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

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

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:
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].

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-

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

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.

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,

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
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

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.

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

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)

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-

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

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

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

ny
Cultura. Spain. M. M. is supported by a predoctoral fellowship of the
Consellerı
´
adeCulturaEducacio
´
iCie
`
ncia, Generalitat Valenciana.
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Ó FEBS 2003 Vitamin A status and proliferative control genes (Eur. J. Biochem. 270) 1501