Shigeki Kuriyama

Glial cell degeneration and hypomyelination caused by overexpression of myelin proteolipid protein gene

Myelin proteolipid protein (PLP), the major myelin protein in the CNS, has been thought to function in myelin assembly. Thus, mutations within the gene coding for PLP (Plp) cause hypomyelination, such as the jimpy phenotype in mice and Pelizaeus-Merzbacher disease in humans. However, these mutants often exhibit premature death of oligodendrocytes, which form CNS myelin. To elucidate the functional roles of Plp gene products in the maturation and/or survival of oligodendrocytes, we produced transgenic mice overexpressing the Plp gene by introducing extra wild-type mouse Plp genes. Surprisingly, transgenic mice bearing 4 more Plp genes exhibited dysmyelination in the CNS, whereas those with 2 more Plp genes showed normal myelination at an early age (3 weeks after birth), but later developed demyelination. Overexpression of the Plp gene resulted in arrested maturation of oligodendrocytes, and the severity of arrest was dependent on the extent of overexpression. Overexpression also led to oligodendrocyte cell death, apparently caused by abnormal swelling of the Golgi apparatus. Thus, tight regulation of Plp gene expression is necessary for normal oligodendrocyte differentiation and survival, and its overexpression can be the cause of both dys- and demyelination.

Development of hepatocytes from ES cells after transfection with the HNF-3beta gene.

We have attempted to generate embryonic stem (ES) cell‐derived hepatocytes expressing liver‐specific functional properties by use of ES cell technology. It was found that ES cells are allowed to differentiate into hepatocytes possessing high metabolic activities when hepatocyte nuclear factor (HNF)‐3β‐transfected ES cells are cultured in α‐MEM medium supplemented with 10% fetal bovine serum (FBS) and fibroblast growth factor (FGF)‐2 in the three‐dimensional cell culture system at 5% CO2. The differentiated cells induced albumin, triacylglycerol, urea, and glycogen synthesis as well as further expression of metabolic proteins and serum factors as markers of hepatocytic differentiation for at least 4 months. The cells differentiated from HNF‐3β‐transfected ES cells also had hepatocyte‐like ultrastructural characteristics, including several endoplasmic reticula, mitochondrion, and glycogen. Our findings indicate that generation of hepatocytes maintaining high metabolic functions developed from mouse ES cells will facilitate the study of the basic mechanism for hepatogenesis and will certainly provide new opportunities for tissue transplantation.

Angiotensin-I-Converting Enzyme Inhibitors May Be an Alternative Anti-Angiogenic Strategy in the Treatment of Liver Fibrosis and Hepatocellular Carcinoma

The renin-angiotensin system (RAS) is frequently activated in patients with chronic liver diseases. Angiotensin-II (AT-II), which is produced by angiotensin-converting enzyme (ACE), has many physiological effects, including strong pro-angiogenic activity. AT-II induces the potent angiogenic factor, vascular endothelial growth factor (VEGF). Recent studies have revealed that angiogenesis is an essential process in many pathological events, such as tumor growth including hepatocellular carcinoma (HCC), and even in liver fibrogenesis. ACE inhibitors are currently widely used as anti-hypertensive agents in clinical practice. Studies have found that the ACE inhibitor, perindopril (PE), which is a potent inhibitor of experimental HCC growth and angiogenesis, is associated with the suppression of VEGF at a clinically comparable dose. PE also markedly suppressed the hepatocarcinogenesis step. In liver fibrogenesis, AT-II is known to stimulate proliferation and production of tissue inhibitor of metalloproteinases-1 (TIMP-1) in activated hepatic stellate cells (Ac-HSC), which play a pivotal role in liver fibrosis development. PE markedly inhibited liver fibrogenesis associated with suppression of Ac-HSC proliferation and TIMP-1 expression via protein kinase-C, which serves as an intracellular signaling pathway. Since ACE inhibitor is used widely in clinical practice without serious side effects, it may provide an alternative new strategy for the treatment of liver fibrosis and HCC.

The copper-chelating agent, trientine, suppresses tumor development and angiogenesis in the murine hepatocellular carcinoma cells

Angiogenesis is now recognized as a crucial process in tumor development, including hepatocellular carcinoma (HCC). Since HCC is known as a hypervascular tumor, anti‐angiogenesis is a promising approach to inhibit the HCC development. Trientine dihydrochloride (trientine) is used in clinical practice as an alternative copper (Cu)‐chelating agent for patients with Wilsons disease of penicillamine intolerance. In our study, we examined the effect of Cu‐chelating agents on tumor development and angiogenesis in the murine HCC xenograft model. Although both trientine and penicillamine in the drinking water suppressed the tumor development, trientine exerted a more potent inhibitory effect than penicillamine. In combination with a Cu‐deficient diet, both trientine and penicillamine almost abolished the HCC development. Trientine treatment resulted in a marked suppression of neovascularization and increase of apoptosis in the tumor, whereas tumor cell proliferation itself was not altered. In vitro studies also exhibited that trientine is not cytotoxic for the tumor cells. On the other hand, it significantly suppressed the endothelial cell proliferation. These results suggested that Cu plays a pivotal role in tumor development and angiogenesis in the murine HCC cells, and Cu‐chelators, especially trientine, could inhibit angiogenesis and enhance apoptosis in the tumor with consequent suppression of the tumor growth in vivo. Since trientine is already used in clinical practice without any serious side effects as compared to penicillamine, it may be an effective new strategy for future HCC therapy.

Particle-mediated gene transfer into murine livers using a newly developed gene gun

Although particle-mediated gene transfer using gene gun technology has been applied for gene transfer into epidermis, applications of this technology to visceral tissues have not been well investigated. Although all helium gas-driven gene gun instruments have used macrocarriers to discharge DNA-coated microprojectiles so far, we used a newly developed gene gun instrument, in which a hammering bullet is used to discharge microprojectiles. With the gene gun, gold particles coated with lacZ expression plasmid were discharged to murine livers. LacZ expression was induced much more profoundly in the liver by particle-mediated gene transfer than by simple plasmid injection and electroporation-mediated gene transfer. LacZ expression was broadly and randomly distributed throughout the bombarded livers, indicating that particle-mediated gene transfer can induce transgene expression even at relatively distant areas from the surface of the bombarded tissue. Furthermore, although transgene expression was at its peak on day 2 after the bombardment, it was still detectable even on day 28. These results indicate that particle-mediated gene transfer with a newly developed gene gun may provide a new approach to gene therapy for human diseases.

Effects of glycyrrhizin on immune‐mediated cytotoxicity

Intravenous administration of glycyrrhizin is known to decrease elevated plasma transaminase levels in patients with chronic viral hepatitis, in which immune‐mediated cytotoxicity by cytotoxic T lymphocytes and tumour necrosis factor (TNF)‐α is considered to play an important pathogenic role. However, the immunological interpretation of the transaminase‐lowering action of glycyrrhizin is not known. Studies were performed to elucidate this action immunologically by assessing the effects of glycyrrhizin on immune‐mediated cytotoxicity using an antigen‐specific murine CD4+ T hybridoma line, which exhibits cytotoxicity against antigen‐presenting cells after stimulation with specific antigen, and a murine TNF‐α‐sensitive fibroblast line. Glycyrrhizin inhibited the cytotoxic activity of the T cells against antigen‐presenting cells and also suppressed TNF‐α‐induced cytotoxicity in the TNF‐α‐sensitive cell line in vitro. These results suggest that the decrease of elevated transaminase levels by glycyrrhizin in patients with chronic viral hepatitis is mediated in part by inhibition of immune‐mediated cytotoxicity against hepatocytes.

Expression of a retrovirally transduced gene under control of an internal housekeeping gene promoter does not persist due to methylation and is restored partially by 5-azacytidine treatment

Although expression of transgenes under the control of a retroviral long terminal repeat (LTR) promoter has been shown not to persist due to methylation, it has been observed that internal promoter may be active even if expression from the LTR promoter is silent. We constructed a retroviral vector carrying the herpes simplex virus thymidine kinase (HSVtk) gene under the control of the albumin gene promoter and transduced the HSVtk gene into hepatocellular carcinoma cells. Three of 14 mice, however, could not eradicate HSVtk-transduced grafts completely despite ganciclovir (GCV) treatment. These GCV-refractory cell lines exhibited resistance to GCV after recultivation. Subsequent Southern blot analysis revealed that the HSVtk gene was not deleted but extensively or completely methylated in GCV-refractory lines. Treatment with 5-azacytidine, a demethylating agent, partially restored the sensitivity of GCV-refractory lines to GCV. These results indicate that expression of retrovirally transduced gene may not persist in vivo due to methylation even when the gene is directed by an internal housekeeping gene promoter. These observations may also have important implications for future clinical applications of retrovirus-mediated gene therapy.

Endothelin-1 plays a major role in portal hypertension of biliary cirrhotic rats through endothelin receptor subtype B together with subtype A in vivo

BACKGROUND/AIMS Endothelin-1 has been suggested to play a key role in cirrhotic portal hypertension, but a role of its receptors in vivo is not fully elucidated. METHODS Biliary cirrhosis was induced by bile duct ligation. Expressions of endothelin-1 and its receptors were evaluated by radioimmunoassay and/or reverse-transcription polymerase chain reaction. Hemodynamics were studied using endothelin receptor agonist or antagonist. RESULTS Portal pressure and hepatic endothelin-1 concentrations progressively increased in parallel after bile duct ligation. Gene expression of hepatic prepro-endothelin-1 and endothelin B receptor enhanced after bile duct ligation, while that of endothelin A receptor was unchanged. Intraportal administration of endothelin-1 or endothelin B receptor agonist sarafotoxin 6c (0.5 nmol/kg, respectively) progressively raised portal pressure in both sham and cirrhotic rats. Portal hypertensive effect of sarafotoxin 6c was more intense in cirrhotic rats than sham animals. Neither endothelin A receptor antagonist FR139317 (1 mg/kg) nor endothelin B receptor antagonist BQ788 (1 mg/kg) alone ameliorated cirrhotic portal hypertension. Only the combined endothelin A and B blockade was associated with a decrease in portal pressure in cirrhotic rats. CONCLUSIONS These results indicate that endothelin-1 plays a major role in cirrhotic portal hypertension through endothelin receptor subtype B together with subtype A in vivo.

Effective and safe gene therapy for colorectal carcinoma using the cytosine deaminase gene directed by the carcinoembryonic antigen promoter

We have recently isolated carcinoembryonic antigen (CEA) promoter regions consisting of 419 bp and 204 bp from CEA-producing human colorectal carcinoma (CRC). We constructed CEA419/CD and CEA204/CD retroviruses carrying the bacterial cytosine deaminase (CD) gene directed by the CEA promoter regions. pCD2 retroviruses carrying the CD gene directed by the retrovirus long terminal repeat promoter were also used. CEA419/CD or CEA204/CD retrovirus-infected CRC cells were found to be susceptible to 5-fluorocytosine (5-FC), while non-CRC cells infected with the same retroviruses were not. CD-transduced CRC xenografts in nude mice were sensitive to 5-FC treatment, resulting in arrest of tumor growth. When mice with intraperitoneally disseminated CRCs were given intraperitoneal injections of CEA419/CD retrovirus-producing cells followed by 5-FC treatment, significantly prolonged survival rates were observed compared with animals injected with pCD2 retrovirus-producing cells followed by 5-FC treatment. Importantly, bone marrow suppression was not observed in animals injected with CEA419/CD retrovirus-producing cells and 5-FC, while profound bone marrow suppression was observed in those injected with pCD2 retrovirus-producing cells and 5-FC. These results indicate that effective and safe in vivo gene therapy for advanced CRC may be feasible by transferring the CD gene controlled by the CEA promoter followed by 5-FC treatment.

Cytosine deaminase/5-fluorocytosine gene therapy can induce efficient anti-tumor effects and protective immunity in immunocompetent mice but not in athymic nude mice

Murine hepatocellular carcinoma cells were retrovirally transduced with the bacterial cytosine deaminase (CD) gene. CD‐transduced cells exhibited more than 120‐fold higher sensitivity to 5‐fluorocytosine (5‐FC) compared with parental cells. When syngeneic immunocompetent mice were inoculated s.c. with parental hepatocellular carcinoma cells containing as little as 5% CD‐transduced cells, significant inhibition of tumor formation was induced by 5‐FC treatment. Furthermore, established solid tumors in immunocompetent mice containing only 5% CD‐transduced cells were infiltrated markedly with CD4+ and CD8+ T lymphocytes and macrophages by 5‐FC treatment, such that significant reduction or even complete regression of tumors was observed. These tumor‐free mice resisted subsequent rechallenge with wild‐type tumor. Conversely, when athymic nude mice were inoculated with a cell mixture containing CD‐transduced cells and parental cells at a ratio of 40:60, all developed tumors despite 5‐FC treatment. Our results indicate that gene therapy using the CD/5‐FC system can induce efficient anti‐tumor effects and protective immunity in immunocompetent mice but not in athymic immunodeficient mice, suggesting that the hosts immunocompetence may be a critical factor for achieving successful gene therapy against cancer. Int. J. Cancer 81:592–597, 1999.