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Possible discrepancy between the dose level required for promoting action, when given after initiation process, and that needed to exert an anticarcinogenic effect when given simultaneously with a carcinogen, of hepatic promoters were investigated in an attempt to obtain a 'practical' threshold dose of promoters. Phenobarbital (PB) and dichlorophenyltrichloroethane (DDT) were used as promoters and 3'-methyl-4-(dimethylamino)-azobenzene (3'-Me-DAB) was used as the carcinogen. Male weanling rats were treated with 600 p.p.m. 3'-Me-DAB for 3 weeks followed by a diet containing a promoter at various dose levels (5-500 p.p.m.), or the animals were treated with a low dose (100 p.p.m.) of 3'-Me-DAB plus a promoter at various dose levels (20-500 p.p.m.). The effects of promoters were measured by scoring size and number of enzyme-altered islands at weeks 12 and 24 of rat age. The promoting effect of PB and DDT was demonstrated in dose-dependent fashion, in the dose range of 10-500 p.p.m. and 20-500 p.p.m., respectively. On the other hand, promoters given simultaneously with a low dose of carcinogen enhanced the carcinogenesis at all the dose levels tested, quite in contrast with their inhibitory effect on carcinogenesis when given together with relatively high doses of carcinogens.

B型肝炎ウイルスcarrierから発症した肉眼分類上はEggelの塊状型肝細胞癌で,採取された複数腫瘍結節間において,異なるウイルスDNAの組み込みパターンが見られた興味ある1例を経験したので報告する.症例は22歳,女性.昭和62年1月に腹部腫瘤を自覚し,某病院へ入院した.腹部超音波画像診断にて多発性肝占拠性病変を指摘され,α-fetoproteinが高値を示したため,肝細胞癌の診断を受けた.昭和62年4月東大病院に転院しLAK, IL-2療法にて治療したが,肝不全が進行し,昭和63年1月に死亡した.剖検時,肝は肉眼的にEggelの塊状型肝細胞癌の像を呈しており,無作為に10個の腫瘍結節を選び,DNAを抽出しHBV-DNAの組み込みを検索したところ,8個については同一の組み込みパターンが見られたが,残りの2個については組み込みが認められなかった.

Two integrated hepatitis B virus (HBV) DNA molecules were cloned from two primary hepatocellular carcinomas each containing only a single integration. One integration (C3) contained a single linear segment of HBV DNA, and the other integration (C4) contained a large inverted duplication of viral DNA at the site of a chromosome translocation (O. Hino, T.B. Shows, and C.E. Rogler, Proc. Natl. Acad. Sci. USA 83:8338-8342, 1986). Sequence analysis of the virus-cell junctions of C3 placed the left virus-cell junction at nucleotide 1824, which is at the 5' end of the directly repeated DR1 sequence and is 6 base pairs from the 3' end of the long (L) negative strand. The right virus-cell junction was at nucleotide 1762 in a region of viral DNA (within the cohesive overlap) which shared 5-base-pair homology with cellular DNA. Sequence analysis of the normal cellular DNA across the integration site showed that 11 base pairs of cellular DNA were deleted at the site of integration. On the basis of this analysis, we suggest a mechanism for integration of the viral DNA molecule which involves strand invasion of the 3' end of the L negative strand of an open circular or linear HBV DNA molecule (at the DR1 sequence) and base pairing of the opposite end of the molecule with cellular DNA, accompanied by the deletion of 11 base pairs of cellular DNA during the double recombination event. Sequencing across the inverted duplication of HBV DNA in clone C4 located one side of the inversion at nucleotide 1820, which is 2 base pairs from the 3' end of the L negative strand. Both this sequence and the left virus-cell junction of C3 are within the 9-nucleotide terminally redundant region of the HBV L negative strand DNA. We suggest that the terminal redundancy is a preferred topoisomerase I nicking region because of both its base sequence and forked structure. Such nicking would lead to integration and rearrangement of HBV molecules within the terminal redundancy, as we have observed in both our clones.

The influence of 3′-methyl-4-(dimethylamino)azobenzene(3′-Me-DAB) on the drug-metabolizing system in the liver was investigated. Feeding of 3′-Me-DAB for 3 weeks at 10, 20 and 600 ppm increased the content of hepatic microsomal cytochrome P-450 slightly (up to 27% raise) but significantly. The feeding effects were also demonstrated in S-9 activity (up to 91% raise) when the mutagenicity of 3-methylcholanthrene (3-MC) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) was assayed in Ames system using S-9 fraction from 3′-Me-DAB-treated rat livers.

The relationship between serological and histochemical markers of hepatitis B virus (HBV) and viral integration in hepatocellular carcinomas (HCCs) in Japan was investigated. Special attention was paid to the exclusion of false-positive results due to bacterial contamination of autopsy materials. Of 85 patients with HCC, 23 were positive for serum HBsAg, 18 were positive for serum HBV antibodies and the remaining 44 were negative for serum HBV markers. Among the 23 HCCs from HBsAg carriers, 19 (82.6%) had integrated HBV DNA in the tumor DNA. In contrast, of the 18 HCCs from HBV antibody-positive patients, only one was positive for HBV DNA integration; this particular HCC was from a long-term HBsAg carrier who became a non-carrier by seroconversion at a later age. Of 44 HCCs from patients negative for serum HBV markers, 3 had integrated HBV DNA. Of these 3 cases, 2 were, however, histochemically positive for HBsAg in non-cancerous portions of the liver. Thus, our present results show a high integration rate of HBV DNA in HCCs of carrier patients and an extremely low rate of viral integration in HCCs of non-carrier patients. The latter finding contrasts strongly with previous results gained from analysing materials from patients in Europe and Africa. No histological differences were apparent between HCCs with or without viral integration.

Integrated hepatitis B virus (HBV) DNA is almost invariably found in hepatocellular carcinomas (HCC) which develop in HBV carriers. Integrated HBV DNAs from two single-integration HCCs (C3 and C4) have been cloned, and the cellular integration sites have been analyzed. Integrated HBV DNA of C3 is present in chromosome 6 and contains a nearly complete linear HBV genome. The HBV DNA integration in tumor C3 was not associated with major rearrangements of cellular DNA. In contrast, the integrated HBV DNA in C4 contains a large inverted repeat of HBV DNA, in which each repeat consists of a linear HBV DNA segment similar to that present in C3. The C4 integration was also accompanied by a cellular DNA translocation at the HBV integration site. The translocation occurred between chromosomes 17 and 18, along with a deletion of at least 1.3 kilobases of chromosome 18 DNA at the translocation site. Our data support a model in which postintegration rearrangement of integrated HBV and cellular DNA results in the generation of chromosomal aberrations. These chromosomal aberrations may function in a multistage mechanism leading to fully malignant HCC.

Recently, it was reported that the product of Birt–Hogg–Dubé syndrome gene (folliculin, FLCN) is directly phosphorylated by 5′-AMP-activated protein kinase (AMPK). In this study, we identified serine 62 (Ser62) as a phosphorylation site in FLCN and generated an anti-phospho-Ser62-FLCN antibody. Our analysis suggests that Ser62 phosphorylation is indirectly up-regulated by AMPK and that another residue is directly phosphorylated by AMPK. By binding with FLCN-interacting proteins (FNIP1 and FNIP2/FNIPL), Ser62 phosphorylation is increased. A phospho-mimic mutation at Ser62 enhanced the formation of the FLCN–AMPK complex. These results suggest that function(s) of FLCN–AMPK–FNIP complex is regulated by Ser62 phosphorylation.Structured summaryMINT-7298145, MINT-7298166: Flcn (uniprotkb:Q76JQ2) physically interacts (MI:0915) with AMPK alpha 1 (uniprotkb:P54645) by anti tag coimmunoprecipitation (MI:0007)MINT-7298267: AMPK alpha 1 (uniprotkb:Q13131) phosphorylates (MI:0217) tsc2 (uniprotkb:P49816) by protein kinase assay (MI:0424)MINT-7298182: FNIP1 (uniprotkb:Q8TF40) physically interacts (MI:0915) with Flcn (uniprotkb:Q76JQ2) by anti tag coimmunoprecipitation (MI:0007)MINT-7298132: AMPK alpha 1 (uniprotkb:Q13131) phosphorylates (MI:0217) Flcn (uniprotkb:Q76JQ2) by protein kinase assay (MI:0424)MINT-7298229: FNIPL (uniprotkb:Q9P278) physically interacts (MI:0915) with Flcn (uniprotkb:Q76JQ2) by anti tag coimmunoprecipitation (MI:0007)

Malignant mesothelioma is a highly aggressive tumor arising from serosal surfaces of the pleura and is triggered by past exposure to asbestos. Currently, there is no widely accepted treatment for mesothelioma. Development of effective drug treatments for human cancers requires identification of therapeutic molecular targets. We therefore conducted a large-scale functional screening of mesothelioma cells using a genome-wide small interfering RNA library. We determined that knockdown of 39 genes suppressed mesothelioma cell proliferation. At least seven of the 39 genes— COPA, COPB2, EIF3D, POLR2A, PSMA6, RBM8A , and RPL18A —would be involved in anti-apoptotic function. In particular, the COPA protein was highly expressed in some mesothelioma cell lines but not in a pleural mesothelial cell line. COPA knockdown induced apoptosis and suppressed tumor growth in a mesothelioma mouse model. Therefore, COPA may have the potential of a therapeutic target and a new diagnostic marker of mesothelioma.

The autopsy findings in a case of hepatocellular carcinoma with hepatitis B virus (HBV)-DNA integration in a Japanese boy, 4 years and 10 month of age, are reported. The boy was an HBV carrier born in a typical familial cluster of HBV infection in Japan. He had been asymptomatic until the sudden manifestation of the liver tumor. Histopathologic examination revealed a well-differentiated, adult-type hepatocellular carcinoma without hepatic cirrhosis. HBV-DNA sequences were detected in the tumor cell DNA by the Southern blot hybridization method. This is the youngest patient with hepatocellular carcinoma with HBV-DNA integration reported to date, suggesting that HBV may manifest its oncogenic properties after a shorter incubation period than generally believed.

One of the earliest events following penetration and uncoating of hepadna viruses is the conversion of the viral DNA into a covalently closed circular (CCC) molecule [1], (Figure 30.1). The CCC DNA accumulates in the nucleus and is believed to serve as the template for synthesis of viral RNAs, including pregenome RNA and mRNA encoding viral proteins. The pregenome RNAs are packaged into particles with viral core protein in the cytoplasm and reverse transcription occurs, leading to the production of a full-length viral DNA minus strand [2]. Plus strand DNA synthesis begins approximately 250 bp from the 5′ end of the minus strand and is primed by an RNA oligonucleotide from the 5′ end of the RNA pregenome [3]. After plus strand DNA synthesis reaches the 5′ end of the minus DNA strand, it “jumps the gap” to the 3′ end of the minus DNA strand, resulting in circularization of the minus strand DNA. For reasons that are unclear, virions are formed and secreted before plus strand DNA synthesis is completed, and therefore, DNA molecules in virions are open circular and contain a single strand region. Completion of the viral DNA plus strand is an early event after the virus infects a permissive host cell (Figure 30.1). Transcription and translation of viral genes proceeds in an undirectional fashion, utilizing all three translational reading frames as the result of the overlap of viral genes [4] (Figure 30.2A).

UNAKAMI, M., KOMIYA, I., ENDO, Y., HARA, M., IKEDA, K., KUMADA, H., YOSHIBA, A., HINO, O. and KITAGAWA, T. Seroconversion from HBs-Ag to Anti- HBs in a Case of Liver Cirrhosis Associated with Hepatocellular Carcinoma. Tohoku J. exp. Med., 1987, 152 (1), 81-86 - This paper reports a case of liver cirrhosis associated with hepatocellular carcinoma (HCC) of a woman who was converted from hepatitis B surface antigen (HBs-Ag) positive to antibody against HBs-Ag (anti-HBs) positive in the serum through an immunoregulatory steroid rebound phenomenon. The histology of the biopsy specimen taken before the seroconversion showed an early stage of liver cirrhosis with moderate infiltration of mononuclear cells. At autopsy about 3 years after the seroconversion, the liver tissue free of the tumor was in an early stage of liver cirrhosis. Fibrosis did not advance as compared with the biopsy specimen. In addition, mononuclear cell infiltration decreased remarkably and piecemeal necrosis disappeared after the seroconversion. The immunohistologic examination of hepatocytes demonstrated that positive stainings for HBs-Ag and for hepatitis B core antigen (HBc-Ag) in the biopsy specimen turned to be negative in the autopsy specimen. These facts indicate that the steroid rebound phenomenon eliminated free hepatitis B virus (HBV) in the hepatocytes in the absence of massive necrosis of hepatocytes. HBV-DNA integration was proved in the genome of HCC by molecular hybridization method.

We produced transgenic mice by microinjecting a partial tandem duplication of the complete hepatitis B virus (HBV) genome into fertilized eggs of C57BL/6 mice. One of eight transgenic mice was a high producer for HBV surface antigen (HBsAg) and HBV e antigen (HBeAg) in the serum. The HBV genomes were transmitted to the next generation and these F1 mice also produced HBsAg and HBeAg. mRNAs of 3.5, 2.1, and 0.8 kilobases were detected in the livers and the kidneys of these mice. In addition, a 0.8-kilobase RNA was detected in the testis. Single-stranded and partially double-stranded HBV DNAs were shown to be produced in the cytoplasm of the liver and kidneys. These HBV DNAs were associated with the core particles, indistinguishable from nucleocapsid produced in an infected human liver. Viral genome DNA was detected in the serum. These results demonstrate that the HBV genome integrated into the mouse chromosome acted as a template for viral gene expression, allowing viral replication. Thus, these transgenic mice should be useful for detailed studies of the replication and expression of HBV and for pathological studies of hepatitis, including the development of hepatocellular carcinoma.

Hepatitis B virus (HBV) DNA in hepatocellular carcinoma (HCC) and/or cirrhotic livers of Japanese patients in Japan was investigated by molecular hybridization utilizing a 32P-labeled cloned HBV DNA of adr subtype. Among 24 HCC cases, 9 (37.5%) were positive for serum HBsAg and 10 (41.7%), including 2 cases which were negative for serum HBV markers, were positive for tissue HBsAg in noncancerous portions of the liver. In the latter 10 cases, integrated HBV DNA was detected in HCC. The restriction pattern of integrated viral DNA was different from one case to another. In the noncancerous portion of these 10 HCC cases, free (3/10), random integration (2/10), or clonal integration (1/10) of viral DNA was demonstrated; no hybridization was detected in the remaining 14 HCC cases, either in cancerous or noncancerous portions. Of 6 cases of liver cirrhosis, 2 were positive for serum and histochemical HBsAg; viral DNA was in free state. Viral DNA was not detected in pancreatic tissue in 5 HBsAg-positive cases. The present results are in agreement with previous reports indicating a high frequency of integrated viral DNA in HBV-associated HCC and heterogeneity in the mode of integration. Our results differ in finding no HBV DNA in HCC cases not associated with HBV markers and in finding clonal populations of hepatocytes with integrated HBV DNA less frequently in the noncancerous portion of the liver of HBsAg carriers.

It seems evident that the development of fully malignant HCC is a multistage process with many variables. One possible mechanism by which many of these variables may interact is as follows. During chronic active hepatitis, viral DNA integration occurs randomly and at a low frequency in hepatocytes. Integration may be stimulated by the increased rate of hepatocyte cell division resulting from liver necrosis and regeneration during chronic disease. The presence of viral integrations in the cellular genome provides focal points for the generation of chromosomal aberrations. One pathway by which these aberrations may be generated involves rearrangement of integrated viral and cellular sequences following viral DNA integration. The rearrangements which occur may include deletion, translocation, transposition or amplification of specific viral and cellular DNA sequences. We and others have directly demonstrated that all of these events are associated with different HBV integrations. The presence of viral integrations in chromosomes may also, by some unknown mechanism, destabilize those chromosomes such that whole chromosomes fail to segregate and are lost from particular cells. Preliminary studies we have conducted using restriction fragment length polymorphisms have revealed the loss of Chromosome 11 alleles in several HCC, indicating that chromosome loss may be a common occurrence in HCC. Our studies with restriction fragment length polymorphisms support such a mechanism involving Chromosome 11 in HCC. Specific chromosomal aberrations associated with all HCCs have not yet been identified.(ABSTRACT TRUNCATED AT 250 WORDS)

We established four cell lines, from the liver cells of a transgenic mouse, constructed with hepatitis B virus DNA that had an inverted repeat structure. The integrated DNA patterns of the four established cell lines were different from one another and from the original pattern. These data show that the instability of integrated hepatitis B virus DNA would also occur in somatic cells during replication, apart from meiosis, which was previously reported.

The promotion effect of diazepam on the hepatocarcinogenesis initiated by 3'-methyl-4-(dimethyl-amino)azobenzene(3'-Me-DAB) in the rat was studied. Weanling rats were fed a diet containing 0.06% 3'-Me-DAB for 3 weeks, followed by either the basal diet or a diet containing 0.05% diazepam. The number and the size of enzyme-altered islands greater than 50 micrometers in diamter were scored at week 16. The feeding of diazepam did not increase the number and the size of enzyme-altered islands significantly, in comparison with those scored in animals fed 3'-Me-DAB alone. It was concluded that diazepam is not a promoter like phenobarbital in hepatocarcinogenesis.

Hepatitis B virus (HBV) DNA integrates into human cellular DNA during long-term persistent infections, implicating integration as one of the steps leading to hepatocarcinogenesis. The present study demonstrates that the integration of HBV DNA can result in or be accompanied by interchromosomal exchange of genomic material containing the integrated DNA. Unique cellular DNA to the left of an HBV DNA integration site cloned from a primary tumor mapped to chromosome 18q (18q11.1–q11.2); right hand flanking DNA mapped to chromosome #17 at a near-terminal region. The presence of chromosomal rearrangements in association with HBV integration may play a role in multistage hepatocarcinogenesis.