Byung-Hwa Hyun

Incidence of hepatocellular carcinoma in transgenic mice expressing the hepatitis B virus X-protein

BACKGROUND/AIMS Chronic infection with hepatitis B virus is a high-risk factor for hepatocellular carcinoma in humans. The HBV X-protein, a multi-functional viral regulator, has been suspected to play a positive role in hepatocarcinogenesis, as demonstrated by the high incidence of hepatocellular carcinoma in HBx-expressing transgenic mice, although it is still controversial. The aim of this study was to generate transgenic mice expressing the HBV X-gene under authentic promoter control and to test whether the gene products can cause hepatic tumors. METHODS Three transgenic mouse lines were generated by microinjecting the X-gene construct into hybrid (C57BL/6 x DBA) eggs. Gene expression was tested by protein and mRNA analyses. During an observation period of 18 months, mice were sacrificed and organs subjected to histologic examinations. RESULTS Grossly defined hepatocellular carcinomas reproducibly were observed in mice expressing the X-protein, which were investigated through six generations from the age of 11 to 18 months. Among 14 transgenic mice investigated from the age of 11 to 18 months, 12 were found to have hepatocellular carcinoma, grossly or microscopically. The lesion of the hepatocellular carcinoma disclosed a significant increase in the proliferating cell nuclear antigen in the nuclei. CONCLUSION The incidence of hepatocellular carcinoma (86%) in our HBV X transgenic mice may be highly significant, since, except for one case, HBV X-gene transgenic mice produced in other laboratories did not develop liver tumor or any other pathologic phenomena.

Mit1/Lb9 and Copg2, new members of mouse imprinted genes closely linked to Peg1/Mest.

Two mouse genes, Mit1/Lb9 and Copg2, linked to Peg1/Mest on mouse chromosome 6, were identified to be imprinted maternally and paternally, respectively. Mit1/Lb9 encoding untranslated transcripts resides within the intron 20 of Copg2. The gene is maternally imprinted in adult mouse brain, partially imprinted in other tissues. Copg2 consists of 24 exons within the >40 kb genomic region, being expressed ubiquitously in mouse tissues with a partial imprinting pattern in embryos, neonates, and adult brain in contrast to maternally imprinted human COPG2. In addition, we identified an antisense transcript of Copg2, Copg2AS, which overlaps 3′‐UTRs of Copg2 and Peg1/Mest. The Copg2AS transcript is maternally imprinted in embryos, neonates, and adult tissues.

An inhibitor of inducible nitric oxide synthase ameliorates experimental autoimmune myocarditis in Lewis rats

We studied the effect of nitric oxide (NO) on experimental autoimmune myocarditis (EAC) in rats. We examined the role of inducible nitric oxide synthase (iNOS), an enzyme that produces NO, on hearts affected with EAC, by testing the effects of aminoguanidine (AG), a selective iNOS inhibitor, on the course of EAC. Western blotting detected iNOS in the affected cardiac tissues, but not in CFA immunized cases. Immunohistochemically, the majority of ED1+ macrophages in the EAC lesions were positive for iNOS and nitrotyrosine. A high dose of AG (200 mg/kg/day) significantly reduced the incidence of EAC (p < 0.05) and ameliorated the histological score for the cardiac inflammation (p < 0.01) compared with the low dose AG (100 mg/kg/day) and vehicle treated groups. The immunoblot analysis showed that a high dose of AG effectively suppressed iNOS in hearts affected with EAC. An iNOS band was barely detected in the high dose AG (200 mg/kg) treated group, while it was distinctively visualized in the vehicle and low dose AG (100 mg/kg) treated groups. These results suggest that iNOS is upregulated in EAC lesions and increased NO production plays an important role in the development of EAC. In addition, selective iNOS inhibitors may have a therapeutic role in treating certain autoimmune diseases including EAC.

Dietary hematein ameliorates fatty streak lesions in the rabbit by the possible mechanism of reducing VCAM-1 and MCP-1 expression

Hematein is a compound isolated from Caesalpinia sappan that has been used in oriental medicine as both an analgesic and an anti-inflammatory agent. In this study, we examined the anti-atherogenic potential of hematein using cholesterol-fed New Zealand White (NZW) rabbits. NZW rabbits were divided into a hematein-supplemented (0.05% in diet) group (n=6), a probucol-supplemented (0.25% in diet) group (n=6), and a control group (n=6). After 8 weeks of treatments, the extent of the atherosclerotic lesions was significantly reduced in the hematein-supplemented group and the probucol-supplemented group without changing plasma lipoprotein levels. Hematein and probucol prevented the up-regulation of the vascular cell adhesion molecule-1 (VCAM-1) expression on the descending aorta induced by cholesterol diet. In culture, hematein also significantly inhibited the secretion of soluble VCAM-1 and of monocyte chemotactic protein-1 (MCP-1) respectively induced by tumor necrotic factor alpha (TNF-alpha) and mildly oxidized low density lipoprotein in human umbilical vein endothelial cell (HUVEC) culture. Also, hematein inhibited monocyte adhesion to endothelial cell and the activation of NF-kappaB in HUVECs stimulated with TNF-alpha. The results of the present study suggest that the anti-atherogenic effect of hematein is not related to control of the plasma lipid profile but probably related to the inhibition of VCAM-1 and MCP-1 expression resulting in an amelioration of lesion development in the rabbit.

Novel Design Architecture for Genetic Stability of Recombinant Poliovirus: the Manipulation of G/C Contents and Their Distribution Patterns Increases the Genetic Stability of Inserts in a Poliovirus-Based RPS-Vax Vector System

ABSTRACT Poliovirus has been studied as a live recombinant vaccine vector because of its attractive characteristics. The genetic instability, however, has hampered recombinant polioviruses (PVs) from being developed as an appropriate vaccine. A variety of different foreign inserts were cloned directly into our poliovirus Sabin 1-based RPS-Vax vector system, resulting in the production of recombinant PVs. The genetic stability of each recombinant PV was examined during 12 rounds of consecutive passage. It was found that the genetic stability of the recombinants was not well correlated with their insert size. Instead, elevated stability was frequently observed in recombinants with inserts of high G/C contents. Furthermore, a comparative study using different constructs of the human immunodeficiency virus env gene revealed that the internal deletion of the unstable insert was seemingly caused by the presence of the adjacent A/T-rich region. The instability of these inserts was completely remedied by (i) increasing the G/C contents and (ii) replacing the local A/T-rich region with the G/C-rich codon without a change of the amino acid. This means that stability is closely associated with the G/C content and the G/C distribution pattern. To see whether these findings can be applied to the design of genetically stable recombinant PV, we have reconstructed the heteromultimeric insert based on our design architecture, including the above-mentioned G/C rules and the template/ligation-free PCR protocol. The heteromultimeric insert was very unstable, as expected, but the manipulated insert with the same amino acid sequence showed complete genetic stability, not only in vitro, but also in vivo. Even though this guideline was established with our RPS-Vax vector system, to some extent, it can also be applied to other live viral vaccine vectors.

Abnormalities in cerebellar Purkinje cells in the novel ataxic mutant mouse, pogo

The pogo mouse is a novel neurological mutant, which was discovered, in an inbred strain (KJR/MsKist) derived from a Korean wild mouse. The pathological manifestations include difficulty in maintaining normal posture, failures of interlimb coordination and the inability to walk straight. The ataxia is first apparent from about 2 weeks of age and progresses throughout life. The mutation is inherited as an autosomal recessive trait. In this report, we describe abnormalities in the pogo/pogo cerebellum. Nissl staining shows that the pogo/pogo cerebellum is normal in size and lobulation. Similarly, immunocytochemical staining for a granule cell marker, 10B5, shows no differences in the thickness of the granular layer between pogo/pogo homozygote and pogo/+ heterozygote littermate controls. By using anti-parvalbumin immunocytochemistry, the cells of molecular layer of the pogo/pogo cerebellum also appeared similar in distribution as compared to normal wild type mouse. In anti-neurofilament immunocytochemistry, the basket cells axons of the pogo/pogo cerebellum appeared normal. Purkinje cell abnormalities were identified by using anti-calbindin D immunocytochemistry. In 120-day-old pogo/pogo mutant mice there was a loss of Purkinje cells throughout the cerebellar vermis. Furthermore, the somata and dendrites were extensively vacuolated in the pogo/pogo Purkinje cells and the primary dendrites were frequently swollen. Focal axonal swellings were commonly observed in the Purkinje cell axons of pogo/pogo mutant mice as they traversed the granular layer. These data suggest that the progressive ataxia seen in pogo mice may be due to a failure of normal Purkinje cell activity.

SINE‐R.C2 (a Homo sapiens specific retroposon) is homologous to CDNA from postmortem brain in schizophrenia and to two loci in the Xq21.3/Yp block linked to handedness and psychosis

We investigated the retroviral/retroposon hypothesis of schizophrenia by generating sequences with PCR primers based on a retroviral sequence recovered by Yee et al. [1998: Schizophr Res 29:92] from a cDNA library from postmortem brain tissue from an individual with psychosis in a genomic region (Xq21.3) that has been tentatively linked to schizophrenia and schizoaffective disorder by Laval et al. [1998: Am. J. Med. Genet. (Neuropsychiatr. Genet.) 81:420-427]. Within the block of homology with Yp that was generated by a transposition between the chimpanzee and Homo sapiens we find two sequences, HS307 and HS408, with a high degree of homology to but not identity with the schizophrenic brain cDNA. The closest match of these three sequences is to a family of retroposons, that has evolved from the HERV-K family of endogenous retroviruses, some members of which (e.g., SINE-R.C2) appear to be specific to the human genome. This element has been reported as a cause of Fukuyama-type muscular dystrophy [Kobayashi et al., 1998: Nature 394:388-392]. Such retroposons, as agents of change in the human genome, provide a strategy for investigating pathogenesis. On account of their genomic location in a region that has been subject to change in the course of hominid evolution, and that may have a relationship to psychosis and/or cerebral asymmetry, we conclude that these particular insertions deserve further investigation.

Molecular Evolution of the Periphilin Gene in Relation to Human Endogenous Retrovirus M Element

HERV-M (human endogenous retrovirus M), related to the super family of HERV-K, has a methionine (M) tRNA primer-binding site, and is located within the periphilin gene on human chromosome 12q12. HERV-M has been integrated into the periphilin gene as the truncated form, 5′LTR-gag-pol-3′LTR. Polymerase chain reaction (PCR) and reverse transcription-polymerase chain reaction (RT-PCR) approaches were conducted to investigate its evolutionary origins. Interestingly, the insertion of retroelements in a common ancestor genome can make different transcript variants in different species. In the case of the periphilin gene, human (10 variants) and mouse (2 variants) lineages show different transcript variants. Insertion of HERV-M (variant 1-3) could affect the protein-coding region. Also, Alusq/x (variant 4-9) and L1ME4a (mammalian-wide subfamilies of LINE-1) (variant 10) in humans and SINE (short interspersed repetitive element) and RLTR15 (the mouse putative long terminal repeat) (variant 2) in mice could be driving forces in transcript diversification of the periphilin gene during mammalian evolution. The HERV-M derived transcripts (variant 1-3) were expressed in different human tissues, whereas they were not detected in crab-eating monkey and squirrel monkey tissues by RT-PCR amplification. Taken together, HERV-M seems to have been integrated into our common ancestor genome after the divergence of simians and prosimians, and then was actively expressed during hominoid evolution.

Phylogenetic Analysis of a Retroposon Family in African Great Apes

Abstract. The SINE-R retroposon family has been identified by its relationship with the long terminal repeats (LTRs) of human endogenous retrovirus class K (HERV-K) as a mobile element that has evolved recently in the human genome. Here we examined the recent evolutionary history of this class of elements by a PCR approach to genomic DNA from the African great apes and by phylogenetic analysis including comparison with the HERV K10 parent sequence. With primers derived from a cDNA sequence from human brain, we identified 27 sequences from the chimpanzee and 16 from the gorilla. Phylogenetic comparisons with previously recognized sequences from the human and from the orangutan and gibbon revealed wide overlap of elements across species, suggesting multiple origins in the course of hominoid evolution. Two human elements SINE-R.C2 and HS307 were the furthest removed from the HERV-K10 sequence but these two elements were closely related to three elements from the chimpanzee and four elements from the gorilla. This group of elements (our clusters 14 and 15) appears to have transposed late in hominoid evolution. One element (Ch-M16) showed 99.1% sequence identity with the SINE-R.C2 element, which is human-specific. Thus the SINE-R family appears to have continued to be active in transposition throughout the course of primate evolution.

Over-expression of urokinase receptor in human epidermoid-carcinoma cell line (HEp3) increases tumorigenicity on chorio-allantoic membrane and in severe-combined-immunodeficient mice.

Using chorio‐allantoic membranes (CAMs) of chick embryos and severe‐combined‐immunodeficient (SCID) mice, we investigated the effects of urokinase‐type plasminogen‐activator receptor (u‐PAR) over‐expression on the process of invasion and tumorigenicity. By the transfection of u‐PAR cDNA, 3 u‐PAR‐over‐expressing clones expressing 1.6‐ to 4.6‐fold more u‐PAR mRNA than parent cells were obtained from a human epidermoid‐carcinoma cell line, HEp3, that expresses urokinase‐type plasminogen activator (u‐PA) and u‐PAR. All the u‐PAR‐over‐expressing clones showed greater invasiveness (13 to 29%) than that of parent HEp3 cells on CAMs. Immunohistochemistry revealed densely stained u‐PAR‐positive cells near the margin of the tumor, where a u‐PAR‐over‐expressing clone, designated SM‐3, was invading thickened fibrous tissue on CAMs. Three u‐PAR‐over‐expressing clones formed larger tumors (>40 mm3) than did parent HEp3 cells on CAMs. Moreover, when the u‐PAR‐over‐expressing clone (SM‐3) was injected s.c. into the back of the SCID mice it produced a larger tumor volume than the control (HEp3) and down‐regulated (AS‐2) clones and significantly shortened the survival of SCID mice. These results demonstrate that increased u‐PAR expression is an important factor in determining the malignant phenotype that makes cancer cells more invasive and tumorigenic. Int. J. Cancer 77:257–263, 1998.© 1998 Wiley‐Liss, Inc.