Hyam L. Leffert

IKKβ Couples Hepatocyte Death to Cytokine-Driven Compensatory Proliferation that Promotes Chemical Hepatocarcinogenesis

IkappaB kinase beta (IKKbeta), required for NF-kappaB activation, links chronic inflammation with carcinogenesis. We investigated whether IKKbeta is involved in chemically induced liver cancer, a model not involving overt inflammation. Surprisingly, mice lacking IKKbeta only in hepatocytes (Ikkbeta(Deltahep) mice) exhibited a marked increase in hepatocarcinogenesis caused by diethylnitrosamine (DEN). This correlated with enhanced reactive oxygen species (ROS) production, increased JNK activation, and hepatocyte death, giving rise to augmented compensatory proliferation of surviving hepatocytes. Brief oral administration of an antioxidant around the time of DEN exposure blocked prolonged JNK activation and compensatory proliferation and prevented excessive DEN-induced carcinogenesis in Ikkbeta(Deltahep) mice. Decreased hepatocarcinogenesis was also found in mice lacking IKKbeta in both hepatocytes and hematopoietic-derived Kupffer cells. These mice exhibited reduced hepatocyte regeneration and diminished induction of hepatomitogens, which were unaltered in Ikkbeta(Deltahep) mice. IKKbeta, therefore, orchestrates inflammatory crosstalk between hepatocytes and hematopoietic-derived cells that promotes chemical hepatocarcinogenesis.

Hepatocyte IKKβ/NF-κB Inhibits Tumor Promotion and Progression by Preventing Oxidative Stress-Driven STAT3 Activation

The NF-kappaB activating kinase IKKbeta suppresses early chemically induced liver tumorigenesis by inhibiting hepatocyte death and compensatory proliferation. To study IKKbetas role in late tumor promotion and progression, we developed a transplant system that allows initiated mouse hepatocytes to form hepatocellular carcinomas (HCC) in host liver after a long latency. Deletion of IKKbeta long after initiation accelerated HCC development and enhanced proliferation of tumor initiating cells. These effects of IKKbeta/NF-kappaB were cell autonomous and correlated with increased accumulation of reactive oxygen species that led to JNK and STAT3 activation. Hepatocyte-specific STAT3 ablation prevented HCC development. The negative crosstalk between NF-kappaB and STAT3, which is also evident in human HCC, is a critical regulator of liver cancer development and progression.

IKKβ Is Required for Prevention of Apoptosis Mediated by Cell-Bound but Not by Circulating TNFα

Abstract IκB kinase β (IKKβ) is required for NF-κB activation and suppression of TNFα-mediated liver apoptosis. To investigate how IKKβ suppresses apoptosis, we generated hepatocyte-specific Ikkβ knockout mice, Ikkβ Δhep , which exhibit little residual NF- κB activity but are healthy with normal liver function. Unexpectedly, Ikkβ Δhep mice are slightly more sensitive than controls to LPS-induced liver apoptosis but are highly susceptible to liver destruction following concanavalin A (ConA)-induced T cell activation. Unlike LPS, a potent inducer of circulating TNFα, ConA exerts cytotoxic effects through cell-bound TNFα, which activates type 1 and 2 TNF receptors (TNFR). While TNFR2 does not contribute to NF-κB activation, it is important for ConA-induced JNK activation, which is augmented by the absence of IKKβ. Using JNK-deficient mice we show that JNK is required for ConA-induced liver damage. Thus, the antiapoptotic function of IKKβ, which is most critical in situations that involve cell-bound TNFα, is mediated partially through attenuation of JNK activity.

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.

GROWTH CONTROL OF DIFFERENTIATED ADULT RAT HEPATOCYTES IN PRIMARY CULTURE

Much of the current knowledge about mammalian cell growth regulation comes from physiological studies of liver regeneration, maturation and carcinogenesis. To simplify the search for cellular and extracellular factors governing these phenomena, we have experimented since 197 1 with highly differentiated primary “monolayer” cultures of fetal and adult rat hepatocytes. These cells do not live in a threedimensional organ structure. They do not “see” gas tensions that normally perfuse the liver. They neither feed from nor excrete into a bathing fluid that continuously exchanges with an uninterrupted nutrient flow. And yet, with simple manipulations, such cells thrive more than a month in culture. This is seen in FIGURE 1 (bottom panel) for adult hepatocytes. Epithelial systems like these are powerful tools because they simulate proliferative and developmental events occurring in the animal.’-5 Work with the adult system suggests that transiently increased Na+ fluxes mediate early mitogenic actions of chemically-defined growth media.6 These fluxes, like those initiating DNA synthesis in fertilized eggs or electrical impulses in neurons, may comprise the first of two parts of a general membrane signalling process controlling eukaryotic cell proliferation7 (see also, Growth Regulation by Ion Fluxes, this Annals, volume 339). The second part, analogous to synaptic events that transduce nerve impulses and facilitated in hepatocytes by the pancreatic peptides glucagon and insulin (FIGURE 2), may involve a Ca++ flux linked to a burst of cyclic AMP synthesis.’ Recent findings of abnormally high Na+ levels in hepatomas implicate a defective Na+ flux system in the pathophysiology of hepatic ~ a n c e r . ~ There is evidence for transformation-related defects in the Ca++/cAMP “couple” as well.” But little is known about the mechanism of liver growth-control loss, which occurs spontaneously with aging or after detectable exposure to environmental carcinogens. In this report, the properties of “normal” adult hepatocytes cultured in this laboratory will be reviewed. New observations linking early ionic signalling events with “membrane potential” (A*) and “intracellular pH” (pHi) changes will be described. Lastly, regarding the problem of chemical carcinogenesis, the results of specific physicochemical and biological interactions of the hepatoprocarcinogen Nacetyl-2-aminofluorene (AAF) with adult cells will be summarized. If parenchymal cells are tumor precursors, these findings may provide information to transform cultured hepatocytes that truly resemble normal liver parenchyma into malignant cells.

[47] Liver cells

Publisher Summary This chapter deals with liver cells. Methods for establishing primary monolayer fetal and adult rat hepatocyte cultures are presented in the chapter. Techniques are emphasized rather than rationale or methodological validation. Fetal livers are obtained from pregnant rats 19–21 days in gestation; 14–19-day-old fetuses can be used but hepatocyte yields are reduced. Adult livers are obtained from rats, 150–300 g body weight, and fed standard Purina Chow and water ad libitum. Fetal bovine serum is purchased from standard suppliers and is pretested to ensure its suitability. Typical differentiated properties of primary monolayer rat liver cell cultures are also summarized in the chapter. The chapter discusses media and digestion buffer construction, cell isolation and plating, and cell counting. Functional properties of primary monolayer rat liver cell cultures are tabulated in the chapter.

Construction of a fusion protein between protein A and green fluorescent protein and its application to Western blotting

Aequorea green fluorescent protein (GFP) and protein A were fused and expressed in Escherichia coli. The fluorescent native fusion protein (PA‐GFP) migrated at 47 kDa in SDS‐PAGE. However, the non‐fluorescent denatured PA‐GFP migrated at 57 kDa which corresponds to the theoretical molecular mass. Although the reason(s) for this mobility shift between fluorescent and non‐fluorescent molecules remains unclear, the small ring structure within the native molecules may affect their mobility. The cell extract, prepared from an E. coli strain producing PA‐GFP, was used in Western and dot blots. The sensitivity and specificity of the PA‐GFP detection were sufficient for rapid and easy screening.

IONIC EVENTS AT THE MEMBRANE INITIATE RAT LIVER REGENERATION

Rat liver regeneration is a prototype for studying the regulation of animal cell proliferation.’ A series of hepatocyte culture and animal experiments has vindicated the longstanding hepatotrophic theory by providing unequivocal evidence that regeneration is hormonally Physiological regulation by at least four, possibly even six blood-borne peptides has been implicatdcg Two originate from the pancreatic islets (insulin and glucagon); and two arise from the thyroparathyroid complex (calcitonin [see Whitfield er al., this volume] and parathyroid hormone). The others belong to a class of “insulin-like” substances produced by the liver (somatomedin-c) and by the gastrointestinal tract (epidermal growth factor-urogastrone-EGF). To understand how peptides might initiate liver regeneration, wc have been studying how their interactions promote the entry of “resting” hepatocytes into the DNA-synthetic (or Sphase) of the cell “cycle.”’* 2. ‘O We also are trying to identify rapidly activated cellular processes that cause the eventual growth response. Our results strongly suggest that, in response to peptides, one (perhaps the earliest) event needed to stimulate liver regeneration involves increased activation of membrane Na+ influx systems. Preldnary accounts of this hypothesis and its supporting evidence have appeared?. ”* ”

Amiloride blocks cell-free protein synthesis at levels attained inside cultured rat hepatocytes

Abstract Amiloride, a passive Na+ influx inhibitor, lowers initial rates and plateau levels of [35S]met uptake into proteins in cell-free rabbit reticulocyte lysates (ID50∽0.4 mM). Isolated hepatocytes take up amiloride through a saturable (Km∽0.02 mM; Vmax∽1.43 nmol/ 106 cells/min) Na+-dependent process. Similar temperature dependent uptake occurs in cultured hepatocyte monolayers. In chemically defined media, under growth reinitiation conditions, amiloride lowers overall rates of cellular protein and albumin synthesis (ID50∽0.4 and ∽0.028 mM, respectively). Amiloride concentrations (0.02 mM) that half-maximally inhibit reinitiation of hepatocyte DNA synthesis reach, within 30 min, cellular levels (∽0.14 mM) that block reticulocyte lysate protein synthesis by 25%. These findings complicate interpretations, from studies in many eukaryotic systems, of cause and effect between mitogen-activated membrane Na+ influxes and the reinitiation of DNA synthesis.

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).