Integrated and free viral DNA in hamster tumors induced

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1 Proc. Ntl. Acd. Sci. USA Vol. 77, No. 2, pp , Ferury 1980 Cell Biology Integrted nd free virl DNA in hmster tumors induced y BK virus (lot-trnsfer hyridiztion/tumor tissues/cultured tumor cells) N. CHENCINER*, G. MENEGUZZIt, A. CORALLINIO, M. P. GROSSIO, P. GRASSIO, G. BARBANTI-BRODANOt, AND G. MILANESIt tlortorio di Genetic Biochimic ed Evoluzionistic CNR, Vi S. Epifnio 14, Pvi, Itly; tistituto di Microiologi, Universiti di Ferrr, Vi L. Borsri 46, Ferrr, Itly; nd *Centre de Biochimie, Fcult6 des Sciences, Prc Vlrose, Nice, Frnce Communicted y L. L. Cvlli-Sforz, Octoer 24, 1979 ABSTRACT BK virus (BKV)induced tumors in hmsters were investigted for the presence of virl DNA y the lottrnsfer hyridiztion technique. Severl virl genomes per cell were found in tumor tissues nd in their derived cell lines nd clones. Most of the detected virl genomes were integrted into the cellulr DNA, ut some tumors lso contined free virl DNA sequences. Integrtion ptterns were different from ech other, nd mny different integrtion sites were ville on the cellulr or on the virl DNA or on oth. Typicl fetures of integrtion ptterns were found in ependymoms, which were the most frequent (72%) mong BKV-induced tumors. Redily detectle virl DNA sequences were only found in neoplstic tissues, ut trces of BKV DNA were lso present in the pprently norml portion of the rin of n niml tht hd developed n ependymom nd in the rin (ut not in the liver) of nother niml 15 dys fter virus inocultion. A cell line nd single-cell clone derived from tumor hd hyridiztion ptterns considerly simpler thn the pttern of the originl tumor, lcking severl integrted virl genomes nd ll free virl sequences. BK virus (BKV) is recently discovered humn ppovvirus originlly isolted y Grdner et l. (1) from the urine of n immunosuppressed ptient. It is relted oth ntigeniclly nd iochemiclly to the ppovvirus simin virus 40 (2-7). Like ll ppovviruses, BKV is oncogenic in rodents, inducing severl types of tumors when injected into neworn hmsters nd mice (8-12). When injected intrcererlly, this virus induced tumors in 88% of inoculted hmsters nd 29% of inoculted mice, nd ll tumors were intrventriculr ependymoms (11). Furthermore, when injected intrvenously into hmsters, BKV induced tumors in 82% of the inoculted nimls, nd 72% of the tumors were cererl ependymoms (12). The two other most frequent types of tumors induced in hmsters inoculted intrvenously with BKV were pncretic insulinoms (12%) nd osteosrcoms (10%). BKV lrge tumor ntigen ws detected in BKVinduced tumors (11, 12), indicting tht t lest portion of the virl genome ws eing expressed in tumor cells. It ws therefore of interest to serch for BKV DNA sequences in tumor tissues nd to nlyze such sequences in order to determine their physicl stte (free or integrted, complete or incomplete virl genomes) nd their mode of integrtion. We present here the results of n nlysis of B5KV DNA sequences in severl BKV-induced hmster tumors y the lottrnsfer hyridiztion technique originlly devised y Southern (13). With this technique, virl DNA sequences re visulized s hyridizle nds fter digestion of tumor cell DNA y restriction endonucleses nd frctiontion y grose gel electrophoresis. The puliction costs of this rticle were defryed in prt y pge chrge pyment. This rticle must therefore e herey mrked "dvertisement" in ccordnce with 18 U. S. C solely to indicte this fct. 975 MATERIALS AND METHODS Virus growth, purifiction nd inocultion into Syrin golden hmsters, pthology of tumors, nd cultivtion of tumor cells hve een descried (12). Cellulr DNA Purifiction. Tissues were minced with scissors, homogenized in TD (25 mm Tris-HCl, ph 7.4/137 mm NCl/5 mm KCI/0.7 mm N2HPO4/5 mm D-glucose) (14) in Dounce homogenizer equipped with loose-fitting piston, nd digested overnight t 37 C with proteinse K (Merck) t 50,ug/ml in 20 mm EDTA/0.5% NDodSO4. The viscous lyste ws then gently extrcted with phenol/chloroform (1:1, vol/ vo1), dilyzed, treted with pncretic RNse (Worthington) nd gin with proteinse K, reextrcted with phenol/chloroform, nd dilyzed (to e descried in detil elsewhere). DNA purifiction from in vitro cultured cells followed the sme procedure ut with the omission of the homogeniztion step. 32P-Leled BKV DNA Proe. Virl DNA ws extrcted from CsCl-purified virions y disrupting the virions in N- DodSO4 t 50 C nd digesting with proteinse K. Superhelicl BKV DNA ws then purified in CsCl/ethidium romide density grdient (15). Leling with deoxyrionucleoside [32P]triphosphtes y nick-trnsltion ws ctlyzed y Escherichi coli DNA polymerse I (Boehringer) nd will e descried in detil elsewhere. The 32P-leled proe hd specific rdioctivity of 1 X 108 to 2 X 108 cpm/,ug of input DNA on the dy of synthesis. Blot-Trnsfer Hyridiztion. Cellulr DNA (25,ug) ws digested with restriction endonucleses (Miles) in the pproprite rection mixtures t 37 C for 3 hr. The rection ws stopped with EDTA, nd DNA ws precipitted with ethnol, dissolved in smll volume of 1:5 diluted electrophoresis uffer (E uffer, 40 mm Tris-HCI, ph 7.8/5 mm sodium cette/1 mm EDTA) (16). Electrophoresis ws performed in 0.8% grose gels (Bio-Rd) for digested DNAs nd 0.6% gels for undigested DNAs, in horizontl sls 0.5 cm thick nd 20 cm long, with 20 Mg of DNA for ech smple. Electrophoresis ws t 1.5 V/cm for 15 hr t room temperture. After electrophoresis, DNA ws dentured in the gel, trnsferred to nitrocellulose memrne, nd hyridized to the 32P-leled BKV DNA proe (to e descried in detil elsewhere). The nitrocellulose sheet ws finlly lid ginst preflshed Kodk Royl X-Omt film in the presence of CWO4 intensifying screen (Ilford Fst Tungstte) (17). Tumors nd Cell Lines. The following hmster tumors induced y BKV injected intrvenously were nlyzed: four ependymoms (nos. 121, 140, 156, nd 158), six osteosrcoms (nos. 208, 239, 275, 284, 297, nd 317), nd three pncretic insulinoms (nos. 208, 209, nd 210). In ddition, nlysis of Arevition: BKV, BK virus To whom reprint requests should e ddressed.

2 976 Cell Biology: Chenciner et l. BKV DNA sequences ws performed on cell lines T117, T1i3, nd T135 derived from three different ependymoms nd on cell line T284 estlished from osteosrcom no. 284, s well s on single-cell clone otined from line T284. RESULTS Ependymoms. Restriction endonuclese EcoRI cuts BKV DNA t single site (18). When DNA from four cererl ependymoms ws digested with EcoRI nd nlyzed y lot-trnsfer hyridiztion to BKV [32P]DNA proe, the ptterns shown in Fig. l were otined. Ech DNA gve rise to mny hyridiztion nds, nd the ptterns from the four tumors were different. Furthermore, most of the EcoRI-generted segments contining BKV sequences were either fster or slower thn liner BKV DNA. This is wht would e expected for virl genomes integrted into cellulr DNA, ecuse digestion with EcoRI would then generte, for one inserted virl genome, two segments ech contining virl sequence joined to cellulr sequence. The length of these two segments would in generl e different from tht of liner virl DNA. Alterntively, ll hyridiztion nds ppering in Fig. l could represent free (nonintegrted) virl DNA molecules smller or lrger thn norml BKV DNA. This possiility ws ruled out y two kinds of experiment. In the first one, uncut DNAs from the sme four tumors were electrophoresed on grose gel nd nlyzed y the sme technique (Fig. 2). No hyridiztion nd ws visile in the.1... i C B L BK FIG. 1. Blot-hyridiztion of 32P-leled BKV DNA to EcoRIdigested DNAs (20,g ech) from neoplstic nd norml tissues of BKV-inoculted nimls. () DNAs from ependymoms nos. 160,156, 140, nd 121; smple 121C ws DNA from the norml portion of the rin from which ependymom 121 ws removed. () B nd L were DNAs from rin nd liver, respectively, of hmster killed 15 dys fter BKV inocultion. BK ws control tht contined 50 pg ech of nicked-circulr (F II) nd liner (F III) BKV DNA, with trces of superhelicl BKV DNA (F I). Molecules migrting hed of F III were defective BKV genomes present in the virl preprtion, which did not contin hmster DNA sequences, s shown y smple L. Autordiogrphs were overexposed in order to revel fint nds in smples from nontumor tissues (rrows). nd represent seprte experiments. The position of control BKV DNA ws determined in ech experiment. A I Proc. Ntl. Acd. Sci. USA 77 (1980).'. c 4 F HI ' BK HB FIG. 2. Blot-hyridiztion of 32P-leled BKV DNA to DNAs from ependymoms. () Uncut DNAs (20.g ech). () HindII-digested DNAs (20,g ech). (c) Pst I-digested DNAs (20,gg ech). BK controls contined 50 pg ech of EcoRI-linerized BKV DNA. nd c were run on the sme gel; represents seprte experiment. The utordiogrphic exposure time ws different in the two experiments. region of the EcoRI-generted nds: insted, ll BKV sequences were found in the region of high moleculr weight DNA, suggesting tht they were integrted into the cellulr genome. Electrophoresis of uncut DNA however, is not the est wy of seprting free virl sequences from cellulr DNA ecuse very lrge DNA could trp the nonintegrted sequences during migrtion. We therefore digested ependymom DNAs with HindIl, n enzyme tht does not cut BKV DNA ut does cut cellulr DNA into segments whose'verge length should exclude ggregtion rtefcts. Agin, prcticlly ll of the hyridiztion ws found in the region of very lrge DNA segments (Fig. 2), indicting tht most virl sequences were indeed integrted in the cellulr DNA. Fint, fster migrting nds were lso visile, which could represent nonintegrted virl DNA or integrted, incomplete virl genomes. Intensities of such nds, however, corresponded to much less thn one genome per cell. A common feture of ll four EcoRI ptterns otined with DNA from ependymoms ws the presence of strong hyridiztion nd in the position corresponding to full-size liner BKV DNA. Considering tht lmost no free virl DNA ws detected in these four tumors, such nds must lso come from integrted virl genomes. Comprison of their intensities with those of known mounts of liner BKV DNA (dt not shown) suggested tht they represent severl virl genome equivlents per cell. The simplest interprettion of this result is tht the full-size liner virl DNA molecules were generted y the cutting of tndemly integrted virl genomes y EcoRI. If this were the cse, then digestion of ependymom DNA with nother enzyme tht cuts BKV DNA t different unique site should gin cleve out liner BKV molecules from tndem integrtions. We chose Pst I, which cuts BKV t single position (6) quite fr from the EcoRI site. A strong hyridiztion nd ws gin visile in ech pttern t the position of full-size BKV liner DNA (Fig. 2c). We conclude tht tndem integrtions of virl genomes into the cellulr DNA did in fct occur in BKV-induced ependymoms. Lne 121c in Fig. l shows hyridiztion to EcoRI-digested DNA from the norml portion of the rin from which ependymom 121 ws removed. Two fint nds were visile fter prolonged utordiogrphic exposure, suggesting tht the presence of BKV DNA ws not rigorously confined to the neoplstic tissue in the ffected niml. However, the very low

3 intensity of the oserved nds compred to the pttern of n equl mount of DNA from the corresponding tumor (no. 121) indictes tht only smll frction of cells in the surrounding rin crried virl DNA. The possile mening of this finding is discussed in conjunction with results from relted experiment (see elow). Osteosrcoms. Fig. S shows the result of n experiment in which DNAs from six osteosrcoms were digested with EcoRI nd nlyzed s ove. Agin, ech pttern consisted of severl nds covering wide rnge of electrophoretic moilities. Comprison with the corresponding EcoRI ptterns of ependymoms (Fig. 1) showed tht some fetures were consistently different in the two types of tumors. The most ovious difference ws the sence, in four of the ptterns from osteosrcoms (nos. 239, 275, 297, nd 208) of hevy hyridiztion nd t the position of liner BKV DNA, wheres this ws lwys the min nd with ependymoms. In fct, only two osteosrcoms gve nds of outstnding intensity: smple 284 nd smple 317. Surprisingly, these strong nds were found to originte from free DNA. Fig. S shows tht uncut DNA from smple 284 gve one mjor nd fster thn liner BKV DNA nd two nds of lower intensity migrting hed. Such nds indicte the presence of free, presumly defective, virl DNA molecules in this smple (see elow). Uncut DNA from smple 317 gve strong hyridiztion nd of free DNA slightly slower thn liner BKV DNA, with moility comprle to tht of nicked circulr BKV DNA (which, in 0.6% grose, migrted.rther close to the liner form). No nds migrting like free DNA were seen in ny of the other four osteosrcoms, with either undigested or HindIl-digested DNA (dt not shown). In n ttempt to chrcterize further the free DNA molecules, we digested DNA from osteosrcoms 284 nd 317 with HindIII, n enzyme tht cuts BKV DNA t four sites (18). Oviously, ecuse integrted virl DNA molecules were present in oth smples together with free virl DNA, such n nlysis cnnot give strightforwrd informtion exclusively on free DNA molecules. Any conclusion must rely on the ssumption tht the most intense nds in the HindIII pttern come from the predominnt nds in EcoRI or uncut-dna ptterns-i.e., free DNA. Fig. Sc shows tht, esides regulr BKV DNA-HindIII segments A nd B, smple 284 gve.-ak.wl A.,.gift w Cell Biology: Chenciner et l. c W W 4A Proc. Ntl. Acd. Sci. USA 77 (1980) 977 strong nd migrting fster thn B. By comprison with intensities of A nd B segments in the control smple, such nd could represent sepnent A crrying deletion, nd this would e consistent with the oservtion tht free DNA molecules in smple 284 migrted fster thn liner BKV DNA (Fig. 3 nd ). Smple 317, on the contrry, showed two "norml" A nd B HindIll segments in lrge mounts, suggesting tht free virl DNA molecules in this tumor were norml in this prt of the genome. It should e rememered tht A covers most of the BKV lte region nd B covers lrge frction of the erly region (6, 18). Anlysis for segments C nd D ws not sufficiently precise, due to their smll size nd, in smple 317, to the presence of degrded mteril migrting in this prt of the gel. Free DNA in smple 284 seemed to lck segment D. All other nds in oth HindIII ptterns presumly cme from the splitting of segments from integrted genomes s result of the integrtion event. Insulinoms. DNAs from three pncretic insulinoms were nlyzed for the presence of BKV sequences. After digestion with EcoRI, they gve rise to reltively smll numer of hyridizle nds compred to ependymoms nd osteosrcoms (Fig. 4). Two of the tumors (nos. 209 nd 210) contined free virl DNA, s indicted y hyridiztion nds seen with uncut DNAs (indicted y rrows in Fig. 4). Unfortuntely, nds were very fint with this type of tumor, nd more precise nlysis could not e performed due to the limited mount of mteril. Presence of BKV DNA Before Tumor Appernce. BKV DNA could e detected in the rin, ut not in the liver, of inoculted hmsters efore the ppernce of tumors. BKV ws injected sucutneously into suckling hmsters nd the nimls were killed fter 15 dys. Orgns from these nimls were exmined histologiclly nd found to e norml. DNA ws purified from liver nd rin of one niml nd nlyzed y lot-trnsfer hyridiztion fter EcoRI digestion. The results of such n experiment re shown in Fig. 1. Three hyridiztion nds were visile in the DNA extrcted from the rin fter prolonged utordiogrphic exposure (Fig. l, lne B), one migrting like liner BKV DNA, one migrting fster, nd one migrting slower. No nds were visile in DNA from the liver 90 F 111I '.i.. U.. U BK BK BK BK04 FIG. 3. Blot-hyridiztion of 32P-leled BKV DNA to DNAs from osteosrcoms. () EcoRI-digested DNAs (20,g ech). () Uncut DNAs from tumors 284 nd 317. (c) HindIII-digested DNAs from tumors 284 nd 317. Bnds A, B, C, nd D indicte hyridiztion to the four BKV DNA-HindIII segments (50 pg totl). The hevy nd mrked y n rrow in c most likely represents defective segment A from the free DNA in tumor 284. nd c represent seprte 0.8% grose gels; represents 0.6% gel. BK BK FIG. 4. Blot-hyridiztion of 32P-leled BKV DNA to DNAs from pncretic insulinoms (tumors 208,209, nd 210). () EcoRIdigested DNAs (20 Mg ech). () Undigested DNAs (20 Mg ech); rrows point to nds of nonintegrted DNA. BK control smples contined 50Mug ech of nicked circulr nd liner BKV DNA () or 50 pg of liner BKV DNA (). The nd migrting hed of F III ws due to defective BKV genomes (see legend to Fig. 1).

4 978 Cell Biology: Chenciner et l. of the sme niml (Fig. l, lne L). Possile implictions of this finding in connection with the tropism of BKV tumorigenic ction for rin tissue re discussed elow. Cultured Tumor Cells. In view of the complexity of hyridiztion ptterns oserved with virus-induced tumors (prticulrly with ependymoms nd osteosrcoms), it is possile tht cells within the sme tumor were heterogeneous with respect to the mode of virl integrtion. To investigte this prolem, cell lines were estlished from three ependymoms (nos. 117, 133, nd 135) nd from osteosrcom 284. DNA ws purified from in vitro cultured cells nd nlyzed s ove. Fig. 5 shows the hyridiztion ptterns otined when 32P-leled BKV DNA ws hyridized to EcoRI-digested DNAs from the three ependymom cell lines. Ptterns were similr to those oserved with tissues from the sme type of tumors (Fig. 1), ll of them showing comprle complexity nd strong nd migrting like liner BKV DNA. Unfortuntely, comprison with the originl tumor tissues ws impossile in this cse ecuse, due to their smll size, the whole tumors were used to estlish the cell cultures. Comprison of originl tumor, cultured tumor cells, nd single-cell clone derived from the estlished line ws possile, however, for osteosrcom 284 nd is shown in Fig. 5. The EcoRI-generted ptterns of the cultivted cells were strikingly simpler thn the pttern of the originl tumor. The hevy nd of free DNA hd disppered in oth ptterns of cultured cells, s well s most of the nds from integrted genomes. Some of the nds seen in line T284 were lso present in the originl tumor, suggesting tht selection of cell types or of chromosomes contining integrted virl genomes hd tken plce. However, this ws proly not the only cuse of vrition ecuse two of the nds of line T284 were not present in detectle mounts in the originl tumor nd t lest one of the nds of clone T284c13 ws not present in the cell line from which the clone ws derived (rrows in Fig. S). Asence of free virl sequences in lines T284 nd T284c13 ws confirmed y the sence of free-migrting nds when uncut DNAs were nlyzed (dt not shown). F III T284 T284 c13 AM,.... m to M 4m e * 1 * FIG. 5. Blot-hyridiztion of 32P-leled BKV DNA to EcoRIdigested DNAs from cultured tumor cells. () DNAs from osteosrcom 284, from cultured cells from the sme tumor (T284), nd from single-cell clone derived from the estlished line (T284c13). () DNAs from cell lines estlished from three ependymoms (nos. 117, 133, nd 125). Controls contined 50 pg of liner BKV DNA (F III), including some defective virl genomes (see legend to Fig. 1). Arrows point to new hyridiztion nds rising during culture (see text). nd represent seprte experiments. Proc. Ntl. Acd. Sci. USA 77 (1980) DISCUSSION We hve investigted the presence nd stte of virl DNA in BKV-induced hmster tumors nd their derived cell lines nd clones. BKV DNA sequences were found in ll tumors exmined; most of them were integrted in the cell genome nd some were in free stte. The precise numer of virl genome equivlents per cell could not e estimted in tumors for severl resons: (i) the reltive mounts of neoplstic nd stroml cells in tumor tissues were not known; (ii) tumor cells could e heterogeneous with respect to the distriution of virl DNA (see elow); nd (iii) the nture of the hyridiztion technique used, involving trnsfer of DNA from grose gels to nitrocellulose nd utordiogrphic exposure, did not llow precise quntittion of hyridizle sequences. However, the presence of mny nds in ll EcoRI-generted hyridiztion ptterns shows tht differently integrted BKV genomes were present in ech tumor. Comprison of nd intensity with hyridiztion nds of known mounts of virl DNA suggested tht, on the verge, ech tumor cell contined t lest one nd in most cses severl integrted virl genomes. Integrtion ptterns oserved were ll different from ech other, nd digestion with EcoRI generted segments tht covered wide size rnge nd contined different lengths of virl DNA (s judged y intensity of hyridiztion nds). All these oservtions suggest tht virl integrtion could tke plce t mny different sites on cellulr nd virl DNA, in greement with wht ws found with cells trnsformed in vitro y simin virus 40 (19-21), polyom (22), nd BKV (unpulished dt). In spite of the gret vriility of hyridiztion ptterns, however, one feture ws common to ll ependymoms exmined nd sent in osteosrcoms nd in insulinoms: ll ependymoms hd tndem integrtions of full-size liner BKV DNA, wheres osteosrcoms nd insulinoms hd ptterns suggesting individul integrtions (or short tndems of defective molecules). The presence of free virl sequences in osteosrcoms nd insulinoms ws unexpected. Becuse hmster cells re nonpermissive for BKV repliction (23, 24), free virl DNA molecules could not e propgted s infectious virl prticles, s supported y the finding tht BKV structurl ntigens were never detected in BKV-induced tumors (12). One possile explntion is tht free virl DNA sequences were ctully replicted s integrted virl genomes nd then occsionlly excised (see ref. 22 for detiled discussion of this possiility). Alterntively, lthough hmster cells do not support extensive BKV DNA synthesis, one could ssume tht limited repliction of free virl DNA sequences occurred in these tumor cells. A rough estimte suggests tht osteosrcom 284 contined, on the verge, one to three copies per cell of n incomplete free BKV genome crrying deletion in the lte region, wheres osteosrcom 317 contined t lest five copies per cell of free, possily norml, virl DNA molecule. Our results, however, do not distinguish etween rndom distriution of free virl DNA mong ll cells in the tumor nd the presence of only few cells producing lrge mounts of free virl DNA, s found in polyom-trnsformed rt cells (25). Virl DNA in redily detectle mounts (i.e., one or more genomes per cell, on the verge) ws found only in neoplstic tissues. However, prolonged utordiogrphic exposure showed tht the norml portion of the rin in which n ependymom hd developed contined smll trces of BKV DNA. Contmintion y neoplstic tissue due to incomplete removl of the tumor ws mde rther unlikely y the comprison of hyridiztion ptterns of ependymom nd surrounding rin (Fig. 1). This finding correltes well with the presence of trces of BKV DNA in the rin of n niml 15 dys fter virus in-

5 Cell Biology: Chenciner et l. jection. Three nds were visile in this cse (Fig. 1), two of them corresponding to n integrted virl genome nd one to either tndemly integrted or free virl genome. No BKV DNA sequences could e detected in the liver of the sme niml (Fig. 1) or in vrious orgns, including rin, of noninoculted nimls (dt not shown). The presence of BKV DNA sequences in pprently norml cererl tissues efore nd fter the ppernce of tumors could led to specultion tht BKV DNA cn persist in the rin of inoculted nimls long fter inocultion, possily ecuse immunologicl protection is less efficient in this orgn. The persistence of virl DNA in rin cells could e one of the cuses of the high incidence of ependymoms mong BKV-induced tumors. Anlysis of the mode of integrtion of virl DNA in virusinduced tumors should offer, in principle, vlule tool to investigte the prolem of the clonl origin of tumor cells. We found tht EcoRI-generted hyridiztion ptterns oserved with DNA from tumor tissues hd more nds thn did ptterns from cultured tumor cells, nd tht single-cell clones isolted from such cultured tumor cells hd even simpler ptterns. These results would e comptile with multiclonl origin of BKV-induced tumors ecuse they suggest tht selection during culture leds to disppernce of certin cell types nd of the corresponding virl genomes. However, considerle cution should e used in evluting these results ecuse other events esides cell selection could cuse loss of virl genomes from tumor cells. Selective loss of chromosomes or rerrngement of integrted virl genomes could lso give rise to new integrtion ptterns, provided tht similr chnges took plce in ll cells of the sme culture. Alterntively, some cells, fter developing n neuploid kryotype or chromosoml errtions during culture, could hve cquired some selective dvntge nd rpidly outgrow other cells. Tht rerrngement of integrted virl sequences could in fct tke plce in cultivted tumor cells ws shown y the ppernce in clone T284c13 of t lest one new nd not present in line T284, from which the clone ws derived. Also, two of the nds of line T284 were pprently not present in the originl tumor. Interestingly, free virl DNA present in osteosrcom 284 ws lost in cultivted tumor cells nd in the derived single-cell clone. In conclusion, we think tht, lthough identicl integrtion ptterns of tumor nd clones would unmiguously prove the monoclonl nture of the tumor, the vritions we oserved re not convincing evidence for multiclonlity. Our findings, however, do show tht loss nd rerrngement of virl DNA sequences cn occur in cultured tumor cells, suggesting tht such cells give only n incomplete ccount of the stte of virl DNA in virus-induced tumors. The excellent technicl ssistnce of W. Rossi-Pusinnti nd C. Mussi is grtefully cknowledged. This work ws supported y Consiglio Proc. Ntl. Acd. Sci. USA 77 (1980) 979 Nzionle delle Ricerche-Progetto Finlizzto Virus Grnts nd /84/ N.C. ws prtilly supported y fellowship from the D6l6gtion GCn6rle l Recherche Scientifique et Technique. G. Meneguzzi ws the recipent of fellowship from the Europen Economic Community. 1. Grdner, S. D., Field, A. M., Colemn, D. V. & Hulme, B. (1971) Lncet i, Penney, J. B. & Nryn, 0. (1973) Infect. Immun. 8, Tkemoto, K. K. & Mullrkey, M. F. (1973) J. Virol. 12, Mullrkey, M. F., Hrusk, J. F. & Tkemoto, K. K. (1974) J. Virol. 13, Khoury, G., Howley, P. M., Gron, C., Mullrkey, M. F., Tkemoto, K. K. & Mrtin, M. A. (1975) Proc. Ntl. Acd. Sci. USA 72, Yng, C. A. & Wu, R. (1978) Proc. Ntl. Acd. Sci. USA 75, Dhr, R., Li, C. J. & Khoury, G. (1978) Cell 13, Shh, K. V., Dniel, R. W. & Strnderg, J. D. (1975) J. Ntl. Cncer Inst. 54, Nse, I. M., Krkkinen, M. & Mntyjrvi, R. A. (1975) Act Pthol. Microiol. Scnd. Sect. B 83, Vn der Noord, J. (1976) J. Gen. Virol. 30, Corllini, A., Brnti-Brodno, G., Bortoloni, W., Nenci, I., Cssi, E., Tmpieri, M., Portolni, M. & Borgtti, M. (1977) J. Ntl. Cncer Inst. 59, Corllini, A., Altvill, G., Cecchetti, M. G., Fris, G., Grossi, M. P., Bloni, P. G., Lnz, G. & Brnti-Brodno, G. (1978) J. Ntl. Cncer Inst. 61, Southern, E. M. (1975) J. Mol. Biol. 98, Smith, J. D., Freemn, G., Vogt, M. & Dulecco, R. (1960) Virology 12, Meneguzzi, G., Pigntti, P. F., Brnti-Brodno, G. & Milnesi, G. (1978) Proc. Ntl. Acd. Sci. USA 75, Shrp, P. A., Sugden, B. & Smrook, J. (1973) Biochemistry 12, Lskey, R. A. & Mills, A. D. (1977) FEBS Lett. 82, Howley, P. M., Khoury, G., Byrne, J. C., Tkemoto, K. K. & Mrtin, M. A. (1975) J. Virol. 16, Ketner, G. & Kelly, T. J. (1976) Proc. Ntl. Acd. Sci. USA 73, Botchn, M., Topp, W. & Smrook, J. (1976) Cell 9, Cmpo, S. M., Cmeron, I. R. & Rogers, M. E. (1978) Cell 15, Birg, F., Dulecco, R., Fried, M. & Kmen, R. (1979) J. Virol. 29, Mjor, E. 0. & DiMyorc, G. (1973) Proc. Ntl. Acd. Sci. USA 70, Portolni, M., Brnti-Brodno, G. & L Plc, M. (1975) J. Virol. 15, Zouzis, D., Prsd, I. & Bsilico, C. (1977) J. Virol. 24,