Nobuyoshi Kawakita

Expression of antigens related to apoptosis and cell proliferation in chronic nonsuppurative destructive cholangitis in primary biliary cirrhosis

The initial injury in primary biliary cirrhosis (PBC) is the destruction of portal bile ducts. Little information is available on apoptosis and cell proliferation in such bile ducts, so we used immunohistochemical techniques to locate molecules related to apoptosis [Fas antigen, Lewis Y antigen (BM1/JIMRO), and bcl-2 protein] and to cell proliferation (proliferating cell nuclear antigen, PCNA) in 21 patients with PBC. In addition, nick-end labelling was done to locate DNA fragmentation. The expression of these molecules in chronic nonsuppurative destructive cholangitis (CNSDC) was examined. Cell death and PCNA expression were both found in portal bile ducts affected by CNSDC in 7 of the 13 CNSDC patients examined. Fas antigen was found on the plasma membrane and rough endoplasmic reticulum of bile-duct cells with CNSDC in the frozen sections of all 6 patients with CNSDC out of the 9 patients inspected, and this antigen was found also in bile-duct cells without CNSDC in 2 of these 9 patients. It was not found in anatomically normal liver (from 2 patients with Gilberts disease). The Lewis Y antigen was found in bile ducts with CNSDC and in proliferated ductules in all 16 patients examined. No bcl-2 protein was found in any bile-duct or ductule cells, but it was found in the cytoplasm of lymphocytes surrounding or invading CNSDC. DNA fragmentation was found in the nuclei of bile-duct cells with CNSDC by nick-end labelling. Our study indicated that Fas-mediated apoptosis might be involved in CNSDC, but that bcl-2 protein seems to participate less than Fas. Although the Lewis Y antigen was found in many bile ducts, the relationship between the antigen and apoptosis remains unknown because there was no evidence that this antigen mediates apoptosis.

Identification and fine structure of proliferating hepatocytes in malignant and nonmalignant liver diseases by use of a monoclonal antibody against DNA polymerase alpha

To analyze the process of liver regeneration and the initiation of hepatocellular carcinoma (HCC), we studied histochemically the morphologic features of proliferating parenchymal cells stained for DNA polymerase alpha (DPA), in 31 patients with various diseases, by use of a monoclonal antibody against DPA. In specimens from patients with acute viral hepatitis with confluent necrosis, most stained hepatocytes were small, with basophilic cytoplasm, and were located next to the necrotic areas. Under electron microscopy, stained granules were seen in the nucleus. Most stained hepatocytes had immature organelles. In specimens from patients with cirrhosis of the liver, the number of stained hepatocytes greatly differed in different pseudolobules. In specimens from patients with adenomatous hyperplasia, stained hepatocytes, mostly small and basophilic, were found diffusely; electron microscopy showed slightly indented nuclei with a few organelles and less condensed chromatin than normal. In specimens from patients with HCC, most stained cancer cells were small and basophilic; electron microscopy showed indented nuclei with a few organelles and less than normal condensed chromatin. Staining showed that during regeneration, immature hepatocytes reentered the cell cycle and repaired a large necrotic area. It was conceivable that in the initiation of HCC, some small hepatocytes with indented nuclei and less condensed chromatin might become HCC cells.

Induction of apoptosis in a human hepatocellular carcinoma cell line by a neutralizing antibody to transforming growth factor-α

A cell line derived from a Japanese man with hepatocellular carcinoma was established in culture and designated OCUH-16. The cell line has the morphological and chromosomal features of hepatocellular carcinoma cells and has a short doubling time (≈33 h). OCUH-16 cells were shown to express transforming growth factor-α, (TGF-α) in addition to albumin, DNA polymerase-α, c-JUN, and the retinoblastoma gene product. Electron microscopy revealed TGF-α immunoreactivity associated with the cell membrane, but TGF-α was not detected in medium conditioned by OCUH-16 cells by enzyme-linked immunosorbent assay. Reverse transcription and polymerase chain reaction analysis revealed the presence of TGF-α messenger RNA in these cells. Culture of OCUH-16 cells in the presence of a neutralizing antibody to TGF-α inhibited cell proliferation and induced many cells to undergo apoptosis (programmed cell death). These observations suggest that endogenous TGF-α is necessary for OCUH-16 cell growth.

Expression of the retinoblastoma gene product in human hepatocellular carcinoma

Using the mouse anti-human retinoblastoma gene product (pRB) monoclonal antibody, PMG3-245, pRB was detected immunohistocytochemically in human hepatocellular carcinoma (HCC) tissues and a human HCC cell line, designated OCUH-16, recently established in our laboratory. This antibody reacted with human pRB and yielded a single band of approximately 110 kd from cultured OCUH-16 cells. The granules that stained for pRB were found mostly in the HCC cell nuclei, with a few granules observed in the rough endoplasmic reticulum by electron microscopy. Most of the stained granules were located in the euchromatin-rich areas. The percentage of OCUH-16 cells that expressed pRB or DNA polymerase alpha (DNA-PA) decreased over time as the number of OCUH-16 cells increased. The number of HCC cells that stained for pRB in the biopsy specimens from 11 patients varied and pRB expression was well maintained in early and advanced HCC. The level of pRB expression did not correlate with the differentiation of HCC cells or the clinical prognosis. The expression of pRB statistically correlated with that of DNA-PA (P < .01; r = .92). Some sinusoidal cells also stained for pRB. These findings imply that large deletions in the pRB gene are rare in the initiation or promotion of HCC. The correlation between pRB and DNA-PA may suggest that stained pRB participates in the proliferation of both HCC and non-HCC cells.

An analysis of proliferating cells in biopsy specimens from patients with small hepatocellular carcinoma

The proliferation of neoplastic and nonneoplastic heap‐tocytes is caused by various humoral growth factors with autocrine and paracrine mechanisms, and the proliferative activity of both hepatocytes and nonhepatocytic cells contributes to neoplastic growth. The authors attempted to detect various kinds of proliferating cells immunohis‐tochemically in small hepatocellular carcinoma (HCC) using a monoclonal antibody against DNA polymerase alpha. Most of the HCC cells that stained for this enzyme were small, had basophilic cytoplasm with poorly developed organelles, and aggregated to form clusters distributed randomly within cancer nests. Nonhepatocytic cells also were stained, including some endothelial cells, Kupffers cells, macrophages, and lymphocytes. Fat‐storing cells were not stained. The number of stained sinusoidal (capillary) cells decreased in this order: Kupffers cells and macrophages, endothelial cells, and fat‐storing cells. Nonhepatocytic cells, including lymphocytes, proliferated more actively in areas with actively growing HCC cells than in those with quiescent cancer cells. The relationship between stained HCC cells and stained sinusoidal cells was clearly defined; the correlation coefficient was 0.97. These findings suggest the possibility of a relationship between the proliferative activity of neoplastic hepatocytes and that of sinusoidal cells, including lymphocytes. Cancer 1992; 69:2433‐4439.

Analysis of proliferating biliary epithelial cells in human liver disease using a monoclonal antibody against DNA polymeraseα

The proliferative activity and ultrastructural characteristics of proliferating biliary epithelial cells were analysed immunohistocytochemically in 39 biopsied liver specimens from patients with acute viral hepatitis, chronic hepatitis and liver cirrhosis using a monoclonal antibody against DNA polymerase α (DNA-PA). In acute viral hepatitis with perivenular confluent necrosis, proliferation of typical bile ducts was found frequently in portal areas. In chronic aggressive hepatitis and cirrhosis, ductular proliferation of both typical and atypical forms was found in enlarged portal and periportal areas and in confluent necrotic areas. The number of proliferating biliary epithelial cells that stained positive for DNA-PA was small. There were very few positively stained cells in atypical bile ducts in confluent necrotic areas of cirrhosis. Atypical bile ducts seen in chronic aggressive hepatitis, cirrhosis and acute hepatitis with confluent necrosis were positively stained for both cytokeratins 8 and 19. In cirrhosis, the number of stained biliary epithelial cells in typical bile ducts was larger than the number of such cells in atypical bile ducts (P< 0.01). By electron microscopy, the cells positively stained for DNA-PA were mostly so-called clear cells with irregular nuclei containing coarse nucleoplasm, and a few small cells with scanty cytoplasm and few organelles.

Detection of the preneoplastic lesions of small hepatocellular carcinoma in cirrhotic livers

To identify the preneoplastic lesions of hepatocellular carcinoma and the fine structure of preneoplastic hepatocytes, we studied proliferative conditions in cirrhosis of the liver. In all, 46 foci of cellular alteration (FCA), three regions of adenomatous hyperplasia (ADH), and 21 small hepatocellular carcinomas (sHCC) were studied by published criteria for sHCC and by the proliferative activity of the lesions as examined with monoclonal antibodies against DNA polymerase alpha and proliferating cell nuclear antigen. The four patients with FCA composed of basophilic hepatocytes were classified by the criteria as having sHCC; cells had features similar to those of sHCC. Two of these four patients with FCA were found to have HCC several years later. The number of hepatocytes stained for proliferating cell nuclear antigen was 72 and 81 per 1000 hepatocyte nuclei in the two patients who developed HCC. In one of the three patients with ADH, a sHCC was found 1 year later, and dysplastic hepatocytes from the region of ADH in this patient had features similar to those of HCC cells by light and electron microscopy. In this patient, the number of hepatocytes stained for DNA polymerase alpha was 452 per 1000 nuclei. Therefore, FCA and ADH might be preneoplastic lesions of sHCC in cirrhosis of the liver. Preneoplastic hepatocytes seem to be small cells with basophilic cytoplasm, with a large nucleus to cytoplasm ratio, finely indented nuclei with a smaller amount of condensed chromatin than normal, and poorly to moderately developed organelles.

A murine model of NKT cell-mediated liver injury induced by alpha-galactosylceramide/d

Natural killer-T (NKT) cells are rich in the liver. However, their involvement in liver injury is not fully understood. We developed here a new murine model of NKT-cell-activation-associated liver injury, and investigated a role of tumor necrosis factor alpha (TNF-alpha) and Fas in pathogenesis. We injected intraperitoneally alpha-galactosylceramide (alpha-GalCer), an NKT-cell stimulant, into D-galactosamine (GalN)-sensitized mice. Survival rate, pathological changes of the liver, and plasma concentrations of cytokines were studied. Alpha-GalCer/GalN administration gave a lethal effect within 7 h, making pathological changes such as massive parenchymal hemorrhage, hepatocyte apoptosis, sinusoidal endothelial cell injury, and close apposition of lymphocytes to apoptotic hepatocytes. Anti-NK1.1 mAb-pretreated mice and Valpha14NKT knock out (KO) mice did not develop liver injury. Tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) were elevated at 4 h in the plasma. These cytokines were produced by hepatic lymphocytes as demonstrated by in vitro stimulation with alpha-GalCer. The lethal effect was suppressed in TNF-alpha KO mice, TNF receptor-1 KO mice, and lpr/lpr (Fas deficient) mice, whereas it was not in IFN-gamma KO mice. These results indicate that the present liver injury is characterized by parenchymal hemorrhage and hepatocyte apoptosis, and mediated by TNF-alpha secretion and direct cytotoxicity of alpha-GalCer-activated NKT cells.

Induction of proto-oncogene c-jun product in rat liver after partial hepatectomy

The product of the proto-oncogene c-jun helps to regulate gene expression by interacting directly with specific target DNA sequences, I and comparison of structural and immunological findings showed that the Fos-associated protein p39 is the c-jun product. 2 We used immunohistochemistry with a polyclonal antibody against the protein coded for by c-jun to find if levels of the gene product increased in rat liver after partial hepatectomy (PH) and, if so, to identify its location. PH was done by the method of Higgins and Anderson. The remaining liver was removed 2, 4, or 6 hr later. Control liver was obtained from rats without PH. The livers were fixed with Zambonis solution, frozen, and sectioned. Rabbit polyclonal antibody against the c-jun product (Oncogene Science) was used as the first antibody. The avidin-biotin peroxidase complex method was used for the immunohistochemical staining. There was little if any staining of control liver (Fig. IA). In liver obtained at 2 hr, the gene product was stained diffusely in the nuclei of the hepatocytes (Fig. IB). Staining for the gene product was less dense at 4 hr, and there was little staining at 6 hr. However, the gene product appeared at slightly different times in each rat after PH. In vitro, mRNA coded for by c-jun is induced in mouse 3T3 cells by serum, platelet-derived growth factor, and fibroblast growth factor. 3 Also, epidermal growth factor stimulates the transcription of c-jun. ~ Our findings showed that the product of c-jun was expressed transiently in the nuclei of hepatocytes during liver regeneration after PH. c-myc and c-ras are transcribed in the liver after PH, also. The relationships among these oncogenes remain unclear, as do their functions.