Katsuhiko Enomoto

Sequential Decrease in Tight Junctions as Revealed by 7H6 Tight Junction-associated Protein during Rat Hepatocarcinogenesis

A sequential decrease in the number of hepatocyte tight junctions during the course of rat hepatocarcinogenesis was demonstrated by immunohistochemistry with a new 7H6 monoclonal antibody generated in our laboratory. Semiqnantitative analysis by confocal laser scanning microscopy revealed that the expression of 7H6 antigen was reduced in hyperplastic foci, hyperplastic nodules and hepatocellular carcinomas (HCC) to 43%, 28% and 25%, respectively, compared to corresponding normal liver tissues. 7H6 antigen was scarce in HCC with a trabecular pattern, whereas it was expressed intensely at the apical and basolateral membrane of HCC with a glandular pattern. Immunoblot analysis of 7H6 expression in hepatocellular carcinomas showed a decrease roughly coincident with that shown by immunohistochemistry. These results indicated, for the first time, that tight junctions decrease progressively during carcinogenesis, leading to disruption of cellular polarity and cellular adhesiveness.

High Sensitivity of LEC Rats with Chronic Hepatitis to Hepatocarcinogenesis: Decreases in Unscheduled and Replicative DNA Synthesis of the Hepatocytes

We carried out the following three experiments to clarify the mechanism of hepatocarcinogenesis in Long‐Evans Cinnamon (LEC) rats. (1) Sensitivity to diethylnitrosamine (DEN): LEC rats (8 and 25 weeks old) without and with hepatitis and age‐matched F344 rats were administered an intraperitoneal injection of a low dose of DEN. Eight weeks after the injection, the numbers of glutathione‐S‐transferase placental‐form (GST‐P)‐positive foci in the 33‐week‐old LEC rat liver were significantly higher than those in the livers of the other three groups of rats. (2) Potential for unscheduled DNA synthesis (UDS): Isolated hepatocytes of 25‐week‐old LEC rats with chronic hepatitis showed about one‐third the level of UDS induced by UV irradiation, as compared to that of age‐matched F344 rats, while no significant difference was found between the UDS of isolated hepatocytes of 8‐week‐old LEC rats and age‐matched F344 rats. (3) Potential for proliferation: Isolated hepatocytes from 8‐week‐old LEC rats responded well to epidermal growth factor (EGF) in culture, to almost the same degree as F344 rat hepatocytes, while a remarkable decrease in the responsiveness of hepatocytes isolated from 25‐week‐old LEC rats to EGF was found. These results suggested that LEC rat hepatocellular carcinoma could be naturally initiated after the onset of hepatitis by carcinogens contaminating food and the environment, probably due to the reduction of DNA repair activity, after which initiated hepatocytes selectively proliferate in response to growth stimuli endogenously produced as a result of continuous loss of hepatocytes (chronic hepatitis), because of a decrease in growth activity of non‐initiated hepatocytes.

High Sensitivity of LEC Rats to Carcinogens Based Upon a Short-Term Carcinogenicity Assay

A high incidence of spontaneous liver tumors has been reported in the LEC rat, a new mutant strain, which has been recently established at the Center for Experimental Plants and Animals of Hokkaido University.

Expression of multiple connexins is differentially modulated during multistage hepatocarcinogenesis

We report changes in expression and localization of multiple connexins (C×) during chemical hepatocarcinogenesis in rats and mice. During rat hepatocarcinogenesis, the expressions of C×26 and C×32 were differentially regulated. In mouse hepatoblastomas, a substantial number of C×43-positive gap junctions were found, whereas the expression of both C×26 and C×32 was greatly reduced.

Replicative and Unscheduled DNA Synthesis of LEC Rat Hepatocytes: Relevance to Natural Development of Hepatocellular Carcinoma

The LEC rat is a new mutant inbred strain which suddenly displays coagulative necrosis of hepatocytes at around 4 months after birth [1], for which a single autosomal recessive gene (hts gene, [2]) is responsible. Those LEC rats surviving with chronic hepatitis eventually develop hepatocellular carcinomas as they become old [1, 3–5]. Since this hepatitishepatocellular carcinoma sequence is quite similar to the development of human liver cancer, LEC rats are expected to serve as an excellent animal model for the study of human hepatocarcinogenesis.

The Multistep Nature of Spontaneous Liver Cancer Development in the LEC Rat: Analysis of Incidence and Phenotype of Preneoplastic and Neoplastic Liver Lesions

The LEC rat has been established and characterized as a new mutant strain which spontaneously develops hepatitis and liver cancer at an extremely high incidence. The LEC rat manifests severe hereditary hepatitis with systemic jaundice and hemorrhagic tendency at around 4 months after birth. About 30%–40% of the rats die during the period of hepatitis because of submassive or massive necrosis of hepatocytes. The remaining rats recover from severe hepatitis and survive more than 1 year. Histopathological examination of the livers of the long-survived LEC rats shows development of preneoplastic and neoplastic lesions at a high incidence with continued hepatocyte death and regeneration (prolonged hepatitis) [1, 2].

Sensitivity of LEC Rats to the Hepatotoxic Effects of D-Galactosamine

Spontaneous hepatitis in the LEC rat is characterized by hyperbilirubinemia, increased levels of serum glutamic-oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) in laboratory examination and spotty coagulative necrosis of single hepatocyte without inflammatory cell response [1, 2]. Although a single autosomal recessive gene (hts) has been shown to be responsible for the hepatitis, the mechanism(s) of hepatocyte necrosis in the LEC rat remains obscure. The histological features of the liver reveal a certain resemblance between the hepatitis of LEC rats and the liver necrosis induced by the administration of D-galactosamine (GalN).

Altered Oncogene Expression in Hepatocellular Carcinomas Developing Spontaneously in LEC Rats

LEC rats spontaneously suffer hepatitis at around 4 months after birth, enter the chronic phase of hepatitis, and spontaneously develop hepatocellular carcinomas (HCCs) between 1 – 1.5 years after birth [1]. Although dominant oncogene activation in chemically induced HCCs in rats is generally understood to be relatively rare, for the LEC rat, in which an endogenous causative agent appears to be involved, the situation remains unclear. Ha-ras activation has been found with a very high frequency in both chemically induced and spontaneously induced liver carcinomas of B6C3 F1 mice [2–5], but, with the exception of those induced by aflatoxin B1 [6, 7], any type of activated ras was only rarely observed in chemically induced rat liver tumors [4, 8, 9]. We examined HCCs in LEC rats for Ha-, Ki- and N-ras gene mutations. The Polymerase chain reaction (PCR) was adopted for amplifying discrete specific DNA fragments [10, 11], and the sequences were determined directly.