Int. J. Cancer: 52, 137-140 (1992) 1992 Wiley-Liss, Inc.

v;I.-

Publicationof the InternationalUnion Against Cancer Publication de I'Union InternationaleContre le Cancer

@ ):

ENHANCEMENT OF HEPATITIS-B SURFACE-ANTIGEN EXPRESSION BY 5-AZACYTIDINE IN A HEPATITIS-B-VIRUS-TRANSFECTED CELL LINE Eiji MIYOSHI~.', Junichi FUJII',Norio HAYASHI?, Keiji UEDA',Takahiro TOWATA*, Hideyuki FUSAMOTO~, Takenobu KAMADA? and Naoyuki TANIGUCHI'.~ 'Department of Biochemistv, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565; and 2FirstDepartment of Internal Medicine, Osaka Universify Medical School, Fukushima, I chome, Fukushima-ku, Osaka 553, Japan. The human hepatoblastoma-derived cell line HB6 I I secretes hepatitis-B surface antigen (HBsAg) and hepatitis-B e antigen (HBeAg) into the medium. Hepatitis-B-virus(HBV) DNA integrated into the cellular genome was found to be hypermethylated. When the cells were treated with 5-azacytidine for 3 days, the level of HBsAg in the medium increased, while the level of HBeAg remained constant. The level of alphafetoprotein (AFP) decreased with the 5-azacytidine treatment. Southern blot analysis of DNA digested with Hpall or Mspl showed that 5-azacytidine treatment resulted in hypomethylation of the integrated HBV DNA, suggesting that 5-azacytidine increased HBsAg production in the cells through hypomethylation of the HBV genomic DNA. 8 1992 Wilq-Liss, Inc.

Methylation of cytosine at the 5 position is the most common base modification in eukaryotic DNA (Vanyushin et al., 1970), and there is evidence that methylation at the dinucleotide CpG is involved in the control of both endogeneous and exogenous gene expression (Doerfler, 1983; Riggs and Jones, 1983). The methylation of specific viral D N A sequences has been shown to play a crucial role in controlling the transcription of several viral genes from herpes virus (Vardimon et al., 1980), adeno virus (Youssoufian et al., 1982), and human T-lymphotropic virus type 1 (Saggioro et al., 1990). There are also some reports which suggest the significance of methylation of hepatitis-B-virus(HBV) DNA in the expression of the viral proteins. Yoakum et al. (1983) showed that expression of the HBV core gene was induced by 5-azacytidine treatment of a human cell line transfected with HBV genomic DNA. Miller and Robinson (1983) suggested that methylation was responsible for the lack of expression of core antigen in PLC/PRF/5 cells. HBV surface antigen (HBsAg) was also induced by 5-azacytidine administration to transgenic mice that carried 3 tandem copies of the HBV genome, but did not express HBrAg (Araki et al., 1989). HB6I 1 , a human hepatoblastoma cell line transfected with cloned HBV DNA, produces both HBsAg and HBeAg (Tsurimoto et al., 1987). We have investigated the effect of 5-azacytidine on methylation of the HBV genome and on viral protein expression in this cell line. We found that 5-azacytidine induced HBsAg but not HBeAg. MATERIAL AND METHODS

Cell culture and cell lines The HB611 cell line, which was established by transfecting 3 tandemly arranged complete HBV D N A into huh6-c-15 cells (Tsurimoto et al., 1987), was kindly provided by Dr. K. Matsubara (Institute for Molecular and Cellular Biology, Osaka University). The cells were grown in Dulbecco's modified Eagle's medium (GIBCO, Grand Island, NY) supplemented with 10% fetal bovine serum (GIBCO), 100 units/ml penicillin (GIBCO), and 200 kg/ml genetigine (Sigma, St Louis, MO) at 37°C in 5% C 0 2 / 9 5 % air. About lo5cells were treated with various concentrations of freshly prepared 5-azacytidine (Sigma). The medium was changed every 3 days. After treatment with 0.5 mg/ml collagenase, cells were stained with 0.3% Trypan blue and were counted on a turk tube through a light microscope.

Quantitatiori of viral and cellularproteins HBsAg and HBeAg in the culture medium were quantitated using an enzyme-linked immunosorbent assay (ELISA) kit from Otsuka (Tokyo). Alpha fetoprotein (AFP) in the medium was quantitated using a radioimmuno assay kit from Otsuka. DNA and R N A preparations High-molecular-weight D N A was purified by proteinase K (Sigma) treatment followed by extraction with phenol/ chloroform (Sambrook et al., 1989). Total R N A was prepared from the cells according to Chomczynski and Sacchi (1987).

Southern blotting D N A (10 kg) was digested with restriction enzyme HpaII or MspI (Toyobo, Osaka) and was fractionated by electrophoresis on 1.5% agarose gel. After alkaline denaturation, the D N A was neutralized and was transferred onto Zeta-probe membrane (BioRad, Richmond, CA) by capillary action (Southern, 1975). Membrane filters were hybridized with 32P-labeled, whole HBV D N A as a probe (Fujiyama et al., 1983) at 42°C in hybridization buffer (Sambrook et al., 1989) containing 50% formamide and 10% dextran sulfate (Nakarai Tesq, Kyouto, Japan), and then washed at 55°C with 2 x SSC and 0.1% SDS for 30 min, and with 0.2 x SSC and 0.1% SDS for 30 min twice. The filters were exposed to X-ray film (Kodak) with an intensifying screen at -80°C for 3 days. RESULTS

The effect of 5-azacytidine on HBsAg and HBeAg levels in the culture niedizim As shown in Figure 1, the level of HBsAg in the medium was increased significantly (p < 0.01) by 3-day incubation with 25 kM or 50 k M 5-azacytidine. In contrast, the HBeAg level was originally high and did not change (data not shown). The elevation of HBsAg was not due to an effect of the reagent on cell growth, since the number of cells after incubation for 3 days was almost the same in the presence and in the absence of 5-azacytidine (Fig. 2). When the levels of HBsAg were calculated as HBsAg production per cell, the number increased up to 10% and 43% of the control level after treatment with 25 k M and 50 FM 5-azacytidine respectively for 3 days. Much more increase in the ratio to the control was observed after 6 and 9 days, although the amount of HBsAg per cell decreased in all concentrations because HBsAg production was inhibited by overgrowth of the cells at longer incubation. In order to avoid a secondary effect from changes in cell growth, the level of HBsAg was measured at various concentrations of 5-azacyti'To whom correspondence and reprint requests should be sent. Fax: 81-6-875-7314. Abhreviufiotzs: HBV, hepatitis-B virus: HBsAg, hepatitis-B surface antigen; HBeAg, hepatitis-B e antigen; HBcAg, hepatitis-B core antigen; AFP, alpha-fetoprotein; ELISA, enzyme-linked immunosorbent assay.

Received: February24, 1992 and in revised form April 21,1992.

MlYOSHl E T A L .

**

*

I

0

0

3

6

9

day

FIGURE1 -Time course of HBsAg production with S-azacyti25 pM dine treatment. About lo5 cells were treated with 0 (O), (m) or 50 p M (B) 5-azacytidine. The HBsAg level in the culture medium was quantitated by ELISA. As purified HBsAg was not available, the levels of HBsAg were expressed as “arbitrary units”. The units were the relative values to cut-off index, which was established for clinical use to find HBV-infected people by measuring serum levels of HBsAg from normal individuals. Bars indicate standard deviation (SD). n = 4, * * : p < 0.01, * : p < 0.05.

3

day

FIGURE3 - Effect of various concentrations of 5-azacytidine on the level of HBsAg. HBsAg levels were quantitated by ELISA after treatment with 0 (0),10 p M (A),25 p M (0),SO p M (A) and 100 p M (H)5-azacytidine for 3 days. The levels of HBsAg are indicated as described for Figure 1. Results represent the mean of experiments in duplicate.

10

0‘

0

3 day

FIGURE4 - Effect of various concentrations of 5-azacytidine on AFP production. The amount of AFP in the medium was quanti10 p M tated by radioimmunoassay after treatment with 0 (0). (A),25 p M (0),SO p M (A) and 100 pM (H)5-azacytidine for 3 days. Results represent the mean of experiments in duplicate.

O’ 0

3

6

9

day

FIGURE2 - Change in cell number after 5-azacytidine treatment. The number of cells was counted after treatment with 0 (0),25 p M (0)and 50 pM (A) 5-azacytidine. Results represent the mean of experiments in triplicate. dine after 3 days (Fig. 3). HBsAg increased in a dosedependent manner up to 50 p M 5-azacytidine. The level of HBsAg was lower at 100 K M 5-azacytidine than at 50 KM 5-azacytidine, probably because 5-azacytidine exerted a cytotoxic effect on cellular activity at this concentration (Cihak, 1974). Northern blot analysis of total R N A showed slightly increased amount of mRNA encoding HBV proteins after 36 hr 5-azacytidine treatment (data not shown).

The efect of 5-azacytidine on AFPproductiori We also measured the amount of AFP secreted from the cells into the medium (Fig. 4). In contrast to the level of viral proteins, the level of AFP in the medium was decreased by all concentrations of 5-azacytidine. Thus, 5-azacytidine seems to specifically induce HBsAg in this cell line.

Southern blot analysis of HBVDNA In order to investigate the mechanism of 5-azacytidine induction of HBsAg in HB611 cells, genomic D N A prepared from 5-azacytidine-treated HB611 cells was digested with the restriction enzymes MspI and HpaII. MspI recognizes and cleaves the CCGG sequence and is not affected by cytosine methylation. HpaII recognizes the same sequence as MspI but does not digest the methylated DNA. Figure 5 shows a typical Southern blot of the digested DNA. There are 6 recognition sites for MspI and HpaII in a single HBV genome, giving 6 fragments (61, 297,353, 463,960, 1080 bp) in total, although 2 of the bands were too small to be seen on this gel. In D N A incubated with HpaII, there was a band of about 10 kb. Since the cell carries 3 tandem copies of HBV DNA, comprising 9.6 kb (Tsurimoto et a/., 1987), the 10-kb band corresponded to whole integrated HBV D N A that was not digested by the enzyme. This higher-molecular-weight band was not seen in the DNAs digested with MspI, which indicates that the CCGG sequences of integrated HBV DNA in the 10-kb fragment were methylated. HpaII digestion of D N A from cells treated with 5-azacytidine yielded much smaller amounts of the 10-kb

139

HEPATITIS-8 SURFACE ANTIGEN AND 5-AZACYTIDINE

DISCUSSION

FIGURE5 - Southern blot analysis of the methylation state of HBV DNA in 5-azacytidine-treated HB611 cells. DNA (10 pg) extracted from the cells was digested with HpaII (lanes 1-3) or Mspl (lanes 4-6). After size fractionation on an agarose gel, the DNAs were blotted onto a Zeta probe membrane and were hybridized with a whole HBV probe (Fujiyama et nl., 1983). The HB611 cells were treated with 0 (lanes 1 and 4), 25 p M (lanes 2 and 5) and SO pM 5-azacytidine (lanes 3 and 6) for 9 days. The hybridized membrane was washed and then exposed to X-ray film for 3 days with an intensifying screen at -80°C. fragment (Fig. 5). The amount of the fragment was inversely related to the length of incubation with Sazacytidine (data not shown).

HBV is one of the viruses whose gene expression is regulated by methylation at cytosine residues in the viral genomic DNAs. Two reports (Yoakum et al., 1983; Miller and Robinson, 1983) have shown that expression of HBV major-core protein (HBcAg) is regulated by D N A methylation, and that cells carrying the integrated HBV genome express HBcAg when the gene is hypomethylated. Another viral protein, HBsAg, is induced by 5-azacytidine in the transgenic mice that carry HBV genomic DNAs (Araki et al., 1989). Transcription of the S gene in transgenic mice is associated with hypomethylation at specific sites and with DNase-I sensitivity (Pourcel et al., 1990). Thus, at least HBsAg and HBcAg expression seem to be regulated by DNA methylation. The HB611 cell line utilized in the present work produces a high level of HBeAg, a secreted form of HBcAg. though the integrated HBV D N A is extensively methylated. Although this cell line contains both integrated copies of the HBV genome and replicative intermediates of the viral DNA, the mRNA species are produced mainly from the integrated HBV DNA (Tsurimoto et al., 1987; Ueda et al., 1989). In the current study, treatment of HB611 cells with 5-azacytidine led to hypomethylation of the integrated HBV DNA and increased the production of HBsAg (Fig. 1) but not HBeAg. In general, 5-azacytidine is known to induce the expression of silent genes (Vardimon et al., 1980; Araki et al., 1989). In this study, we have shown that the S gene, which was already being expressed, was further activated by 5-azacytidine through hypomethylation of HBV genomic DNA. Unlike secretion of the viral proteins, the secretion of AFP into the medium decreased upon treatment with various concentrations of 5-azacytidine. Jeffer and Jen-Fu (1983) showed that 5-azacytidine markedly decreased AFP synthesis in neonatal rat liver, while having little effect on albumin production. Although this suppression of AFP production was in normal hepatocytes, the HB611 cells in the present study were derived from hepatoblastoma cells. Thus, 5-azacytidine suppressed AFP-gene expression both in normal hepatocytes and in hepatoblastoma cells. The enhancement of HBsAg expression by 5-azacytidine treatment of HB611 cells adds to the evidence that DNA methylation serves as a regulatory mechanism for HBV gene transcription. The degree of hypomethylation appears to be directly related to the level of HBsAg production in this cell line. ACKNOWLEDGEMENTS

We thank Dr. K. Matsubara for giving us the HB611 cell line and the HBV probe. This work was supported in part by grants-in aid for Cancer Research from the Ministry of Education, Science and Culture, Japan.

REFERENCES

ARAKI. K., MIYALAKI,J., TSURIMOTO, T.. INOUE, T., IWANAGA, T., MA'I'SUBARA, K. and YAMAMURA. K., Demethylation by 5-azacytidine results in the expression of hepatitis-B-virus surface antigen in transgenic mice. Jap. J. Cancer Res., 80,295-298 (1989). CHOMCZYNSKI. P. and SACCHI,N., Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroformextraction. .4nal. Biochem.. 162, 156-159 (1987). CIHAK, A,, Biological effects of 5-azacytidine in eukaryotes. Oncology,

JEFFER,R.C. and JEN-Fu, C., Effect of 5-azacytidine on rat liver alpha-fetoprotein gene expression. Biochetn. Biophys. Res. Commun., 116,939-944 (1983).

M I L L ~ RR.H. , and ROBINSON, W.S., Integrated hepatitis-B-virusDNA

sequences specifying the major viral core polypeptide are methylated in PLCIPRFIS cells. Proc. nut. Acad. Sci. (Wash.), 80, 2534-2538 (1983).

POURCEL, C., TIOLLAIS, P. and FARZA, H., Transcription of the S gene in transgenic mouse is associated with hypomethylation at specific sites gene activity. Ann. Rev. Bio- and with DNase-I sensitivity.J. Virology, 64,931-935 (1990). FUJIYAMA,A., MIYANOHARA, K., NOZAKI,C., YONEYAMA. T.. OHTOMO, RIGGS,A.D. and JONES,P.A., 5-methylcytosine,gene regulation, and N. and MATSUBARA, K., Cloning and structural analyses of hepatitis-B- cancer.Adv. Cancer Rex, 40, 1-30 (1983). virus DNAs. subtype adr. Nucleic Acids Res., 11,4601-4609 (1983). SAGGIORO, D., PANOZZO,M. and CHECO-BIANCHI, L., Human T-lym30,405-422 (1Y74). DOLRFLER. W.. DNA methylation and chem.. 52,93-124 (1983).

140

MIYOSHI E T A L .

photropic-virus type I transcriptional regulation by methylation. Cancer Res., 50,49684973 (1990). SAMBROOK, J., FRITSCH. E.F. and MANIATIS, T., Molecular cloning: a laboratory manual (second edition), Cold Spring Harbor Laboratory, New York (1989). SOUTHERN, E.M., Detection of specific sequences among fragments separated by gel electrophoresis. J. mol. B i d , 98,503-517 (1975). TSURIMOTO, T.. FUJIYAMA, A. and MATSUBARA, K.. Stable expression and replication of hepatitis-B-virus genome in an integrated state in a human hepatoma cell line transfected with the cloned viral DNA. Proc. nut. Acad. Sci. (Wash.),84,444448 (1987). UEDA,K., TSURIMOTO, T., NAGAHATA, T., CHISAKA, 0. and MATSUBARA, K., An in vifro system for screening anti-hepatitis-B-virus drugs. Virology, 169,213-216 (1989).

VANYUSHIN. B.F., TKACHEVA, S.G. and BELOZERSKY, A.N., Rare bases in animal DNA. Nature (Lond.), 225,948-949 (1970). VARDIMON, L., NEUMANN, R., KUHLMANN, I., SURER, D. and DOERFLER, W., DNA methylation and viral gene expression in adenovirustransformed and infected cells. Nzrcleic Acids Res., 8, 2461-2473 (1980). YOAKUM, G.H., KORBA,B.E., LECHNER, J.F.. TOKIWA, T., GAZDAR, A.F., SEELY,M., LEEMAN, L.. AUTRUP, H. and HARRIS,C.C., Highfrequency transfection and cytopathology of the hepatitis-B-virus core antigen gene in human cells. Science. 222,385-389 (1983). YOUSSOUFIAN, H.. HAMMER, S.M.. HIRSCH,M.S. and MULDER,C., Methylation of viral genome in an in vifro model of herpes simplex virus latency. Proc. nut. Acad. Sci. (Wash.),79,2207-2210 (1982).

Enhancement of hepatitis-B surface-antigen expression by 5-azacytidine in a hepatitis-B-virus-transfected cell line.

The human hepatoblastoma-derived cell line HB611 secretes hepatitis-B surface antigen (HBsAg) and hepatitis-B e antigen (HBeAg) into the medium. Hepat...
495KB Sizes 0 Downloads 0 Views