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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.

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.

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.

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.

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.

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.

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.