Vol. 61, No. 12JOURNAL OF VIROLOGY, Dec. 1987, p. 3848-38540022-538X/87/123848-07$02.00/0Copyright C) 1987, American Society for Microbiology

Chromosomal Translocation and Inverted Duplication Associatedwith Integrated Hepatitis B Virus in Hepatocellular Carcinomas

TAKASHI TOKINO,' SHINICHI FUKUSHIGE,' TAKAAKI NAKAMURA,' TSUTOMU NAGAYA,'tTOMOAKI MUROTSU,' KIYOSHI SHIGA,2 NAOTO AOKI,2 AND KENICHI MATSUBARAl*

Institute for Molecular and Cellular Biology, Osaka University, Yamada-oka, Suita, Osaka 565,' and Department ofPathology, University of Tokyo, Faculty of Medicine, Hongo, Bunkyo-ku, Tokyo 113,2 Japan

Received 25 June 1987/Accepted 31 August 1987

Integrated hepatitis B virus (HBV) DNA is found in hepatocellular carcinomas which develop in HBVcarriers. Presented here are the results of analyses of four integrants that show chromosomal rearrangementsassociated with the integrated HBV DNA. Two clones (p4 and C15) were found to have large inverted repeatingstructures, each consisting of HBV genome along with flanking cellular sequences. The structure must havearisen by duplication of the primary integrant, including the flanking cellular DNA, followed by recombinationwithin the viral DNA. One of the two viral arms in each clone joins to the other viral arm at the "cohesive endregion." Two clones (DA2-2 and DA2-6) were found to have integrated HBV sequences, each flanked bycellular DNAs from different chromosomes (chromosome X joined to 17 and chromosome 5 joined to 9). Theymust be the products of cellular DNA translocations using the integrated HBV DNA as the switch point. Theviral DNA in each clone is a continuous stretch of a single virus genome with one end in the cohesive end region.These complex structures seem to have been produced by activation of the cohesive end of an integrant viralgenome, followed by its recombination with another chromosomal DNA.

Hepatitis B virus (HBV) is a causative agent of humanhepatitis that affects some 200 million people in the world.Epidemiological studies as well as DNA analyses haverevealed a strong correlation between HBV infection and theincidence of hepatocellular carcinoma (HCC). A high ratio ofHCCs from patients in endemic areas carry integrated HBVDNA in cellular DNA, suggesting that the integration or theresults thereof are connected with cancer development (14).

In a previous report (T. Nagaya, T. Nakamura, T. Tokino,T. Tsurimoto, M. Imai, T. Mayumi, K. Kamio, K.Yamamura, and K. Matsubara, Genes Develop., in press),we analyzed the structures of 19 virus genomes integrated inchromosomes ofhuman hepatomas and showed that in abouthalf of the studied cases, the virus DNA was joined tocellular DNA at its "cohesive end region," where most ofthe viral transcriptions and replication start or terminate(Fig. 1). We also showed that almost all the integrated virusgenomes have a deletion(s) in or around the cohesive endregion which makes the integrants unable to act as templatesfor the virus replication. No particular cellular DNA se-quence has been found that is used as a direct target for virusDNA integration (19). The complexity of the integrants ingenomic structure could only be explained by assuming insome cases that the virus DNA had induced chromosomaltranslocations or, in other cases, that viral-cellular DNAjoints must have been amplified first, followed by rearrange-ments. Because changes in chromosome structures are oftenconnected with the development of cancer, we were inter-ested in investigating the structure of such rearranged DNAsto obtain insights into the general features and implicationsof the rearrangements.

In this study we report four cases, including two chromo-somal translocations associated with HBV integration and

* Corresponding author.t Present address: Institute for Bioscience, Nippon Zeon Co.,

Yako 1-2-1, Kawasaki 210, Japan.

two cases in which amplification of the integrated HBVDNA took place first, followed by rearrangement.

MATERIALS AND METHODS

Human HCC tissues, cloning, and DNA analyses. Twosamples of HCC tissue (HCC7, HCC21) were obtained atautopsy, and one sample of HCC tissue (PLC342) wasobtained as a mass propagated in nude mice. Table 1 lists theHCC tissues used in this work and the A clones obtained thatcarry the integrated HBV DNA with flanking cellular DNAs.Preparation of these X clones has been described previously(Nagaya et al., submitted). DNA extraction from tumors,genome mapping, and Southern blot analyses (12) were doneas described elsewhere (Nagaya et al., in press).Chromosomal assignment of the flanking cellular DNA.

Chromosomal assignments were made according to proce-dures described previously (4). A karyotypically normalhuman lymphoblastoid cell line, GM0131, and human fibro-blast cell lines GM3463 and GM2971, each carrying recipro-cal chromosomal translocations, were obtained from theHuman Genetic Mutant Cell Repository (Camden, N.J.).Condensed chromosome suspensions from these cells wereprepared by the polyamine method of Sillar and Young (11)and sorted into eight fractions by using a fluorescence-activated cell sorter (CHROSS-1; Japan Spectroscopic Co.,Ltd., Tokyo, Japan), and the DNA from each sorted chro-mosome (5 x 105 chromosomes) was digested with a restric-tion endonuclease, followed by electrophoresis and South-ern blotting. The blots were hybridized to unique DNAsequences prepared from cellular DNA flanking the inte-grated HBV DNA. When necessary, DNAs from human-mouse hybrid cells that carry selected human chromosomeswere prepared for precise assignments. Hybridizations weredone under stringent conditions with 32P-labeled DNAprobes by using a Multiprime DNA labeling system(Amersham Japan Corp., Tokyo, Japan).

3848

INTEGRATED HBV DNA AND CHROMOSOMAL REARRANGEMENTS 3849

-~~~0

Rtegion X

FIG. 1. Structure and genetic organization of the HBV genome.

Numbers represent nucleotide numbers starting from the hypothet-

ical EcoRI site in subtype adr (3). This numbering system was

chosen to coordinate the map with other systems (14). Broad

arrows, Open reading frames; C and S, core and surface antigen

genes, respectively; X and P, unidentified coding frames. DR1 and

DR2 at positions 1820 and 1590, respectively, are the 11-base-pair

direct repeats. The region between DRi and DR2 is the cohesive end

region. The pregenome RNA synthesis starts at DR1, proceeding

clockwise, and terminates at DR1, with some overlaps (2). The first

DNA strand synthesis starts at DR1, proceeds counterclockwise,

and terminates just in front of DR1 (10, 17). The second DNA strand

synthesis starts at DR2, proceeds clockwise, and terminates at

around 0-10OO. Some restriction sites in the adr4 genome (3) are

also displayed.

RESULTS

HCC7, HCC21, and PLC342 each carry multiple copies of

integrated HBV DNAs as revealed by Southern blot analy-

ses with HindIII or EcoRI digestion, which does not cause

cleavage inside of the HBV subtype adr genome (see Fig. 1).

The unintegrated form of the virus DNA was not observed in

HCC7 or HCC21. The DNA from each HCC was cleaved

with EcoRI to construct a library, and from each library,

recombinant phages carrying the integrated HBV DNA were

obtained. Clones p4, DA2-2 and DA2-6, and Ci5 were

obtained from HCC7, HCC21, and PLC324, respectively

(Table 1). The insert from each clone was subjected to

restriction enzyme mapping, and subfragments carrying the

viral-cellular DNA junctions were sequenced. The structural

organization of integrated HBV DNA was determined

mostly by nucleotide sequencing (9) and partly by restriction

mapping.

Structure of clones p4 and C15. Lambda clones p4 and

Ci5, derived from different HCCs, were found to have a

large inverted repeating structure consisting of portions of

the HBV genome along with flanking cellular sequence.

Clone p4 contains 2.8 kilobases (kb) of integrated HBV

DNA, composed of a 1.7-kb segment and a 1.1-kb segment

(Fig. 2). The former segment consists of a virus genome

covering the preS2 region (54) to the 3' end of the X gene

(1795), and the latter segment covers the preS2 region (54) tothe 3' end of the P gene (1177). These two segments arelinked in inverted fashion by the joining of the 1795 end ofthe former and the 1177 end of the latter. A remarkablefeature of this clone lies in the fact that its two viral-cellularDNA junctions are identical, and its flanking cellular se-quences, 3.5 kb each, are also identical, as shown by thecleavage map.

Clone C15 is similar in structure, consisting of two HBVDNA segments linked in inverted fashion (Fig. 2). Onesegment (2.5 kb) covers the virus genome from the 3' end ofthe X gene (1756) to the 3' end of the P gene (1073), andanother segment (1.4 kb) covers the genome from the 5' endof the preSl region (2840) to the 3' end of the P gene (1073).The former segment carries a small 156-base-pair deletion(2148 to 2305). Again, the two viral-cellular DNA junctions,appearing at position 1073, are identical, and the flankingcellular DNAs are identical.

It must be stressed that in both clones p4 and C15, one ofthe two viral arms uses the cohesive end region to form theviral-viral DNA junction (1795 in P4 and 1753 in C15). Theinverted repeating structure of p4 is "head to head," in thesense that transcriptions of the original two viral genomesproceed divergently. The inverted repeat structure in cloneC15, by the same definition, is "tail to tail."To know from which chromosome the flanking cellular

DNAs are derived, single-copy DNA fragments were ex-tracted (marked "probe" in Fig. 2) and hybridized to South-ern blots of DNAs from flow-sorted chromosomes (Fig. 3).The same probes were hybridized to human-mouse hybridcell DNAs (Table 2). The results show that cellular DNA ofp4 originates from chromosome 11 and that that of C15originates from chromosome 3.

Structure of the clones DA2-2 and DA2-6. The integratedvirus genome in clone DA2-2 is 2.8 kb and covers a regionfrom 1816 to 1407 via 1/3214. DNA mapping and nucleotidesequencing studies showed that the viral DNA is a continu-ous stretch of a single viral genome with no interruption orrearrangement, except for a small 33-base-pair deletion thatlies between 3046 and 3078. The 1816 end is in the cohesiveend region. Clone DA2-6 carries a 2.8-kb integrated HBVDNA that covers a region from 2255 to 1791 via 1/3214. Theintegrated HBV sequence is again a continuous stretch of asingle virus genome with no deletion or rearrangement. The1791 end lies in the cohesive end region.To assign flanking cellular sequences to chromosomes,

single-copy DNAs were obtained from left and right flankingcellular DNAs and designated DA2-2L and DA2-2R andDA2-6L and DA2-6R, respectively (shown in Fig. 2). Theywere hybridized to Southern blots of DNA from flow-sortedchromosome fractions prepared from a human lymphoblas-toid cell line, GM0131, which carries a normal karyotype.The probes DA2-2L and DA2-2R were hybridized to DNA infractions c and f, which carry chromosomes 6, 7, 8, and X

TABLE 1. HCC tissues and X clones used in this study

Tissuea Copy no.b bCtaoned

HCC7 4 p4HCC21 6 DA2-2, DA2-6PLC342 10 C15

a HCC7 and HCC21 were obtained by surgical operations. PLC342 was alsoobtained by operation and was then propagated in nude mice (7). The threepatients bearing HCC were hepatitis B surface antigen positive.bThe number of integrated HBV genomes as shown by the number of bands

in Southern blot analysis of HindIII-digested HCC DNA.

VOL. 61, 1987

3850 TOKINO ET AL.

p4E X G G X I B D X

.IG GI I

X EI I

probe* *CTCCTGGGhTCCTGCT.

54**0GTCTCTGd1TATG*1177 1195

* *AGCCCCAGGAG654

G G X D XG B- . I I I

probe % %% %

* *GAmCTCITACATGC**1073

DA2-2B

I *-CT

X G G

UCCCATWGTTAAAG T**GTATG R TC*2846 1756 1073

X BA a

G BI I

I I I

-PrObe \Lprobe

- IA I PAWY% 1e * 1-.-1816

X B

I Rprobe

140G7ATCCTMAGAA-1407

B

Lprobe'* GTGTTCT GGTGTC'*

2255

_ It probe

* *TAGGCATCATCATT*-1791

FIG. 2. Structures of integrated HBV DNA and flanking cellular sequences. Open bars and lines represent integrated virus DNA andcellular DNA, respectively. Restriction endonuclease cleavage sites: B, BamHI; D, DraI; E, EcoRI; G, BgIIl; X, XhoI. Sequences at thecellular-viral and viral-viral DNA junctions are shown under each map. The HBV DNA sequences are boxed, and the nucleotide numbersat junctions are displayed. Solid bars represent the regions of cellular DNA that are nonrepetitious and were used as probes for Southern blotanalyses.

and chromosomes 16, 17, and 18, respectively (Fig. 3). TheDA2-2R probe was assigned to chromosome 17 by using asmall set of human-mouse hybrid cells (Table 2). The probeDA2-2L was found to be derived from chromosome 8 or Xby using a small set of human-mouse hybrid cells (data notshown). Next, human chromosomes from a fibroblast cellline, GM2971, which carries t(Xp22;13ql2) reciprocaltranslocations were flow sorted and subjected to Southernblot analysis using DA2-2L as a probe. This probe hybrid-ized to DNAs in fractions c and h (Fig. 4A). Fraction ccarries chromosomes 6, 7, and 8 and an untranslocated X asnoted above, and fraction h carries chromosomes 21 and 22along with the translocated chromosome t(Xp22;13ql2) car-

rying Xpter-Xp22. Thus, the DA2-2L probe must be locatedon this translocated portion of chromosome. Therefore, we

concluded that the DA2-2 clone has one arm from chromo-some 17 and the other arm from chromosome Xpter-Xp22.Similarly, the probes DA2-6L and DA2-6R were assigned toflow-sorted chromosome DNA fractions d and b, whichcarry chromosomes 9, 10, 11, and 12 and chromosomes 3, 4,and 5, respectively (Fig. 3). The probe DA2-6L was sug-

gested to be located on chromosome 9 by a discordant assay(Table 2) and was assigned to chromosome 9pter-9p22 bySouthern blot analysis with flow-sorted chromosomes from afibroblast cell line, GM3463, which carries t(9p22;14ql3)reciprocal translocations. This probe hybridized to fractionsd and h, the latter of which carries the translocated portionof chromosome 9 (Fig. 4B). The probe DA2-6R was assignedto chromosome 5 by using a small set of human-mousehybrid cells (Table 2). Therefore, we concluded that theclone DA2-6 has one arm from chromosome 9pter-9p22 andthe other arm from chromosome 5. As the integrant viralDNAs in both cases were in the continuous form, thesestructures seem to be produced by chromosomal transloca-tions mediated by the viral DNA. Cloning artifacts were notlikely, because Southern blotting profiles of the originaltumor DNA cleaved with EcoRI displayed the correspond-ing bands (data not shown).Chromosomal rearrangements in HCC21. An interesting

question is whether the original tumor cells in HCC21carried the normal counterpart of the translocated chromo-somes. To examine this point, DNA extracted from this

C15E GX V XI I I a

DA2-6E GI I

I I I I I I

q

--T-

-1---

I I

J. VIROL.

I

INTEGRATED HBV DNA AND CHROMOSOMAL REARRANGEMENTS 3851

12-9

18-16 -1

B Probe

h g f e d c b

p4

hg fe c d c b a

0

h g f e d c

C15

a t

b a t

4 0

h g f e d c b a t

7.5

"' L 0 ` 70

h g f e d c b a

4

DA2-6

,oRIt

0 .0

4* -- 25

FIG. 3. Assignments of the flanking cellular DNA sequences to chromosomes. (A) Flow histogram of human chromosomes from alymphoblastoid cell line, GM0131, that carries a normal diploid karyotype. Numbers in the histogram indicate the chromosome numbers.Chromosomes were sorted into eight fractions, a through h, as described in Materials and Methods. (B) Southern profiles. DNA from eachflow-sorted chromosome fraction was purified and digested with PvuII (p4 sample) or EcoRI (other samples) and subjected to Southern blotanalyses, using as probes the flanking cellular DNAs whose names are shown on the left of each panel. For the origins of these probes, see

Fig. 2. L and R in the autoradiograms indicate signals detected by corresponding probes. Sizes of the positive bands are given in kilobases.Lanes a through h, DNA from each sorted chromosome fraction; lane t, total human lymphocyte DNA (1 ,ug).

tissue was subjected to Southern blot analyses, using thesame set of cellular DNAs as probes. The DA2-2L probehybridized to a 7-kb EcoRI fragment from normal humanDNA (Fig. 5, lane b) and to an 8-kb EcoRI fragment from thetumor DNA. No normal counterpart DNA was detected in

this case because the DA2-2L probe originates from the Xchromosome, which was not diploid in the male patient. Theweak 7-kb band in lane a of Fig. 5 represents carryover of a

small amount of normal tissue in the original tumor speci-men. On the other hand, the DA2-2R probe hybridized to a

A

kb

0 - 2

1.5

DA2-2

t

_ 7.5

VOL. 61, 1987

3852 TOKINO ET AL.

TABLE 2. Chromosome assignments of flanking cellular DNA probes, using small sets of human-mouse hybrid cellsa

Hybrid Human chromosome Probecell line 3 4 5 9 10 11 12 16 17 18 C15 DA2-6R p4 DA2-6L DA2-2R

HM31 - + -HM06 - + - - + + - - + + - + +HM66A - + -HM23 - - +HM76 - + - +HM76D - + - +HM09 - + - +TAlA - + - - + - + + - + - - - - -

TA3A + - - + _III'27910 - - + - - + - +III'2 - + + _ + + _ + +p23-7-10-13 - - + - + -p23-5 - - +

a Mouse-human hybrid cells were obtained from Y. Kaneda at Osaka University, except for TAlA and TA3A, which were provided by N. Shimizu at KeioUniversity. Relevant chromosome karyotypes were examined by Giemsa and trypsin staining and by assaying enzyme markers. The results from both assays weremutually confirmatory. DNA from each hybrid cell was extracted, cleaved with EcoRI, and subjected to Southern blot analyses, using as probes the uniquecellular DNA segments shown in Fig. 2. (+) and (-) indicate, respectively, the presence and absence of positive bands in the blots.

7.5-kb EcoRI fragment from normal humanlane d), whereas the tumor DNA producedand an 8-kb band (lane c).

Similar treatment with the DA2-6L prot

ProbeA h g f e d c b a

DA2-2L v

B h g f e d c b a

DA2-6L 0

FIG. 4. Assignments of cellular sequences DA2respectively, to the short arm of chromosome X aof chromosome 9. (A) Chromosomes from a humline, GM2971, carrying the reciprocal translocati,were flow-sorted into eight fractions, a througcontains chromosomes 6, 7, and 8 and an untransloh contains chromosomes 21 and 22 along withchromosome t(X;13) carrying Xpter-p22. DNA Mgested with EcoRI, and subjected to Southern blDA2-2L as a probe. Lanes a through h, DNA i

chromosome fraction; lane t, total human lympho(Lanes c, h, and t showed positive signals. Thus, Elocated on Xpter-p22. Sizes of the positive batkilobases on the panels. (B) Chromosomes from a

cell line, GM3463, carrying the reciprocal tran14q13) were flow-sorted and treated as for panel Aprobe used was DA2-6L. Fraction d carries a:chromosome 9 along with 10, 11, and 12. Frachromosomes 21 and 22 along with the translocat(9;14) carrying 9pter-9p22. Lanes a through h,sorted chromosome fraction; lane t, total human 1

(1 jig). Lanes d, h, and t showed positive sigDA2-6L should be located on 9pter-p22.

i DNA (Fig. 5, bands in the tumor sample, 6.5 and 3 kb in size (Fig. 5, lanethe 7.5-kb band e), the former being an EcoRI fragment generated from the

HBV-integrated chromosome and the latter being the normal)e showed two counterpart, as judged from the appearance of the 3-kb band

in normal human DNA (lane f). Similarly, the DA2-6R probehybridized to a 7.5-kb EcoRI fragment in normal humanDNA (Fig. 5, lane h), whereas the 7.5-kb band and a 6.5-kb

t band appeared in the tumor DNA. Apparently, therefore,the rearrangement occurred in one chromosome withoutaffecting its normal counterpart (Fig. 6). Using the C15 probe

_ 7 kb (see Fig. 2), we also looked for the normal counterpart ofchromosomal DNA in EcoRI-digested DNA from thePLC342 clone that has the invertedly repeating DNA struc-ture. Whereas only a 1.5-kb band was seen in the normalsample (Fig. 5, lane j), a 6-kb "rearranged" band appeared inthe tumor sample, in addition to the 1.5-kb band (Fig. 5, lanei), showing again that the normal counterpart sequence was

L left intact. Thus, these observations demonstrate that exceptfor DA2-2L, which is located on chromosome X, transloca-tion of chromosomes, including formation of an inverted

22.5 kb repeating structure, takes place on one of the two alleles,leaving another allele intact. The normal counterpart chro-mosome remains intact even after the progeny cells havedeveloped into tumors. Another tumor, HCC7, from which

'-2L and DA2-6L, clone p4 originates, was not examined because of the limitedand the short arm amount of tumor DNA.ian fibroblast cellon t(Xp22;13ql2) DISCUSSION,h h. Fraction c DSUSOtcated X. Fraction We have demonstrated rearrangements of cellular DNAthe translocated associated with HBV DNA integration. Comparison of

lot analysis using Southern blotting profiles of HCC DNAs with X-cloned:rom each sorted DNAs showed that such rearrangements are not cloning arti-cyte DNA (1 ,ug). facts, but reflect events that have occurred in the liver cell.)A2-2L should be Two clones, both from HCC21 tissue, had chromosomalnds are given in translocations, and two other clones, from PLC342 andhuman fibroblast HCC7, had invertedly duplicated structures that includedislocation t(9p22; viral-cellular DNA junctions. Though not examined in thisk, except that the paper, clone C6, originating from PLC342, also had an

.ction h contains invertedly duplicated structure. Thus, altogether 5 rear-

ted chromosome ranged samples have been found among 19 analyzed samplesDNA from each which had been prepared from seven HCC tissues (Nagayalymphocyte DNA et al., in press). Recently, Hino et al. (5) found a similargnals. Therefore, translocation product by employing chromosomes 17 and 18.

Mizusawa et al. (8) described an example of invertedly

J. VIROL.

INTEGRATED HBV DNA AND CHROMOSOMAL REARRANGEMENTS 3853

probe DA2-2L R

a b c d

DA2-6L R

e f g h

C15

i J

8- - _87-

0

-7.5 ._ _ -7.5

6.5-- -6.5

2.5- E _a

1.5- -

FIG. 5. Southern blot analyses of DNAs isolated from tissues of HCC21 (lanes a, c, e, and g), PLC342 (lane i), and normal humanlymphocytes (lanes b, d, f, h, and j). DNAs were extracted from these tissues or cells, digested with EcoRI, and subjected to Southernanalyses using flanking cellular DNA probes as shown in Fig. 1. The probes used are shown at the top. Numerals represent the position andsize (in kilobases) of the bands. Arrowheads represent the bands resulting from the translocations or the inverted duplications.

DA2-2L probe

E - E

EDA2-2 13B

E

DA2-2R probe

DA2-6L probe

E E

E

I E

DA2-6R probe

FIG. 6. Schematic representations of DNAs in clonDA2-6. In both cases, the integrated HBV DNA icellular DNA from different chromosomes. The unocDNA fragments in EcoRI digests of normal human gare also displayed. Black bars represent DNAs from cl

(panel A) and chromosome 9 (panel B). Open b-chromosome 17 (panel A) and chromosome 5 (paunique-sequence DNA probes used in this report are

duplicated DNA structure. Apparently the frequency ofencountering these complex structures is fairly high, incontrast to the original expectations, and demonstrates theimportance of studies with chromosomal rearrangements

7kb after HBV integration. Despite interest in the karyotype oftumor cells having translocations or invertedly duplicated

E DNA, no data are yet available; autopsy samples or resected8kb samples were not sufficient for these tests.

HBV DNA integration is not an obligatory reaction forreplication of the virus genome, unlike that of retroviruses

E (16). However, it occurs at a certain frequency among theJ 7.5kb population of infected liver cells. The current model for

integration (18) assumes that the replicative intermediate ofHBV DNA, having at least one end terminating within one ofthe two direct repeats or within the cohesive end region, usesthe end for recombination with cellular DNA. No longsequence homology seems to be needed in this process. Inthe primary product of integration, one of the two viral-cellular DNA junctions may represent the once-active viralDNA end. How the other junction which appears randomly

2.5kb along the viral genome was produced is not known at present.The complex integrant forms we have analyzed can only

be explained by assuming that this primary integrationproduct was involved in subsequent rearrangement(s). The

6.5kb invertedly duplicated samples must have arisen by duplica-tion of the primary integrant, including the flanking cellularDNA, followed by recombination within the viral DNA. In

7.5kb both p4 and C15, one of the viral DNA arms has a cohesiveend sequence at the viral-viral DNA junction, suggestingstrongly that this cohesive end in the primary integrant mayhave been activated again and may have led to initiation of a

ies DA2-2 and second round of integration into its sister virus genome.is flanked by The chromosomal translocation samples, examined with:cupied target particular caution, showed that each of the viral DNArenomic DNA integrants is a continuous stretch of virus DNA; i.e., thehromosome X viral DNAs show no record of rearrangement or deletion,ars represent except for a small one (33 base pairs) in the clone DA2-2. Wealso shown. conclude that the two viral DNAs are in intact form. Since

we do not have reason to believe that the chromosomal

A

Ch.X

Ch. 17

B

Ch.9

DA2-6

Cli.5

I

VOL. 61, 1987

3854 TOKINO ET AL.

translocation samples were made by integration of an HBVright at the junction point of rearrangement, we proposeinstead that the cohesive end region of a primary integrant isactivated and then recombined to DNA in another chromo-some. An alternative possibility that cannot be ruled out isthat homologous recombination takes place between twointegrated HBV DNAs, each in different chromosomes. Butif this is the case, the question arises as to how heterologouschromosomes each having an integrant(s) could have foundeach other.

Besides the four integrants described here, a significantfraction of the integrant HBV genomes thus far analyzedhave inverted structures. Thus, among the 19 clones ana-lyzed, 8 have such a structure (Nagaya et al., in press), andamong 13 reported cases, 4 were in the inverted structure (1,5, 6, 8, 18, 19). (Note that the inverted structure in thiscontext does not imply that the two viral-cellular DNAjunctions are identical.) Among the 12 integrants that were inthe inverted structure, 7 (including the two cases analyzed inthis paper) carried the cohesive end sequence at either theviral-viral junction or the viral-cellular DNA junction. Al-though further proof is needed, we can hypothesize that suchstructures were produced by the same cohesive end-activating mechanism, followed by recombination of theactivated HBV end with another viral DNA or with thesecond chromosomal DNA. To clarify these problems, fur-ther analyses of flanking sequences are necessary.The occasional activation at the cohesive end region of an

integrated HBV genome, if it occurs as suggested, may becarried out by the viral replication machinery. Such activa-tion may be a rare event in the infection process of HBV,because in infected liver cells the majority of the replicativeintermediates are trapped inside the viral capsid along withthe DNA polymerase (13, 15). Thus, generation of thecomplex molecules may be limited by time and by the specialphysiological conditions of the cell.The complex structures we have observed are in HCC

tissues that have experienced many cycles of cellular selec-tions. Although the specific functions of the rearrangedchromosomes are not understood, they may have playedsome role in the multistage process leading to generation andselection of malignant cells.One could argue that such chromosomal rearrangements

simply reflect a general recombination of cellular DNAs thatoccurs in the developing neoplastic cells. If this is the case,then one wonders why so many junctions associated with therearrangements are found within the HBV sequence. Forprecise evaluation of the idea that the rearrangements arecorrelated with viral functions, cultured cells that allow repli-cation and recombination of the HBV genome are needed.

ACKNOWLEDGMENTS

We thank Y. Kaneda at Osaka University and N. Shimizu at KeioUniversity for the kind gifts of human-mouse hybrid cells.

This work was supported by a Grant-in-Aid for Special ProjectResearch in Cancer-BioScience from the Ministry of Education,Science and Culture of Japan.

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3. Fujiyama, A., A. Miyanohara, C. Nozaki, T. Yoneyama, N.Ohtomo, and K. Matsubara. 1983. Cloning and structural anal-ysis of hepatitis B virus DNAs, subtype adr. Nucleic Acids Res.11:4601-4610.

4. Fukushige, S., T. Murotsu, and K. Matsubara. 1986. Chromo-somal assignment of human genes for gastrin, thyrotropin(TSH)-p subunit and c-erbB-2 by chromosome sorting com-bined with velocity sedimentation and Southern hybridization.Biochem. Biophys. Res. Commun. 134:477-483.

5. Hino, O., T. B. Shows, and C. E. Rogler. 1986. Hepatitis B virusintegration site in hepatocellular carcinoma at chromosome 17;18 translocation. Proc. Natl. Acad. Sci. USA 83:8338-8342.

6. Koch, S., A. Freytag von Loringhoven, R. Kahmann, P. H.Hofschneider, and R. Koshy. 1984. The genetic organization ofintegrated hepatitis B virus DNA in the human hepatoma cellline PLC/PRF/5. Nucleic Acids Res. 12:6871-6886.

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