JOURNAL OF SURGICAL RESEARCH 73, 97–100 (1997)ARTICLE NO. JR975182

Detection of Hepatitis B Virus x Transcripts in HumanHepatocellular Carcinoma Tissues1

Susumu Kobayashi, M.D., Ken-ichi Saigoh, M.D., Tetsuroh Urashima, M.D.,Takehide Asano, M.D., and Kaichi Isono, M.D.

Second Department of Surgery, Chiba University School of Medicine, 1-8-1 Inohana, Chuoh-ku, Chiba 260, Japan

Submitted for publication June 17, 1997

noma (HCC) patients; therefore, HBV or HCV has beenBackground: The hepatitis B virus (HBV) or hepati- considered as the major putative cause of HCC in Ja-

tis C virus (HCV) sequences were frequently detected pan [1, 2]. Approximately 70% of HCC patients werein hepatocellular carcinoma (HCC) tissues by the seropositive for anti-HCV testings, another 20% beingmethod of polymerase chain reaction (PCR). However, detected for serum HBsAg. Several works have beenthe expression levels of HBV and HCV in HCC tissues addressed to the mechanism of HCC development un-remain to be documented. This study evaluates the der the influence of these hepatitis viruses. Unlike ret-mRNA expression levels of those virus sequences in rovirus, HCV has no DNA intermediate through thethe human HCC tissues through Northern blot hybrid- reverse transcription, which makes it more difficult toization. Methods: We performed Northern blot hybrid- prove HCV as the cause of HCC [3], whereas HBV asization to identify the HBV x mRNA and HCV RNA in

the DNA virus is regarded as a nonselective mutagenicHCC tissues of 14 cases excised at Chiba Universityinsertional agent and the identification of a viral regu-Hospital. Results: The HBV x transcripts could be iden-latory gene HBV x suggests the direct involvement oftified in the tumor tissues from all three serum HBsAg-HBV in oncogene [4]. The HBV x protein has been dem-positive cases and two cases among 11 HBsAg-negativeonstrated to transactivate viral and cellular genespatients. One HBsAg-negative case showed the mostthrough transcription regulatory factors such as AP-1,intensive signal in those five cases. However, HCV RNAAP-2, NF-k B, CREB, etc. [5]. Although, as shown incould not be identified by this method. The HBV x genethese reports, more analytical data have been accumu-transcript is predicted to have a size of about 0.7 kb;lated about HBV-related carcinogenesis for HCC thanhowever, the molecular weight of HBV x specific

mRNA is not consistent and is distributed between ap- about HCV; the more frequent detection of serum anti-proximately 2.0 and 9.0 kb. The hybridization signal HCV has induced clinical researchers to study theintensity is also variable according to the cases. Con- HCV-related mechanism of HCC development. How-clusion: These observations suggest that HBV x gene ever, we have already shown that we could detect themay be transcribed with flanking cellular sequences HBV x sequences more frequently than HCV RNA, ifand the carcinogenic activity may be different ac- we perform polymerase chain reaction (PCR) detectioncording to the expressed levels. The identification of for the liver tissues [6]. These data make it obscure, inHBV x transcripts in HCC tissues on the Northern blot which the hepatitis virus is clinically more importantlevel also suggests that HBV x mRNA expression might for the development of HCC. Therefore, we report herebe significant and concerned with the development of on the expression of oncogenic HBV x gene and HCVhuman HCC even in HBsAg-negative cases. q 1997 by Northern blot hybridization in surgically excised hu-Academic Press man HCC tissues including those of HBsAg-negativeKey Words: Northern blot hybridization; mRNA; cases and discuss whether it is possible to determinePCR; RT-PCR.

the major oncogenic hepatitis virus for the developmentof human HCC through mRNA expression.

INTRODUCTIONMATERIALS AND METHODS

Hepatitis B virus surface antigen (HBsAg) or anti- The subjects studied were 14 HCC cases including 3 HBsAg-posi-hepatitis C virus antibody (anti-HCV) can be detected tive cases and 11 negative cases who had undergone hepatectomy

at the Second Department of Surgery of Chiba University Hospital.in the sera of more than 90% of hepatocellular carci-The excised liver tissues were stored at 0807C immediately aftersurgical resection through liquid nitrogen freezing.

1 This study was financially supported by a Grant-in-Aid from the The sera were obtained on admission for the detection of HBsAgand anti-HCV. Serum HBsAg was detected by enzyme immunoassayMinistry of Health and Welfare for a New 10-Year Strategy for Can-

cer Control in Japan. (EIA) kits (Dinabot, Tokyo, Japan). Serum anti-HCV testing was

97 0022-4804/97 $25.00Copyright q 1997 by Academic Press

All rights of reproduction in any form reserved.

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98 JOURNAL OF SURGICAL RESEARCH: VOL. 73, NO. 2, DECEMBER 1997

carried out using a first-generation HCV-EIA kit (C100-3 Ab) (Ortho cases were positive for HBsAg and the remaining 11Diagnostic, Westwood, MA) for cases 2, 13, and 14 indicated in Table cases were negative for HBsAg. Anti-HCV antibody1. A second-generation HCV-passive hemagglutination (PHA) kit could be detected in 2 HBsAg-negative patients among(Dinabot) was used for the other cases [7].

8 available cases.PCR for HBV x gene and RT-PCR for HCV RNA. HBV DNA wasdetected by PCR assay for the x gene region, after extraction of thetissue DNA by the phenol/chloroform procedure [6, 12]. We synthe- PCR for HBV x Gene and RT-PCR for HCV RNAsized two sets of oligonucleotide primers for the region [outer sense,

PCR detection in the liver tissues revealed that HBV5*-TGTGCTGCCAACTGGATCCT-3 * (nt 14–33); outer antisense, 5*-AGCCTCCTAGTACAAAGACC-3 * (nt 371–390); inner sense, 5*- x sequences could be identified in 8 of 9 cases for tumorCCCCGTCTGTGCCTTCTCATC-3 * (nt 174–194); and inner anti- tissues and 6 of 7 cases for nontumor tissues. Figure 1sense, 5*-CCTAATCTCCTCCCCCAACTC-3 * (nt 344–364)]. The first shows the HBV x sequences of cases 2, 5, and 13 asPCR was carried out for 30 cycles using the external primers under

211-bp bands, whereas RT-PCR revealed that the HCVThermus aquaticus (Taq) DNA polymerase (Perkin–Elmer Cetus,RNA could be detected in 8 tumor and nontumor tis-Norwalk, CT), and the PCR product was identified as size 396 bp.

For the purpose of confirming the first PCR product as a component sues of 14 cases. Figure 2 shows the RT-PCR productsof x gene, subsequent PCR was performed for 30 cycles to use internal of HCV RNA from cases 4, 5, and 8 as 145-bp bands.primers within the first PCR product. The second PCR product was The results of HBV x were not compatible with thealso confirmed as 211 bp stained by ethidium bromide.

serum test such as HBsAg; however, the data wereHCV RNA was detected by the RT-PCR method [13] followingconsistent between serum anti-HCV testing and RT-extraction of RNA by the guanidium/CsCl method. HCV RNA was

converted to complementary DNA by Moloney murine leukemia virus PCR detection of HCV RNA in tissues.(M-MLV) reverse transcriptase (Life Technologies, Inc., Gaithers-burg, MD) and amplified by a two-stage PCR with the pairs of exter- Northern Blot Hybridization for HBV x Gene andnal and internal (nested) primers deduced from the 5*-noncoding

HCV RNAregion of HCV RNA [outer sense, 5*-CTGTGAGGAACTACTGTC-TTC-3 * (nt 28—48); outer antisense, 5*-AACACTACTCGGCTA- Northern blot analysis revealed that HBV x tran-GCAGT-3 * (nt 229–248); inner sense, 5*-TTCACGCAGAAAGCG-

scripts in tumor tissues could be identified in all 3TCTAG-3 * (nt 46–65); and inner antisense, 5*-GTTGATCCAAGA-AAGGACCC-3 * (nt 171–190)]. The first and second PCR were HBsAg-positive (cases 1, 2, and 3) and 2 (cases 4 andperformed for 35 and 30 cycles, and the PCR products were confirmed 5) of 11 HBsAg-negative cases (Table 1). The especiallyas size 221 bp in the first stage and 145 bp in the second stage intensive signals of HBV x transcripts were detectedto avoid nonspecific results. During the whole procedure, extensive

in tumor tissues of cases 1 and 4. As shown in Fig. 3,precaution and high stringency were taken to avoid contaminationthe molecular weight levels of expressed HBV x RNAwith physical separation and pipets.were variable and ranged between 2.0 and 9.0 kb. TheNorthern blot hybridization for the HBV x gene and HCV RNA.

Total RNA was obtained from the frozen liver tissues following the signal intensity was also apparently different ac-guanidium/cesium chloride (CsCl) procedure [8]. The tissues were cording to the cases. Case 4 in Fig.3 was negative formixed with guanidinium thiocyanate solution and homogenized at serum HBsAg; however, its signal showed the highest47C. Each 1-ml homogenate was added to 0.4 g of CsCl and layered

intensity of all cases. The signal of HCV RNA couldonto a 0.6-ml cushion of 5.7 M CsCl with 0.1 M EDTA. The solutionnot be detected on the Northern blot level in any HCCwas centrifuged at 45,000 rpm for 12 hr at 207C, and then RNA

pellets were dissolved in a buffer of 10 mM Tris–HCl, 5 mM EDTA, tissues of these 11 cases (Fig. 3).1% SDS and then purified thorough the procedure of chloroform/n-butanol and ethanol.

DISCUSSIONNorthern blot hybridization was prepared with 10 mg total RNAobtained from tumor and nontumor tissues. The RNA was electro-phoresed on 1.0% agarose gels prepared in Mops buffer (0.02 M mor- The frequent detection of serum anti-HCV in HCCpholinopropanesulfonic acid, 5 mM sodium acetate, and 1 mM EDTA, patients suggests that the major putative cause of HCCpH 7.0) containing 2.2 M formaldehyde [9, 10]. After identification may be HCV in Japan and has induced many research-of 28S and 18S of RNA stained by ethidium bromide, the denatured

ers to investigate the carcinogenesis mechanism con-RNA was transferred to nylon membrane filters (Amersham Interna-cerning HCV. We have demonstrated that HCV couldtional plc., Amersham, UK).

The HBV x gene probe was prepared as an inner sequence of 396 replicate in the transformed hepatocytes through itsbp by a PCR cloning strategy using HBV DNA subtype adv [11]. The minus-strand form; however, the direct mechanism ofprobe for HCV RNA was constructed through the RT-PCR for the its transformation effect remains to be documented [2].5*-noncoding region of HCV RNA and cloned. The filters were incu-

Unlike retrovirus, HCV as RNA virus has no DNA in-bated with this 32P-labeled probe for 15 hr in a solution containing50% formamide, 51 SSC, 51 Denhardt’s solution, and 20 mg per termediate through the reverse transcription [3]. Asmilliliter denatured salmon sperm DNA after prehybridization. The we already reported, however, we could detect the HBVfilter was washed in 21 SCC containing 0.1% SDS at 607C and then sequences in HCC tissues more frequently than HCVexposed to X-ray film. RNA, if PCR detection is applied for the surgically ex-

cised specimens. The analysis of HCC carcinogenesisRESULTS by HBV has progressed more than HCV and many data

have been accumulated. HBV as DNA virus is regardedClinical Features and Serological Testsas a nonselective mutagenic insertional agent, and theHBV x protein, acting as a transcriptional transactiva-The clinical data of the 14 patients were shown in

Table 1. The pathological diagnoses were determined tor, may alter the host gene expression [4]. These factssuggest that HBV is a main causative factor for HCCas chronic hepatitis (CH) for 9 cases and cirrhosis for

the remaining 5 cases. Serum testing revealed that 3 development in more cases than HCV.

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99KOBAYASHI ET AL.: HBV x TRANSCRIPTS IN HEPATOCELLULAR CARCINOMA

TABLE 1

Relationship between Northern Blot Hybridization for HBV x in Liver Tissuesand Other Markers for HBV and HCV

Liver tissue Liver tissue

Diagnosis Serum PCR for NBH for Serum RT-PCR forNo. Age/Sex (HCC with) HBsAg HBV x HBV x anti-HCV HCV RNA

T N T N T N1 66/F CH / / / / / 0 0 02 29/F CH / / / / 0 0 0 03 65/M CH / / / / / 0 0 04 67/F CH 0 / NA / / / / /5 66/M CH 0 / / / 0 / / /6 78/M CH 0 / NA 0 0 NA / /7 60/M CH 0 NA NA 0 0 NA / /8 58/M Cirrhosis 0 NA NA 0 0 NA / /9 66/M Cirrhosis 0 NA NA 0 0 NA / /

10 62/M Cirrhosis 0 NA NA 0 0 NA / /11 56/M Cirrhosis 0 / / 0 0 NA / /12 40/M Cirrhosis 0 NA NA 0 0 0 0 013 41/M CH 0 / / 0 0 0 0 014 74/F CH 0 0 0 0 0 0 0 0

3/14 8/9 6/7 5/14 3/14 2/8 8/14 8/14

Note. CH, chronic hepatitis; NA, not available; T, tumor; N, nontumor; NBH, Northern blot hybridization.

In a current study, we performed Northern blot hy- major cause for its development of HCC, although HCCdevelopment had been considered to have little relationbridization by which the detected transcript must be

of a much greater amount and could be more significant with HBV because of being negative for serum HBsAg.It was reported that HBV x transcripts could be de-than RT-PCR. The results of this method reveal that

there could be an RNA message of HBV x in the tumor tected as various sizes in the HBsAg positive cases,using biopsy specimens or cell lines [14, 15]. In a cur-tissues of cases 1, 2, 3, 4, and 5 and nothing in the

5*-noncoding region of HCV existing in tumor tissues rent study, we could also detect the HBV x transcriptsof various sizes in the surgically excised HCC tissuesamong all cases. Since HCV exists in HCC cells as HCV

virus and has no DNA intermediates, no detection of including HBsAg-negative cases. It was reported thatthe transgenic mice of HBV x gene with a promoterthe 5*-noncoding region of HCV RNA indicates that it

is impossible to identify sequence of HCV RNA by this region produced the 0.7-kb transcripts in their livers,and most of those mice developed HCC [16]. However,method. These results indicate that HBV x is signifi-

cantly expressed in HCC cells, whereas HCV RNA is the transcripts were detected as larger molecules be-tween 2.0 and 9.0 kb in this study than those of thein very small quantity and can be identified only by

RT-PCR. Therefore, HBV x might be more important transgenic mice. The genomic DNA of HBV is knownto have only one transcription termination site and thefor HCC development than HCV in cases 1, 2, 3, 4, and

5 in this study. This might be a fact in cases 4 and 5, HBV sequences are integrated in the host liver DNAwith changing of the insertional sites and those sizes.despite serological tests of HBsAg-negative and HCV

Ab-positive results. Especially, case 4 showed the high- If the integrated sequences had no transcription termi-est signal of HBV x transcripts and HBV might be a

FIG. 1. PCR detection of HBV x gene in the tumor tissues and FIG. 2. RT-PCR detection of HCV RNA in the tumor tissues andnontumor tissues of cases 4, 5, 8, and 14. In cases 4, 5, and 8, HCVnontumor tissues of cases 2, 5, 13, and 14. In cases 2, 5, and 13,

HBV x sequences were detected as 211-bp bands. RNA was detected as 145-bp bands.

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