Research Article
Differential Role of Transcription-Coupled Repair in UVB–Induced
Response of Human Fibroblasts and Keratinocytes
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Mariarosaria D’Errico, Massimo Teson, Angelo Calcagnile, Tiziana Nardo, Naomi De Luca,
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Chiara Lazzari, Silvia Soddu, Giovanna Zambruno, Miria Stefanini, and Eugenia Dogliotti
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Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Viale Regina Elena, Rome, Italy; 2Laboratory of
Molecular and Cell Biology, Istituto Dermopatico dell’Immacolata, IRCCS, Via dei Monti di Creta, Rome, Italy; 3Istituto di Genetica
Molecolare, Consiglio Nazionale delle Ricerche, Via Abbiategrasso, Pavia, Italy; and 4Molecular Oncogenesis Laboratory, Department of
Experimental Oncology, Regina Elena Cancer Institute, Rome, Italy
which selectively repairs the transcribed strand of active genes
(reviewed in refs. 1–3). The dramatic consequences of a defect in
NER are reflected in individuals with the inherited syndromes,
xeroderma pigmentosum (XP) and Cockayne syndrome (CS). XP
patients are sun-sensitive and show a dramatic increase of UVinduced skin cancer incidence. Cell fusion studies have identified
seven XP complementation groups (XP-A through XP-G). The XP
gene products are all involved in specific steps of the NER process
(reviewed in refs. 4–6). More specifically, cells from patients with XP
belonging to the A, B, D, F, and G groups are defective in both TCR
and GGR, whereas XP-C and XP-E cells are defective only in GGR
playing a role as sensors proteins for DNA damage in nontranscribed sequences.
Like patients with XP, patients with CS show hypersensitivity to
sunlight but have no predisposition to skin cancer. Their distinctive
features are severe developmental and neurologic abnormalities as
well as premature aging. CS is caused by mutations in either the
CSA or the CSB genes that lead to defects in only one pathway, TCR
(reviewed in refs. 6, 7). However, additional roles outside NER have
been suggested for CS proteins. CSB is involved in chromatin
remodeling (8), in general transcription (9–12) and in rRNA
synthesis (13). Additionally, CSB seems to be implicated in TCR
(14) and GGR (15, 16) of oxidative DNA damage. Like CSB, CSA has
been reported to physically interact with TFIIH (17), thus
suggesting an additional role in general transcription, but less is
known about its function (reviewed in ref. 5). Recently, the CSBdependent translocation of the CSA protein to the nuclear matrix
after DNA damage has been described (18).
The characterization of the response to UV light of healthy
subjects as well as patients with XP and CS have been mainly done
by using skin fibroblasts. We have recently shown that the response
to UVB damage of normal human keratinocytes differs significantly
from that of fibroblasts from the same donors (19), supporting the
idea that the response to DNA damage is cell type–specific (20). In
addition, evidence has been accumulated that within UV radiation,
different types of DNA damage and repair are induced depending
on the wavelength (21).
In order to learn more about the response to solar radiation of
the target cells for skin cancer, we established primary cultures of
keratinocytes from normal, XP-C, and CS-A individuals, and we
compared UVB-induced effects in these cells with those caused in
fibroblasts obtained from the same biopsy. Here, we show that the
very efficient repair of UVB-induced damage by keratinocytes (19)
is due to a more efficient GGR in this cell type as compared with
that of fibroblasts. We also show that a defect in the CSA gene
leads to massive apoptosis and lack of p53-mediated mdm2
transactivation in fibroblasts but not in keratinocytes. The efficient
GGR of keratinocytes might operate as a back-up system to remove
Abstract
Most solar radiation–induced skin cancers arise in keratinocytes. In the human epidermis, protection against cancer is
thought to be mediated mainly by nucleotide excision repair
(NER) of UVB-induced cyclobutane pyrimidine dimers, and by
elimination of the damaged cells by apoptosis. NER consists of
two subpathways: global genome repair (GGR) and transcription-coupled repair (TCR). Here, we investigate the impact of
defects in NER subpathways on the cellular response to UVBinduced damage by comparing primary human keratinocytes
and fibroblasts from normal, XP-C (GGR-defective), and CS-A
(TCR-defective) individuals. We show that human keratinocytes are more resistant to UVB killing than fibroblasts and
present higher levels of UVB-induced DNA repair synthesis due
to a more efficient GGR. The CS-A defect is associated with a
strong apoptotic response in fibroblasts but not in keratinocytes. Following an UVB dose of 1,000 J/m2, no p53-mediated
transactivation of mdm2 is observed in CS-A fibroblasts,
whereas the p53-mdm2 circuit is fully activated in CS-A
keratinocytes. Thus, in fibroblasts, the signal for apoptosis
originates from DNA photoproducts in the transcribed strand
of active genes, whereas in keratinocytes, it is largely TCRindependent. This study shows that the response to UVB
radiation is cell type–specific in humans and provides the first
evidence that a deficiency in TCR has a different impact depending on the cell type. These findings have important implications for the mechanism of skin cancer protection after
UVB damage and may explain the lack of skin cancer in
patients with Cockayne syndrome. (Cancer Res 2005; 65(2): 432-8)
Introduction
Chronic exposure to sunlight is a causative factor in skin cancer
development. Modifications of DNA and other cellular components
by the higher-energy shorter solar wavelengths comprising the UVB
spectra (290-320 nm) are the most damaging to the skin. Sunlightinduced cancer develops from damaged epidermal cells as a result
of a multistep process initiated by DNA damage. To counteract the
catastrophic effects of UV-induced DNA damage, cells possess a
repair system which removes UV photoproducts, the nucleotide
excision repair (NER) mechanism. NER can operate via two
pathways: the global genome repair (GGR), which repairs damage
over the entire genome, and the transcription coupled repair (TCR),
Requests for reprints: Eugenia Dogliotti, Department of Environment and Primary
Prevention, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy.
Phone: 39-064990-2580; Fax: 39-064990-3650; E-mail: dogliott@iss.it.
I2005 American Association for Cancer Research.
Cancer Res 2005; 65: (2). January 15, 2005
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UVB Response of Human Keratinocytes
by terminal transferase-mediated dUTP-biotin nick end labeling (TUNEL)
assay using the in situ cell death detection kit (Roche Molecular
Biochemicals, Mannheim, Germany) according to the supplier’s instructions. At least 100 cells were scored per experimental point. Apoptosis of
normal keratinocytes were also analyzed by a fluorimetric assay for
detection of caspase-3 using the Apoalert Caspase-3 Fluorescent Kit (BD
Biosciences, Clontech, Palo Alto, CA).
Unscheduled DNA Synthesis Analysis. The response to UVB irradiation
was analyzed by measuring Unscheduled DNA Synthesis (UDS) as described
previously (29). Briefly, cells were exposed to UVB (500-2,000 J/m2),
incubated in medium containing 10 ACi/mL [3H]-thymidine (3H-TdR,
specific activity 35 Ci/mmol; ICN, Irvine, CA, USA) and fixed 1 or 3 hours
later. UDS was determined on autoradiographic preparations by counting
the number of grains on at least 50 non–S-phase cells.
Western Blotting. Whole-cell extracts were prepared by lysing the cells
in radioimmunoprecipitation assay buffer 0.5% NP40 in 50 mmol/L Tris-HCl,
150 nmol/L NaCl (pH 7.4) containing protease inhibitors. Total proteins (100
Ag) were resolved by SDS-PAGE (12.5% or 8% gels), transferred into
polyvinylidene difluoride membranes (Millipore, Bedford, MA), and blocked
with 5% non fat dry milk in TBS-T. Membranes were incubated with primary
antibodies: anti-p53 and p21 (Santa Cruz Biotechnology, Santa Cruz, CA,
USA) and anti-mdm2 monoclonal antibodies (Ab-1; Oncogene Research
Products, Boston, MA, USA). In order to normalize the expression levels of
the proteins of interest in the different samples, the membranes were
incubated with anti-h actin (Santa Cruz Biotechnology) or anti-a tubulin
monoclonal antibodies (TU-01) (Immunological Sciences, Rome, Italy) in the
case of fibroblasts, and with anti–14-3-3 ~ (Santa Cruz Biotechnology) in the
case of keratinocytes. Detection was through enhanced chemiluminescence
(Amersham; Amersham Bioscience, Buckinghamshire, United Kingdom).
Quantitation of protein bands was done by using Gel Doc 2002 analysis
program (Bio-Rad, Hercules, CA, USA).
Indirect Immunofluorescence. Cells (1 104) were cytocentrifuged on
slides, fixed with 2% formaldehyde in PBS, permeabilized with 0.25% Triton-X
100 in PBS, and subjected to indirect immunofluorescence with the mouse
anti-MDM2 monoclonal antibodies (Ab1; Oncogene Research Products), and
FITC-conjugated anti-mouse serum (Cappel, West Chester, PA). Nuclei were
counterstained with 1 Ag/mL Hoechst 33258 dye (Sigma).
transcription-blocking lesions, thus providing keratinocytes from
patients with CS of an efficient protection from skin cancer.
Materials And Methods
Cell Cultures and UVB Irradiation. Primary human fibroblast and
keratinocyte cultures were established from biopsies of unaffected skin
obtained from two patients, XP26PV and CS6PV, affected by XP and CS,
respectively, and from two age-matched controls, coded KN1RO and
KN2RO. By complementation analysis, XP26PV was assigned to the XP-C
group (22), whereas CS6PV was assigned to the CS-A group. 5 Some of
the experiments were carried out with primary fibroblasts obtained from
another CS-A patient, coded CS4PV (23). Fibroblasts were grown in F10
medium supplemented with 10% fetal calf serum. Keratinocytes were
cultivated on a feeder-layer of lethally irradiated 3T3-J2 fibroblasts (a gift
from H. Green, Harvard Medical School, Boston, MA) and passaged at
the stage of subconfluence as previously described (24). Cells were
cultured at 37jC in a 10% carbon dioxide atmosphere. All experiments
were carried out with cells at passages two to four. Cells were irradiated
with UVB (TL20W12 sunlamps, Philips, Monza, Italy) and the doses were
determined using a DM-300HA radiometer (Spectronics Corporation,
Westbury, NY, USA).
Plasmids and Transfections. Cells (5 105) were plated on 60 mm
Petri dishes and transfected for 6 hours by the BES-modified calcium
phosphate method (25) with 10 Ag of pcDNA3 plasmid (Invitrogen, Life
Technologies, SRI, Milan, Italy), or with the same vector in which the
cDNA of the human MDM2 gene was subcloned (pcMDM2) and 1 Ag of
pBabe-puro, carrying the puromycin resistance gene (26). Cells were
selected with 2 Ag/mL of puromycin (Sigma-Aldrich, St. Louis, MO) for
24 hours to transiently enrich for MDM2-expressing cells.
Cytotoxicity Assay. For clonal analysis, subconfluent cell cultures were
exposed to UVB (50-1,000 J/m2) and then plated at increasing density as a
function of UVB dose (500 to 105 cells and from 500 to 8 103 cells per 100
mm dish in the case of keratinocytes and fibroblasts, respectively). Colonies
were fixed 14 days later, stained and scored under microscope. The number
of colonies in the irradiated samples were expressed as percentages of
those in unirradiated samples. Keratinocyte colonies were scored as
progressively growing or aborted, as described in ref. 27.
Kinetics of Removal of UVB-Induced DNA Photoproducts. Primary
human fibroblasts and keratinocytes were irradiated with 1,000 J/m2 of UVB
and harvested after different post irradiation incubation times. DNA was
extracted with the Qiagen kit (Genenco, Florence, Italy). The level of
photoproducts were measured in microtiter plates, coated with protamine
sulfate [10-30 ng for cyclobutane pyrimidine dimers (CPD) and 100-400 ng
for 6-4 pyrimidine-pyrimidone photoproducts (PP)] using TDM-2 and 6-4
M2 monoclonal antibodies (a kind gift from O. Nikaido, Division of Radiation
Biology, Kanazawa University, Kanazawa, Japan) in a standard ELISA
technique as previously described (28). Briefly, after extensive washing with
0.05% Tween 20/PBS, DNA were incubated with the antibodies for 90
minutes at 37jC, washed again with 0.05% Tween 20/PBS, and then
incubated with biotinylated F(abV)2 fragment of anti-mouse IgG (Zymed, San
Francisco, CA; 1:1,000 in PBS) for 90 minutes at 37jC. After washing with
0.05% Tween 20/PBS, DNAs were incubated with horseradish peroxidasestreptavidin (Zymed; 1:10,000 in PBS) for 90 minutes at 37jC, washed with
citrate-phosphate buffer, and incubated with 0.4 mg/mL o-phenylene
diamine buffer and 0.007% hydrogen peroxide/citrate-phosphate buffer
(pH 5) for 30 minutes at 37C. The reaction was stopped with 2 mol/L sulfuric
acid.
Detection of Apoptosis. The induction of apoptosis was measured in
normal and defective cells following exposure at different UVB doses
(250-1,500 J/m2). Apoptotic keratinocytes and fibroblasts were detected
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Results
Global Genome Repair Is the Main Determinant for UVB
Survival in Human Keratinocytes. Primary cultures of normal,
XP-C, and CS-A keratinocytes and fibroblasts were exposed to UVB
(50-1,000 J/m2) and cell survival was determined by measuring
colony-forming ability. As shown in Fig. 1A, in general, UVB induced
a decrease of cell viability in all cell strains but, at equal dose
exposure, keratinocytes were more resistant to the lethal effects of
UVB than fibroblasts from the same skin biopsy. XP-C and CS-A
defects conferred marked hypersensitivity to UV in both fibroblasts
and keratinocytes. The survival hierarchy was the same in both cell
types (i.e., normal>CS-A>XP-C). These data indicate that the
capacity to perform GGR is the main determinant for UVB survival
in keratinocytes, as previously reported for fibroblasts.
To verify whether the differential sensitivity to UVB of the two
cell types might be affected by differences in the level of DNA
damage, monoclonal antibodies directed either against CPD or 6-4
PP were used to detect UV photoproducts in cellular DNA.
Fibroblasts and keratinocytes were exposed to 1,000 J/m2 of UVB
and the amount of CPD and 6-4 PP was determined on the
extracted DNA by ELISA using the specific antibodies. The yield of
both DNA lesions was approximately 1.5-fold higher in fibroblasts
than in keratinocytes but the ratio of CPD to 6-4 PP was similar in
the two cell types (Fig. 1B). Although keratinocytes present a lower
level of UV photoproducts than fibroblasts, this difference might
only partially account for their UVB resistance.
M. Stefanini and T. Nardo, unpublished results.
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Figure 1. A, cell survival after UVB irradiation of human primary fibroblasts (open symbols ) and keratinocytes (closed symbols ) from normal (4, E), XP-C (o, .),
and CS-A (5, n) donors. Survival was determined by colony formation assay. The reported values are the mean of at least two independent experiments each done in
triplicate with standard error (SE) always < 10%. The values for keratinocytes include both proliferative and aborted colonies. In all keratinocyte strains, the relative
number of proliferative colonies was slightly decreased and that of aborted colonies increased after UVB (data not shown) as previously reported [Otto et al. (30)].
B, induction of UV photoproducts in human primary fibroblasts (4) and keratinocytes (E). The levels of CPD and 6-4 PP lesions present on DNA isolated immediately
after UVB irradiation (1,000 J/m2) from fibroblasts and keratinocytes were measured by ELISA. Genomic DNAs were monitored for the presence of UV photoproducts
by using either anti-CPD (TDM-2) or anti-6-4 PP (6-4M-2) antibodies. The data are the mean of four independent experiments. Bars, SE.
Global Genome Repair Is Responsible for the Efficient
Repair of UVB Damage by Human Keratinocytes. The speed and
efficiency of repair might also play a role in UVB resistance. In a
previous study (19), accelerated removal of UV photoproducts, as
detected by using specific antibodies, was observed in keratinocytes as compared with fibroblasts. To gain insight into the
mechanism of DNA repair, the capacity to perform DNA repair
synthesis (UDS) following UVB irradiation was analyzed at single
cell level by autoradiography in normal and NER-defective
keratinocytes and fibroblasts. UDS is an efficient measure of the
overall repair (GGR), whereas the contribution of TCR is low
because it occurs in a minor part of the genome. Accordingly, TCRdefective CS fibroblasts (CS-A and CS-B) were characterized by
UDS levels similar to those of normal cells. UDS levels observed 1
hour (data not shown) and 3 hours (Fig. 2) after UVB exposure
were time-dependent, but the pattern of response in each cell type
was substantially the same. In both fibroblasts and keratinocytes,
the UDS levels were UVB dose–dependent (Fig. 2A and B). In
normal keratinocytes, the UDS values were higher than those
measured in fibroblasts. The significance of the increased UDS
levels of keratinocytes is strengthened by the observation that, after
the same UVB dose, keratinocytes present lower levels of UV
photoproducts than fibroblasts (Fig. 1B; ref. 30). Similarly to CS-A
fibroblasts, the UDS levels of CS-A keratinocytes approached those
observed in normal cells. Therefore, also in the absence of TCR,
the repair efficiency of keratinocytes was higher than that of
fibroblasts. In contrast, GGR-defective XP-C cells showed a drastic
reduction of UDS levels in both cell types. These findings show that
an efficient GGR is responsible for the more efficient CPD removal
(19) and DNA repair capacity (this study) displayed by keratinocytes as compared with fibroblasts.
A Defect in Transcription Coupled Repair Is Associated with
a Strong Apoptotic Response in Human Fibroblasts but not in
Keratinocytes. A primary mechanism to remove UVB-damaged
skin cells is apoptosis. Apoptosis was measured by the TUNEL
assay in both normal and NER-defective cells at different times
after exposure to a UVB dose of 1,000 J/m2 (Fig. 3A). Massive
Cancer Res 2005; 65: (2). January 15, 2005
apoptosis was observed in CS-A fibroblasts already at 12 hours
post irradiation and 65% apoptotic cells were scored at 24 hours
post-UVB. Similar results were obtained with another CS-A
fibroblast strain (CS4PV) that showed 52% apoptotic cells 24
hours post–1,000 J/m2 UVB. Under the same irradiation regimen,
no apoptosis was detected in normal fibroblasts that need higher
doses to activate the apoptotic program (Fig. 3A). These findings
provide further evidence that TCR-defective fibroblasts trigger the
apoptotic pathway in response to UV light (31–33). In contrast
with fibroblasts, normal keratinocytes exposed to 1,000 J/m2 UVB
undergo apoptosis (Fig. 3A), in agreement with previous
observations (19). The activation of an apoptotic response was
observed in two primary keratinocyte cell lines and confirmed by
fluorimetric detection of caspase-3 (data not shown). Surprisingly,
the frequency of apoptotic cells in CS-A keratinocytes was
significantly lower than that observed in fibroblasts from the
same donor. An anticipation of the apoptotic response was
observed (maximum level at 6 hours post irradiation) but the
level of apoptotic cells at longer post irradiation times only
slightly exceeded that observed in normal keratinocytes. XP-C
keratinocytes (Fig. 3A) and fibroblasts (data not shown) showed
the same apoptotic pattern observed in the corresponding cell
type from the normal donor, confirming that UVB damage
repaired by GGR is not a signal for apoptosis (23, 31, 34).
To further characterize the apoptotic response of keratinocytes
to UVB, the induction of apoptosis was analyzed after exposure
to UVB doses ranging between 50 and 1,000 J/m2 (Fig. 3B).
Normal keratinocytes showed apoptosis at doses as low as 150
J/m2 of UVB, whereas fibroblasts did not activate this process in
the analyzed dose range (this study; refs. 19, 33). A higher frequency
of apoptotic cells was detected in CS-A keratinocytes when
compared with normal cells, particularly at low UVB doses.
However, the number of apoptotic cells in CS-A keratinocytes is
clearly lower compared with that in CS-A fibroblasts after equal
UVB exposure and even more after doses inducing similar amounts
of damage (see Fig. 1B). Besides confirming the crucial role of TCR
in inducing apoptosis in fibroblasts, these findings provide clear
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UVB Response of Human Keratinocytes
the higher DNA repair capacity of these specialized epidermal
cells (19). In XP-C cells, the p53/p21/mdm2 induction profile was
very similar to that of the corresponding normal cell type
(Fig. 4B), indicating that a defect in GGR does not significantly
affect the p53-associated pathway neither in fibroblasts nor in
keratinocytes. In the case of CS-A (Fig. 4C), the kinetics of p53
and p21 induction in fibroblasts were similar to those of normal
fibroblasts, whereas a lack of mdm2 increase was detected
following UVB-induced p53 stabilization. Unexpectedly, in the CSA keratinocytes, the p53-mdm2 circuit was unaffected and mdm2
expression followed the kinetics of p53/p21 induction with a
consistent increase after 6 hours. Although caution should be
taken when Western blotting is used for quantitative analysis, it is
interesting to notice that the levels of p21 in UVB-treated CSA
keratinocytes were higher and persisted longer than in normal
cells. Because of the pleiotropic role of this protein in
keratinocyte survival, cell cycle arrest and differentiation
(reviewed in ref. 36) this observation deserves further analysis.
These data indicate that the CS-A defect has a cell type–
specific effect on p53-associated pathway in response to UVB:
the absence of TCR does not affect the p53/mdm2 circuit in
keratinocytes as it does in fibroblasts.
Figure 2. UVB-induced DNA repair synthesis expressed as mean number of
autoradiographic grains/nucleus of fibroblasts (A) and keratinocytes (B ).
Fibroblasts (open symbols ), keratinocytes (closed symbols ) from normal (4, E),
XP-C (o, .), and CS-A (5, n) donors.
evidence that in human keratinocytes, the persistence of DNA
lesions in the transcribed strand of active genes contributes but is
not the only mechanism that triggers apoptosis.
Lack of p53-Mediated mdm2 Transactivation after UVB
Exposure in CS-A Fibroblasts but not in Keratinocytes. UV
light activates the p53 tumor suppressor gene which controls
DNA damage response by transactivating several downstream
genes, such as p21 and mdm2 (reviewed in ref. 35). We analyzed
the kinetics of induction of the stress response genes p53, p21,
and mdm2 after UVB irradiation of NER-defective fibroblasts and
keratinocytes and we compared the gene response with that of
normal cells.
As shown in Fig. 4A, in normal fibroblasts following 1,000 J/m2
of UVB, p53 started to accumulate 2 hours after irradiation and
reached a plateau at 12 to 24 hours postirradiation. The p53
response of normal keratinocytes was significantly different from
that of fibroblasts. In keratinocytes, p53 levels reached a
maximum at 6 hours postirradiation and then drastically
decreased to background levels at 12 hours. In both cell types,
the time course of p21 induction paralleled that observed for p53,
whereas mdm2 induction was shifted in time, reaching the
highest level at 24 and 12 hours post-UVB in fibroblasts and
keratinocytes, respectively. The accelerated p53 response in
keratinocytes as compared with fibroblasts is consistent with
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Figure 3. Apoptosis of human primary fibroblasts (open symbols ) and
keratinocytes (closed symbols ) from normal (4, E, y), XP-C (.), and CS-A
(5, n) donors. Frequency of apoptosis was measured by the TUNEL assay.
A, cells were analyzed at different times after irradiation with 1,000 J/m2 UVB.
The data relative to two primary keratinocyte cell lines from normal donors are
shown for comparison (E, y). B, cells were analyzed 24 hours after
irradiation with different UVB doses. The reported values are the mean of
three independent experiments with SE always < 10%.
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induction, typically observed in CS fibroblasts following UV
exposure, is due to their defect in TCR (i.e., in the removal of
transcription blocking lesions). Conversely, the normal p53mdm2 response in UVB-exposed CS keratinocytes supports the
hypothesis that this cell type ‘‘tolerates’’ the defect in TCR
because of an efficient GGR back-up system.
Discussion
Cell Survival after UVB Irradiation. UVB is responsible for the
most deleterious effects of solar radiation including erythema,
immunosuppression, and skin cancer. The majority of skin cancers
originate from epidermal keratinocytes. These specialized cells,
which are the cellular target of solar radiation, display a higher
clonal cell survival following UVB irradiation than the
corresponding fibroblasts which are located in the skin in the
underlying dermis. As in the case of fibroblasts, UVB survival of
keratinocytes seems to reflect mainly the capacity to perform GGR
because TCR-defective keratinocytes (CS-A) are less sensitive to
UVB damage than GGR-defective keratinocytes (XP-C). It should be
taken into account that significantly lower levels (1.5-fold to 2-fold)
of UV photoproducts are induced in keratinocytes as compared
with fibroblasts under the same UVB dose regimen (ref. 30; this
study). The presence of keratin might act as natural shielding
against UVB damage thus contributing to keratinocyte UVB
resistance. Cell cycle control mechanisms might also be involved
because cell cycle progression of keratinocytes is unaltered after
UVB doses that determine an abrupt G1-S arrest in fibroblasts (19).
Figure 4. Human primary fibroblasts (open symbols ) and keratinocytes
(closed symbols ) from normal (A), XP-C (B), and CS-A (C ) donors were
UVB-irradiated and quantitative analysis of the level of expression of p53, p21,
and mdm2 were done. Protein lysates were obtained from unirradiated cells
and after 2, 6, 12, and 24 hours post-UVB exposure to 1,000 J/m2. The samples
were probed with anti-p53, anti-p21, and anti-mdm2 antibodies. Appropriate
housekeeping genes (h-actin and 14-3-3 ~ for fibroblasts and keratinocytes,
respectively) were measured simultaneously. The Western blotting analysis
was carried out in parallel on keratinocytes and fibroblasts derived from the
same individual; therefore, a comparison of the intensity of the signals is
allowed only between keratinocytes and fibroblasts of the same subject.
In order to explore the mechanism behind the lack of mdm2
transactivation in CS-A fibroblasts, we decided to verify whether
the p53-mdm2 circuit is fully functional in this cell type. CS-A
fibroblasts were either transfected with an expression vector
carrying the human mdm2 cDNA (pcMDM2) or with an empty
vector (pcDNA.3) and then enriched for plasmid-containing cells
by puromycin selection. CS-A fibroblasts transfected with
pcMDM2 vector expressed high levels of mdm2 as shown by
both immunofluorescence and Western blotting (Fig. 5). If the
p53-mdm2 circuit of CS-A fibroblasts is functional, the high
levels of exogenous mdm2 should lead to inhibition of UVBinduced p53, and therefore to decreased apoptosis. Indeed, at 24
and 48 hours post-1,000 J/m2 UVB, the CS-A fibroblasts
expressing mdm2 showed a reduction (approximately 50%) in
the frequency of apoptotic cells as compared with fibroblasts
transfected with the empty vector (Fig. 5). The restoration of a
normal p53-mdm2 circuit in CS-A fibroblasts overexpressing
mdm2 provides the first direct evidence that the lack of mdm2
Cancer Res 2005; 65: (2). January 15, 2005
Figure 5. CS-A human fibroblasts were transiently transfected with an
expression vector encoding for the human mdm2 protein (pcMDM2) or with the
empty vector (pCDNA.3). The cell population, enriched for mdm2 expressing
cells by 24 hours puromycin selection, was exposed to 1,000 J/m2 UVB and the
frequency of apoptotic cells was measured by TUNEL. Cells transfected with
pcMDM2 and pCDNA.3 were analyzed by immunofluorescence with anti-mdm2
antibodies (top ) and the cell extracts analyzed by Western blot with the same
antibodies and with anti-tubulin antibodies as a control (bottom, left). The
histogram (bottom, right ) reports the frequency of apoptotic cells in the cell
population expressing mdm2 and in that transfected with the empty vector at 24
and 48 hours post-UVB. The frequency of apoptotic cells in the untreated cells
(mock) is shown for comparison. A representative experiment, out of the three
independent experiments done, is shown.
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UVB Response of Human Keratinocytes
keratinocytes were more sensitive to UVB-induced apoptosis than
fibroblasts but, similarly to fibroblasts, the XP-C defect did not affect
the apoptotic response as compared with normal keratinocytes. A
defect in TCR was associated with an anticipation of the apoptotic
response after UVB and higher levels of apoptosis at low UVB doses.
Similar results have been reported for one strain of CS keratinocytes
(33). However, in contrast with fibroblasts, the level of apoptosis in
keratinocytes is only partially affected by the lack of TCR.
p53 Regulatory Pathway after UVB. What are the factors that
govern the apoptotic response in human skin cells? In a previous
study, Conforti et al. (23) reported that in CS fibroblasts exposed to
UVC, the p53 increase was not followed by mdm2 induction and this
phenomenon was associated with the appearance of apoptosis. In
this study, we have observed the same response in CS-A fibroblasts
following UVB irradiation. mdm2 expression was detected at basal
levels but it was not induced by UVB doses that triggered a strong
apoptotic response. Unexpectedly, mdm2 induction was detected in
CS-A keratinocytes after UVB injury (Fig. 4). These keratinocytes
showed significantly lower UVB-induced apoptosis than the
corresponding fibroblasts (Fig. 2). Is there a causal link between
lack of mdm2 transactivation ( following p53 stabilization) and
apoptosis? We provide strong evidence that this is the case. mdm2overexpressing CS-A fibroblasts were less prone to UVB-induced
apoptosis than vector-transfected fibroblasts (Fig. 5). This finding
shows that mdm2 gene regulation ensures the elimination of heavily
damaged cells by apoptosis and that the persistence of transcription-blocking UVB damage in CS-A fibroblasts is responsible for the
lack of mdm2 transactivation. Recently, it has been shown that
induction of UV-induced gene expression in human cells is subject to
a strong gene size constraint (37). mdm2, as well as other large-sized
genes encoding negative regulators of p53 would act as molecular
dosimeters of irreparable transcription-blocking DNA damage.
In the case of keratinocytes, we have shown that apoptosis is
GGR-independent and is only partially affected by a defect in TCR.
Our findings support the notion that DNA damage is not the
only mediator causing UVB-induced apoptosis in keratinocytes
(reviewed in ref. 38). UVB directly activates death receptors,
including CD95 (39) and tumor necrosis factor receptors (40), and
the inhibition of this activation leads to a partial reduction in UVBinduced apoptosis (39). Additionally, UVB-induced intracellular
formation of reactive oxygen species accompanied by mitochondrial dysfunction and cytochrome c release, was shown to be
involved in the apoptotic program in this cell type (41). Finally, it is
important to mention that UV-induced apoptosis can be influenced
by cytokines. These mediators are specifically released by
keratinocytes during UV exposure and affect apoptosis in a diverse
manner. For instance, IL-1 enhances apoptosis (42), whereas IL-12
protects from apoptosis by inducing DNA repair (43).
Cell Type–Specific Response to UVB and Skin Cancer. One of
the most puzzling disparities between patients with CS and CS
mouse models is the difference in cancer incidence. The mouse
models are photosensitive and skin cancer–prone (44, 45), whereas
patients with CS have photosensitive skin but do not develop skin
cancer (7). In particular, CS mouse fibroblasts (45) and keratinocytes (46–48) are UV-sensitive, do not repair CPD in the
transcribed strand of active genes, and are extremely sensitive to
UV-induced apoptosis. It has been shown that in vivo in mouse
keratinocytes, the signal for p53 induction and sunburn formation
originates from DNA damage of actively transcribed genes (48). In
this study, we show that, as in the rodent situation, CS-A
fibroblasts are extremely sensitive to UV-induced apoptosis. This
Figure 6. A model for cell type–specific signaling pathways involved in
UVB-induced response in humans. Following UVB damage to DNA, p53 is
up-regulated and induces the expression of its primary regulator, mdm2. The
p53-mdm2 autoregulatory loop is involved in the control of apoptosis. In
fibroblasts (left ), the signal for UVB-induced apoptosis involves DNA
photoproducts in actively transcribed genes (TCR-dependent), whereas in
keratinocytes (right ) this pathway has a minor role due to the presence of
an efficient GGR that eventually repairs most CPD from both strands (GGR
back-up system). The other pathways that are known to contribute to
UVB-induced apoptosis in keratinocytes, including activation of death
receptors like CD95 and induction of reactive oxygen species, are indicated.
DNA Repair of UVB Damage. In a previous study, we have
shown that the repair of CPD, as measured by specific antibodies, is
accelerated in keratinocytes as compared with fibroblasts (19). Only
20% of residual CPD were detected on keratinocyte DNA 24 hours
after irradiation, whereas 60% of CPD were still present on fibroblast
DNA. In this study, by measuring UDS, we confirmed the higher
efficiency of keratinocytes in repairing UVB damage when compared
with fibroblasts and we were able to ascribe this phenomenon to a
more efficient GGR. The skin cells from the XP-C donor showed a
similar drastic decrease of UDS levels independently on the cell type,
whereas a defect in TCR (CS-A cells) was associated with UDS levels
higher in keratinocytes than in fibroblasts. Altogether, our data show
that the increased UVB-induced DNA repair synthesis of keratinocytes reflects a more efficient damage recognition/excision from the
genome overall.
Apoptosis after UVB Exposure. UVB-mediated apoptosis is a
highly complex process in which a variety of signaling pathways is
involved. Induction of nuclear DNA damage seems to be the
predominant pathway. Upon UVB irradiation, p53 is up-regulated
proportionally to UV damage inferred to DNA. Accordingly, a rapid
decrease in the levels of p53/p21/mdm2 was observed in
keratinocytes which repair UV photoproducts more efficiently than
fibroblasts (19). p53 is critically involved in the apoptotic signaling in
human fibroblasts that, in the absence of TCR (CS-A and CS-B
fibroblasts), show p53 induction and massive apoptosis at UVC
doses which are significantly lower than those required for normal
cells (23, 31, 34), whereas in the absence of GGR (XP-C fibroblasts),
the apoptotic response is similar to that of normal cells (23). We
confirm these findings after UVB irradiation showing that the
absence of TCR (CS-A fibroblasts) determines massive apoptosis at
doses where no apoptosis is detected in normal and XP-C fibroblasts.
When the apoptotic response of keratinocytes was analyzed,
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Cancer Research
Fig. 6. Altogether, our findings strongly suggest that in humans,
protection from skin carcinogenesis does not rely upon a
functional TCR. This would explain the lack of skin cancer in
patients with CS and account for the disparity in skin cancer rates
in patients with CS and CS mouse models.
finding confirms that in human fibroblasts, the signal for p53
induction and apoptosis involves DNA photoproducts in actively
transcribed genes. Conversely, in human keratinocytes, this
pathway does not (or only to a minor extent) contribute to
apoptosis because GGR repairs eventually most CPD from both
strands. Therefore, in human keratinocytes, the mechanism for
elimination of heavily UVB-damaged cells must rely on signaling
pathways that are largely TCR/p53-independent. Indeed, it is well
known that in keratinocytes UVB-induced apoptosis is mediated
by several pathways that do not involve DNA damage as already
discussed. A model summarizing the different responses of human
keratinocytes and fibroblasts to UVB irradiation is reported in
References
Acknowledgments
Received 5/21/2004; revised 10/30/2004; accepted 11/11/2004.
Grant support: MIUR/FIRB (RBNE01RNN7), Italian Ministry of Health (Ricerca
Finalizzata) and Associazione Italiana per la Ricerca sul Cancro (AIRC).
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
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Differential Role of Transcription-Coupled Repair in UVB−
Induced Response of Human Fibroblasts and Keratinocytes
Mariarosaria D'Errico, Massimo Teson, Angelo Calcagnile, et al.
Cancer Res 2005;65:432-438.
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