°for up to 300 hr. Tubes were removed at desired intervals and frozenat 20Â°until processed. Percentage of hybridization of single-stranded[â€˜H]DNAto the nonlabeled single-stranded DNA was determined byapplying each sample to an individual 1-cm water-jacketed columncontaining 2 cm of hydroxyapatite maintained at 60'. The samples wereeluted with 16 successive 1-mi fractions ofO.12 Mphosphate buffer followedby 16 successive l-ml fractions of0.48 Mphosphate buffer. The radioactivity of each fraction was counted by liquid scintillation. Data are plotted aspercentage of single strands of [3H]DNA versus C,t.

lower than that of cellular DNA, is high enough for part ofit to carry information of several genes.

The general AT:GC ratio is the same for cellular andextracellular DNA, as shown by CsCl gradients, apparently excluding the possibility of a mitochondrial origin ofthe released DNA.

The C0: hybridization curves show that the newly synthesized cellular [3H]DNA and the released [3H]DNA are verysimilar, at least until C0t V2. There is a strong complementarity between both released and cellular [3H]DNA towardtotal cellular DNA, since there is already about 20%hybridization at low C0:. The hybridization curve appears to follow a 2nd-order kinetics with a high â€˜/@C0t value,indicating that both released and cellular [3H]DNA arehighly complex and are not composed of numerous copies ofsimple sequences. The fact that the hybridization curve ofthe extracellular [3H]DNA reaches a plateau before thecellular [3H]DNA, whether the mass ratio of labeled DNAto unlabeled DNA is 1:100 or 1:400, seems to indicate thatabout 50% of the sequences of the released DNA are nothomologous to cellular sequences. Another explanationcould be that 50% of the released DNA consists ofsequences representing a small proportion of the cellularDNA (perhaps 1% or less, like ribosomal DNA), with thesequences being amplified during expulsion. The specificityof the extracellular DNA is demonstrated by the ab

.otlo_1 1 10

SEPTEMBER 1975 2381



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1975;35:2375-2382. Cancer Res Philippe Anker, Maurice Stroun and Pierre A. Maurice

Systemin VitroShown in an Spontaneous Release of DNA by Human Blood Lymphocytes as

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