In vivo studies of altered expression patterns of p53 and proliferative control genes in chronic vitamin A deficiency and hypervitaminosis Elisa Borra ´ s, Rosa Zaragoza ´ , Marı ´ a Morante, Concha Garcı ´ a, Amparo Gimeno, Gerardo Lo ´ pez-Rodas, Teresa Barber, Vicente J. Miralles, Juan R. Vin ˜ a and Luis Torres 1 Departamento de Bioquı ´ mica y Biologı ´ a Molecular, Facultades de Medicina y Farmacia., Universidad de Valencia, Valencia, Spain Several clinical trials have revealed that individuals who were given b)carotene and vitamin A did not have a reduced risk of cancer compared to those given placebo 2,3 ; 2,3 rather, vita- min A could actually have caused an adverse effect in the lungs of smokers [Omenn, G.S., Goodman, G.E., Thorn- quist, M.D., Balmes, J., Cullen, M.R., Glass, A., Keogh, J.P., Meyskens, F.L., Valanis, B., Williams, J.H., Barnhart, S. & Hammar, S. N. Engl. J. Med (1996) 334, 1150–1155; Hennekens, C.H., Buring, J.E., Manson, J.E., Stampfer, M., Rosner,B.,Cook,N.R.,Belanger,C.,LaMotte,F.,Gazi- ano, J.M., Ridker, P.M., Willet, W. & Peto, R. (1996) N.Engl.J.Med. 334, 1145–1149]. Using differential display techniques, an initial survey using rats 4 showed that liver RNA expression of c-H-Ras was decreased and p53 increased in rats with chronic vitamin A deficiency 5 .These findings prompted us to evaluate the expression of c-Jun, p53 and p21 WAF1/CIF1 (by RT-PCR) in liver and lung of rats. This study showed that c-Jun levels were lower and that p53 and p21 WAF1/CIF1 levels were higher in chronic vitamin A deficiency. Vitamin A supplementation increased expression of c-Jun, while decreasing the expression of p53 and p21 WAF1/CIF1 . Western-blot analysis demonstrated that c-Jun and p53 showed a similar pattern to that found in the RT-PCR analyses. Binding of retinoic acid receptors (RAR) to the c-Jun promoter was decreased in chronic vitamin A deficiency when compared to control hepatocytes, but contrasting results were found with acute vitamin A sup- plementated cells. DNA fragmentation and cytochrome c release from mitochondria were analyzed and no changes were found. In lung, an increase in the expression of c-Jun produced a significant increase in cyclin D1 expression. These results may explain, at least in part, the conflicting results found in patients supplemented with vitamin A and illustrate that the changes are not restricted to lung. Furthermore, these results suggest that pharmacological vitamin A supplementation may increase the risk of adverse effects including the risk of oncogenesis. Keywords: vitamin A; retinoic acid; p53; cyclin D1; c-Jun 6 . Vitamin A (retinol) is an essential nutrient that is metabo- lized in mammalian cells to retinal and retinoic acid. The latter shares some of the activities of retinol but is unable to support processes such as vision (11-cis-retinal). Retinoids exert their effects by binding to specific receptors that comprise two subfamilies, RARs (retinoic acid receptors) and RXRs (retinoid X/cis RAR) [1,2]. A variety of studies have shown that vitamin A is necessary for normal growth and development through control of gene expression [3–9]. Vitamin A has other important effects; it can function as a pro-oxidant or as an antioxidant. The antioxidant properties of vitamin A have been shown both in vitro and in vivo [10–12]. Vitamin A deficiency causes oxidative damage to liver mitochondria in rats that can be reversed by vitamin A supplementation [13]. However, the addition of retinol and retinal to cultures of HL-60 cells causes cellular DNA cleavage and an increased formation of 8-oxo-7,8- dihydro-2¢-deoxyguanosine via superoxide generation [14]; moreover, in vitro, it has been shown that b-carotene cleavage products induce oxidative stress by impairing mitochondrial respiration [15]. Therefore, maintaining the vitamin A concentration within the physiological range is critical to normal cell function because either a deficiency or an excess of vitamin A induces oxidative stress. This study was undertaken to identify genes that may be regulated by vitamin A. Liver and lung were evaluated in normal, chronic vitamin A deficient and vitamin A supple- mented rats by the technique of differential display. As expression of c-H-Ras was found to be lower and that of p53 7 was higher in chronic vitamin A deficiency and c-H-Ras and p53 play an inverse role in the control of cellular prolifer- ation, related genes were studied by RT-PCR. The results presented here emphasize the importance of vitamin A in controlling the expression of p53 and related genes that are essential for maintaining the integrity of tissues. Materials and methods Rats Wistar rats (Charles River Lab., Barcelona, Spain) 8,9,10 were given a vitamin A deficient, solid diet 8,9,10 . 8,9,10 Pregnant rats were housed in individual cages in a room maintained at 22 °C Correspondence to J. R. Vin ˜ a, Departamento de Bioquı ´ mica y Biologı ´ a Molecular, Facultad de Medicina, Universidad de Valencia, Avenue. Blasco Iban ˜ ez 17, Valencia-46010, Spain. E-mail: Juan.R.Vina@uv.es Abbreviations: RAR, retinoic acid receptors. (Received 24 October 2002, revised 14 January 2003, accepted 20 January 2003) Eur. J. Biochem. 270, 1493–1501 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03511.x with a 12-h light : 12-h dark cycle. Rats were cared and handled in conformence with the Guiding Principles for Research Involving Animals and Humans, approved by the Council of the American Physiological Soceity. The School of Medicine Research Committee approved this study. One day after pup birth, dams were fed either a control diet or vitamin A-deficient diet. Milk production was evaluated during lactation in both groups. After weaning, the rats were fed the same corresponding diet until 50 days old [13]. A third group of rats received a daily peritoneal injection of vitamin A (200 000 IU all trans-retinol palmitate per kg body weight) over a period of 5 days. The trans-retinol palmitate (type VII: synthetic Sigma) was dissolved in NaCl 0.15 M . The control rats for the latter group were injected with NaCl, 0.15 M over a period of 5days. Retinoid determination The experiments were performed between 10.00 h and 12.00 h. Rats were anesthetized with Pentothal (50 mgÆkg )1 body weight, intraperitoneally). Blood was collected from the aorta in heparinized syringes and then liver and lung samples were taken and processed immediately. Retinol and retinyl palmitate were measured in serum and tissues following the method described by Barua and Olson [16]. Differential display analysis Liver RNA was isolated by extraction with the guanidinium thiocyanate method followed by centrifugation in a cesium chloride solution as described previously [17]. A 25-lg amount of total RNA was incubated for 30 min at 37 °C with 10 U of Dnase I/RNase-free (Roche Molecular Bio- chemicals) in 10 m M Tris/HCl pH 7.5, 10 m M MgCl 2 ,then samples were phenol/chloroform (1/1) 11 extracted and etha- nol precipitated in the presence of 0.3 M sodium acetate. RNA was redissolved in sterile nuclease-free water. Differential display was performed using oligo(dT) anchored primers [17,18] with the Hieroglyph mRNA Profile Kit (Genomyx, Beckman Instruments, Fullerton, CA, USA) following the manufacturer’s instructions with some modifications. First strand cDNA synthesis was performed with 2 lL of DNase-treated total RNA (0.1 lgÆlL )1 ), 2 lL of oligo(dT) anchored primer (2 l M ) and 2 lLdNTPmix(250l M ) (1 : 1 : 1 : 1) (v/v/v/v) 12 using SuperScript TM RNase H–Reverse Transcriptase (Gibco- BRL Life Technologies) in 50 m M Tris/HCl pH 8.3, 75 m M KCl, 20 m M dithiothreitol in a Perkin-Elmer Gene- Amp 9700 thermal cycler at 25 °C(10min),50°C (60 min) and 70 °C (15 min). The PCR was started by adding 2 lL of the cDNA solution to a mixture containing 9.95 lL of sterile nuclease-free water, 1.2 lLMgCl 2 (25 m M ), 1.6 lLdNTPmix(250l M )(1:1:1:1)(v/v/v) (Roche Molecular Biochemicals), 2 lL5¢-arbitrary primer (2 m M ), 2 lL AmpliTaq Buffer[10 · ], 0.2 lL AmpliTaq enzyme (5 UÆlL )1 ) (Perkin-Elmer, Branchburj, NJ, USA), and 0.25 lL[a- 33 P]dATP (10 lCiÆlL )1 ). Thermal cycling parameters using a GeneAmp 9700 thermal cycler were as follows: 95 °C (2 min), four cycles at 92 °C(15s),50°C (30 s) and 72 °C (2 min), 30 cycles at 92 °C(15s),60°C (30 s), and 72 °C (2 min), and an additional final extension step at 72 °C for 7 min. Reactions were performed with each cDNA solution in duplicate. Control reactions were set using sterile nuclease-free water or each DNase I-treated RNA instead of the cDNA solution. Following differential display PCR, radiolabeled cDNA fragments were electrophoretically separated on 4.5% polyacrylamide gels under denaturing conditions in a Genomix LR DNA sequencer (Genomix, Beckman). Gels were dried and exposed to produce an autoradiograph. Bands of interest were excised from the gel, and the gel slides were placed directly into PCR tubes and covered with 40 lL of PCR mix (24.4 lL sterile nuclease-free water, 3.2 lLdNTPmix,4lL T7 promoter 22-mer primer (2 l M ), 4 lL M13 reverse 24-mer primer (2 l M ), 2.4 lL MgCl 2 (25 m M ), 4 lL AmpliTaq PCR Buffer (10 ·), and 0.4 lL AmpliTaq enzyme (5 UÆlL )1 ). PCR was performed as follows: 95 °C (2 min), four cycles at 92 °C(15s),50°C (30 s) and 72 °C (2 min), 30 cycles at 92 °C(15s),60°C (30 s), and 72 °C (2 min), and an additional extension step at 72 °C for 7 min. Amplified fragments was sequenced in both directions using M13 reverse (-24) primer and T7 promoter forward (-22) primer. Nucleotide sequence homology search analysis of the EMBL [19] and GenBank [20] databases were performed using the BLAST program [21]. RNA isolation and Northern blot analysis Total RNA from the different tissues used was isolated by the guanidinium thyocianate method [22]. Aliquots (20 lg) of total RNA were size-fractioned by electrophoresis in a 1% agarose gel under denaturing conditions. RNAs were then blotted and fixed to Nytran membranes (Schleicher and Schuell, Keene, NH). Prehybridization and hybridiza- tion were performed as described previously [23]. Probes were the isolated clones from differential display (0.8-kb of mRNA p53 gene and 0.9-kb of mRNA c-H-Ras gene). Equal loading of the gels was assessed using ethidium bromide staining of the gel. The probes were labeled with [a 32 P]dCTP (3000 CiÆmmol )1 ) by random priming using the rediprime TM II DNA labeling system (Amersham Pharma- cia Biotech). Specific activity was  5 · 10 8 c.p.m.Ælg )1 of DNA. Quantitation was performed by densitometry of the X-ray films. Analysis of mRNA expression by RT-PCR RT-PCR was performed in one step with an Enhanced Avian RT-PCR Kit following the instructions of the manufacturer (Sigma). c-Jun expression levels were deter- mined using the following primers (5¢-TGAGTGCA AGCGGTGTCTTA-3¢ (forward) and 5¢-TAGTGGTGA TGTGCCCATG-3¢ (reverse); primers for p21 WAF1/CIF1 : 5¢-ACAGCGATATCGAGACACTCA-3¢ (forward) and 5¢-GTGAGACACCAGAGTGCAAGA-3¢ (reverse); pri- mers for p53:5¢-CACAGTCGGATATGAGCATC-3¢ (forward) and 5¢-GTCGTCCAGATACTCAGCAT-3¢ (reverse) and primers for cyclin D1:5¢-TGTTCGTGGC CTCTAAGATGA-3¢ (forward) and 5¢-GCTTGACTCCA GAAGGGCTT-3¢ (reverse); primers for 18S rRNA: 5¢-GAGTATGGTCGCAAGGCTGAA-3¢ (forward) and 5¢-GCCTCCAGCTTCCCTACACTT-3¢ (reverse). 18S 1494 E. Borra ´ s et al. (Eur. J. Biochem. 270) Ó FEBS 2003 rRNA was simultaneously amplified and used as an internal control. Routinely, RNA concentration curves were performed to verify that the RT-PCR was quantitative. Reactions were resolved using a 2% agarose gel stained with ethidium bromide and quantified using the Gene Genius System Tools analysis software ( SYNGENE ). Antibodies Monoclonal (mouse) anti-p53 was purchased from Calbiochem (Ab-3 op29), anti-c-Jun Ig and anti-RAR (a,b,c) Ig, were purchased from Santa Cruz Biotechnology, Inc. Immunoblot analysis Tissues were homogenized in 10 mLÆg )1 of tissue of ice- cold buffer A [10 m M Hepes, pH 7.9, 10 m M KCl, 2 m M MgCl 2 ,0.5m M dithiothreitol, 1 m M phenylmethane- sulfonyl fluoride, 5 m M NaF, 0.5 m M Na 3 VO 4 and 0.1% Triton X-100 in the presence of protease inhibitor (5 lLÆmL )1 P8340, Sigma)] 13 . The resulting homogenate was centrifuged at 14 000 r.p.m. 14 for 15 min at 4 °C. To obtain the nuclear proteins, the sediment was re-sus- pended in 3 mLÆg )1 of tissue in 20 m M Hepes pH 7.9, 25% glycerol, 0.42 M NaCl, 1.5 m M MgCl 2 ,0.2m M EDTA, 0.5 m M dithiothreitol, 1 m M phenylmethanesulfonyl fluoride, 5 m M NaF, 0.5 m M Na 3 VO 4 in the presence of protease inhibitors. The suspension was incubated for 30 min on ice and centrifuged at 17 089 g 15 for 10 min, at 4 °C. The supernatant was diluted with 500 lLof20m M Hepes pH 7.9, 20% glycerol, 50 m M KCl, 1.5 m M MgCl 2 , 0.2 m M EDTA, 0.5 m M dithiothreitol, 0.5 m M phenyl- methanesulfonyl fluoride, 5 m M NaF, 0.5 m M Na 3 VO 4 .To obtain the cytosolic proteins, the original supernatant was centrifuged at 15 868 g 16 for 10 min at 4 °Cinorderto remove mitochondria. Samples were subjected to 10% SDS/PAGE to study the high molecular mass protein or a gradient (10–15%) SDS/ PAGE to study the low molecular mass protein. In any case, after electrophoresis, the proteins were electroblotted onto nitrocellulose membranes (Schleicher and Schuell). Immu- nodetection of specific proteins was made with the respective antibody. Blots were incubated in blocking solution (5% w/v nonfat dry milk with 0.05% v/v Tween 20), for 1 h at room temperature with shaking; following three washes with TTBS (25 m M Tris/HCl, pH 7.5, 0.15 M NaCl and 0.1% v/v Tween 20), blots were incubated with primary antibodies in TTBS for 1 h at room temperature with gentle agitation. Blots were washed again with TTBS and incubated with the secondary antibody conjugates to horseradish peroxidase (Bio-Rad) for 20 min. Finally, blots were washed with TTBS and developed with a chemiluminescence kit (Lumi-Light Western Blotting Substrate, Roche). Immunoprecipitation of RAR-DNA complexes (chip assay) Liver cells were isolated from control, chronic vitamin A- deficient and hypervitaminic rats by the method of Berry and Friend as modified by Romero and Vin ˜ a [24]. Cells were treated with 1% formaldehyde in Krebs–Ringer buffer under gentle agitation for 10 min at room temperature in order to crosslink the transcription factors to DNA. The cells were collected by centrifugation at 190 g for 5 min, washed twice in 40 mL of NaCl/P i pH 7.4, once in solution I (10 m M Hepes, pH 7.5, 10 m M EDTA, 0.5 m M EGTA, 0.75% Triton X-100) and once in solution II (10 m M Hepes pH 7.5, 200 m M NaCl, 1 m M EDTA, 0.5 m M EGTA). The cells were resuspended in 500-lLof lysis buffer (25 m M Tris/HCl pH 7.5, 150 m M NaCl, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate) supplemen- tedwithproteaseinhibitorsandthensonicatedonicefor10 steps of 10 s at 30% output in a Branson 250 Sonicator (with microtip). The samples were centrifuged at 19 000 g for 2 min to clear the supernatants. The supernatants were transferred to an eppendorf tube and centrifuged at 19 000 g for 10 min. The lysates were diluted tenfold in lysis buffer and stored at )20 °C in aliquots of 1 mL (sample named as ÔinputÕ). The immunofractionation of RAR–DNA complexes was performed by addition of 10 lgÆmL )1 of RARc antibody (Santa Cruz Biotechnology, sc-773) and incubation at 4 °C overnight (on a 360° rotator). The inmunocomplexes were incubated with 10 mg of protein A Sepharose, prewashed with lysis buffer for 4 h at 4 °C under gentle rotation. The immunocomplexes were collected by centrifugation (6500 g, 1 min). The supernatant was stored at )20 °C. This fraction was named the Ôunbound fractionÕ. Antibody- bound fraction was washed once with RIPA buffer (50 m M Tris/HCl pH 8.0, 150 m M NaCl, 0.5% deoxycholate, 0.1% SDS, 1% NP-40, 1 m M EDTA), once with high salt buffer (50 m M Tris HCl pH 8.0, 500 m M NaCl, 0.1% SDS, 0.5% deoxycholate, 1% NP-40, 1 m M EDTA) once with LiCl buffer (50 m M Tris/HCl pH 8.0, 1 m M EDTA, 250 m M LiCl, 1% de NP-40, 0.5% deoxycholate) and twice with Tris/EDTA 17 buffer pH 8.0. The cross-links were reversed by heating the samples at 65 °C overnight. The DNA from all fractions (input, bound and unbound) were extracted with phenol/chloroform (1/1) 18 and quantified by fluorescence with PicoGreen dye (Molecular Probes). Analysis of immunoprecipitated DNA To check that the immunoprecipitation contains the c-Jun promoter among the pull of DNA, the different DNA samples (input, bound and unbound) were analyzed by PCR using the primers 5¢-TGTAACCTCTACTCCCA CCCA-3¢ (forward) and 5¢-TCTGAGTCCTTATCCAGC CTG-3¢ (reverse) corresponding to a region of the c-Jun promoter that expands between the start transcription site and the )504 position. Statistics In the experiment shown in the Table 1, a two-way ANOVA was performed; in the other experiments, a one-way ANOVA was performed. The homogeneity of the variances was analyzed by the Levene test; in those cases in which the variances were unequal, the data were adequately transformed before the ANOVA . The null hypothesis was accepted for all the values of these sets in which the F-value was nonsignificant at P > 0.05. The data for which the F-value was significant were examined by the Tukey’s test at P < 0.05. Values in the text are means ± SEM. Ó FEBS 2003 Vitamin A status and proliferative control genes (Eur. J. Biochem. 270) 1495 Results Physiological parameters and all- trans retinol and all- trans retinyl palmitate concentrations in plasma and tissues After delivery, dams were fed either a control diet or a vitamin A-deficient diet for 50 days. Milk production was evaluated at the peak of lactation in both groups, and no difference was found. After weaning, the pups either ate the control diet or the vitamin A-deficient diet; no statistical difference in body weight or food intake was found between groups. In control rats injected daily with all-trans retinyl palmitate over a period of 5 days, food intake was also not different from control rats. All-trans retinol plasma concentration was decreased significantly in chronic vitamin A deficiency when com- pared to control rats (Table 1). In the liver of vitamin A-deficient rats, all-trans retinol and all-trans retinyl palmi- tate were decreased significantly when compared to control livers. In lung from vitamin A-deficient rats, all-trans-retinyl palmitate was decreased significantly when compared to control lungs (Table 1). When rats were injected daily over a period of 5 days with retinyl palmitate, the concentration of this compound was significantly higher in plasma, liver and lung when compared to controls. Liver gene expression evaluated by differential display in control and in chronic vitamin A-deficiency Liver differential display analysis was performed in control and chronic vitamin A-deficient rats (50 days). Several bands were differentially expressed in chronic vitamin A- deficiency when compared to control liver. Two of the bands selected for analysis, whose expression were differ- entialy expressed in chronic vitamin A-deficiency, were excised from the gel, amplified and sequenced (Fig. 1). The first clone of  0.8 kb had 100% homology to part of the rat p53 cDNA and could hybridize to a 1.6-kb mRNA in total cellular RNA from rat liver. The band detected in Northern blot by the first clone corresponded in size to that reported for rat p53 and was up-regulated fivefold when compared with control rat liver, thus confirming the up-regulation detected in the differential display gel. The second clone of about 0.9 kb had 100% homology to part of the rat c-H-Ras cDNA and hybridized with a 1.7-kb mRNA in total RNA from rat liver. This gene was down- regulated sixfold when compared with control rat liver (Fig. 1), showing an opposite expression pattern to that of the p53 gene. RT-PCR analysis of c-jun , p53 and p21 WAF1/CIF1 in liver and lung of control, chronic vitamin A-deficiency and hypervitaminosis rats Based on the results found in the differential display study and taking into account the role that p53 plays in the control of cellular proliferation, the expression levels of p53, c-Jun (a negative regulator of p53)andp21 WAF1/CIF1 (positively regulated by p53) were evaluated in liver by RT-PCR analysis. The mRNA levels in liver (Fig. 2) and in lung (Fig. 3) of p53 and of p21 WAF1/CIF1 were significantly increased in vitamin A-deficient rats when compared to controls but the expression of c-Jun was significantly lower when compared to controls. When retinyl palmitate was injected daily over a period of 5 days, the expression levels of p53 and p21 WAF1/CIF1 were significantly lower than in controls while the expression level of c-Jun was signifi- cantly higher than in control (Figs 2 and 3). Western blot analysis in liver and lung of control, chronic vitamin A-deficiency and hypervitaminosis rats Liver and lung samples were electrophoresed and immuno- blotted with specific antibodies. The amount of p53 protein was significantly higher in chronic vitamin A-deficient rats than in controls, while the amount of c-Jun was significantly lower in chronic vitamin A-deficiency as compared to controls. In hypervitaminosis the amount of p53 was significantly lower than in controls, whereas the amount of c-Jun was significantly higher than in controls (Fig. 4). These changes in the pattern levels followed the pattern of p53 and c-Jun gene expression showed in Figs 2 and 3. Immunoprecipitation of complex RAR–DNA To elucidate the mechanism of this pattern of expression, the binding of RAR to c-Jun promoter was studied using the immunoprecipitation of complex RAR–DNA with a polyclonal antibody reactive to RARa,RARb and RARc. Table 1. Retinol concentrations in plasma and tissues from control, vitamin-A deficient and hypervitaminosis rats. Values are means ± SEM, with the numbers of animals indicated in parentheses. Different superscript letters within a row indicate significant differences, P <0.05.ND,notdetected. Control Vitamin A-deficient Hypervitaminosis PLASMA (l M ) All-trans retinol 2.99 ± 0.48 (6) a 0.46 ± 0.10 (4) c 1.61 ± 0.12 (5) b All-trans retinyl palmitate ND ND 1.60 ± 0.22 (5) TISSUES (lg/g) Liver All–trans retinol 2.88 ± 0.48 (6) b 0.18 ± 0.03 (2) c 78.08 ± 15.40 (3) a All–trans retinyl palmitate 64.66 ± 10.97(6) b 5.60 ± 1.66 (4) c 4276 ± 1334 (5) a Lung All–trans retinol 0.23 ± 0.02 (3) b ND 44.14 ± 6.05 (5) a All–trans retinyl palmitate 2.99 ± 0.32 (4) b 0.64 ± 0.11 (4) c 262.74 ± 35.55 (5) a 1496 E. Borra ´ s et al. (Eur. J. Biochem. 270) Ó FEBS 2003 DNA was extracted from the input, bound and unbound fractions; equal amounts from each fraction were analyzed using primers that amplify the c-Jun promoter region between the start transcription site and the )504 position. The amplified DNA was resolved by agarose gel electro- phoresis. The specific binding was determined by the relative intensity of ethidium bromide fluorescence when compared to the input control. Our data show that the RAR binding was almost undetectable in vitamin A-deficiency when compared to controls, while in acute hypervitaminosis the binding was significantly higher. These changes in the RAR–DNA binding is not due to different levels of the retinoic acid receptor induced by the treatment, as RAR expression was similar in control, vitamin A-deficiency and hypervitaminosis (Fig. 5) rats. An antibody against a protein unrelated to vitamin A was used as a mock control and binding was not observed (results not shown). Discussion Using differential display analysis, it has been shown in the liver of chronic-vitamin A deficient rats that the expression of p53 was significantly higher when compared to control rats. It was also found that expression of c-H-Ras was significantly lower in chronic vitamin A-deficient rats than in controls. Based on these findings, c-Jun, a proto-oncogene, that encodes a component of the mitogen-inducible immediate early transcription factor, AP-1 and has that been implicated as a positive regulator of cell proliferation Fig. 2. Expression of c-Jun, p5 3 and p21 WAF1/CIF1 in liver. (C) control rats (D) vitamin A-deficient and (H) hypervitaminosis. Total RNA was isolated for each condition amplified by RT-PCR using specific primers for p53, c-Jun, p21 and for 18S rRNA as described in Mate- rials and methods. *P < 0.05. Fig. 1. Detection of differential gene expression induced by chronic vitamin A-deficiency in rats. Panel A, sequencing gel electrophoresis of PCR amplified cDNAs performed in duplicate, from control (C) and vitamin A-deficient rats (D). A differentially displayed fragment (arrow) was detected, isolated, and identified as a 0.8-kb fragment of p53 cDNA. Northern blot analysis of total RNA from control and vitamin A-deficient rats with p53 cDNA fragment confirmed its dif- ferential expression. Panel B, another differentially displayed fragment was detected, isolated and identified as a 0.9-kb fragment of c-H-Ras cDNA. Northern blot analysis of total RNA confirmed its differential expression. Ó FEBS 2003 Vitamin A status and proliferative control genes (Eur. J. Biochem. 270) 1497 and G 1 –S phase progression [24,26], was analyzed by RT-PCR. As expected, in chronic-vitamin A-deficiency, c-Jun was significantly lower than in control animals as it negatively regulates transcription of p53 by binding directly to a variant AP1 site in the p53 promoter [25]. As p21 WAF1/CIP1 encodes a cyclin dependent kinase inhibitor that is a critical target of p53 in facilitating G 1 arrest, we also studied the expression of p21 WAF1/CIP1 , and found that it is increased in chronic-vitamin A deficiency when compared to control rats. Using Western blot analysis it was also found that p53 protein was increased and c-Jun protein was decreased in liver chronic-vitamin A deficient rats when compared to controls. No apoptosis was found in liver of chronic-vitamin A deficiency because the typical ladder- type DNA pattern of nuclear apoptosis was not observed and also no cytochrome c was detected in the cytosol (data not shown). All these results found in chronic vitamin A deficient rats suggest that the increase of p53,resultsin arrest of progression through the cell cycle [27]. In rats Fig. 4. Western blot analysis of p53 and c-Jun in liver and lung. Total protein extracts were obtained as described in experimental proce- dures. A single band of about 53 kDa was detected in liver and lung indicating that the amount of p53 protein was significantly increased in the liver and lung from vitamin A-deficient rats that in controls. c-Jun protein was significantly decreased in vitamin A-deficient rats. In the liver of rats with hypervitaminosis the results showed a decrease in theamountofp53andanincreaseofthec-Jun.Thefigureshows that the expression of p53 is time course dependent. C, control; D, vitamin A-deficient rats; H, hypervitaminosis. Fig. 3. Expression of c-Jun, p53 and p21 WAF1/CIF1 in lung. (C) control rats (D) vitamin A-deficient rats (H) hypervitaminosis. Total RNA was isolated for each condition amplified by RT-PCR using specific primers for p53, c-Jun, p21 and for 18S rRNA as described in Mate- rials and methods. *P < 0.05. 1498 E. Borra ´ s et al. (Eur. J. Biochem. 270) Ó FEBS 2003 injected with a high-dose of vitamin A over a period of 5days,c-jun was increased and p53 and p21 WAF1/CIP1 were significantly lower when compared to controls. Overexpression of c-Jun alters cell cycle parameters and increases the proportion of cells in S, G 2 and M relative to G 0 phases of the cell cycle. The role of c-Jun in promoting cell growth has been highlighted from studies of c-Jun deficient mouse embryonic fibroblasts [27]. Fibroblasts lacking c-Jun exhibit a severe proliferation defect, because these cells accumulate the tumor suppressor, p53 and its downstream target, the cyclin-dependent kinase inhibitor p21 WAF1/CIF1 , suggesting that one particular function of c-Jun is the negative regulation of p53 transcription. The fact that the binding of RAR to the c-Jun promoter is affected by the vitamin A status and that no changes in the RAR amount were observed, suggests that c-Jun is under direct control of retinoic acid. However, c-Jun expression can be also regulated by Ras by means of the MAPkinase pathway [28] and this mechanism could explain in part the decreased expression of c-Jun found in chronic- vitamin A-deficient rats 19 . The molecular mechanism that explains the decrease in C-H-Ras expression found in chronic-vitamin A deficient rats is unknown at the present time. The role of vitamin A status in the expression of genes related to the regulation of cell proliferation has been studied in vivo and in vitro, and shows that retinoids play an important role in proliferation and differentiation. Using mice deficient in retinaldehyde dehydrogenase-2 it has been shown that retinoic acid synthesized by the postimplanta- tion mammalian embryo is an essential developmental hormone whose absence leads to early embryonic death [7]. In rat Sertoli cells, a significant up-regulation in c-Jun (beginning at 30 min and reaching a fourfold peak over controls at 1 h) has been observed [9]. Our results, in an in vivo model, are in agreement with these observations because c-Jun, p53 and p21 WAF1/CIF1 are modulated in liver by the vitamin A status. Moreover, this modulation in part can be produced by the control that the retinoic acid exerts on c-Jun expression. All the results found in liver were reproduced in lung, which can explain in part the conflicting results found in adults and children given b-carotene or vitamin A [29]. Moreover, in lung of rats injected with high-dose of vitamin A over a period of 5 days, the overexpression of c-Jun, produced a significant increase in the cyclin D1 expression, a positive regulator of G 1 –S phase transition (Fig. 6). These results and the fact that the transcription of p53 and p21 were significantly decreased as well as the levels of the p53 protein may indicate that the exposure to b-carotene can increase the carcinogenesis risk. Epidemio- logic studies in humans suggest that high consumption of fruits and vegetables is associated with a reduced risk of chronic diseases including cancer and cardiovascular disease [30–33]. Recently, it has been shown in two cohort studies that a-carotene and lycopene intakes were significantly Fig. 5. Immunoprecipitation of complex RAR-DNA and c-Ju n promoter detection. Panel A, the binding of RAR to c-Jun promoter was studied using the immunoprecipitation of complex RAR-DNA with a polyclonal antibody reactive to RARa, RARb and RARc, as described in Experimental procedures. DNA was extracted from the input, bound and unbound fractions; equal amounts from each fraction were analyzed using primers that amplify the c-Jun promoter region between the start transcription site and the )504 position, the amplified DNA were resolved by agarose electrophoresis. The intensity of fluorescent dye, ethydium bromide, relative to the intensity from the input gives the enrichment generated by the antibodyselection.PanelB:detectionofRARbyWesternblotting.C,control;D,vitaminA-deficientrats;H,hypervitaminosis. Ó FEBS 2003 Vitamin A status and proliferative control genes (Eur. J. Biochem. 270) 1499 associated with a lower risk of lung cancer, and the intakes of b-carotene, lutein and b-cryptoxanthin were also associ- ated with a lower risk but it was not significant. Even in smokers, a significant reduction in cancer risk was noted in association with increased lycopene intake [34]. However, The a-Tocopherol b-Carotene (ATBC) trial [35] and the b-Carotene and Retinol Efficacy Trial (CARET) [36] revealed that individuals that were given b-carotene and vitamin A received no protection from cancer and there may even have been an adverse effect on the incidence of lung cancer (and on the risk of death from lung cancer) and due to any cause in smokers and workers exposed to asbestos. 20 In another trial (Physician’s Health Study), the supplementation with b-carotene to male physicians during a 12-year-span produced neither benefits nor harm in terms of the incidence of cancer, cardiovascular disease, or death from all causes [37] 21 . The failure of these studies to demon- strate that large b-carotene supplement has a protective role has been explained by several factors: (a) the high tissue b-carotene concentrations (as much as 50-fold higher than those observed in a normal population that eat large amounts of fruits and vegetables) may had adverse effects and interactions that were not observed at the lower con- centrations obtained with diet [38]; (b) individual variation in serum response to administration among subjects given an identical dose of b-carotene [38]; (c) interference with the uptake, transport, distribution, and/or metabolism of other nutrients; (d) high levels of carotene and the products of its oxidation may act as prooxidants; (e) the alcohol intake of the subjects 25 studied [35,39,40] and (f) the different bioavail- ability found when a single high dose is used when compared to the mixtures found when fruit and vegetables are eaten [38]. All these facts emphasize that fruit and vegetable intakes are more convenient than an increased intake of a single Ôdrug-likeÕ chemopreventive carotenoid [41]. In ferrets, the hazard association of high-dose b-carotene supplementation and tobacco smoking is asso- ciated with elevated carotene oxidation products in lung tissues, significantly lower concentrations of retinoic acid and reductions (18–73%) in bRAR gene expression. Ferrets given a diet supplemented 22 with carotene and exposed to tobacco smoke had an increased expression of c-Jun and c-Fos genes [41,42]. Our work provides a mechanism that may explain in part the regulation of control of proliferative genes that cause an increased incidence of cancer in smokers supplemented with b-carotene. This mechanism (Fig. 7) consists of the direct regulation that exerts the retinoic acid on c-Jun expression 23 . The increased levels of c-Jun induce a positive effect in the expression of the cyclin D1 gene and the down-regulation of p53. Our results explain the importance of keeping vitamin A status within the normal range, because p53 tumor suppressor protein, a transcription factor involved in maintaining genomic integrity by controlling cell cycle progression and cell survival, changes its expression at different vitamin A levels. Acknowledgements The authors thank Luis Franco for advice and valuable comments. This work has been supported by FIS 99/1157, BFI2001-2842, CICYT- Comisio ´ n Europea (1FD97-1336), Generalitat Valenciana and PB97- 1368 from DGICYT, Ministerio de Educacio ´ n y Cultura (Spain). Projectes Precompetitius Universitat de Vale ` ncia (68200) and Redes de Investigacion Cooperation Instituto Carlos III (RC03-08). R. Z. is supported by a predoctoral fellowship of the Ministerio de Educacio ´ ny Cultura. Spain. M. 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In vivo studies of altered expression patterns of p53 and proliferative control genes in chronic vitamin A deficiency and hypervitaminosis Elisa Borra ´ s,. p21 WAF1/CIF1 : 5¢-ACAGCGATATCGAGACACTCA-3¢ (forward) and 5¢-GTGAGACACCAGAGTGCAAGA-3¢ (reverse); pri- mers for p53: 5¢-CACAGTCGGATATGAGCATC-3¢ (forward) and