The protein expression and interactions of the ErbB receptors were examined in different liver proliferation models in vivo and in vitro, including ontogeny and regeneration following partial hepatectomy.

Expression and tyrosine phosphorylation status of specific ErbB proteins were studied by immunologic methods.

The epidermal growth factor receptor, ErbB2, and ErbB3 were the only ErbB proteins detected in the liver parenchyma on embryonic day 19. ErbB2 disappeared by the third week after birth and could not be appreciably induced in the adult animal by partial hepatectomy. ErbB2 was also detected in multipotent stem (RLE) and hepatoma (H4IIe) cell lines as well as in fetal, but not adult, hepatocyte cultures. Only epidermal growth factor receptor and ErbB3 were detected in adult liver, and both showed circadian variation in protein expression. ErbB4 was not detected in any model. Patterns of ligand-induced ErbB phosphorylation differed between fetal and adult hepatocytes.

Complex and independent programs regulate the ErbB receptors, with implications for differential cell signaling in hepatic development and regeneration.

We examined the effects of various peroxisome proliferators (PPs) such as the hypolipidaemic agents clofibric acid (CLO), bezafibrate (BEZA), ciprofibrate (CIPRO) and nafenopin (NAFE) and the plasticizer di-(2-ethylhexyl)phthalate (DEHP) on peroxisomal enzyme activities, apoptosis and DNA synthesis in rat FaO and human HepG2 hepatoma cell lines. Both growing and confluent cultures were treated with PPs (250 microM) for 48 or 72 h. In accordance with our previous observations in PP-treated primary hepatocyte cultures of rat and human origin, the various PPs increased peroxisomal enzyme activities in rat FaO cells but not in human HepG2 cells. PPs strongly induced apoptosis in FaO cells. They did not affect TGFbeta-induced apoptosis, with the exception of DEHP and NAFE, respectively blocking and increasing induced apoptosis in confluent cultures. Moreover, PPs produced a minor, but significant, decrease in DNA synthesis in FaO cells. PPs also decreased DNA synthesis in growing HepG2 cells, and CLO, CIPRO and NAFE induced apoptosis in confluent HepG2 cultures. This is in opposition with the effects of PPs on primary hepatocyte cultures, i.e. inhibition of both spontaneous and TGFbeta-induced apoptosis and increases in DNA synthesis in rat hepatocytes, and unchanged mitosis-apoptosis balance in human hepatocytes.

The liver clears circulating plasma-kallikrein through a receptor-mediated endocytosis process: an initial fast phase is followed by a slow exponential phase.

To determine whether the clearance rate of plasma-kallikrein is affected during liver regeneration, we perfused isolated rat livers with rat plasma-kallikrein (rPK) at 0, 1, 2, 3 and 7 days after partial hepatectomy or sham operation.

Liver regeneration was followed by the expression of the proliferating-cell nuclear antigen (PCNA) labeling index. The serum concentration of alpha2-macroglobulin, an acute phase protein in rats, was measured. At day 1, the fast phase of rPK clearance rate increased in hepatectomized rats when compared with day 0 (4.9+/-0.4 and 3.7+/-0.4 mU/g liver min, p<0.05). However, at day 2, the rPK fast phase clearance rate dropped significantly (2.6+/-0.2, p<0.05), when compared with day 1. No difference was found among the sham groups at different days of hepatectomy. These changes seem to be independent of the acute phase reaction. The regenerative liver weight increased continuously during the observation period. PCNA expression increased significantly after hepatectomy, with maximal PCNA-labeling indices at days 1 and 2, declining thereafter.

The rPK fast phase clearance rate changes during liver regeneration, with a zenith occurring when PCNA labeling index is maximal (day 1) and a nadir occurring at the mitotic phase (day 2).

We previously reported that CO2 laser incisions in carcinogen-initiated fields promoted cancer development and caused release of growth factors. Here we examined the quantitative and additive properties of this tumor-promoting event and examined whether this promotion could be nullified by treatment with a bombesin antagonist, which down-regulates epidermal growth factor receptors. The model used for cancer promotion was the hamster buccal cheek pouch that had been treated with a carcinogen (9,10-dimethyl-1,2-benzanthracene) for 6 weeks, producing premalignant lesions. These lesions would evolve into a cancer eventually without further treatment. Promotion was measured both by increased fluorescence in response to systemically administered Photofrin, measured noninvasively using an in vivo fluorescence photometer, and by the timing of appearance of clinical tumors. Laser incisions (0-3) were made into the hamster cheek 1 week apart, or three incisions were done 1 day apart. Another group of animals received bombesin antagonist RC-3095 for 4 weeks during the time incisions were made, again measuring promotion. Laser incisions 1 week apart produced additive promotion, whereas three incisions 1 day apart were not statistically different from the group receiving only one incision. RC-3095 treatment completely eliminated the promoting effects of incision and totally stopped promotion for the 4-week period of treatment. After discontinuing treatment with RC-3095, lesion progression resumed at the untreated control rate. This work confirms that the promoting event of a laser incision follows a comparable time course to release of growth factors after such an incision and that it can be eliminated by treatment with bombesin antagonists.

We have shown previously that approximately 1 in 10,000 primary hepatocytes isolated from untreated rats undergo clonal growth in soft agar in vitro in response to the synergistic action of nafenopin, a peroxisome proliferator (PP) and epidermal growth factor (EGF), a naturally occurring liver growth regulator. Here, we demonstrate that prior treatment of the animals with the genotoxic hepatocarcinogen diethylnitrosamine (DEN) caused a dose-dependent increase in soft agar colony numbers formed in vitro. These data suggest that the colony assay may offer a method of detecting in vitro hepatocytes transformed in vivo by DEN. It is known that rats treated with DEN develop enzyme altered foci prior to the development of tumours. The majority of these foci express high levels of gamma-glutamyl transpeptidase (GGT). However, foci promoted by PPs do not show this increased enzyme activity. In the present study, the colonies we have generated in vitro mimicked this pattern since the majority (approximately 80%) of the spontaneous colonies expressed GGT whereas colonies promoted by the synergistic action of nafenopin and EGF were mainly (75%) GGT negative. The proportion of colonies positive for GGT were similar using either hepatocytes isolated from control or from DEN-initiated rats. Further studies are required to assess if the hepatocytes selected for clonal expansion by this EGF/nafenopin regime reflect the presumed pre-neoplastic cells induced by genotoxin in vivo and associated with an increased propensity to cancer.

The influence of the tumor promoter 2-acetylaminofluorene (2-AAF) on cell proliferation and on the epidermal growth factor receptor (EGFR) system was assessed in normal and nodular rat livers. DNA replication in vivo was inhibited below the detection level after 8d of dietary 2-AAF treatment of previously unexposed rats. The 2-AAF-induced growth inhibition was accompanied by downregulation of the number of epidermal growth factor (EGF)-binding sites and decreased levels of EGFR transcripts, whereas no changes in the transforming growth factor-alpha (TGF-alpha) mRNA levels were observed. The persistent liver nodules generated by intermittent 2-AAF-feeding had a 30- to 35-fold higher replicating cell fraction than normal liver. Treatment with 2-AAF in vivo reduced the replicating cell fraction to one third in nodules after 14 d of 2-AAF treatment. The initial EGFR mRNA levels and number of EGF binding sites in nodules before 2-AAF administration was about 605 that of control livers and was slightly reduced by 2-AAF feeding. The levels of EGFR mRNA after 14 d of 2-AAF feeding were thus similar in the nodules and in the 2-AAF-treated control livers, whereas the fraction of proliferating cells in nodules after the 2-AAF treatment was much larger than in normal liver. The TGF-alpha mRNA level in the nodules was found to be 1.4-fold and in malignant hepatomas 1.7-fold the level in normal liver. Primary hepatocytes isolated from control livers were four to five times more sensitive to replicative stimulation with EGF than with TGF-alpha, whereas nodular cells responded at lower concentrations than control cells and equally well to both EGF and TGF-alpha. We conclude that the decreased amounts of EGFR in the nodular cells with respect to proliferative stimulation could be more than compensated for by elevated synthesis of TGF-alpha combined with an increased TGF-alpha sensitivity. Collectively, these changes implicate TGF-alpha in sustaining cell proliferation during chemically induced rat liver carcinogenesis.

Biochemical membrane alterations appearing during the process of chemical carcinogenesis are described. Emphasis is put on membrane composition, structure, and biogenesis. In this presentation the knowledge gained from experimental studies of liver and skin in the process of cancer development is acknowledged. Important biochemical changes have been reported in lipid composition, fatty acid saturation, constitutional enzyme expression, receptor turnover and oligomerization. Functional consequences of the altered membrane structure is discussed within the concepts of regulation of cell proliferation, regulation of membrane receptor expression, redox control, signal transduction, drug metabolism, and multidrug resistance. Data from malignant tumours and normal tissue are addressed to evaluate the importance of the alterations for the process and for the eventual malignant transformation.

Liver growth during regeneration is controlled by several growth factors which may be involved in the triggering, progression and termination of hepatocyte replication. It is likely that liver regeneration involves both circulating factors and those produced in hepatic tissue during the growth response. TGF alpha is an autocrine stimulator of hepatocyte proliferation which increases transiently in replicating hepatocytes both in vivo and in vitro. Constitutive TGF alpha overexpression in young transgenic mice causes liver hypertrophy and enhanced proliferation that progress to hepatic tumor development in the great majority of animals after 12 months of age. In contrast, HGF is present in normal blood in humans and animals and plasma concentrations increase after partial hepatectomy, liver injury and fulminant hepatic failure. In liver tissue, levels of HGF and its mRNA correlate better with the extent of injury than with the degree of proliferative activity. The factor is produced by nonparenchymal cells and presumably acts on hepatocytes through paracrine or endocrine mechanisms. A transient increase of TGF beta 1 in regenerating liver may promote the formation of extracellular matrix components and signal the end of hepatocyte proliferation. Prolonged overexpression of the factor in nonparenchymal cells causes liver fibrosis both in humans and experimental animals. The liver contains TGF beta 1,2 and 3, all of which inhibit hepatocyte DNA synthesis. Their mRNAs increase in the regenerating liver but with very different kinetics. Despite the enormous progress achieved in understanding the mechanisms that regulate liver regeneration, it is not known whether HGF, TGF alpha and TGF beta interact with each other or with other factors or hormones during the growth process. Further, it remains to be established how the effect of these factors may relate to the sequential changes in proto-oncogene expression that occur after partial hepatectomy.