0270-9139/87/0704-0753$02.00/0

Copyright 0 1987 by the American Aasociat ion !or the Study of Liver Diseases HEPATOLOGY Vol. 7. No. 4, pp. 753-757, 1987

Printed in 11. S.A.

RNA Transcripts of Hepatitis B Virus in Hepatocellular Carcinoma

FUMIO IMAZEKI, KATSLJYUKI YAGINUMA, MASAO OMATA, KUNIO OKUDA, MIDORI KOBAYASHI AND KATSURO KOIKE

Department of Gene Research, Cancer Institute, Tokyo 170, Japan and First Department of Medicine, Chiba University School of Medicine, Chiba 280, Japan

The states of hepatitis B virus DNA, RNA transcripts and antigens (HBsAg and HBcAg) were studied in the liver with hepatocellular carcinoma by blot hybridiza- tion and immunohistological methods. We used whole hepatitis B virus DNA and gene specific probes (S, C and X genes) for hybridization. Integrated viral DNA was found in all five tumors, but episomal DNA was not detected in the neoplastic tissue. In the nonneoplastic cirrhotic liver, episomal DNA was found in four cases and integrated viral DNA in four cases. A large amount of hepatitis B virus-specific RNA transcripts was dem- onstrated in nonneoplastic liver of all five cases. Two major transcripts, 2.4 and 3.4 kb in length, were iden- tified. The former hybridized with the S gene probe and the latter with the C gene probe, suggesting that they were messenger RNAs for HBsAg and HBcAg, respec- tively. In contrast to nonneoplastic liver, RNA tran- scripts were found in the neoplastic portion in only two cases, small in quantity; they primarily hybridized with S and X, but not with C genes. Novel species of 4.0 and 3.9 kb transcripts were found in the neoplastic and the nonneoplastic portions, respectively, in one case. They may represent fusion transcripts of integrated viral DNA and cellular flanking sequences.

The state of hepatitis B virus (HBV) DNA in various liver diseases, including hepatocellular carcinoma (HCC), has been studied by Southern blot hybridization (1-5), and the structure of integrated viral DNA has been analyzed in detail by molecular cloning (6-11). HBV- related transcripts from hepatoma cell lines were also analyzed by Northern blot hybridization (2, 3, 12), but as yet, little is known about the state of HBV-related transcripts in clinical materials. We have analyzed HBV transcripts and DNA from neoplastic and nonneoplastic portions of the liver from five HBsAg seropositive pa- tients with HCC by the blot hybridization techniques.

Received May 12, 1986; accepted January 2, 1987. This work was supported in part by a Grant-in-Aid from the Min-

istry of Health and Welfare for a Comprehensive 10-Year Strategy for Cancer Control and by Grants-in-Aid for Cancer Research from the Ministry of Education, Science, and Culture, Japan.

Address reprint requests to: Katsuro Koike, Ph.D., Head and Mem- ber, Department of Gene Research, Cancer Institute, JFCR, Kami- Ikebukuro, Toshima-ku, Tokyo 170, Japan.

MATERIALS AND METHODS Five HBsAg seropositive patients with

HCC were studied. They ranged from 28 to 51 years of age. Two of these patients were positive for HBeAg in serum, and all had cirrhosis in the nonneoplastic portion of the liver (Table 1). Liver samples were taken at the time of tumor resection, frozen immediately and stored at -70°C until use.

Cellular DNA was extracted from the tumor and the cirrhotic portion as described previously (4). Briefly, about 200 mg of tissue sample were homogenized in 10 ml ice-cold buffer containing 10 mM Tris-HC1 (pH 7.4), 10 mM NaCl and 2 mM EDTA, followed by the addition of 0.1 ml proteinase K (50 mg per ml) and 2 ml 10% sodium dodecyl sulfate (SDS). The mixture was incubated overnight at 37°C. Nucleic acids were extracted with 1 volume phenol twice and 1 volume chloroform/isoamyl alcohol (24/1) once. The aqueous phase was then precipitated wit,h two volumes of ethanol. The nucleic acids were digested with 50 pg per ml RNase A for 1 hr at 37"C, followed by extraction with phenol and chloroform/ isoamyl alcohol. DNA was precipitated with 2 volumes ethanol, dissolved in 20 mM Tris-HC1 buffer (pH 7.6) with 0.1 mM EDTA and stored at 4°C.

Aliquots of 10 pg DNA were digested overnight with 20 units Hind111 and BamHI separately at 3 7 T , subjected to 1.0% agarose gel electrophoresis, and the DNA was transferred to a nitrocellulose filter by the method of Southern (13). Prehybrid- ization was performed at 65°C for 2 hr in a solution of 6X standard saline citrate (SSC), Ix Denhardt's solution (0.02% polyvinyl pyrrolidone, 0.02% Ficoll 400, 0.02% bovine serum albumin) and 0.5% SDS. The DNA filter was hybridized with radiolabeled HBV DNA at 65°C overnight in a solution of 6X SSC, l x Denhardt's solution, 0.5% SDS and 100 rg per ml denatured salmon sperm DNA and washed at room temperature with 2~ SSC, and then at 56°C with 0.1X SSC three times, followed by autoradiography.

For the S'P-labeled HBV DNA probe, whole HBV DNA (subtype adr) was prepared from pHBV 1-1 (14) and purified by agarose gel electrophoresis. HBV DNA was labeled with [a- '"PldCTP (ca. 5,000 Ci per mmole, New England Nuclear, Boston, Mass.) by nick translation (15), and the specific activity of the probe was ca. 2 to 3 x lo8 cpm per pg.

Total RNA was extracted from the neoplastic and nonneoplastic portion by the guanidinium/ cesium chloride method (16). Liver samples were homogenized in a buffer containing 6 M guanidinium isothiocyanate, 5 mM sodium citrate, 0.1 M P-mercaptoethanol and 0.5% sarkosyl. The homogenate was placed over a 5.7 M CsC1/0.1 M EDTA cushion and centrifuged at 20°C for 12 hr at 35,000 rpm in a Beckman SW 50.1 rotor. The pellet thus obtained was dissolved

Liver Samples.

Detection of RBV DNA.

Detection of HB V RNA.

IMAZEKI ET AL. HEPATOLOGY 754

TABLE 1. The state of HBV-related transcripts and DNAs in liver samples from five HBsAg seropositive patients with HCC

HBV DNA HBV RNA HBeAg

serum Tumor CaSe Age Sex in Tumor Nontumor

F" I b F I Nontumor

1 46 M + + + + 2.4 kb 3.4 kb 2.4 kb 3.4 kb 2.4 kb 2 51 M

- - - - - + + - -

3 50 F + 13.4 kb - 3.4 kb 2.4 kb 14.0 kb 2.6 kb 3.9 kb 2.6 kb

+ + + -

2.4 kb - 2.4 kb

- - - + + + + +

- - 4 51 F 5 28 M - - - - -

F = Free viral DNA. ' I = Integrated viral DNA.

in a buffer of 10 mM Tris-HC1 (pH 7.4), 5 mM EDTA, 1% SDS and extracted with 1 volume chloroform/l-butanol (4/1) fol- lowed by precipitation with 2.5 volumes ethanol. Total RNA was dissolved in distilled water and stored at -20°C after precipitation with ethanol.

Aliquots of 20 pg total RNA were incubated at 50°C for 1 hr with 1 M glyoxal, 50% dimethyl sulfoxide, 10 mM sodium phosphate buffer (pH 7.0) and subjected to a 1.0% agarose gel electrophoresis. Following this, the RNA was blotted onto nitrocellulose filter by the method of Thomas (17). As a size marker, Hind111 fragments of h-phage DNA were endlabeled with [CX-~*P]TTP using T4-polymerase, denatured and electro- phoresed. HBV-related RNA was detected by hybridization using a 32P-labeled HBV DNA probe. The filter was incubated with the probe at 42°C overnight in a solution containing 50% formamide, 5X SSC, l x Denhardt's solution, 0.5% SDS and 100 pg per ml denatured salmon sperm DNA. Our hybridization technique allowed detection of as little as 0.2 viral genome equivalent DNA per haploid cell when 10 pg cellular DNA was analyzed.

Preparation of Gene-Specific Probes. Subgenomic viral DNAs corresponding to the surface antigen gene (S gene), the X gene and the core antigen gene (C gene) were used as probes to characterize the HBV-specific transcripts. A HindIII/ AccI fragment (0.61 kb) covered the S gene, a BamHI/RsaI fragment (0.37 kb) corresponded to the X gene and a BglII fragment (0.44 kb) was included in the C gene (14, 18). These subgenomic viral DNAs were subcloned into pBR322, and insert DNAs were purified by agarose gel electrophoresis.

Demonstration of HBsAg and HBcAg. Immuno- histological demonstration of HBsAg and HBcAg was per- formed by indirect immunoperoxidase method, as we previously described (19).

RESULTS Integrated viral DNA was

found in all five tumors. However, free viral DNA was detected in none of them (Table 1). In the nonneoplastic portion of the liver, both free and integrated viral DNAs were found in three cases, free viral DNA alone in one and an integrated form alone in one. The sizes of discrete high molecular bands after enzyme digestion appeared different in these five tumors. In four cases in which integration of viral DNA was observed both in the neo- plastic and nonneoplastic portion, none of the bands were common between the two different portions (Figure 1). These data indicate that integration has occurred both in the neoplastic and nonneoplastic portions at different sites of the genome.

State of HBV DNA.

State of HBV RNA. In nonneoplastic liver, two major RNA transcripts, 3.4 and 2.4 kb in length, were found (Table 1) (Figure 2). The 3.4 kb band, slightly larger than the HBV genome of 3.2 kb, hybridized with all of the S, X and C gene-specific probes (Figure 3). The 3.4 kb transcript was found in the nonneoplastic liver portion in three cases with free viral DNA and/or serum HBeAg, but was undetectable in two cases negative for serum HBeAg (Table 1). The 2.4 kb band hybridized with both the S gene and X gene probes but not with the C gene probe. These data suggest that the 3.4 kb band contains a message for HBcAg production (C-messenger RNA) and that the 2.4 kb band primarily contains the message for HBsAg synthesis (S-messenger RNA). The 2.4 kb transcript was found in the nonneoplastic portion in all five cases. Besides the two major transcripts, two other transcripts (3.9 and 2.6 kb) were identified in the nonneoplastic portion of the liver of Case 3. Both hy- bridized with the S and X gene probes, but failed to do so with the C gene probe (Figure 3).

In the neoplastic portion of the liver, HBV RNA transcripts were found in two cases. In Case 3, 4.0, 3.4 and 2.6 kb transcripts were identified (Figure 3). The three bands hybridized with the X and S gene probes (Figure 3). Only the 3.4 kb band hybridized with the C gene probe, and the other two bands did not hybridize with it (Figure 3). In another case (Case l), only the 2.4 kb band was detected. This band hybridized with the S and X gene probes, but not with the C gene probe.

State of HBsAg and HBcAg. HBsAg and HBcAg were demonstrated in the liver tissue by the immunohis- tological method. No viral antigens were detected in the neoplastic area of the liver. HBsAg was demonstrated in the nonneoplastic portion in all five cases, whereas HBcAg was found in the nonneoplastic liver in all three cases that showed the 3.4 kb RNA transcript.

DISCUSSION HBV-specific DNA, RNA transcripts and protein

products were studied in the liver samples of five HBsAg seropositive patients with HCC. The 2.4 kb RNA was found in the nonneoplastic portion of the liver in all HBsAg-positive cases, preserving the sequences of the S and X gene regions, but not those of the C gene region. These data support the earlier contention that the 2.4 kb RNA transcript corresponds to the messenger RNA

Vol. 7, No. 4,1987 HBV TRANSCRIPTS IN HCC 755

FIG. 1. Southern blot hybridization of neoplastic (Lanes 1 to 3) and nonneoplastic (Lanes 4 to 6) portions of the liver in three cases [(a) Case 1. (b) Case 2. (c) Case 31. High molecular signals were seen in the neoplastic portion in all three cases (Lanes 1 to 3). Similarly, high molecular signals were identified in the nonneoplastic portion in Cases 1 and 3, but not in Case 2. Free viral DNA was seen in the nonneoplastic portion of the liver in the three cases, but not in the neoplastic portion in any of them. Lanes I and 4 = uncut; Lanes 2 and 5 = HindIII cut; Lanes 3 and 6 = BamHI cut. HindIII-cleaved X DNA fragments were run as the size markers.

756 IMAZEKI ET AL. HEPATOLOGY

for HBsAg protein (20). In this study, HBsAg was im- munohistologically demonstrated in the nonneoplastic portion of all five livers with the 2.4 kb RNA transcript. The 3.4 kb RNA transcript, larger than the full length of HBV genome (3.2 kb), was found to contain the sequences of C, S and X genes. This transcript was found in the cases having free viral DNA in the liver and HBeAg in the serum. These findings are consistent with the notion that the 3.4 kb RNA is transcribed from the entire genome, most likely from free viral DNA, and apparently acts not only as a messenger RNA for the HBcAg/HBeAg protein, but also serves as a potential template for the reverse transcriptase (pregenome) in the replicative cycle of the virus (20, 21). The 3.4 kb RNA seems to play a key role both in protein synthesis and DNA replication. In fact, HBcAg was demonstrated in the liver with the 3.4 kb RNA transcript.

HBV RNA transcripts comparable to our 2.4 and 3.4 kb HBV-specific RNAs have been observed in the livers of infected chimpanzees (22), Pekin ducks (23), ground squirrels (24) and woodchucks (25) with active virus replication. However, little has been known about the state of HBV RNA transcripts in clinical samples of HCC. In an established cell line (PLC/PRF/5), it was shown that very little HBV-specific RNA transcript was expressed (ca. 0.05% of total mRNA in the cell) (26). Recent cloning and sequence analyses of integrated viral

1 2 3 4 5 6

FIG. 2. Northern blot hybridization of RNA, extracted from neo- plastic (Lanes 1,3 and 5) and nonneoplastic (Lanes 2 ,4 and 6 ) portions of the liver in three cases (Case 1 = Lanes 1 and 2 ; Case 4 = Lanes 3 and 4; Case 5 = Lanes 5 and 6). Hybridization was performed with a whole HBV DNA probe. Twenty micrograms of RNA extracted from the liver were electrophoresed on a 1.0% agarose gel and blotted onto nitrocellulose filter.

T N T T N T T N T T N T origin-

kb

2.4'

1 2 3 4 FIG. 3. Northern blot hybridization of HBV-related RNA from Case

3. Twenty micrograms of RNA extracted from the neoplastic (T) and nonneoplastic (NT) portions of the liver were electrophoresed on a 1.0% agarose gel and blotted onto nitrocellulose filter. The filter was hybridized with the whole HBV DNA and three gene probes. Lane I = whole HBV DNA; Lane 2 = C gene probe; Lane 3 = X gene probe; Lane 4 = S gene probe. HindIII-cleaved X DNA fragments were run as the size markers.

DNA in HCC have shown that integrated viral DNA is often incomplete (all or part of the C gene is deleted very frequently) or extensively rearranged (6-11). Thus, it was presumed that RNA transcription was impaired because of the rearranged and deleted DNA template in HCC. In this study, we substantiated this assumption by demonstrating that RNA transcripts were scarce in the neoplastic tissue in contrast to the amount detected in the nonneoplastic liver, and that they contained scant amounts of C gene-specific signal.

Interestingly, some species of HBV-specific RNA tran- scripts other than 3.4 and 2.4 kb transcripts were iden- tified a 4.0 kb RNA in the neoplastic and a 3.9 kb RNA in nonneoplastic liver tissue (Case 3). Although these transcripts were longer than the length of the HBV genome (3.2 kb), they did not hybridize with the C gene probe (Figure 3), suggesting that they contained nonviral sequences. They could be novel species of RNA tran- scripts that consist of HBV-specific and adjacent cellular sequences. This possibility is yet to be determined in individual clinical materials by further analysis of tran- scripts or their complementary DNA. Fusion sequences of viral and cellular DNA have already been documented in woodchuck hepatitis virus infection (25). There is a possibility that a fusion protein plays a role in hepato- carcinogenesis.

REFERENCES 1. Brechot C, Pourcel C, Louise A, et al. Presence of integrated

hepatitis B virus DNA sequences in cellular DNA of human hepatocellular carcinoma. Nature 1980; 286533-535.

2. Chakraborty PR, Ruiz-Opazo N, Shouval D, et al. Identification of integrated hepatitis B virus DNA and expression of viral RNA in an HBsAg-producing human hepatocellular carcinoma cell line. Nature 1980; 286531433.

3. Edman J, Gray P, Valenzuela P, et al. Integration of hepatitis B virus sequences and their expression in a human hepatoma cell. Nature 1980; 286535-538.

4. Koike K, Kobayashi M, Mizusawa H, et al. Rearrangement of the surface antigen gene of hepatitis B virus in the human hepatoma

Vol. 7, No. 4,1987 HBV TRANSCRIPTS IN HCC 757

cell lines. Nucleic Acids Res 1983; 11:5391-5402. 5. Marion PL, Salazar FH, Alexander J J , et al. State of hepatitis B

viral DNA in a human hepat,oma cell line. J Virol 1980; 33795- 802.

6. Dejean A, Brechot C, Tiollais P, et al. Characterization of inte- grated hepatitis B viral DNA cloned from a human hepatoma and the hepatoma-derived cell line PLC/PRF/5. Proc Natl Acad Sci

7. Mizusawa H, Taira M, Yaginuma K. et al. Inversely repeat.ing integrated hepatitis B virus DNA and cellular flanking sequence in the human hepatoma-derived cell line huSP. Proc Natl Acad Sci USA 1985; 82208-212.

8. Yaginuma K, Kobayashi M, Yoshida E, et al. Hepatitis B virus integration in hepatocellular carcinoma DNA: duplication of cel- lular flanking sequences a t the integration site. Proc Natl Acad Sci

9. Koshy R, Koch S, Freytag von Loringhoven A, et al. Integration of hepatitis B virus DNA: evidence for integration in the single- stranded gap. Cell 1983; 34915-223.

10. Dejean A, Sonigo P, Wain-Hobson S, et al. Specific hepatitis R virus integration in hepatocellular carcinoma DNA through a viral 11-base-pair direct repeat. Proc Natl Acad Sci USA 1984; 81 5354.

11. Ziemer M, Garcia P, Shaul Y , et a]. Sequence of hepatitis B virus DNA incorporated into the genome of a human hepatoma cell line. J Virol 1985; 532385-892.

12. Cattaneo R, Will H, Hernandez N. et al. Signals regulating hepzi- titis B surface antigen transcription. Nature 1983; :305:336-338.

13. Southern EM. Detection of specific sequences among DNA frag- ments separated by gel electrophoresis. J Molec Biol 1975; 9850% 517.

14. Kobayashi M, Koike K. Complete nucleotide sequence of hepatitis B virus DNA of subtype adr and its conserved gene organization. Gene 1984; 30:227-232.

15. Rigby PW, Dieckmann MA, Rhodes C, et al. Labelling deoxyribo- nucleic acid to high specific activity in uitro by nick translation with DNA polymerase I. J Molec Biol 1977: 113:237-251.

USA 1983; 80:2505-2509.

USA 1985,824458-4462.

16. Chirgwin JM, Przybyla AE, MacDonald RJ, e t al. Isolation of biologically active ribonucleic acid from sources enriched in ribo- nuclease. Biochemistry 1979; 18:5294-5299.

17. Thomas P. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci USA

18. Koike K, Yaginuma K, Mizusawa H, et. al. Structure of integrated HBV DNA in human hepatomas. In: Blumberg BS, Nishioka K, Koike K, et al., eds. Hepatitis viruses and hepatocellular carcinoma: approaches through molecular biology and ecology. New York: Academic Press, 1985: 99-115.

19. Omata M, Aschcavai M, Liew CT, et a]. Hepatocellular carcinoma in the IJ.S.A., et,iologic consideration. Localization of hepatitis B antigens. Gastroenterology 1979; 76:279-287.

20. Yokosuka 0, Omata M, Imazeki F, et al. Hepatitis B virus RNA transcripts and DNA in chronic liver disease. N Engl J Med 1986;

21. Summers d, Mason WS. Replication of the genome of a hepatitis B-like virus by reverse t,ranscription of an RNA intermediate. Cell

22. Cattaneo R, Will H, Shaller H. Hepatitis B virus transcription in the infected liver. EMBO J 1984: 32191-2196.

23. Buscher M, Reiser W-, Will H, et al. Transcripts and the putative RNA pregenome of duck hepatitis B virus: implication for reverse transcription. Cell 1985; 40:717-724.

24. Enders GH, Ganem D, Varmus H. Mapping the major transcripts of ground squirrel hepatitis virus: the presumpt,ive temp1at.e for reverse transcriptase is t,erminally redundant. Cell 1985; 42:297- 308.

25. Moroy T, Etiemble J , Trepo C, et al. Transcription of woodchuck hepatitis virus in the chronically infected liver. EMBO J 1985; 1: 1507-1514.

26. Rutter WJ, Ziemer M, Ou J, et al. Transcription units of hepatitis B virus genes and expression of integrated viral sequences. in: Vyas GN, Dienstag JL, Hoofnagle JH, eds. Viral hepatitis and liver disease. New York: Grune & %ratton, 1984: 67-86.

1980; 775201-5205.

3151187-1 192.

1982; 29:409-415.