Multiple rearrangements and activated expression of c-myc induced by woodchuck hepatitis virus integration in a primary liver tumour

(~) INSTITUTPASTEUR/ELsEVIER Paris 1992

Res. Virol.

1992, 143, 89-96

Multiple rearrangements and activated expression of c - m y c induced by woodchuck hepatitis virus integration in a primary liver tumour Y. Wei (l), A. Ponzetto (2), p. Tiollais (~) and M.-A. Buendia (j) (*) (I) Unit# de Recombinaison et Expression g#n#tique, INSERM-U. 163, Institut Pasteur, 75724 Paris Cedex 15, and ¢2) Divisione di Gastroenterologia, Ospedale Molinette, Torino (Italy)

SUMMARY

Woodchuck hepatitis virus (WHV) is a small, partially double-stranded DNA virus. Like the related human hepatitis B virus (HBV), WHV induces acute and chronic hepatitis and hepatocellular carcinoma (HCC) in its natural host. WHV DNA integration into c-myc and N-myc, resulting in deregulated expression of these genes, has been described previously in woodchuck HCC. We have analysed a woodchuck liver tumour in which WHV DNA was integrated in the c-myc gene. The virus insertion provoked multiple alterations in one c-myc allele, probably involving secondary deletions and mutations. Integrated viral DNA, including promotor and enhancer sequences, acted as an insertional mutagen, leading to enhanced expression of heterogenous c-myc transcripts ranging from 7.2 to 14 kb in size, strikingly longer than normal 2.3-kb c-myc RNA. These results provide an additional exemple in which the oncogenic activation of a myc gene by cisacting effect of WHV insertion may play a critical role in virus-induced woodchuck HCC. Key-words: Oncogenesis, Hepatitis, Proto-oncogene, Woodchuck hepatitis virus, DNA, Hepatocellular carcinoma; c-myc gene, Rearrangement, Activation, Hybridization, Enhancer.

INTRODUCTION

The proto-oncogene c-myc was initially identified as the cellular homolog to the viral oncogene carried by the avian sarcoma virus MC29 (Sheiness and Bishop, 1979). Although the exact function of c - m y c remains elusive, it is thought to play an important role in regulation of normal cellular proliferation and differenti-

Submitted January 31, 1992, accepted February 24, 1992. (*) Correspondingauthor.

ation (for reviews, see Li~scher and Eisenman, 1990; DePinho et al., 1991). For example, the expression of c-myc increases in the response of quiescent cells to mitogenic stimuli during the cell cycle (Greenberg and Ziff, 1984; Levine et aL, 1986) and shows cell type and developmental stage specificity during embryogenesis (Downs et al., 1989). Moreover, the c-mycencoded oncoprotein possesses a leucine zipper

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motif and helix-loop-helix/basic region element which are considered to mediate protein/protein interactions and bind to specific DNA sequences (Ransone et ai., 1989; Schuermann et aL, 1989). This suggests that the c-rayc product may act as a direct regulator of DNA replication and of transcription of certain genes. Therefore, it is not surprising that deregulated c-myc expression has been implicated in vast range of malignant diseases (for reviews, see Cole, 1986; Spencer and Groudine, 1991). Proviral insertion resulted in activation of the c-myc locus in a number of avian, feline and murine B or T lymphoid tumours (Hayward et al., 1981 ; Neil et al., 1984; Selten et al., 1984). The aberrant expression of c-myc is strongly linked to neoplasia in many different cell types. The woodchuck hepatitis virus (WHV), a member of the Hepadna virus family (including human hepatitis B virus (HBV), ground squirrel hepatitis virus (GSHV) and Peking duck hepatitis B virus (DHBV)), is a small, partially double-stranded DNA virus (Summers et al., 1978; for reviews, see Tiollais et al., 1985; Ganem and Varmus, 1987). The WHV-infected woodchucks develop acute and chronic hepatitis with similar pathology to that of humans infected with HBV (Summers et al., 1978 ; Korba et al., 1989, 1990). Like HBV, WHV is a causal agent of hepatocellular carcinoma (HCC) in its host (Popper et aL, 1981 ; Snyder and Summers, 1980; Popper et al., 1987). WHV shares with such oncogenic viruses as HBV and non-acute retroviruses the ability to integrate the host genome with high frequency (Ogston et al., 1982; Rogler and Summers, 1984; Hsu et al., 1990). Previous studies in our laboratory have demonstrated that WHV DNA integration occurs in m y c family genes, including c-myc, N - m y c l and N-myc2, a woodchuck retroposon derived from N-myc, in more than 50 °70 of 50 woodchuck HCC analysed (M6r6y et al., 1986; Hsu et al., 1988; Fourel et al., 1990; Wei

DHBV GSHV HBV

= = =

(Peking) duck hepatitis B virus.

ground squirrel hepatitis virus. {human) hepatitis B virus.

et al., manuscript in preparation). Rearrangements of c-rayc have been implicated in three tumours. In two, WHV sequences were integrated either upstream of the first exon or in the exon 3 non-coding region of c-myc (Hsu et al., 1988). A chromosomal translocation occurred in the third tumour (M6r6y et ai., 1986). The genetic aberration caused either by proviral insertion or chromosomal translocation resulted in deregulated expression of c-rayc. The tumours produced enhanced levels of c-myc transcripts of abnormal size, indicating that the c-myc transcriptional unit was disrupted by virus insertion or chromosomal exchange. Inappropriate c-myc overexpression may be critically responsible for neoplastic malignancy in these tumours.

Here, we report c-myc rearrangements induced by WHV DNA integration in a new woodchuck Liver tumour. The viral insertion provoked complex structural alterations in the c-myc gene and induced activated expression of c-myc RNA of abnormal sizes.

MATERIALS

AND METHODS

Animal

Woodchuck 899 was a wild caught animal purchased from Northeastern Wildlife, Inc., and kept in the laboratory animal facility at the Molinette Hospital (Torino, Italy). The woodchuck was chronically infected with WHV, as judged by WHV surface antigen and WHV DNA in serum. The animal was sacrificed at 3 years of age. One tumour mass and surrounding liver tissues were resected, quickly frozen in liquid nitrogen and stored at -70°C. Southern blot analysis

Genomic DNA was extracted from W899 HCC and surrounding liver tissue as described (Hsu et al., 1988). DNA was analysed by Southern blotting (20 ~.g per lane) using alkaline transfer on HybondN ÷ membrane (Amersham). The Hinf and XBg

HCC WHV

= =

hepatocellular carcinoma. woodchuck hepatitis virus.

REARRANGEMENTS

AND

O F C-MYC B Y

ACTIVATION

WHV

INSERTION

91

a c-myc

H

Ba X

probes

Bg Ba

BgPv

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2

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Fig. l. Rearrangement of c-myc in a woodchuck tumour. (a) Restriction map of the normal woodchuck c-myc gene. Three exons were boxed and coding regions were blacked. The fragments Hinf and XBg specific for exon 1 and exon 2, respectively, were shown. Ba = B a m H l ; Bg=Bg/ll; H = H i n d l l I ; Pv = P v u l l ; S = Sacl and X = X b a l . (b) Southern blot hybridization of 5 different woodchuck tumour DNA digested by HindllI with the c-myc Hinf fragment. T = tumour; NT = surrounding liver tissue.

fragments, specific for c - m y c exon 1 and exon 2, respectively, isolated from a woodchuck c - m y c clone (M6r6y et al., 1986), as well as cloned W H V D N A (Ogston et al., 1982) and the WHV subgenomic clone EnI (a 514-bp A p a I - P v u I fragment) were used as hybridization probes. Hybridizations were carried out with 7 % SDS, 0.5 M phosphate pH 7, l mM EDTA and 100 g.g/ml salmon sperm DNA at 65°C, using 32p-labelled probes. Blots were washed twice in 2 × S S C , 0 . 1 % SDS for 15 min and once in 0.1 × S S C , 0 . 1 % SDS for 15 min at 65"C. Filters were exposed to " K o d a k X - O m a t - A R - 5 " films at - 8 0 ° C with intensifying screens.

Northern blot analysis Total RNA was extracted from a W899 t u m o u r and corresponding liver tissue using a hot phenol procedure (Hsu et al., 1988). RNA was loaded on agarose gels (40 Ixg per lane) after denaturation in 50 % DMSO, 1 M deionized glyoxal and 20 m M phosphate p H 7 and transferred to H y b o n d - N + membrane in 0.05 N N a O H . Hybridizations were performed under the same conditions as for D N A analysis, using the same probe. Membranes were washed in 1 °7o SDS, 0.04 M phosphate p H 7, 1 m M E D T A , at 650C for 45 min.

92

Y. W E / E T

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I-

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-,23,.-

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2

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Fig. 2. Hybridization of PvulI-digested 899T and 899NT genomic DNA with (a) c-myc Hinf fragment and (b) WHV DNA. Diagonal arrows indicate c-myc- and WHV-specific bands migrating at the same position. The marker was positioned. The 5-kb bands which appear in both T and NT lanes in (a) might be due to cross-hybridization with another cellular locus, as also shown in murine DNA (Dedieu et al., 1988).

AL.

tumours and surrounding liver tissues was digested with HindlII for Southern blot analysis. This enzyme was chosen since it does not cut inside the c-myc locus (fig. la). Hybridization with the c-myc exon-l-specific probe (fig. 1a) showed an additional band in 899T (fig. lb, lane 5), but not in the other tumours, indicating that c-myc was rearranged in this tumour. To further examine the link between virus insertion and c-myc rearrangement, we performed Southern blot analysis of PvulI-digested DNA (this enzyme has no site in the WHV genome). Hybridization with W H V DNA, as shown in figure 2b, revealed that 899T contains both integrated and free replicative forms of WHV, corresponding, respectively, to two discrete bands of high molecular weight and intense smears beneath the linearized 3.3-kb genome. Hybridization of the same blot with the c-myc exon-l-specific probe (fig. la) also detected an additional band which comigrated with one of the WHV-specific bands (fig. 2a, lane 1). These results indicate that the c-myc rearrangement was directly induced by WHV integration. Moreover, Southern hybridization with a subgenomic fragment of WHV covering the virus enhancer (EnI) proved the presence of virus enhancer sequences in WHV DNA integrated in the c-myc locus (data not shown). The strong hybridization signal obtained with the WHV probe at a large PvulI fragment (18-kb) (fig. 2b) suggests that a long viral sequence was integrated into c-myc in this tumour.

The c-myc gene in 899T contains more than one structural alteration RESULTS

Rearrangement of c-myc resulted from W H V integration

Previous studies showing frequent activation of myc family genes by WHV DNA insertion in woodchuck HCC (MfrSy et al., 1986; Hsu et al., 1988; Fourel et aL, 1990) prompted us to analyse the state of c-myc in a new panel of 19 tumours. Genomic DNA from woodchuck

To more precisely localize the virus integration site in c-myc, we carried out a series of Southern blot analyses using genomic DNA digested with different restriction endonucleases, such as BgllI, BamHI, HindlII, SacI and XbaI, which present different sites distributed throughout the c-myc gene (fig. la). Southern blot hybridization with subcloned c-myc fragments from either exon 1 or exon 2 revealed c-myc rearrangements for all enzymes used. Both exon 1 and exon 2 regions appeared to be mutated

REARRANGEMENTS

AND ACTIVATION

OF C-MYC BY WHV INSERTION

93

b

8 1

2

3

4

5

6

7

8

9

10

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2

3

4

5

6

7

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10

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m

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1 Fig. 3. Determination of complex rearrangements in W899. Genomic DNA was digested by BamHI (lanes l and 2), BglII (lanes 3 and 4), HindIII (lanes 5 and 6), SacI (lanes 7 and 8) and Xbal (lanes 9 and 10). The same blot was hybridized with (a) c-myc Hinf fragment and (b) c-myc XBg fragment. The arrows in the tumour lanes (odd-numbered lanes) indicate rearranged bands, whereas normal profiles are seen in the surrounding tissues (even-numbered lanes).

(fig. 3), indicating that the c-myc gene in 899T bears complex mutations. The precise site of WHV DNA integration could not be deduced from restriction mapping. The presence of a cm y c band of normal size in all DNA digests excluded involvement of both alleles. This suggested that WHV insertion in one c-myc allele in 899T was probably accompanied by other genetic changes.

Expression of c-myc was activated by WHVintegrated enhancer

Proviral insertion in proto-oncogenes usually resulted in aberrant expression of these genes (Hayward et al., 1981 ; Payne et al., 1982; Selten

et al., 1984). We searched for the effect of WHV integration effect on c-myc expression in 899T by Northern blot analysis. The c-myc gene was

abnormally expressed in this tumour, both by the longer size of c-myc RNA, ranging from 7.2 to 14 kb, as compared to the normal 2.3-kb cm y c RNA, and by increased levels of c-myc transcripts (fig. 4a, lane 1) as compared to nontumoral liver and to the other tumours. Northern hybridization with a WHV probe showed high levels of WHV-specific RNA of normal size produced during viral replication (fig. 4b, lane 1). In addition, at least one RNA species produced in 899T was identical in size with a c-myc transcript, suggesting viral-cellular cotranscription. It is difficult to discern the linkage between viral RNA and other abnormal transcripts of c-myc.

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Y. W E I E T A L .

q. 28s..

188,,-

288

: ,.:,,

,-|

18S~

i 1

1

i 2

3

4

1

2

3

4

Fig. 4. RNA analysis by Northern hybridization with (a) c-myc Hinf fragment and (b) WHV probe. The positions of 18S and 28S ribosomal RNA are indicated. Actin hybridization is shown for loading control.

DISCUSSION

We have characterized a woodchuck liver tumour carrying a c-myc rearrangement caused by WHV integration. The virus insertion induced complex recombinations in one c-myc allele, probably involving secondary deletions and mutations. WHV integration was associated with elevated level of c-myc transcripts with abnormal sizes. The transcriptional enhancement of c-myc by WHV is probably due to the insertion of viral regulatory sequences, including promoters and/or enhancers. The complex pattern of recombination at the c-myc locus, as judged by Southern analyses using various enzymes and different probes, did not enable determining the location of virus promoters nor their configuration with respect to c-myc. However, we cannot exclude the possibility that transcription started at a virus promoter inserted upstream of the gene, which subverted the expression of c-myc by supplying a more efficient promoter.

Such a mechanism has already been described in chicken B-cell lymphomas induced by avian leukosis virus (Hayward et al., 1981). This was also the case in two human HCC in which expression of the retinoic acid receptor gene (Dejean et al., 1986) and cyclin A gene (Wang et al., 1990) was enhanced by HBV promoter insertion. In contrast, in woodchuck tumours bearing c-myc or N - m y c rearrangements, we systematically observed a mechanism of enhancer activation by viral integrated sequences (Hsu et al., 1988; Fourel et al., 1990; Wei et al., manuscript in preparation). In the woodchuck tumour 899T, hybridization with a WHV subgenomic fragment showed the presence of the virus enhancer in the WHV sequences integrated in c-myc. The WHV enhancer may activate normal c-myc promoters, resulting in increased c-myc expression, as observed in murine and chicken lymphomas in which murine leukaemia virus or avian leukosis virus was located in the vicinity of c-myc at an opposite orientation (Payne et al., 1982; Corcoran et al., 1984). Thus, we suggest that c-myc activation in 899T was probably achieved by virus enhancer present in adjacent integrated WHV DNA. The WHV insertion in c-myc produced more than one alteration to the gene. A similar situation has also been observed occasionally in Burkitt lymphoma (Taub et al., 1984) and murine plasmacytoma (Greenberg et ai., 1985), showing duplication of part of the translocated c-myc gene joined to the immunoglobulin light chain locus, or additional insertion of an intracisternal A particle. Since the woodchuck tumour we analysed here showed unique rearranged patterns for all enzymes used, the neoplastic cells with c-myc rearrangement probably derived from the same clonal origin. It seems likely that the parental cell harbouring virus insertion and secondary rearrangements in c-myc gene acquired a selective growth advantage, leading to rapid proliferation and eventually to malignant transformation. Moreover, recent unpublished data from our laboratory showed that the c-myc gene, together with adjacent integrated WHV sequences from a woodchuck liver tumour, can induce HCC in transgenic mice (Etiemble et al., manuscript in preparation).

REARRANGEMENTS

AND ACTIVATION

These results p r o v i d e a t h i r d case o f a w o o d c h u c k H C C in which W H V D N A was integrated into the c - m y c locus a n d activated expression o f the gene. In a recent analysis o f 50 d i f f e r e n t w o o d c h u c k t u m o u r s , we observed the insertional a c t i v a t i o n o f c - m y c by W H V D N A in three cases, a n d that o f N - m y c genes in 22 other cases. T h u s it n o w a p p e a r s t h a t W H V , like n o n - a c u t e retroviruses, is integrated into m y c f a m i l y genes as p r e f e r e n t i a l targets a n d causes t u m o r i g e n e s i s by activating expression o f the genes. H o w e v e r , p r e f e r r e d i n t e g r a t i o n sites for H B V h a v e never been described in h u m a n H C C , i n d i c a t i n g t h a t different m e c h a n i s m s are involved in h u m a n a n d rodent hepatocarcinogenesis.

Acknowledgements

We are grateful to G. Fourel and C. Transy for helpful discussions, and C.-A. Renard for technical assistance. This work was supported in part by grants from the European Economic Community (Number TS2.0189F EDB) and from the Association pour la Recherche contre le Cancer (Number 6550). Y.W. is supported by the Fondation M6rieux.

Recombinaison et activation de c-myc par I'int~gration du virus de I'h~patite de la marmotte dans un h~patocarcinome

Nous avons &udi6 plusieurs h6patocarcinomes (HCC) de marmotte par la technique de transfert et d'hybridation. Dans une tumeur, nous avons observ6 des r~arrangements de l'oncog~ne c-myc induits par l'int6gration du virus de l'h6patite de la marmotte (WHV). Le g~ne a subi plusieurs 6v6nements recombinants comprenant l'int6gration virale ainsi que des d616tions et des mutations. L'expression du g6ne cm y c a 6t6 fortement activ6e par des ~l~ments r6gulateurs viraux (activateurs ou promoteurs) pr~ents dans les s6quences int6gr6es, conduisant/l la synth~se de plusieurs transcripts de taille anormale. Ces r6sultats sugg~rent que I'activation des oncog~nes cellulaires par int6gration virale pourrait jouer un r61e important dans l'h6patocarcinogen~se associ6e /~ l'h6patite B chez la marmotte. M o t s - c l 6 s : Oncogen6se, H6patite, P r o t o oncog6ne, Virus de l'h6patite B de la marmotte, ADN, H6patocarcinome; G~ne c-myc, R6arrangement, Int6gration virale, Activateur.

O F C-MYC B Y W H V I N S E R T I O N

95

References

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