Induction of hepatocyte proliferation by retinoic acid
G.M. Ledda-Columbano1,
M. Pibiri,
F. Molotzu,
C. Cossu,
L. Sanna,
G. Simbula,
A. Perra and
A. Columbano
Department of Toxicology, Oncology and Molecular Pathology Unit, University of Cagliari, Italy
1 To whom correspondence should be addressed Email: gmledda{at}unica.it
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Abstract
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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.
Abbreviations: BrdU, bromodeoxyuridine; L.I., labelling index; M.I., mitotic index; RAR, retinoic acid receptor; RXR, retinoic X receptor; tRA, all-trans retinoic acid.
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Introduction
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Retinoids, which are natural and synthetic vitamin A derivatives, have marked effects on cellular proliferation, differentiation and on the immunosystem (1,2). The pleiotropic effects of retinoids are mediated by two classes of receptors, retinoic acid receptors (RARs: RAR
, RARß and RAR
) (36), and retinoid X receptors (RXRs: RXR
, RXRß and RXR
), both of which are members of the steroid/thyroid receptor superfamily of ligand-dependent transcriptional factors (79). The RARs-dependent signalling pathway is mainly transduced through heterodimeric complexes of RAR/RXR promoted by ligand binding to RAR (10). All-trans retinoic acid (tRA), a natural metabolite of vitamin A, among its several biological effects has been used as a chemotherapeutic agent because of its strong anti-proliferative activity against certain types of cancer (1113). As far as the liver is concerned, only limited and somewhat conflicting information are available regarding the effects of tRA on hepatocyte proliferation and carcinogenesis. It was shown that tRA given shortly prior to 2/3 partial hepatectomy (PH) inhibited DNA synthesis in the regenerating rat liver (14). In the same study, it was suggested that inhibition of liver regeneration could probably occur via repression of the expression of immediate early genes such as c-fos and c-jun. Although the latter study, together with other reports in different cellular types (1113), supports the hypothesis that tRA may exert an antitumoral effect through inhibition of cell proliferation, other studies have shown that tRA, while inhibiting the number and size of preneoplastic lesions induced in rat liver by a choline-deficient diet, does not exert any inhibitory effect on the growth of lesions induced by the genotoxic nitroso compound, diethylnitrosamine (DENA) (15). In addition, other studies have shown that tRA enhanced development of liver tumors following initiation with DENA in B6D2F1/Hsd mice (16).
Due to the conflicting results about the role of tRA on liver carcinogenic process, and since surprisingly no data are available on the effect of tRA on normal hepatocyte turnover, the main aim of this study was to investigate the effect of a short-term administration of tRA on liver cell proliferation in intact mice.
The need for studies aimed at clarifying the effect of tRA on normal hepatocyte turnover, stems also from the finding that several other ligands of nuclear receptors have been shown to be mitogenic for the liver. Indeed, peroxisome proliferators, thyroid hormone and the halogenated hydrocarbon TCPOBOP are potent primary mitogens for hepatocytes in rats and mice (1722); interestingly, these agents induce proliferation in the absence of modifications of transcription factors (AP-1, NF-
B, STAT3), immediate early genes (c-fos, c-jun, c-myc) and cytokines (interleukin-6, tumor necrosis factor-
), commonly believed to be essential prerequisite for liver regeneration after partial surgical resection of the liver or chemically induced liver cell necrosis (2328), indicating that the mitogenic activity of ligands of nuclear receptors involves a signal transduction pathway different from that activated in compensatory liver regeneration (21,29).
Our results show clearly that tRA is a powerful inducer of hepatocyte proliferation and that recruitment of hepatocytes into the cell cycle occurs in the absence of any significant evidence of liver cell damage, thus supporting the notion that ligands of nuclear receptors of the superfamily of steroid/thyroid nuclear hormone receptors possess a direct mitogenic activity for rodent hepatocytes.
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Materials and methods
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Animals
Female CD-1 mice (78 weeks old) purchased from Charles River (Milano, Italy) were used in these experiments. The animals were fed a laboratory chow diet provided by Ditta Mucedola (Settimo Milanese, Italy) and had free access to food and water. We followed Guidelines for the Care and Use of Laboratory Animals during the investigation. All experiments were performed in a temperature-controlled room with alternating 12 h dark/light cycles. tRA-supplemented diet (150 mg/kg) and bromodeoxyuridine (BrdU, 1 mg/ml in drinking water) were purchased from Mucedola (Settimo Milanese, Italy) and Sigma Chemical (St Louis, MO), respectively. Hematoxylin and eosin were from Carlo Erba Reagenti (Milano, Italy).
Experimental protocol I
To determine the effect of tRA on normal resting liver, CD-1 mice were fed a tRA-supplemented diet (150 mg/kg) for 1, 2 and 4 weeks. Controls received a basal diet and were killed at the same time points. For determination of hepatocyte proliferation, 1 week before death, mice were given BrdU, dissolved in drinking water (1 mg/ml).
Experimental protocol II
To determine the kinetics of proliferation after tRA feeding, mice were fed a tRA-supplemented diet for 5 days and were killed at 24, 48, 72, 96 and 120 h. BrdU dissolved in drinking water, was given for 24 h before each death. Controls received a laboratory chow diet and were killed at the same time points. To establish whether the increased DNA synthesis observed in the livers of tRA-fed animals was due to liver damage, additional groups of mice were fed tRA for 1, 2, 3, 4, 5 or 7 days. Five animals were killed at each time point. Controls were killed at the same time points.
Histology and immunohistochemistry
Immediately after death, liver sections were fixed in 10% buffered formalin and processed for staining with hematoxylineosin or immunohistochemistry. The remaining liver was snap-frozen in liquid nitrogen and kept at 80°C until use.
For determination of hepatocyte proliferation, mouse monoclonal anti-BrdU antibody was obtained from Becton Dickinson (Becton Dickinson, San Jose, CA) and the peroxidase method was used to stain BrdU-positive hepatocytes. Peroxidase goat anti-mouse Immunoglobulin was obtained from Dako (Dako
Peroxidase Mouse, Dako, Carpinteria, CA). Four-micron thick sections were deparaffinized, treated with 2 N HCl for 1 h, then with 0.1% trypsin type II (crude from porcine pancreas, Sigma, Milano, Italy) for 20 min, and treated sequentially with normal goat serum 1:10 (Dako), mouse anti-BrdU 1:100 and Dako
Peroxidase Mouse ready-to-use. The sites of peroxidase binding were detected by 3,3'-diaminobenzidine. The labelling index (L.I.) was expressed as number of BrdU-positive nuclei/100 nuclei. The mitotic index (M.I.) was expressed as number of mitoses/1000 hepatocyte nuclei. Results are expressed as means ± SE of 45 mice/group. At least 2000 hepatocyte nuclei per liver were scored.
The incidence of apoptotic bodies was determined by scoring 15002000 hepatocytes/liver. Four to five mice per group were analysed. Only apoptotic bodies containing nuclear fragments were recorded.
Serum glutamate pyruvate transaminases determination
The activity of serum glutamate pyruvate transaminases (S-GPT) was determined according to a GP-Transaminase Kit (Sigma Diagnostics, St Louis, MO).
Determination of hepatic DNA content
After death, the livers were frozen at 80°C. Total hepatic DNA content was quantitatively assayed by Burton's diphenylamine method (30).
Western blot analysis
Total cell extracts were prepared from frozen livers powdered in liquid nitrogen-cold mortar. Equal amounts of powder from different animals were resuspended in 1 ml Triton lysis buffer (1% Triton X-100, 50 mM TrisHCl pH 7.4, 135 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 10 mM NAF, 5 mM iodoacetic acid, 10 µg/ml each of aprotinin, pepstatin and leupeptin). Several protease inhibitors were added to the isolation buffer to minimize protein degradation during the isolation protocol. Extracts were incubated for 30 min on ice, centrifuged at 12 000 r.p.m. at 4°C and the supernatants recovered. All inhibitors used were purchased from Boheringer Mannheim GmbH (Germany) with the following exception: PMSF, NaF and DTT were purchased from Sigma Chemical and iodoacetic acid from ICN Biomedicals (Irvine, CA). The protein concentration of the resulting total extracts was determined according to Bradford (31) using bovine serum albumin as standard (DC Protein Assay, Bio-Rad Laboratories, USA). Nuclear extracts were prepared for the analysis of pRb, p107, p130, p21, p27 and H3. For immunoblot analysis equal amounts (from 100 to 200 µg/lane) of proteins were electrophoresed on SDS-12% or -8% polyacrylamide gels. Acrylamide and bis-acrylamide were purchased from ICN Biomedicals, Irvine, CA. After gels electrotransfer onto nitrocellulose membranes (MSI), to ensure equivalent protein loading and transfer in all lanes, the membranes and the gels were stained with 0.5% (wt/vol) Ponceau S red (ICN Biomedicals) in 1% acetic acid, and with Coomassie Blue (ICN Biomedicals) in 10% acetic acid, respectively. Before staining, gels were fixed in 25% (v/v) isopropanol and 10% (v/v) acetic acid (Sigma Chemicals). After blocking in TBS containing 0.05% Tween 20 (Sigma) and 5% non-fat dry milk, membranes were washed in TBS-T and then incubated with the appropriate primary antibodies diluted in blocking buffer. Whenever possible, the same membrane was used for detection of the expression of different proteins. Depending on the origin of primary antibody, filters were incubated at room temperature with either anti-mouse, anti-rabbit or anti-rat horseradish peroxidase conjugated IgG (Santa Cruz Biotechnology, Santa Cruz, CA). Immunoreactive bands were identified with a chemiluminescence detection system, as described by the manufacturer (Supersignal Substrate, Pierce). When necessary, antibodies were removed from filters by 30 min incubation at 60°C in stripping buffer (100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM TrisHCl pH 7.6) and the membranes reblotted as above.
Antibodies
For immunoblotting experiments mouse monoclonal antibody directed against Cyclin D1 (72-13 G, Santa Cruz Biotechnology), p21 (Powerclonal, Upstate Biotech, Lake Placid, NY), and p27 (Kip1-p27, Transduction Laboratories, Lexington, KY) were used; rabbit polyclonal antibodies against Cyclin A (C-19), Cyclin E (M-20), p130 (C-20), H3 (FL-136) and the goat polyclonal antibody against p107 (C-18) were from Santa Cruz. The rabbit polyclonal antibody against phospho-Rb (Ser 780) was from Cell Signaling Technology (Beverly, MA).
Statistical analysis
Comparison between treated and control groups were performed by Student's t test.
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Results and discussion
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To evaluate the effect of tRA on normal hepatocyte turnover, experiments were performed wherein CD-1 mice were fed a basal diet supplemented with 75, 150 and 300 mg/kg of tRA for 5 days. To determine the L.I., tRA-fed mice and controls were given BrdU (1 mg/ml, dissolved in drinking water) all throughout the experimental period. The results showed that the three tRA concentrations induced an almost similar proliferative response (L.I. was 9.5 ± 2.7, 8 ± 1.0 and 11 ± 4.1 versus 0.2 ± 0.06 of controls). Analysis of serum transaminase levels (S-GPT), at the time of death, did not show any significant change compared with control values (IU/l were 12 ± 2, 9 ± 1 and 13 ± 2, respectively, versus 12 ± 1 of controls), clearly demonstrating the absence of liver necrosis. Histological examination of the livers did not reveal any significant increase in the number of apoptotic bodies compared with controls.
As the diet supplemented with 300 mg of tRA caused a 10% loss of body weight, the next experiments, aimed to further characterize the mechanisms of tRA-induced hepatocyte proliferation, were performed using a diet supplemented with 150 mg/kg. This concentration was chosen also in view of its anticarcinogenic effect in the absence of severe toxicity in a rat model of liver carcinogenesis (15). As shown in Figure 1A, the tRA-supplemented diet resulted in increased mouse hepatocyte proliferation, as monitored by BrdU incorporation into hepatic DNA; the effect of tRA was maximal during the first week (L.I. of 16.5% against 1.7% of control mice; Figure 2A). At this time point, most of the labelled cells were localized in the midzonal area of the liver lobule with the areas around zone III being relatively unaffected. Non-parenchymal cells were also labelled although at a lower degree. The L.I. was still significantly higher than control values at 2 weeks, with an almost complete return to the values of age-matched controls after 4 weeks (Figure 2A); as expected, an increase in mitotic activity, with a peak at 1 week after tRA treatment, was associated with the increased L.I. (Figures 1B and 2B).

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Fig. 1. Representative microphotography that illustrates the effect of tRA on BrdU incorporation (A) or mitoses (B) in mouse hepatocytes. Mice were fed a tRA-supplemented diet (150 mg/kg) or a basal diet for 1, 2 or 4 weeks. Immediately after the administration of tRA in the diet, mice were given BrdU (1 mg/ml) in drinking water for 7 days, during the first, the second or the fourth week (x200, sections counterstained with hematoxylin). CO, Controls; tRA, all-trans-retinoic acid.
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Fig. 2. L.I. and M.I. of mouse hepatocytes. Mice were treated as described in legend to Figure 1. (A) L.I. was expressed as number of BrdU-positive hepatocyte nuclei/100 nuclei. At least 2000 hepatocyte nuclei per liver were scored. (B) M.I. is expressed as number of cells undergoing mitosis/1000 hepatocytes. Results are expressed as means ± SE of 45 mice/group.
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To determine the timing of tRA-induced hepatocyte entry into DNA synthesis, further studies were performed where mice were given BrdU in drinking water every 24 h for the first 5 days of tRA feeding. Results shown in Figure 3 indicated that DNA synthesis began between 24 and 48 h (L.I. 2.54 versus 0.31% of controls), with a much higher BrdU incorporation between the fourth and fifth day (L.I. 9.75 versus 0.23% of controls).

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Fig. 3. Effect of tRA on L.I. of mouse hepatocytes. Mice fed a tRA-supplemented diet were given BrdU (1 mg/ml) in drinking water from 1 to 24 h, 24 to 48 h, 48 to 72 h, 72 to 96 h and 96 to 120 h, respectively. At least 2000 hepatocyte nuclei per liver were scored. L.I. was expressed as number of BrdU-positive hepatocyte nuclei/100 nuclei. Results are expressed as means ± SE of 45 mice/group.
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Next, we asked the question whether the proliferative effect induced by tRA could be the consequence of tRA-induced liver damage and compensatory regeneration or, rather, a direct mitogenic effect. Results indicate that no increase in S-GPT occurred between day 1 and day 7, the values ranging from 5.3 to 7.5 IU/l in tRA-fed mice versus 6.0 to 7.2 IU/l in control mice. Since apoptotic cell death may occur in the absence of changes in the levels of serum transaminases (32), the apoptotic index was determined in liver sections of mice given tRA for 1, 2, 3, 4 and 5 days. Results did not reveal any significant change in the number of hepatocytes undergoing apoptosis compared with controls (apoptotic index in treated and control animals ranged from 0.02 to 0.07%).
Treatment with tRA for 1 week resulted in an increase in liver weight and, most important, in total hepatic DNA content (21.11 ± 0.63 versus 15.67 ± 0.88 mg DNA/100 g body wt of controls), further establishing that the increased proliferative activity of the hepatocytes after tRA feeding is a direct mitogenic effect.
The increased BrdU incorporation seen in the liver of tRA-fed mice was associated with increased protein levels of cyclin A, a cell cycle regulatory protein specifically expressed during S phase; indeed, as shown in Figure 4, cyclin A levels were induced at a significantly higher level in tRA-fed mice starting from day 2 with maximal expression between day 4 and day 5. It has been shown previously that entry of hepatocytes into the cell cycle following treatment with ligands of nuclear receptors is not associated with changes in cytokines, such as tumor necrosis factor-
and interleukin-6, immediate early genes or transcription factors (2224), generally observed during liver regeneration after 2/3 PH (2528); on the other hand, nuclear receptor-mediated proliferation is accompanied by an early increase in the expression of the G1 cyclin, cyclin D1 (22,23). Therefore, we determined the expression of cyclin D1 protein in the liver of tRA-treated mice. The results shown in Figure 4 indicate that the hepatic levels of cyclin D1 protein were almost undetectable in three out of four untreated mice; 2 days after tRA feeding a strong induction of cyclin D1 protein levels was seen, its expression being elevated over control values also at days 3, 4 and 5; levels of another G1 cyclin, namely cyclin E, were also increased in tRA-fed mice although the peak of maximal expression was somewhat different (Figure 4). Together with its partners CDK4 and CDK6, cyclin D1 is thought to stimulate entry into S phase by phosphorylating pRb and causing the release of components of the E2F family of transcription factors (33,34). Therefore, we have examined changes in the phosphorylation state of pRb and in the expression of its family members p107 and p130 on liver nuclear extracts of tRA-fed mice. Results shown in Figure 5, demonstrated that p107 was enhanced in tRA-fed mice, with no major changes in the phosphorylative state of pRb and p130 expression. Finally, to determine whether the increased expression of p107 depends entirely on enhanced cyclin-associated CDK activities or could also be due to inhibition by tRA of the CDKs inhibitors p27 and p21, we measured the level of these proteins. As shown in Figure 5, no significant difference in p21 was observed between untreated and tRA-fed mice, while p27 protein content was found to be higher than that of controls; these results clearly indicate that the increased p107 expression is not due to inhibition of the CDKs inhibitors.

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Fig. 4. Western blot analysis of cyclin A, D1 and E in liver of female mice fed a tRA-supplemented diet for 1, 2, 3, 4 and 5 days. Protein extracts (100 µg/lane) were prepared from the livers and western analysis was performed as described in the Materials and methods. Appropriate loading was confirmed by staining the gel with Coomassie Blue and efficiency of transfer was monitored by staining the membranes with Ponceau S red. Each lane represents pool of at least three livers.
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Fig. 5. Western blot analysis of pRb, p130, p107, p21 and p27 in mice killed 1, 2, 3, 4 and 5 days after treatment with tRA. Nuclear extracts for p130, pRb, p107, p21 and p27 (100200 µg/lane) were prepared from the livers and western analysis was performed as described in the Materials and methods. Appropriate loading was confirmed by staining the gel with Coomassie Blue and using H3 antibody as loading control; efficiency of transfer was monitored by staining the membranes with Ponceau S red. Each lane represents pool of three livers. CO, controls.
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The present study demonstrates that tRA exerts a clear stimulatory effect on hepatocyte proliferation in mice. The mitogenic effect exerted by tRA is not associated with liver injury but is a primary event leading to an increase in hepatic DNA content. Previous studies addressed to determine the effect of tRA on cell proliferation have produced conflicting results. Indeed, while tRA was shown to inhibit cell cycle progression in MCF-7 human breast cancer cells, most probably by down-regulating cyclin D3/CDK4, pRb and E2F1 (35), an increased number of normal hamster epithelial cells was observed after treatment with physiological concentrations of retinoic acid (36). As to the liver, studies in rats have shown that tRA given shortly prior to 2/3 PH inhibited DNA synthesis in regenerating rat liver (14), and that fast growing hepatomas showed depletion of retinoids (37). Whether the different effect observed in the present study is due to the different species used (mouse versus rat) is unknown and deserves further studies.
There are two main points arising from this study: (i) the finding that tRA, together with thyroid hormone, peroxisome proliferators and the halogenated hydrocarbon TCPOBOP, ligands of the nuclear receptors RARs, TRs, PPARs and CAR, respectively, are all strong mitogens for rodent liver, supports the notion that nuclear receptors are, among several other biological effects, strongly involved in activating signal transducing pathways leading to cell cycle entry; (ii) the hepatomitogenic activity of tRA is in contrast to several previous studies suggesting a possible chemotherapeutic role of tRA in many types of cancer, based on its cytostatic effect. However, the finding of enhanced hepatocyte proliferation by tRA as demonstrated in this study, does not necessarily indicate that tRA has no antitumor effects. It is known that the great majority of preneoplastic nodules, after reaching a certain size, through replicative cell cycles, spontaneously undergo re-differentiation to normal-appearing liver (remodelling), with a few remaining as precursor for the further development to cancer (38). Although the mechanisms associated with re-differentiation of preneoplastic nodules are unknown, it is possible that agents capable of stimulating hepatocyte proliferation may also modulate genes involved in the re-differentiation programme, thus favouring the regression of preneoplastic lesions. In this respect, it is of interest the finding that another ligand of nuclear receptors, T3, accelerates regression of preneoplastic hepatic nodules and exerts an inhibitory effect on the development of hepatocellular carcinoma; these effects occur in spite of its mitogenic effect on normal and preneoplastic liver cells (39), suggesting that T3 may induce a re-differentiation programme in preneoplastic hepatocytes. By virtue of the great differentiating capacity of RA, its possible induction of preneoplastic hepatocyte proliferation coupled with induction of a greater degree of re-differentiation of the nodules, might theoretically decrease the incidence of liver cancer. Thus, the experimental model of tRA-induced hepatocyte proliferation may serve useful for future analyses of the role of tRA on liver carcinogenesis.
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Acknowledgments
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Supported by Associazione Italiana Ricerca sul Cancro (AIRC), Ministero Università e Ricerca Scientifica (PRIN ex 40 and 60%) and Fondazione Banco di Sardegna, Italy.
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References
|
---|
- Bollag,W. (1983) Vitamin A and retinoids: from nutrition to pharmacotherapy in dermatology and oncology. Lancet, 1, 860863.[Medline]
- Ellis,C.N. and Voorhes,J.J. (1987) Eretrinate therapy. A. J. Am. Acad. Dermatol., 16, 267291.[ISI][Medline]
- Giguere,V., Ong,E., Segui,P. and Evans,R.M. (1987) Identification of a receptor for the morphogen retinoic acid. Nature, 330, 624629.[CrossRef][ISI][Medline]
- Brand,N., Petkocic,M., Krust,A., Chambon,P., de The,H., Marchio,A., Tiollais,P. and Dejan,A. (1988) Identification of a second human retinoic acid receptor. Nature, 332, 850853.[CrossRef][ISI][Medline]
- Krust,A., Kastener,P., Petkoviv,M., Kastner,P. and Chambon,P. (1989) A third human retinoic receptor, hRARg. Proc. Natl Acad. Sci. USA, 86, 53105314.[Abstract]
- Zelent,A., Krust,A., Petkovic,M., Kastner,P. and Chambon,P. (1989) Cloning of murine a and b retinoid acid receptors and a novel receptor g predominantly expressed in skin. Nature, 339, 714717.[CrossRef][ISI][Medline]
- Mangelsdorf,D.J., Ong,E., Dyck,J.A. and Evans,R.M. (1990) Nuclear receptor that identifies a novel retinoic acid response pathway. Nature, 345, 224229.[CrossRef][ISI][Medline]
- Leid,M., Kastner,P., Lyons,R. et al. (1992) Purification, cloning and RXR identity of the HeLa cell factor with which RAR and TR heterodimerizes to bind target sequences efficiently. Cell, 68, 377395.[ISI][Medline]
- Mangelsdorf,D.J., Bormeyer,U., Heyman,R.A., Zhou,J.Y., Ong,E., Oro,A.E., Kakizuka,A. and Evans,R.M. (1992) Characterization of three RXR genes that mediate the action of cis-retinoic acid. Genes Dev., 6, 329344.[Abstract]
- Xiao,J.H., Durand,B., Chambon,P. and Voorhes,J.J. (1995) Endogenous retinoic acid receptor (RAR)-retinoid X receptor (RXR) heterodimers are the major functional forms regulating retinoid-responsive elements in adult human keratinocytes. Binding of ligands to RAR only is sufficient for RAR-RXR heterodimers to confer ligand-dependent activation of hRAR beta2/RARE (DR5). J. Biol. Chem., 270, 30013011.[Abstract/Free Full Text]
- Bollag,W. (1971) Effects of vitamin A on transplanted and chemically induced tumors. Cancer Chemother. Rep., 55, 5358.[ISI][Medline]
- Smith,M.A., Parkinson,D.R., Cheson,B.D. and Friedman,M.A. (1992) Retinoids in cancer therapy. J. Clin. Oncol., 10, 839864.[Abstract]
- Lacroix,A., Doskas,C. and Bhat,P. (1990) Inhibition of growth of established N-methyl-N-nitrosourea-induced mammary cancer in rats by retinoic acid and ovariectomy. Cancer Res., 50, 57315734.[Abstract]
- Ozeki,A. and Tsukamoto,I. (1999) Retinoic acid represses the expression of c-fos and c-jun and induced apoptosis in regenerating rat liver after partial hepatectomy. Biochim. Biophys. Acta, 1450, 308319.[CrossRef][ISI][Medline]
- Tamura,K., Nakae,D., Horiguchi,K. et al. (1997) Inhibition by N-(4-hydroxyphenyl)retinamide and all-trans-retinoic acid of exogenous and endogenous development of putative preneoplastic glutathione S-transferase placental form-positive lesions in rat liver. Carcinogenesis, 18, 21332141.[Abstract]
- McCormick,D.L., Hollister,J.L., Bagg,B.J. and Long,R.E. (1990) Enhancement of murine hepatocarcinogenesis by all-trans-retinoic acid and two synthetic retinamides. Carcinogenesis, 11, 16051609.[Abstract]
- Short,J., Brown,R.F., Husakova,A., Gilbertson,J.R., Zemel,R. and Lieberman,I. (1972) Induction of deoxyribonucleic acid synthesis in the liver of intact animal. J. Biol. Chem., 247, 17571766.[Abstract/Free Full Text]
- Francavilla,A., Carr,B.I., Azzarone,A., Polimeno,L., Wang,Z., Van Diehe,D.H., Subbotin,V., Prelich,J.G. and Starzl,T.E. (1994) Hepatocyte proliferation and gene expression induced by triiodothyronine in vivo and in vitro. Hepatology, 20, 12371241.[ISI][Medline]
- Rao,M.S. and Reddy,J.K. (1987) Peroxisome proliferators and hepatocarcinogenesis. Carcinogenesis, 8, 631636.[ISI][Medline]
- Marsman,D.S., Cattley,R.C., Conway,G. and Popp,J.A. (1988) Relationship of hepatic peroxisome proliferation and replicative DNA synthesis to the hepatocarcinogenicity of the peroxisome proliferators di(2-ethylhexyl)phthalate and [4-chloro-6-(2,3-xilidino)-2-pirimidynilthio]acetic acid (WY14643) in rats. Cancer Res., 48, 67396744.[Abstract]
- Ledda-Columbano,G.M. and Columbano,A. (2003) Mitogenesis by ligands of nuclear receptors: an attractive model for the study of the molecular mechanisms implicated in liver growth. Cell Death Differ., 10, S19S21.[CrossRef][ISI][Medline]
- Ledda-Columbano,G.M., Pibiri,M., Loi,R., Perra,A., Shinozuka,H. and Columbano,A. (2000) 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. Am. J. Pathol., 56, 9197.
- Pibiri,M., Ledda-Columbano,G.M., Cossu,C., Simbula,G., Menegazzi,M., Shinozuka,H. and Columbano,A. (2001) Cyclin D1 is an early target in hepatocyte proliferation induced by thyroid hormone (T3). FASEB J., 15, 10061013.[Abstract/Free Full Text]
- Ledda-Columbano,G.M., Curto,M., Piga,R. et al. (1998) In vivo hepatocyte proliferation is inducible through a TNF and IL-6-independent pathway. Oncogene, 17, 10391044.[CrossRef][ISI][Medline]
- Michalopoulos,G.K. and DeFrances,M.C. (1997) Liver regeneration. Science, 276, 6066.[Abstract/Free Full Text]
- Yamada,Y., Kirillova,I., Peschon,J.J. and Fausto,N. (1997) Initiation of liver growth by tumor necrosis factor: deficient liver regeneration in mice lacking type I tumor necrosis factor receptor. Proc. Natl Acad. Sci. USA, 94, 14411446.[Abstract/Free Full Text]
- Cressmann,D.E., Greenbaum,L.E., DeAngelis,R.A., Ciliberto,G., Furth,E.E., Poli,V. and Taub,R. (1996) Liver failure and defective hepatocyte regeneration in interleukin-6 deficient mice. Science, 274, 13791383.[Abstract/Free Full Text]
- Akerman,P., Cote,P., Yang,S.-Q., McClain,C., Nelson,S., Bagby,G.J. and Diehl,A.M. (1992) Antibodies to tumor necrosis factor-
inhibit liver regeneration after partial hepatectomy. Am. J. Physiol., 263, G579G585.[ISI][Medline]
- Columbano,A. and Shinozuka,H. (1996) Liver regeneration versus direct hyperplasia. FASEB J., 10, 11181128.[Abstract/Free Full Text]
- Burton,K. (1956) A study of the conditions and mechanisms of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. J., 62, 315323.[ISI]
- Bradford,M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Anal. Biochem., 72, 248254.[CrossRef][ISI][Medline]
- Columbano,A., Ledda-Columbano,G.M., Coni,P., Liguori,C., Santa Cruz,G. and Pani,P. (1984) Occurrence of cell death (apoptosis) during the involution of liver hyperplasia. Lab. Invest., 52, 670675.[ISI]
- Herwig,S. and Strauss,M. (1997) The retinoblastoma protein: a master regulator of cell cycle, differentiation and apoptosis. Eur. J. Biochem., 15, 581601.
- Sherr,C.J. (1996) Cancer cell cycles. Science, 274, 16721677.[Abstract/Free Full Text]
- Zhu,W.-Y., Jones,C.S., Kiss,A., Matsukuma,K., Amin,S. and De Luca,L.M. (1997) Retinoic acid inhibition of cell cycle progression in MCF-7 human breast cancer cells. Exp. Cell. Res., 234, 293299.[CrossRef][ISI][Medline]
- Lancillotti,F., Darwiche,N., Celli,G. and De Luca,L.M. (1992) Retinoid status and the control of keratin expression and adhesion during the histogenesis of squamous metaplasia of tracheal epithelium. Cancer Res., 52, 61446152.[Abstract]
- De Luca,L.M., Brugh,M. and Silverman-Jones,C.S. (1984) Retinyl palmitate, retinyl phosphate, dolichyl phosphate of postnuclear membrane fraction from hepatoma, host liver and regenerating liver: marginal vitamin A status of hepatoma tissue. Cancer Res., 44, 224232.[Abstract]
- Enomoto,K. and Farber,E. (1982) Kinetics of phenotypic maturation of remodelling of hyperplastic nodules during liver carcinogenesis. Cancer Res., 42, 23302335.[Abstract]
- Ledda-Columbano,G.M., Perra,A., Loi,R., Shinozuka,H. and Columbano,A. (2000) Cell proliferation induced by triiodothyronine in rat liver is associated with nodule regression and reduction of hepatocellular carcinomas. Cancer Res., 60, 603609.[Abstract/Free Full Text]
Received December 27, 2003;
revised June 17, 2004;
accepted June 22, 2004.