Báo cáo khoa học: In vivo studies of altered expression patterns of p53 and proliferative control genes in chronic vitamin A deficiency and hypervitaminosis pot

Using differential display techniques, an initial survey using rats RNA expression of c-H-Ras was decreased and p53 increased in rats with chronic vitamin A deficiency findings prompted us

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

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

RNA expression of c-H-Ras was decreased and p53

increased in rats with chronic vitamin A deficiency

findings prompted us to evaluate the expression of c-Jun, p53

and p21WAF1/CIF1(by RT-PCR) in liver and lung of rats This

study showed that c-Jun levels were lower and that p53 and

p21WAF1/CIF1 levels were higher in chronic vitamin A

deficiency Vitamin A supplementation increased expression

of c-Jun, while decreasing the expression of p53 and p21WAF1/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

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 p53play 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) were8,9,10

given a vitamin A deficient, solid diet.Pregnant rats were8,9,108,9,10

housed in individual cages in a room maintained at 22C

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)

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 0.15M The control rats for the latter

group were injected with NaCl, 0.15 M over a period of

5 days

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 37C

with 10 U of Dnase I/RNase-free (Roche Molecular

Bio-chemicals) in 10 mMTris/HCl pH 7.5, 10 mMMgCl2, then

samples were phenol/chloroform (1/1)

etha-nol precipitated in the presence of 0.3M 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 lM)

and 2 lL dNTP mix (250 lM) (1 : 1 : 1 : 1) (v/v/v/v)

SuperScriptTM RNase H–Reverse Transcriptase

(Gibco-BRL Life Technologies) in 50 mMTris/HCl pH 8.3, 75 mM

KCl, 20 mM dithiothreitol in a Perkin-Elmer

Gene-Amp 9700 thermal cycler at 25C (10 min), 50 C

(60 min) and 70C (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 lL MgCl2

(25 mM), 1.6 lL dNTP mix (250 lM) (1 : 1 : 1 : 1) (v/v/v)

(Roche Molecular Biochemicals), 2 lL 5¢-arbitrary primer

(2 mM), 2 lL AmpliTaq Buffer[10· ], 0.2 lL AmpliTaq

enzyme (5 UÆlL)1) (Perkin-Elmer, Branchburj, NJ, USA),

and 0.25 lL [a-33P]dATP (10 lCiÆlL)1) Thermal cycling

parameters using a GeneAmp 9700 thermal cycler were as

follows: 95C (2 min), four cycles at 92 C (15 s), 50 C

(30 s) and 72C (2 min), 30 cycles at 92 C (15 s), 60 C

(30 s), and 72C (2 min), and an additional final extension step at 72C 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 lL dNTP mix, 4 lL T7 promoter 22-mer primer (2 lM), 4 lL M13 reverse 24-mer primer (2 lM), 2.4 lL MgCl2 (25 mM), 4 lL AmpliTaq PCR Buffer (10·), and 0.4 lL AmpliTaq enzyme (5 UÆlL)1) PCR was performed

as follows: 95C (2 min), four cycles at 92 C (15 s), 50 C (30 s) and 72C (2 min), 30 cycles at 92 C (15 s), 60 C (30 s), and 72C (2 min), and an additional extension step

at 72C 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 theBLASTprogram [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 [a32P]dCTP (3000 CiÆmmol)1) by random priming using the rediprimeTMII DNA labeling system (Amersham Pharma-cia Biotech) Specific activity was 5 · 108c.p.m.Ælg)1of 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 p21WAF1/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

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)1of tissue of

ice-cold buffer A [10 mM Hepes, pH 7.9, 10 mMKCl, 2 mM

MgCl2, 0.5 mM dithiothreitol, 1 mM

phenylmethane-sulfonyl fluoride, 5 mM NaF, 0.5 mM Na3VO4 and 0.1%

Triton X-100 in the presence of protease inhibitor

(5 lLÆmL)1 P8340, Sigma)]

was centrifuged at 14 000 r.p.m

To obtain the nuclear proteins, the sediment was

re-sus-pended in 3 mLÆg)1of tissue in 20 mMHepes pH 7.9, 25%

glycerol, 0.42M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA,

0.5 mM dithiothreitol, 1 mM phenylmethanesulfonyl

fluoride, 5 mM NaF, 0.5 mM Na3VO4 in the presence of

protease inhibitors The suspension was incubated for

30 min on ice and centrifuged at 17 089 g

4C The supernatant was diluted with 500 lL of 20 mM

Hepes pH 7.9, 20% glycerol, 50 mMKCl, 1.5 mMMgCl2,

0.2 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM

phenyl-methanesulfonyl fluoride, 5 mMNaF, 0.5 mMNa3VO4 To

obtain the cytosolic proteins, the original supernatant was

centrifuged at 15 868 g

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 mMTris/HCl, pH 7.5, 0.15MNaCl 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/Pi pH 7.4, once in solution I (10 mMHepes, pH 7.5, 10 mMEDTA, 0.5 mM

EGTA, 0.75% Triton X-100) and once in solution II (10 mM Hepes pH 7.5, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA) The cells were resuspended in 500-lL of lysis buffer (25 mM Tris/HCl pH 7.5, 150 mMNaCl, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate) supplemen-ted with protease inhibitors and then sonicasupplemen-ted on ice for 10 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 input)

The immunofractionation of RAR–DNA complexes was performed by addition of 10 lgÆmL)1of RARc antibody (Santa Cruz Biotechnology, sc-773) and incubation at 4C overnight (on a 360 rotator) The inmunocomplexes were incubated with 10 mg of protein A Sepharose, prewashed with lysis buffer for 4 h at 4C 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 unbound fraction Antibody-bound fraction was washed once with RIPA buffer (50 mM

Tris/HCl pH 8.0, 150 mMNaCl, 0.5% deoxycholate, 0.1% SDS, 1% NP-40, 1 mMEDTA), once with high salt buffer (50 mMTris HCl pH 8.0, 500 mMNaCl, 0.1% SDS, 0.5% deoxycholate, 1% NP-40, 1 mM EDTA) once with LiCl buffer (50 mM Tris/HCl pH 8.0, 1 mM EDTA, 250 mM

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 65C overnight The DNA from all fractions (input, bound and unbound) were extracted with phenol/chloroform (1/1)

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-wayANOVA

was performed; in the other experiments, a one-wayANOVA

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

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 ofc-jun, p53 and p21WAF1/CIF1in 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) and p21WAF1/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 p21WAF1/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 p21WAF1/CIF1were 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, not detected.

PLASMA (l M )

All-trans retinol 2.99 ± 0.48 (6) a 0.46 ± 0.10 (4) c 1.61 ± 0.12 (5) b

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 retinyl palmitate 2.99 ± 0.32 (4) b 0.64 ± 0.11 (4) c 262.74 ± 35.55 (5) a

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, p53 and p21WAF1/CIF1in 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 bychronic

vitamin A-deficiencyin 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.

and G1–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

p21WAF1/CIP1 encodes a cyclin dependent kinase inhibitor

that is a critical target of p53 in facilitating G1arrest, we also

studied the expression of p21WAF1/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, results in

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 the amount of p53 and an increase of the c-Jun The figure shows 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.

injected with a high-dose of vitamin A over a period of

5 days, c-jun was increased and p53 and p21WAF1/CIP1were

significantly lower when compared to controls

Overexpression of c-Jun alters cell cycle parameters and

increases the proportion of cells in S, G2and M relative to

G0phases 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

p21WAF1/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

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 p21WAF1/CIF1are 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 G1–S phase transition (Fig 6) These results and the fact that the transcription of p53and 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-Jun 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 antibody selection Panel B: detection of RAR by Western blotting C, control; D, vitamin A-deficient rats; H, hypervitaminosis.

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 drug-like 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

tobacco smoke had an increased expression of c-Jun and

c-Fosgenes [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´n y Cultura Spain M M is supported by a predoctoral fellowship of the Consellerı´a de Cultura Educacio´ i Cie`ncia, Generalitat Valenciana. References

1 Chambon, P (1996) A decade of molecular biology of retinoic acid receptors FASEB J 10, 940–954.

2 Evans, T.R.J & Kayne, S.B (1999) Retinoids; present role and future potential Br J Cancer 80, 1–8.

3 Chen, Y., Derguini, F & Buck, J (1997) Vitamin A in serum is a survival factor for fibroblasts Proc Natl Acad Sci 94, 10205– 10208.

4 Mason, K.E (1935) Foetal death, prolonged gestation and diffi-cult parturition in the rat as a result of vitamin A deficiency Am J Anat 57, 303–349.

Fig 6 Induction of cyclin D1 in lung Total RNA was isolated for each

condition amplified by RT-PCR using specific primers for cyclin D1

and for 18S rRNA as described in Materials and methods *P < 0.05.

C, control rats; H, rats with a high-dose of vitamin A over a period of

5 days.

Fig 7 Diagrammatic representation of the changes induced in the control of proliferative genes byvitamin A Within the nucleus, retinoic acid binds to RAR/RXR (heterodimeric retinoic acid-receptors) The ligand-attached receptors then bind to the retinoic acid-receptor ele-ment of the c-Jun promoter, thereby activating its expression The increased levels of c-Jun up-regulate cyclin D 1 expression and inhibit the transcription of p53 and p21.

5 Hale, F (1937) The relation of maternal vitamin A deficiency to

microphthalmia in pigs Texas State J Med 33, 228–232.

6 Zile, M.H (2001) Function of vitamin A in vertebrate embryonic

development J Nutr 131, 705–708.

7 Niederreither, K., Subbarayan, V., Dolle´, P & Chambon, P.

(1999) Embryonic retinoic acid synthesis is essential for early

mouse post-implantation development Nature Genet 21, 444–

448

24

8 Batourina, E., Gim, S., Bello, N., Shy, M., Clagett-Dame, M.,

Srinivas, S., Costantini, F & Mendelsohn, C (2001) Vitamin A

controls epithelium/mesenchymal interactions through Ret

expresion Nature Genet 27, 74–78.

9 Page, K.C., Heitzman, D.A & Chernin, M.I (1996) Stimulation

of c-jun and c-myb in rat Sertoli cells following exposure to

retinoids Biochem Biophys Res Comm 225, 595–600.

10 Monagham, B.R & Schmitt, F.O (1932) The effects of carotene

and of vitamin A on the oxidation of linoleic acid J Biol Chem.

96, 387–395.

11 Ciaccio, M., Valenza, M., Tesoriere, L., Bongiorno, A., Albiero,

R & Livrea, M.A (1993) Vitamin A inhibits doxorubicin-induced

membrane lipid peroxidation in rat tissues in vivo Arch Biochem.

Biophys 90, 103–108.

12 Palacios, A., Piergiacomi, V.A & Catala´, A (1996) Vitamin A

supplementation inhibits chemiluminescence and lipid

peroxida-tion in isolated rat liver micrososmes and mitochondria Mol Cell

Biochem 154, 77–82.

13 Barber, T., Borra´s, E., Torres, L., Garcı´a, C., Cabezuelo, F.,

Lloret, A., Pallardo´, F.V & Vin˜a, J.R (2000) Vitamin A

defi-ciency causes oxidative damage to liver mitochondria in rats Free

Rad Biol Med 29, 1–7.

14 Murata, M & Kawanishi, S (2000) Oxidative DNA damage by

vitamin A and its derivate via superoxide generation J Biol.

Chem 275, 2003–2008.

15 Siaems, W., Sommerburg, O., Schild, L., Augustin, W., Langhans,

C.D & Wiswedel, I (2002) b-Carotene cleavage products induce

oxidative stress in vitro by impairing mitochondrial respiration.

FASEB J 16, 1289–1291.

16 Barua, A.B & Olson, J.A (1998) Reversed-phase gradient high

performance liquid chromatographic procedure for simultaneous

analysis of very polar to non-polar retinoids, carotenoids and

tocopherols in animals and plant samples J Chromatogr B 707,

69–79.

17 Otero, G., Avila M.A., De la Pen˜a, L., Emfietzoglou, J.,

Cansado, G.F & Popescu, and Notario, V (1997) Altered

pro-cessing of precursor transcripts and increased levels of the subunit

I of mitochondrial cytochorme c oxidase in syrian hamster fetal

cells initiated with ionizing radiation Carcinogenesis 18, 1569–

1775.

18 Liang, P & Pardee, A.B (1992) Differential display of eukaryotic

messenger RNA by means of the polymerase chain reaction.

Science 257, 967–971.

19 Hamon, G.H & Cameron, G.N (1986) The EMBL data library.

Nucleic Acids Res 14, 5–10.

20 Bilofsky, H.S., Burks, C & The GenBank data bank (1988) Nucl

Acids Res 16, 1861–1864.

21 Pearson, W.R & Lipmann, D.J (1988) Improved tools for

bio-logical sequences comparison Procl Natl Acad Sci USA 85,

2444–2448.

22 Chomczynski, P & Sacchi, N (1987) Single-step method for RNA

isolation by acid guanidium thiocyanate-phenol-chloroform

extraction Anal Biochem 162, 156–159.

23 Thomas, P.S (1980) Hybridization of denatured RNA and small

DNA fragments transferred to nitrocellulose Proc Natl Acad.

Sci USA 162, 156–159.

24 Romero, F.J & Vin˜a, J (1983) A simple procedure for the

pre-paration of isolated liver cells Biochem Educ 11, 135–136.

25 Schreiber, M., Kolbus, A., Piu, F., Szabowski, A., Mo¨hle-Stein-lein, U., Tian, J., Karin, M., Angel, P & Wagner, E.F (1999) Control of cell cycle progression by c-jun is p53 dependent Genes Dev 13, 607–619.

26 Wisdom, R., Johnson, R.S & Moore, C (1999) c-Jun regulates cell cycle progression and apoptosis by distinct mechanisms EMBO J 18, 188–197.

27 Griffiths, J.K (2000) The vitamin A paradox J Pediatr 137, 604– 607.

28 Mechta-Grigoriou, F., Gerald, D & Yaniv, M (2001) The mammalian Jun proteins: redundancy and specificity Oncogene

20, 2378–2389.

29 Peto, R., Doll, R., Buckley, J.D & Sporn, M.B (1981) Can dietary beta carotene materially reduces human cancer rates? Nature 290, 201–208.

30 Block, G., Patterson, B & Subar, A (1992) Fruit, vegetables and cancer prevention: a review of the epidemiologic evidence Nutr Cancer 18, 1–29.

31 Ames, B.N., Gold, L.S & Willett, W.C (1995) The causes and prevention of cancer Proc Natl Acad Sci USA 92, 5258–5265.

32 Ames, B.N & Gold, L.S (1997) Environmental pollution, pesti-cides, and the prevention of cancer: misconceptions FASEB J 11, 1041–1052.

33 Michaud, D.S., Feskanich, D., Rimm, E.B., Colditz, G.A., Spe-izer, F.E., Willett, W.C & Giovannuci, E (2000) Intake of specific carotenoids and risk of lung cancer in 2 prospective US cohorts.

Am J Clin Nutr 72, 990–997.

34 Albanes, D., Heinonen, O.P., Taylor, P.R., Virtamo, J., Edwards, B.K., Rauthalathi, M., Hartman, A.M., Palmgren, J., Freedman, L.S., Haapakoski, J., Barrett, M.J., Pietinen, P., Malila, N., Liipo, K., Salomaa, E.R., Tangrea, J.A., Teppo, L., Askin, F.B., Erozan, Y., Greenwald, P & Huttunen, J.K (1996) Tocopherol and carotene supplements and lung cancer incidence in the alpha tocopherol, beta carotene cancer prevention study: effects of base line characteristics and study compliance J Natl Cancer Inst 88, 1560–1570.

35 Omenn, G.S., Goodman, G.E., Thornquist, M.D., Balmes, J., Cullen, M.R., Glass, A., Keogh, J.P., Meyskens, F.L., Valanis, B., Williams, J.H., Barnhart, S & Hammar, S (1996) Effects of a combination of beta-carotene and vitamin A on lung cancer and cardiovascular disease N Engl J Med 334, 1150–1155.

36 Hennekens, C.H., Buring, J.E., Manson, J.E., Stampfer, M., Rosner, B., Cook, N.R., Belanger, C., LaMotte, F., Gaziano, J.M., Ridker, P.M., Willet, W & Peto, R (1996) Lack of effect of long-term supplementation with beta-carotene on the incidence of malignant neoplasma and cardiovascular disease N Engl J Med.

334, 1145–1149.

37 Pryor, W.A., Stahl, W & Rock, C.L (2000) Beta-carotene: from biochemistry to clinical trials Nutr Rev 58, 39–53.

38 Bowen, P.E., Mobarhan, S & Smith, J.C Jr (1993) Carotenoid absorption in humans Methods Enzymol 214, 3–16.

39 Omenn, G.S., Goodman, G.E., Thornquist, M.D., Balmes, J., Cullen, M.R., Glass, A., Keogh, J.P., Meyskens, F.L., Valanis, B., Williams, J.H., Barnhart, S., Cherniack, M.G., Brodkin, C.A & Hammar, S (1996) Risk factors for lung cancer and for inter-ventions effects in CARET, beta-carotene and retinol efficacy trial.

J Natl Cancer Inst 88, 1550–1559.

40 Heber, D (2000) Colorful cancer prevention: alpha carotene, lycopene and lung cancer Am J Clin Nutr 72, 901–902.

41 Wang, X.D., Liu, C., Bronson, B.T., Smith, D.E., Krinsky, N.I & Russell, R.M (1999) Retinoid signaling and activator protein-1 expression in ferrets given carotene supplements and exposed to tobacco smoke J Natl Cancer Inst 91, 60–66.

42 Wolf, G (2002) The effect of low and high doses of b-carotene and exposure to cigarette smoke on lungs of ferrets Nutrition Rev 60, 88–90.