Stewart Sell

Molecular pathogenesis of hepatocellular carcinoma in hepatitis B virus transgenic mice

Transgenic mice that overproduce the hepatitis B virus large envelope polypeptide and accumulate toxic quantities of hepatitis B surface antigen (HBsAg) within the hepatocyte develop severe, prolonged hepatocellular injury that initiates a programmed response within the liver, characterized by inflammation, regenerative hyperplasia, transcriptional deregulation, and aneuploidy. This response inexorably progresses to neoplasia. The incidence of hepatocellular carcinoma in this model corresponds to the frequency, severity, and age of onset of liver cell injury, which itself corresponds to the intrahepatic concentration of HBsAg and is influenced by genetic background and sex. Thus, the inappropriate expression of a single structural viral gene is sufficient to cause malignant transformation in this model. These results suggest that severe, prolonged cellular injury induces a preneoplastic proliferative response that fosters secondary genetic events that program the cell for unrestrained growth.

Liver Cancer Stem Cells

In an effort to review the evidence that liver cancer stem cells exist, two fundamental questions must be addressed. First, do hepatocellular carcinomas (HCC) arise from liver stem cells? Second, do HCCs contain cells that possess properties of cancer stem cells? For many years the finding of preneoplastic nodules in the liver during experimental induction of HCCs by chemicals was interpreted to support the hypothesis that HCC arose by dedifferentiation of mature liver cells. More recently, recognition of the role of small oval cells in the carcinogenic process led to a new hypothesis that HCC arises by maturation arrest of liver stem cells. Analysis of the cells in HCC supports the presence of cells with stem-cell properties (ie, immortality, transplantability, and resistance to therapy). However, definitive markers for these putative cancer stem cells have not yet been found and a liver cancer stem cell has not been isolated.

Cellular events during hepatocarcinogenesis in rats and the question of premalignancy.

The cellular, biochemical, and genetic changes that occur in the liver of rats exposed to chemical hepatocarcinogens are reviewed. Multiple new cell types appear in the liver of carcinogen-treated rats including foci, nodules, ducts, oval cells, and atypical hyperplastic areas. The application of phenotypic markers for these cell types suggests that hepatocellular carcinomas may arise from more than one cell type, including a putative liver stem cell that proliferates following carcinogen exposure. Study of DNA, RNA, and proteins produced by hepatocellular carcinomas and putative premalignant cells has so far failed to identify a gene or gene product clearly associated with the malignant or premalignant phenotype. Understanding the cellular lineage from normal cell through putative premalignant cell to cancer is critical to understanding the process of carcinogenesis. Application of new immunological (monoclonal antibody, transplantation) and molecular biological (gene cloning, oncogene identification) approaches to this problem holds promise that the process of hepatocarcinogenesis will be better known in the near future.

RELATIONSHIP OF THE BIOSYNTHESIS OF α‐FETOPROTEIN, ALBUMIN, HEMOPEXIN, AND HAPTOGLOBIN TO THE GROWTH STATE OF FETAL RAT HEPATOCYTE CULTURES*

AFP and albumin are produced by arginine-synthesizing fetal rat hepatocytes in vitro. AFP and hemopexin production are coupled to hepatocellular proliferation, whereas albumin and haptoglobin production are not. During the cell cycle, AFP is synthesized prior to S and released prior to M. AFP may play a role in regulation of hepatocellular growth through estradiol binding and modulation of the intracellular concentration of lipoprotein (VLDL).

Glutathione-S-transferase A3 knockout mice are sensitive to acute cytotoxic and genotoxic effects of aflatoxin B1

Aflatoxin B1 (AFB1) is a major risk factor for hepatocellular carcinoma (HCC) in humans. However, mice, a major animal model for the study of AFB1 carcinogenesis, are resistant, due to high constitutive expression, in the mouse liver, of glutathione S-transferase A3 subunit (mGSTA3) that is lacking in humans. Our objective was to establish that a mouse model for AFB1 toxicity could be used to study mechanisms of toxicity that are relevant for human disease, i.e., an mGSTA3 knockout (KO) mouse that responds to toxicants such as AFB1 in a manner similar to humans. Exons 3-6 of the mGSTA3 were replaced with a neomycin cassette by homologous recombination. Southern blotting, RT-PCR, Western blotting, and measurement of AFB1-N(7)-DNA adduct formation were used to evaluate the mGSTA3 KO mice. The KO mice have deletion of exons 3-6 of the mGSTA3 gene, as expected, as well as a lack of mGSTA3 expression at the mRNA and protein levels. Three hours after injection of 5 mg/kg AFB1, mGSTA3 KO mice have more than 100-fold more AFB1-N(7)-DNA adducts in their livers than do similarly treated wild-type (WT) mice. In addition, the mGSTA3 KO mice die of massive hepatic necrosis, at AFB1 doses that have minimal toxic effects in WT mice. We conclude that mGSTA3 KO mice are sensitive to the acute cytotoxic and genotoxic effects of AFB1, confirming the crucial role of GSTA3 subunit in protection of normal mice against AFB1 toxicity. We propose the mGSTA3 KO mouse as a useful model with which to study the interplay of risk factors leading to HCC development in humans, as well as for testing of additional possible functions of mGSTA3.

Cell kinetics of repair after allyl alcohol-induced liver necrosis in mice.

The cellular kinetics of repair and scarring which occurs after induction of periportal necrosis in mice by allyl alcohol were examined by histology and immunohistochemistry. Thirty‐six six‐week‐old female C57Bl/6J mice were injected intraperitoneally with two doses of allyl alcohol on day 0 and tissue sections were taken at various times and stained by haematoxylin and eosin or immunostained for proliferating cell nuclear antigen (PCNA), bile duct/oval cell marker A‐6, and DNA fragments (apoptosis). Within 6 hours, periportal necrosis was seen extending to produce large zones of confluent, pan‐acinar irregular necrosis, predominantly in the right and medial lobes with sparing of the left and caudate lobes. Restoration of liver mass was accomplished mainly by proliferation of mature hepatocytes in the surviving lobes of the liver (hyperplasia). In the right and medial lobes where necrosis was limited to the periportal zone, there was some, but much less, proliferation of small, oval periportal cells. The large necrotic zones in the right and median lobes shrank and were replaced by granulomatous inflammation. This cellular contribution of liver regeneration in the mouse was different from that previously reported in the rat and provides a means of inducing only a small proliferation of oval cells.

Topography of lymphocyte surface immunoglobulin using scanning immunoelectron microscopy.

The topographical distribution of the surface immunoglobulin (Ig) of rabbit blood lymphocytes has been determined using immunoelectron microscopic labels and scanning electron microscopy (SEM). Direct and indirect immunolatex labeling on aldehyde fixed cells reveals a loosely clustered pattern of surface Ig; a diffuse labeling pattern is not observed. Much of the immunolatex label appears to be specifically associated with surface microvilli; areas of smooth unlabeled membrane are conspicuous. Dense patchlike aggregations of surface Ig occur with multivalent labeling reagents on live cells at 0°C, but a sparse and loosely clustered pattern is observed on aldehyde fixed cells. It is postulated that the loosely clustered pattern observed represents the natural arrangement of surface immunoglobulin.

In vitro studies of the rabbit immune system: II. Functional characterization of rabbit T and B populations separated by adherence to nylon wool or lysis with anti-thymocyte serum and complement

Abstract Rabbit spleen lymphocytes with functions analogous to murine T and B cells have been separated by treatment with goat anti-rabbit thymocyte serum plus complement (ATS + C) or passage over columns of nylon wool (NW). ATS + C lysis of spleen cells from sheep erythrocyte (SRBC)-immunized rabbits removes 40–60% of the total cells, but 80–100% of the fully differentiated B cells (PFC) are recovered. Conversely, the primed spleen cells which are nonadherent to NW contain ~5–20% of the applied cells but only 0.05–1.0% of the applied PFC. Neither cell population is capable of producing an anti-SRBC PFC response when cultured individually. However, the two cell types complement each other and act synergistically to reconsitute the in vitro anti-SRBC PFC response. Thus, the rabbit spleen contains separable lymphocyte populations with functions equivalent to T and B cells as defined in the murine system.

In vitro studies of the rabbit immune system. IV. Differential mitogen responses of isolated T and B cells.

Abstract The mitogenic responses of separated rabbit lymphocyte populations functionally analogous to mouse T and B cells have been tested in vitro . Purified T cells were prepared by passage over nylon wool (NW) and purified B cells prepared by treatment with antithymocyte serum and complement (ATS + C). ATS + C kills 70% of peripheral blood lymphocytes (PBLs) and 50% of the spleen cells while passage over NW yields 40% of the applied PBLs and 5–23% of the applied spleen cells. NW-purified T cells from the spleen or PBLs respond fully to concanavalin A (Con A) but have a reduced response to phytohemaglutinin (PHA) and little or no response to goat anti-rabbit immunoglobulin (anti-Ig). PBLs that survive ATS + C (B cells) are stimulated by anti-Ig but not by Con A or PHA. B cells purified from spleen do not respond to Con A or PHA but will respond to anti-Ig under appropriate conditions. A full spleen B-cell response to anti-Ig required removal of Ig produced by the cultures that blocked anti-Ig stimulation. It is concluded that, for rabbit lymphocytes, Con A and PHA are primarily T-cell mitogens and that anti-Ig is primarily a B-cell mitogen. However, the mitogen response of unfractionated PBL or spleen cell populations indicates an overlap in reactivity. This could be due to cells sharing T and B properties, alteration of cell populations by the fractionation procedures used, or recruitment of one population in the presence of a mitogenic response of the other population.

Rat α-fetoprotein—III. Refinement of radioimmunoassay for detection of 1 ng rat α1F

Abstract The conditions for a competitive binding radioimmunoassay of rat α 1 -fetoprotein, sensitive and reproducible at the nanogram level, are described. Careful selection of the antiserum dilution and incubation times permits approximately a 10-fold increase in the sensitivity of the radioimmunoassay. The mean serum α 1 F concentration for normal adult male Buffalo rats is 0·026 ± 0·0012 (SEM) μ g/ml.