ARTICLE
Auteur(s) : Moetaz M. EL‐DOMYATI1, Sameh K.
ATTIA1, Fatma Y. SALEH1, Hesham M.
AHMAD1,2, Frances P. GASPARRO2, Jouni J.
UITTO2
1 Department of Dermatology, Faculty of Medicine,
Al‐Minya University, Cairo, Egypt. 2 Department of
Dermatology and Cutaneous Biology, Jefferson Medical College and
Jefferson Institute of Molecular Medicine, Thomas Jefferson
University, Philadelphia, Pennsylvania, USA.
Reprints: M.M. El‐Domyati Fax: (+1) 202‐738‐3485 E‐mail:
m_domyatihotmail.com
Article accepted on 25\12\2002
P53 is a nuclear phosphoprotein which serves as a tumor
suppressor. In its natural form (wild‐type) p53 can bind to DNA and
prevent cells from entering the S (synthetic) phase of the cell
cycle so as to allow time for DNA repair. Alternatively, p53
dependent events can eliminate the cells by sending them down to an
irreversible apoptotic pathway [1]. Thus p53 allows the DNA either
to be repaired or ultimately destroyed before replication renders
the damage permanent [2]. Ultraviolet (UV) radiation produces
damage in DNA molecules [3], and skin responds to such UV induced
DNA damage with a p53 dependent response [2]. Although the cells
contain robust systems to repair DNA, cell replacement may be a
preferred alternative to DNA repair, particularly when damage is
extensive. Such replacement requires that the damaged cells first
die by apoptosis and are then replaced by division of nearby
functional cells ultimately derived from the stem cell pool [4].
Thus programmed cell death or apoptosis is an important cellular
process that may play a critical role in cutaneous aging as well as
in maintaining proliferative homeostasis within the skin [5‐8]. The
present study is a pilot project to evaluate changes that occur in
the expression of p53 in vivo, as a regulator of the process
of apoptosis, following the use of some therapeutic modalities that
are used in the treatment of photoaged facial skin.
Materials and methods
The present study was conducted on 20 patients attending
the dermatology outpatient clinic of Al‐Minya University Hospital,
Al‐Minya, Egypt, for treatment for signs of photoaging, as well as
12 control volunteers undergoing cosmetic and dermatosurgical
procedures for other causes. Of these patients, 5 (25 %) were
males and 15 (75 %) females. The age of the patients ranged
from 23 years to 77 years with a mean age and standard
deviation (SD) of 37.17 ± 13.2. Punch biopsies were taken
from the facial skin before and after treatment using 0.05 %
topical tretinoin (Retin A cream, Janssen‐Cilag) (11 cases),
10‐30 % superficial TCA chemical peeling (5 cases) and
dermabrasion (4 cases). In the topical tretinoin group the
mean age was 30.7 ± 9.7, with only one male and
10 females. However, in the TCA peeling group the mean age was
28.0 ± 4.3, 2 males and 3 females. Whereas, in
the dermabrasion group the mean age was
60.5 ± 15.6 years, 2 males and 2 females.
Patients within the tretinoin group were classified into
3 subgroups. Biopsies were obtained after a mean duration of
3.3 months (subgroup 1), 5.5 months (subgroup 2) and
9.7 months (subgroup 3). In TCA treated patients biopsies were
obtained after clinical improvement of signs of photoaging, within
3 months from the first session, usually after
3‐5 sessions of TCA peeling with increasing concentration. On
the other hand, in the dermabrasion group biopsies were obtained
after 3 weeks and then after 3 months of dermabrasion.
Skin samples (24 specimens) were also obtained from the facial
(sun exposed) and abdominal (sun protected) skin of 12 control
volunteers from the same age groups as the patients included in
each treatment group. The mean age of the control subjects
(3rd and 4th decade of age), matching with
the TCA and retinoic acid treated patients, was
31.5 ± 7.2 years (4 females and 2 males),
whereas the mean age of the other 6 control subjects
(6th and 7th decade), matching with
dermabrasion patients, was 58.4 ± 6.2 years
(3 males and 3 females). Skin biopsies were fixed in
formalin (10 %), embedded in paraffin and sectioned into
5 µm sections. These sections were used for
immunohistochemical staining.
Immunohistochemical staining
The following protocol was used for immunohistochemical staining
of p53: after overnight incubation at 37 °C, tissue sections
were deparaffinized in xylene 101 and rehydrated in ascending
grades of alcohol. Endogenous peroxidase activity was exhausted by
incubation of tissue sections in 0.3 % H2O2 for
30 minutes at room temp. Tissue sections were then treated
with DAKO retrieval solution (DAKO® cat # S1699).
20 % rabbit serum in TBS was used for blocking. The monoclonal
antibody DO‐7 (DAKO® #M7001) in 1:200 dilution in
2 % rabbit serum was used to stain p53. It was incubated with
the samples overnight. Biotinylated rabbit anti‐mouse IgG
(DAKO® cat # E0354) was used as a secondary antibody.
All tissue sections were stained under similar conditions to ensure
equal staining quality. Squamous cell carcinoma was used as a
positive control. In the negative control samples, the primary
antibody was not added.
Scoring of p53 immunoreactivity
The level of p53 expression is evaluated according to the
scoring system that was set forth by Liang et al. (1999)
[2]. Each p53 score represents the mean value of different fields
from 3 sections of each specimen. This system evaluates the
degree of positivity and intensity of staining in only those
specimens demonstrating a dispersed pattern, i.e., the
"wild‐type" of p53 expression. This system results in a score
ranging from 0 to 3 for both the degree of positivity (%
of positively stained nuclei of epidermal cells) and the degree of
intensity of staining (the relative intensity of color of the
positively stained nuclei from faint‐brown for score 1 to
deep‐brown for score 3). The sum of the two scores is used as a
representative of the level of p53 expression (Table I). The pattern of expression, whether
compact or dispersed, has been determined according to the system
reported by Ren et al. (1996) [9] (Table
II).
Table I. Scoring method for quantifying the
level of p53 expression [2]
| GRADE |
POSITIVITY |
GRADE |
INTENSITY |
| 0 |
Less than 1 % |
0 |
Negative |
| 1 |
1‐10 % |
1 |
Weak |
| 2 |
10‐50 % |
2 |
Moderate |
| 3 |
More than 50 % |
3 |
Strong |
.
Table II. Characteristics of compact and
dispersed patterns of p53 immunoreactivity [9]
|
COMPACT
PATTERN |
DISPERSED PATTERN |
| Definition |
An uninterrupted row of at least 10 strongly and uniformly
immunopositive basally
located nuclei. |
Immunopositive cells are randomly dispersed through the
epidermis and\or hair follicle epithelium
‐‐ there are negative nuclei dispersed among the positive
cells. |
| Lateral borders |
Distinct |
Absent |
| Variability |
No internuclear
variation |
Significant internuclear variation |
A dispersed pattern could be also considered even if there is a row
of 10 consecutive positively stained nuclei if the positivity
is not confined to a sharply demarcated area and when the hair
follicles also have positive cells (i.e. not bypassed) [9].
.
Statistical analysis
The significance of the differences was determined by Student‘s
two‐tailed t‐test. All p values were two‐tailed, and differences
were considered significant when the p value was less than or equal
to 0.05. Summary data are expressed as mean ± standard
error of the mean (SEM).
Results
Immunohistochemical staining for p53 in the epidermis of facial
skin revealed a dispersed pattern of staining in all patients and
controls.
Evaluation of p53 expression after topical tretinoin
treatment
Evaluation of p53 expression in facial skin before and after
treatment with topical tretinoin revealed the tendency of topical
tretinoin to induce an initial significant decrease followed by a
significant increase in the level of p53 expression. The mean value
of p53 expression in the whole tretinoin group (11 cases)
decreased from 3.79 ± 1.14 before treatment to
2.43 ± 0.73 after tretinoin therapy. However, the score
of p53 expression significantly decreased from
3.8 ± 0.8 before treatment to 2.8 ± 0.7
after 3.3 months (p ∓ 0.05) (Fig. 1) and significantly
decreased from a score of 4.05 ± 0.5 to
0.9 ± 0.6 after 5.5 months
(p ∓ 0.04). This is followed by a statistically
significant increase from 3.4 ± 0.2 before treatment
to 4.6 ± 0.2 after 9.7 months
(p ∓ 0.005) (Table III;
Fig 2).
.
Table III. Score of p53 expression in facial
skin before and after topical tretinoin
Cases and duration
of treatment |
Score of p53 expression
before treatment |
Score of p53 expression
after treatment |
| 1 |
3 months |
5.25 |
4.25 |
| 2 |
3 months |
2.5 |
2.0 |
| 3 |
4 months |
3.5 |
2.25 |
| Mean |
3.3 |
3.8 |
2.8 |
| SEM |
0.58 |
0.8 |
0.7 |
| 4 |
5 months |
5.0 |
0.0 |
| 5 |
5 months |
5.0 |
0.0 |
| 6 |
5.5 months |
3.25 |
0.0 |
| 7 |
6 months |
2.5 |
3.0 |
| 8 |
6 months |
4.5 |
1.5 |
| Mean |
5.5 |
4.05 |
0.9 |
| SEM |
0.5 |
0.5 |
0.6 |
| 9 |
8 months |
3.75 |
5.0 |
| 10 |
10 months |
3.0 |
4.25 |
| 11 |
11 months |
3.5 |
4.5 |
| Mean |
9.7 |
3.4 |
4.6 |
| SEM |
1.53 |
0.2 |
0.2 |
P53: p ∓ 0.05 "After 3.3 months"; p ∓ 0.005 "After 5.5
months"; p ∓ 0.04 "After 9.7 months"
.
.
Evaluation of p53 following TCA peeling
Evaluation of p53 expression in facial skin before and after TCA
peeling revealed that the level of expression increased in
4 out of 5 cases and remained unchanged in one case.
However, these changes were statistically insignificant
(p ∓ 0.06) (Table IV; Fig
1).
Table IV. Score of p53 expression in facial
skin before and after TCA peeling
| Case Number |
Score of p53 expression before treatment |
Score of p53 expression
after treatment |
| 1 |
3.0 |
3.25 |
| 2 |
3.75 |
5.0 |
| 3 |
1.0 |
1.5 |
| 4 |
4.0 |
4.0 |
| 5 |
4.0 |
5.0 |
| Mean |
3.15 |
3.75 |
| SEM |
0.6 |
0.7 |
P53: p value ∓ 0.06
.
Evaluation of p53 following dermabrasion
Evaluation of p53 expression in facial skin before and after
dermabrasion revealed a significant decrease in the level of
expression in biopsies obtained after complete re‐epithelialization
followed by a significant increase. The score of p53 expression
initially showed a significant decrease from a mean of
5.0 ± 0.5 before treatment to a mean of
0.75 ± 0.4 in biopsies obtained after complete
re‐epithelialization (3 weeks) (p ∓ 0.02). This is
followed by a significant increase in the score of expression to a
mean of 2.5 ± 0.2 (after 3 months)
(p ∓ 0.04). However, this score was still significantly
lower than the pre‐treatment level (Table V;
Fig 1, 3).
Table V. Score of p53 expression in facial skin
before and after dermabrasion
| Cases |
Score of p53 before treatment |
Score of p53 after 3 weeks |
Score of p53 after 3 months |
| 1 |
4.0 |
1.5 |
3.0 |
| 2 |
6.0 |
0,0 |
2.0 |
| 3 |
4.25 |
1.5 |
2.5 |
| 4 |
5.75 |
0.0 |
2.5 |
| Mean |
5.0 |
0.75 |
2.5 |
| SEM |
0.5 |
0.43 |
0.2 |
P53: p value ∓ 0.02 "after complete
re‐epithelialization" and 0.04 within 3 months after
treatment.
.
.
Evaluation of p53 in control cases
The degree of p53 expression in facial skin significantly
increased (p < 0.05) from a mean score of
2.3 ± 0.9 in control specimens from the
3rd and 4th decade to a mean of
4.3 ± 0.3 in the 6th and 7th
decades, while no significant changes (p > 0.05) are
detected in abdominal skin. However, p53 expression is
significantly (p < 0.05) higher in facial sun exposed
skin than sun protected abdominal skin (Table
VI, Fig. 4).
However, no significant difference (p > 0.05) was
encountered as regards p53 expression in facial skin of patients
when compared to their age‐matched controls.Table
VI. Score of p53 expression (mean) in facial and abdominal
skin in control cases
| Decade |
Number of subjects |
Facial skin
Score
SEM |
Abdominal skin
Score
SEM |
| 3rd & 4th |
6 |
2.3
0.9 |
0.8
0.5 |
| 6th &7th |
6 |
4.3
0.3 |
1.1
0.6 |
. .
Discussion
The p53 gene, located on the short arm of chromosome 17, acts as
a tumour suppressor gene [10]. The p53 protein product is a
393‐amino acid phosphoprotein which localizes to the nucleus. There
is increasing evidence that mutations of the p53 gene are among the
most common genetic alterations in human malignancies [10], and
they have been implicated as an important factor in the
pathogenesis of ultraviolet light‐induced skin cancer [11]. Since
cellular death by apoptosis plays an important role in the process
of cutaneous aging [5‐8], disturbance of p53 function as a
regulator of apoptosis may also play an important role in the
process of skin aging.
In the present study we evaluate the effect of topical retinoic
acid, TCA peeling and dermabrasion on the expression of p53 in
vivo in order to clarify one of the possible mechanisms through
which these modalities could act. To the best of our knowledge no
previous publications have reported the effect of retinoic acid,
TCA peeling or dermabrasion on the expression of p53 in the
skin.
Evaluation of p53 expression in facial skin before and after
treatment with topical tretinoin revealed a tendency of topical
tretinoin to induce an initial significant decrease followed by a
significant increase in the level of p53 expression. This suggests
that the effect of topical tretinoin on the epidermis may be
through adjustment of the proliferation\apoptosis balance. However,
this change is transient and tends to return back to the
pre‐treatment level with continued treatment. This observation is
consistent with the previous report of Bhawan et al. (1995)
[12]. By measuring the epidermal thickness before and after
treatment with topical tretinoin, they observed that the epidermal
thickness initially increased and then returned back to the
pretreatment thickness despite continued treatment. They suggested
that certain growth regulatory mechanisms functioning in the skin
prevent the infinite epidermal hyperplasia in response to tretinoin
treatment [12]. However, evaluation of p53 expression in facial
skin before and after superficial TCA peeling (10‐30 %)
revealed a mild increase in 4 out of 5 cases but this
change was statistically insignificant. However, a larger sample of
patients, more treatment sessions or higher concentrations of TCA
may be needed to demonstrate any statistically significant
effect.
On the other hand, evaluation of p53 expression in facial skin
before and after dermabrasion revealed a significant decrease in
the level of expression in early biopsies obtained after complete
re‐epithelialization, whereas late biopsies, obtained after
3 months of dermabrasion, revealed a significant increase in
the level of p53 expression. However, the score of p53 expression
was still significantly lower than the pretreatment level. This
suggests that epidermal changes induced by dermabrasion are
reversible by time.
Irradiation of the skin with a light source emitting a broad band
of UVB, UVA and near infrared radiation has been shown to result in
p53 accumulation in a dispersed pattern [13]. Meanwhile, p53
positivity has been reported in chronically sun exposed skin
[13‐15].
In the control group of the present study, the level of p53
expression is significantly higher in sun exposed than sun
protected skin, which agrees with our recent reports [14, 15].
However, no significant difference was observed as regards the
score of p53 expression in facial skin of patients when compared to
their age matched controls.
On the other hand, as recently reported [14, 15], p53 expression in
facial skin is higher in the older age group of controls and
patients (dermabrasion group). This age related increase may
reflect an accumulation over the years (cumulative insult)
[14].
Age dependent p53 accumulation in photodamaged skin may be
partially explained on the basis of altered differentiation of the
damaged keratinocytes. It has been noted that the degree of nuclear
expression of p53 is inversely correlated with the degree of
keratinocyte differentiation [10]. Thus, it is possible that
accumulation of wild type p53 in photoaged skin is partially
related to altered keratinocyte differentiation.
In conclusion, some of the modalities that are currently used in
the treatment of facial skin may affect the level of expression of
p53. This may play a role in mediating the effects of such
modalities on the epidermis. Meanwhile, since the tumour suppressor
protein p53 has an important role in the regulation of cellular
proliferation, changes in its level of expression may have a
potential role in the prevention of actinic neoplasia by adjusting
alteration in the proliferation\apoptosis balance normally observed
in photoaged facial skin. Future studies on a larger group of
patients, and implying specific stains for the detection of
apoptotic cells, such as TUNEL technique, is mandatory to confirm
these preliminary findings.
References
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