Sequential analysis of multistage hepatocarcinogenesis reveals that miR-100 and PLK1 dysregulation is an early event maintained along tumor progression.

MicroRNAs (miRNAs) have an important role in a wide range of physiological and pathological processes, and their dysregulation has been reported to affect the development and progression of cancers, including hepatocellular carcinoma (HCC). However, in the plethora of dysregulated miRNAs, it is largely unknown which of them have a causative role in the hepatocarcinogenic process. In the present study, we first aimed to determine changes in the expression profile of miRNAs in human HCCs and to compare them with liver tumors generated in a rat model of chemically induced HCC. We found that members of the miR-100 family (miR-100, miR-99a) were downregulated in human HCCs; a similar downregulation was also observed in rat HCCs. Their reduction was paralleled by an increased expression of polo like kinase 1 (PLK1), a target of these miRNAs. The introduction of miR-100 in HCC cells impaired their growth ability and their capability to form colonies in soft agar. Next, we aimed at investigating, in the same animal model, if dysregulation of miR-100 and PLK1 is an early or late event along the multistep process of hepatocarcinogenesis. The obtained results showed that miR-100 downregulation (i) is already evident in very early preneoplastic lesions generated 9 weeks after carcinogenic treatment; (ii) is also observed in adenomas and early HCCs; and (iii) is not simply a marker of proliferating hepatocytes. To our knowledge, this is the first work unveiling the role of a miRNA family along HCC progression.

Triiodothyronine stimulates hepatocyte proliferation in two models of impaired liver regeneration.

OBJECTIVES: Liver regeneration is attenuated in old age and is substantially slower after 90% than after 70% partial hepatectomy (PH). We have previously demonstrated that the proliferative response to a primary mitogen is intact in aged mice, indicating that impaired liver regeneration is not due to loss of proliferative capacity. Here, we have investigated whether mitogenic effects of triiodothyronine (T3) could reverse the impaired regeneration of ageing or 90% hepatectomy, in the rat. MATERIALS AND METHODS: T3 (20 microg/100 g body weight) was administered to 14-month-old rats subjected to 70% PH or to young rats subjected to 90% PH. Cell-proliferative capacity was determined by bromodeoxyuridine incorporation and microscopy and changes of cell cycle-related proteins were analysed by Western blot analysis. RESULTS: Treatment of old intact rats with T3 increased cyclin D(1) expression that was followed by an enhanced proliferative response, the labelling index (LI), being 7.8% versus 1.3% of controls. T3 given before 70% PH stimulated regenerative response (LI was 10.8% versus 2.28%), and expression of cyclin D(1) and proliferating cell nuclear antigen (PCNA) 24 h after PH. Pre-treatment with T3 also improved the regenerative response of the liver after 90% hepatectomy (LI was 27.9% versus 14.2%). CONCLUSIONS: These findings show in principle that mitogen-induced hyperplasia could be applied to human therapy in patients with reduced regenerative capacity or massive loss of hepatocytes.

Increased ROS generation and p53 activation in alpha-lipoic acid-induced apoptosis of hepatoma cells.

Alpha-lipoic acid (alpha-LA) is an antioxidant used for the treatment of a variety of diseases, including liver cirrhosis, heavy metal poisoining, and diabetic polyneuropathy. In addition to its protective effect against oxidative stress, alpha-LA induces apoptosis in different cancer cells types. However, whether alpha-LA acid induces apoptosis of hepatoma cells is unknown. Herein, we investigated whether alpha-LA induces apoptosis in two different hepatoma cell lines FaO and HepG2. The results showed that alpha-LA inhibits the growth of both cell lines as indicated by the reduction in cell number, the reduced expression of cyclin A and the increased levels of the cyclin/CDKs inhibitors, p27(Kip1) and p21(Cip1). Cell cycle arrest was associated with cell loss, and DNA laddering indicative of apoptosis. Apoptosis was preceded by increased generation of reactive oxygen species, and associated with p53 activation, increased expression of Bax, release of cytochrome c from mitochondria, caspases activation, decreased levels of survivin, induction of pro-apoptotic signaling (i.e JNK) and inhibition of anti-apoptotic signaling (i.e. PKB/Akt) pathways. In conclusion, this study provides evidence that alpha-LA induces apoptosis in hepatoma cells, describes a possible sequence of molecular events underlying its lethal effect, and suggests that it may prove useful in liver cancer therapy.

Induction of pancreatic acinar cell proliferation by thyroid hormone.

Thyroid hormone is known to elicit diverse cellular and metabolic effects in various organs, including mitogenesis in the rat liver. In the present study, experiments were carried out to determine whether thyroid hormone is able to stimulate cell proliferation in another quiescent organ such as the pancreas. 3,5,3'-L-tri-iodothyronine (T3) added to the diet at a concentration of 4 mg/kg caused a striking increase in nuclear bromodeoxyuridine (BrdU) incorporation of rat acinar cells 7 days after treatment (the labeling index was 46.7% in T3-treated rats vs 7.1% in controls). BrdU incorporation was limited to the acinar cells, with duct cells and islet cells being essentially negative. The increase in DNA synthesis was accompanied by the presence of several mitotic figures. Histological examination of the pancreas did not exhibit any sign of T3-induced toxicity. Determination of the apoptotic index, measurement of the serum levels of alpha-amylase and lipase, and glycemia determination did not show any increase over control values, suggesting that the enhanced proliferation of acinar cells was a direct effect induced by T3 and not a regenerative response consequent to acinar or beta-cell injury. Additional experiments showed that DNA synthesis was induced as early as 2 days after T3 treatment (the labeling index was 9.4 vs 1.9% in controls) and was associated with increased protein levels of cyclin D1, cyclin A and proliferating cell nuclear antigen, with no substantial differences in the expression of the cyclin-dependent kinase inhibitor p27. The mitogenic effect of T3 on the pancreas was not limited to the rat, since extensive acinar cell proliferation was also observed in the pancreas of mice treated with T3 for 1 week (the labeling index was 28% in T3-treated mice vs 1.8% in controls). Treatment with three other ligands of nuclear receptors, ciprofibrate, all-trans retinoic acid and 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene, induced little or no pancreatic cell proliferation. These results demonstrated that T3 is a powerful inducer of cell proliferation in the pancreas and suggested that pancreatic acinar cell proliferation by selected agents may have potential for therapeutic use.

Induction of hepatocyte proliferation by retinoic acid.

Retinoids have been shown to exert an anticarcinogenic effect through suppression of the cell cycle, induction of apoptosis and/or differentiation. In rat liver, in particular, retinoic acid has been shown to inhibit regeneration after partial hepatectomy, most probably through repression of the expression of c-fos and c-jun. Surprisingly enough, in spite of the proposed therapeutic effects of all-trans retinoic acid (tRA) no data are available on its effect on normal adult liver. Here, we show that tRA administration in the diet (150 mg/kg) increased DNA synthesis in mouse liver, at 1 and 2 weeks, with a return to control values at 4 weeks (labelling index was 16.5, 8.3 and 3.3%, respectively, versus control values of 1.4, 1.3 and 2.5%). Increase in mitotic index paralleled that of bromodeoxyuridine incorporation. Kinetic studies showed that entry into S phase began between 24 and 48 h, with a peak between 96 and 120 h. Histological observation of the liver and biochemical evaluation of the levels of serum glutamate-pyruvate transaminases did not reveal any evidence of cell death demonstrating that increased DNA synthesis was not due to tRA-induced liver damage and regeneration, but rather the consequence of a direct mitogenic effect. In addition, analysis of total hepatic DNA content after a 7-day treatment showed a significant increase in tRA-fed mice compared with controls (21.11 mg/100 g body wt in tRA-fed mice versus 15.67 mg/100 g body wt of controls). Hepatocyte proliferation in tRA-fed mice was associated with increased hepatic levels of cyclin D1, E and A, and enhanced expression of the member of pRb family, p107. In conclusion, the results showed that tRA induces hepatocyte proliferation in the absence of cell death, similarly to other ligands of steroid/thyroid hormone nuclear receptor superfamily. The mitogenic effect of tRA cautions about its possible use for antitumoral purposes in liver carcinogenesis.

Peroxisome proliferator-activated receptor-alpha mice show enhanced hepatocyte proliferation in response to the hepatomitogen 1,4-bis [2-(3,5-dichloropyridyloxy)] benzene, a ligand of constitutive androstane receptor.

Previously, we have suggested that liver cell proliferation induced by certain mitogens is dependent on their binding and activation of nuclear receptors of the steroid/thyroid superfamily. More recently, it was shown that absence of the nuclear receptors peroxisome proliferator-activated receptor-alpha (PPARalpha) and constitutive androstane receptor (CAR) completely abolishes the proliferative response of hepatocytes to the mitogenic stimulus exerted by their specific ligands, peroxisome proliferators (PPs) and 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP), respectively. Here we show that deletion of the PPARalpha gene accelerates and enhances the proliferative response evoked by the xenobiotic 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP), a powerful mouse-liver mitogen and a ligand of the nuclear receptor CAR. Indeed, the number of hepatocytes entering S phase 24 hours after mitogen treatment was much greater in PPARalpha(-/-) mice compared with that of wild type mice (labeling indices 21.4% and 7.5%, respectively). Labeling index of hepatocytes from PPARalpha(-/-) mice was found to be higher than that of wild type mice up to 36 hours after treatment, indicating that lack of PPARalpha not only accelerated but also enhanced the overall proliferative response of the liver. The accelerated entry into S phase observed in hepatocytes from PPARalpha(-/-) mice was associated with a very rapid induction of cyclin D1. No major differences between TCPOBOP-treated PPARalpha(-/-) and wild type mice were observed in the expression of the 2 inhibitors of cyclin/CDKs complexes, p27 and p21. The results suggest that PPARalpha may play a role in modulating CAR-signaling pathways in the cell, in particular those leading to hepatocyte proliferation.

Cyclin D1 is an early target in hepatocyte proliferation induced by thyroid hormone (T3).

The thyroid hormone (T3) affects cell growth, differentiation, and regulates metabolic functions via its interaction with the thyroid hormone nuclear receptors (TRs). The mechanism by which TRs mediate cell growth is unknown. To investigate the mechanisms responsible for the mitogenic effect of T3, we have determined changes in activation of transcription factors, mRNA levels of immediate early genes, and levels of proteins involved in the progression from G1 to S phase of the cell cycle. We show that hepatocyte proliferation induced by a single administration of T3 to Wistar rats occurred in the absence of activation of AP-1, NF-kappa B, and STAT3 or changes in the mRNA levels of the immediate early genes c-fos, c-jun, and c-myc. These genes are considered to be essential for liver regeneration after partial hepatectomy (PH). On the other hand, T3 treatment caused an increase in cyclin D1 mRNA and protein levels that occurred much more rapidly compared to liver regeneration after 2/3 PH. The early increase in cyclin D1 expression was associated with accelerated onset of DNA synthesis, as demonstrated by a 20-fold increase of bromodeoxyuridine-positive hepatocytes at 12 h after T3 treatment and by a 20-fold increase in mitotic activity at 18 h. An early increase of cyclin D1 expression was also observed after treatment with nafenopin, a ligand of a nuclear receptor (peroxisome proliferator-activated receptor alpha) of the same superfamily of steroid/thyroid receptors. T3 treatment also resulted in increased expression of cyclin E, E2F, and p107 and enhanced phosphorylation of pRb, the ultimate substrate in the pathway leading to transition from G1 to S phase. The results demonstrate that cyclin D1 induction is one of the earlier events in hepatocyte proliferation induced by T3 and suggest that this cyclin might be a common target responsible for the mitogenic activity of ligands of nuclear receptors.

Ciprofibrate and triiodothyronine do not suppress in vivo induction of placental glutathione S-transferase expression in rat hepatocytes.

Studies on hepatocyte primary cultures have suggested that loss of expression of the placental form of glutathione S-transferase in peroxisome proliferator (PP)-induced hepatocarcinogenesis is due to inhibition of glutathione S-transferase P (GSTP) transcription by the PPs. In the present study, we have analyzed the effect of a PP, ciprofibrate, and of another ligand of nuclear receptors, 3,3', 5-triiodo-L-thyronine (T3), on GSTP mRNA and protein levels in an in vivo model where GSTP expression was induced in Wistar rats by pre-treatment with a single dose of lead nitrate. Results indicate that administration of ciprofibrate or T3, immediately after lead nitrate treatment, did not exert any inhibitory effect on GSTP mRNA and protein levels, as revealed by both Western and immunohistochemical analysis. The results indicate that PPs do not inhibit hepatocyte GSTP expression induced in vivo by lead nitrate and suggest that inhibition of GSTP expression by PPs may not necessarily be the cause for the rapid disappearance of GSTP-positive preneoplastic lesions observed after a short term exposure to these agents.

Cell proliferation induced by triiodothyronine in rat liver is associated with nodule regression and reduction of hepatocellular carcinomas.

Previous studies have demonstrated that short-term treatment with peroxisome proliferators decreased the size and number of gamma-glutamyl transpeptidase or placental glutathione S-transferase (GSTP)-positive hepatic hyperplastic lesions. In this study, we have examined the effect of the hormone triiodothyronine (T3), which, similarly to peroxisome proliferators, is a strong liver mitogen and a ligand of nuclear receptors, on the growth of GSTP-positive nodules generated by the resistant hepatocyte model and on the development of hepatocellular carcinoma. Hepatic hyperplastic nodules were induced in male Fischer rats by a single dose (150 mg/kg) of diethylnitrosamine, followed by a 2-week exposure of the animals to 2-acetylaminofluorene and partial hepatectomy. Nine weeks after diethylnitrosamine administration, rats were switched to a diet containing 4 mg/kg T3 for 1 week (experiment 1) and sacrificed during T3 feeding or were exposed to seven cycles of T3-supplemented diet (1 week/month per 7 months), and sacrificed 6 months after the last cycle (experiment 2). Results showed that T3 treatment for 1 week caused a 70% reduction in the number of GSTP-positive nodules (14/cm2 in T3-fed rats versus 44/cm2 of control animals), as well as GSTP-positive area (12% versus 43% of controls). Reduction in the number of GSTP-positive nodules observed 1 week after T3 feeding was associated with a strong increase in the labeling index of enzyme-altered nodules compared with that of controls (labeling index was 64 and 31%, respectively). No significant differences in the apoptotic index were observed between the two groups. Results from experiment 2 did reveal that although rats treated with diethylnitrosamine + 2-acetylaminofluorene developed 100% hepatocellular carcinoma and 33% of them showed lung metastasis, only 50% of rats exposed to repeated cycles of triiodothyronine developed hepatocellular carcinoma with no lung metastasis. This study indicates that cell proliferation per se might not necessarily represent a promoting condition for putative preneoplastic lesions and demonstrates an anticarcinogenic effect of T3.

Early increase in cyclin-D1 expression and accelerated entry of mouse hepatocytes into S phase after administration of the mitogen 1, 4-Bis[2-(3,5-Dichloropyridyloxy)] benzene.

We have previously demonstrated that hepatocyte proliferation induced by the mitogen 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) is independent of changes in cytokines, immediate early genes, and transcription factors that are considered to be necessary for regeneration of the liver after partial hepatectomy (PH) or necrosis. To further investigate the differences between mitogen-induced mouse hepatocyte proliferation and liver regeneration after PH, we have measured the expression of cyclin D1, cyclin D3, cyclin E, and cyclin A and of the cyclin-dependent kinases CDK2, CDK4, and CDK6. The involvement of the cyclin-dependent kinase inhibitors p21 and p27 and of the oncosuppressor gene p53 was also examined at different times after stimulation of hepatocyte proliferation. Results showed that a single administration of TCPOBOP caused a very rapid increase in the levels of cyclin D1, a G1 protein, when compared with two thirds PH (8 hours versus 30 hours). The early increase in cyclin D1 protein levels was associated with a faster onset of increased expression of S-phase-associated cyclin A (24 hours versus 36 hours with PH mice). Accordingly, measurement of bromodeoxyuridine (BrdU) incorporation revealed that, although approximately 8% of hepatocytes were BrdU-positive as early as 24 hours after TCPOBOP, no significant changes in BrdU incorporation were observed at the same time point after two thirds PH. The expression of other proteins involved in cell cycle control, such as cyclin-dependent kinases (CDK4, CDK2, CDK6), was also analyzed. Results showed that expression of CDK2 was induced much more rapidly in TCPOBOP-treated mice (2 hours) than in mice subjected to PH (36 hours). A different pattern of expression in the two models of hepatocyte proliferation, although less dramatic, was also observed for CDK4 and CDK6. Expression of the CDK inhibitors p21 and p27 and the oncosuppressor gene p53 variably increased after two thirds PH, whereas basically no change in protein levels was found in TCPOBOP-treated mice. The results demonstrate that profound differences in many cell cycle-regulatory proteins exist between direct hyperplasia and compensatory regeneration. Cyclin D1 induction is one of the earlier events in hepatocyte proliferation induced by the primary mitogen TCPOBOP and suggests that a direct effect of the mitogen on this cyclin may be responsible for the rapid onset of DNA synthesis observed in TCPOBOP-induced hyperplasia.

Cell proliferation induced by 3,3',5-triiodo-L-thyronine is associated with a reduction in the number of preneoplastic hepatic lesions.

Previous studies have suggested that liver cell proliferation is fundamental for the growth of carcinogen-initiated cells. To gain further information on the association between cell proliferation and hepatocarcinogenesis, we have examined the effect of the hormone 3,3',5-triiodo-L-thyronine (T3), a strong liver mitogen, on the growth of diethylnitrosamine (DENA)-induced hepatic lesions positive for the placental form of glutathione S-transferase (GSTP). Two weeks after a single initiating dose of DENA (150 mg/kg), cycles of liver cell proliferation were induced in male Fischer rats by feeding a T3-supplemented diet (4 mg/kg) 1 week/month for 7 months. Rats were killed at the end of the seventh cycle or 1 month later. Results indicate that, in spite of an increased labelling index, a 70% reduction in the number/cm(2) of GSTP-positive minifoci occurred in T3-treated rats. A decrease in the number of GSTP-positive foci was also observed in T3-treated rats killed 1 month after the last exposure to the hormone (40, versus 67 foci/cm(2) in controls), indicating that the reduction was not due to an inhibitory effect on GSTP exerted by the concomitant presence of T3. In a second series of experiments where DENA-treated rats were fed T3 for 1 week and then subjected to the resistant hepatocyte (RH) model, it was found that T3 treatment prior to promotion resulted in a decrease in the number of GSTP-positive foci (16 GSTP(+) foci/cm(2) in T3-fed animals versus 45 in the control group). The results indicate that cell proliferation associated with T3 treatment: (i) reduces the number of carcinogen-induced GSTP-positive lesions; (ii) does not exert any differential effect on the growth of the remaining foci; (iii) inhibits the capacity of putative DENA-initiated cells to be promoted by the RH model. Data suggest that cell proliferation may not necessarily represent a stimulus for the growth of putative preneoplastic lesions.

In vivo hepatocyte proliferation is inducible through a TNF and IL-6-independent pathway.

Recent studies in mice harboring a targeted disruption of genes encoding TNF receptor 1 (TNFR-1) or Interleukin 6 (IL-6) suggested a critical role for TNF and IL-6 in initiation of liver regeneration after 2/3 partial hepatectomy. However, hepatocyte proliferation can also occur following treatment with agents that do not induce tissue loss (primary mitogens). To determine whether the above cytokines could also be involved in mitogen-induced liver cell proliferation, we studied the hepatocyte proliferative response after treatment with primary mitogens in mice knock-out for TNFR-1 or IL-6. Our results showed no difference in the proliferative response of the liver between the wild type and the knock-out mice following treatment with the mitogens 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), or the peroxisome proliferator, ciprofibrate, suggesting that TNF or IL-6 may not play a major role in this type of proliferation. Gel shift assay indicated that TCPOBOP-induced hepatocyte proliferation is not associated with activation of STAT3 transcription factor, a major target of IL-6 and other growth factors/cytokines. Our results thus indicate that hepatocyte proliferation can be induced by at least two different pathways; compensatory regeneration being TNF and IL-6-dependent, and mitogen-induced direct hyperplasia which does not require TNF or IL-6.

Liver cell proliferation induced by nafenopin and cyproterone acetate is not associated with increases in activation of transcription factors NF-kappaB and AP-1 or with expression of tumor necrosis factor alpha.

Our previous studies have shown a different pattern of immediate early gene and growth factor gene expression between compensatory liver regeneration occurring after cell loss/death and direct hyperplasia induced by primary mitogens. In the present study, modifications in the activation of two transcription factors, NF-kappaB and AP-1; steady-state levels of tumor necrosis factor alpha (TNF-alpha) messenger RNA (mRNA); and induction of the inducible nitric oxide synthase (iNOS) were examined in rat liver during different types of cell proliferation. Compensatory regeneration was induced in male Wistar rats by partial hepatectomy of two thirds (PH) or a necrogenic dose of CCl4 (2 mL/kg), whereas direct hyperplasia was induced by a single administration of the primary mitogens lead nitrate (LN, 100 micromol/kg), cyproterone acetate (CPA, 60 mg/kg), or nafenopin (NAF, 200 mg/kg). Liver regeneration after treatment with CCl4 was associated with an increase in steady-state levels of TNF-alpha mRNA, activation of NF-kappaB and AP-1, and induction of iNOS. A strong and prolonged activation of NF-kappaB but not of AP-1 was observed in LN-induced hyperplasia. LN also induced an increase in hepatic levels of TNF-alpha and iNOS mRNA. On the other hand, direct hyperplasia induced by two other primary mitogens, NAF and CPA, occurred in the complete absence of modifications in the hepatic levels of TNF-alpha mRNA, activation of NF-kappaB and AP-1, or induction of iNOS, although the number of hepatocytes entering S phase 18 to 24 hours after NAF was similar to that seen after PH. These results add further support to the hypothesis that cell proliferation occurring in the absence of cell loss/death may be triggered by unknown signaling pathways different from those responsible for the transition of hepatocytes from G0 to G1 after PH or cell necrosis.

Induction of cellular DNA synthesis in the pancreas and kidneys of rats by peroxisome proliferators, 9-cis retinoic acid, and 3,3',5-triiodo-L-thyronine.

We recently suggested that peroxisome proliferators (PPs), 3,3',5-triiodo-L-thyronine (T3), and 9-cis retinoic acid (9-cis RA) induce hepatocyte proliferation in rats through the activation of their nuclear receptors, PP-activated receptors, T3 receptors, and retinoid X receptors. To test whether nuclear hormone receptor-mediated cell proliferation can be observed in organs other than liver, we examined the effects of these agents on the pancreas and kidneys of male Wistar rats using BrdUrd immunohistochemistry. A single s.c. injection of T3 (2 mg/kg) and single intragastric administration of 9-cis RA (40 mg/kg) or 4-chloro-6-(2, 3-xylidino)-2-pyrimidinylthio-(N-beta-hydroxyethyl) acetamide (200 mg/kg) induced a wave of DNA synthesis in the pancreatic acinar cells and in the proximal tubular epithelial cells of the kidneys, peaking after 24 h. No stimulation of DNA synthesis was observed in ductal or islet cells of the pancreas and in glomeruli of the kidneys. All-trans-retinoic acid, a ligand for retinoic acid receptor, at a dose (200 mg/kg) that induced hepatocyte proliferation, had no effects on cell proliferation of the pancreas and the kidneys. The results suggest that T3, 9-cis RA, and PP activate genes that regulate cell proliferation in target cells through receptor-mediated pathways and initiate cellular DNA synthesis.

Increased expression of c-fos, c-jun and LRF-1 is not required for in vivo priming of hepatocytes by the mitogen TCPOBOP.

The notion that an increased expression of immediate early genes such as c-fos and c-jun is an absolute requirement for the G0-G1 transition of the hepatocytes has recently been challenged by the finding that rat liver cell proliferation induced by primary mitogens may occur in the absence of such changes (Columbano and Shinozuka, 1996). To further investigate the relationship between immediate early genes and hepatocyte proliferation, we have compared the hepatic levels of c-fos, c-jun and LRF-1 transcripts during mouse liver cell proliferation in two conditions: (i) direct hyperplasia induced by the non-genotoxic hepatocarcinogen 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene, and (ii) compensatory regeneration caused by a necrogenic dose of carbon tetrachloride. The results show striking differences in the activation of early genes. In spite of a rapid stimulation of S phase by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (approximately 8% of hepatocytes were BrdU-positive as early as 24 h after mitogen treatment versus 1% of labelled hepatocytes after 2/3 partial hepatectomy), no changes in the expression of c-fos, c-jun and LRF-1 could be observed. Moreover, no change in steady state mRNA hepatic levels of IGFBP-1 (a gene highly expressed in rat liver following partial hepatectomy), and only a slight increase in c-myc and PRL-1, was found after mitogen administration. On the contrary, a rapid, massive and transient increase in the hepatic mRNA levels of all these genes was observed during carbon tetrachloride induced regeneration. The results indicate that increased expression of immediate early genes may be dependent upon the nature of the proliferative stimulus, and it may not be a prerequisite in certain in vivo conditions such as proliferation induced in the absence of liver tissue damage.

Possible roles of nonparenchymal cells in hepatocyte proliferation induced by lead nitrate and by tumor necrosis factor alpha.

A single intravenous injection of lead nitrate (LN) to rats induces liver cell proliferation without causing cell necrosis (direct hyperplasia). We suggested that liver cell proliferation in this model may be triggered by the induction of liver tumor necrosis factor alpha (TNF-alpha). Because administration of TNF-alpha in vivo has been shown to induce proliferation of both parenchymal and nonparenchymal cells of the liver, we analyzed the temporal sequences of DNA synthesis in both cell populations following LN and recombinant TNF-alpha treatment by 5-bromo-2-deoxyuridine (BrdU) immunohistochemistry. The patterns of cell proliferation induced by these agents were further compared with those induced by a single dose of nafenopin (NAF), a direct mitogen which does not induce liver TNF-alpha messenger RNA (mRNA). In male Wistar rats given a single dose of LN (100 micromol/kg), BrdU incorporation of hepatocytes and nonparenchymal cells (Kupffer cells, endothelial cells and periportal nondescript cells) became evident 12 hours after the treatment. The labeling of all cell types reached a peak after 36 hours and declined thereafter. Rats given a single intravenous injection of human recombinant TNF-alpha (46 microg/rat) showed an increase of BrdU labeling in nonparenchymal cells after 24 hours, whereas the labeling of hepatocytes became evident at 36 hours. A single intragastric administration of NAF resulted in a rapid increase in the number of labeled hepatocytes with no substantial labeling of nonparenchymal cells. These results add further support to the notion that LN-induced liver cell proliferation is mediated by TNF-alpha, and suggest that different cell populations are involved in the initial proliferative response of the liver to mitogens, depending on the capacity of the mitogens to stimulate TNF-alpha production.

Effects of cell proliferation and cell death (apoptosis and necrosis) on the early stages of rat hepatocarcinogenesis.

An experiment was performed to investigate whether, during regression of the liver hyperplasia induced by a direct mitogen, apoptosis differentially affects replicated and non-replicated hepatocytes. After a single dose of the direct mitogen lead nitrate (LN), male Wistar rats were given repeated injections of tritiated thymidine, and were killed either 3 days (time of maximal hepatic DNA increase) or 15 days (complete regression of the hyperplasia) after mitogen treatment. Determination of liver DNA radioactivities and labelling indices (LIs) at the two time points revealed an approximately 40% loss in total liver DNA radioactivity, a 20% decrease in the specific activity of DNA, and a 20% reduction in the cell LI. Three days after LN administration 64% of the apoptotic bodies contained thymidine grains in their nuclear fragments. The results indicated that apoptosis affects both hepatocytes that replicated, and those that did not replicate, the former being slightly more sensitive. A second experiment was then performed to investigate whether and to what extent different types of cell death (apoptosis versus necrosis) influence the growth of hepatocytes initiated by a chemical carcinogen. Male Wistar rats were given a single dose of diethylnitrosamine, and 2 weeks thereafter either a single dose of LN, or a necrogenic dose of carbon tetrachloride (CCl4). Bromodeoxyuridine was next infused for 5 days, and some of the animals were killed at this time point, and others after an additional 3 weeks. Administration of CCl4 resulted in an increase in both the average size and the percentage area occupied by placental glutathione S-transferase-positive lesions. In contrast, administration of lead nitrate resulted in a strong reduction (50%) in the number of positive lesions with no remarkable change in the percentage area occupied by them. These differential effects occurred even though comparable LIs were observed in rats treated with the two agents. The results suggest that lead nitrate leads to a loss of initiated hepatocytes, due to the apoptosis that occurs during regression of the LN-induced hyperplasia.

Hepatocyte proliferation induced by a single dose of a peroxisome proliferator.

In compensatory hyperplasia after partial hepatectomy or liver cell injury, hepatocyte proliferation is triggered by coordinated actions of growth factor such as hepatocyte growth factor and transforming growth factor-alpha and -beta. Initiation of hepatocyte DNA synthesis is preceded by the activation of the set of early growth response genes mediated by enhanced nuclear factor-kappa B binding to DNA. Using an experimental model to induce hepatocyte DNA synthesis in vivo by a single dose of a peroxisome proliferator, which does not induce liver cell necrosis (direct hyperplasia), we investigated whether peroxisome proliferator-induced hepatocyte proliferation involved an induction of known growth factors, an activation of early growth response genes, and nuclear factor-kappa B. A single intragastric administration of 250 mg/kg BR931 (4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio-(N-beta-hydroxyethyl) acetamide) to male wistar rats induced a wave of hepatocyte DNA synthesis starting after 12 hours and peaking at approximately 24 to 36 hours. The response was dose dependent. The treatment also induced the expression of the mRNA for the peroxisomal bifunctional enzyme, one of the peroxisome-related fatty acid beta-oxidation enzymes. Pretreatment of rats with dexamethasone (2 mg/kg) inhibited both hepatocyte DNA synthesis and the induction of the peroxisomal bifunctional enzyme gene. Northern blot analyses of liver RNA during a period preceding the onset of DNA synthesis revealed no induction of hepatocyte growth factor, transforming growth factor-alpha, or tumor necrosis factor-alpha mRNAs. No induction of early growth response genes, liver regeneration factor-1, or c-myc was detected. Furthermore, gel mobility shift assays showed no enhanced nuclear factor-kappa B binding to its DNA consensus sequence after BR931 treatment, whereas control studies demonstrated a distinct increase in binding after partial hepatectomy or lead nitrate treatment. The results suggest that peroxisome-proliferator-induced hepatocyte proliferation may be triggered by signal transduction pathways different from those after partial hepatectomy and that the binding of peroxisome proliferators to their nuclear receptors may play a role in stimulation of DNA synthesis and peroxisome proliferation.

Cell proliferation, cell death and hepatocarcinogenesis.

The carcinogenic process in the liver is a multistep process, characterised by an altered ratio between cell proliferation and cell death. In the last few years, we have undertaken studies aimed at determining the possible differences exhibited by two different types of cell proliferation, namely compensatory regeneration and direct hyperplasia at a molecular and cellular level. These two types of proliferative stimuli appear to play different roles in liver carcinogenesis. The scope of this article is to summarise the present knowledge about the differences in the expression of genes involved in the entry of liver cells into cell cycle, between liver regeneration following cell loss and/or cell death and direct hyperplasia induced by primary mitogens.