Differential repair of UVB-induced cyclobutane pyrimidine dimers in cultured human skin cells and whole human skin

d n a r e p a i r 7 ( 2 0 0 8 ) 704–712

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Differential repair of UVB-induced cyclobutane pyrimidine dimers in cultured human skin cells and whole human skin St´ephane Mouret a , Marie Charveron b , Alain Favier a , Jean Cadet a , Thierry Douki a,∗ a

Laboratoire “L´esions des Acides Nucl´eiques”, Service de Chimie Inorganique et Biologique UMR-E 3 CEA-UJF, CEA/DSM/D´epartement de Recherche Fondamentale sur la Mati`ere Condens´ee, CEA-Grenoble, 38054 Grenoble Cedex 9, France b Institut de Recherche Pierre Fabre, Laboratoire de Biologie Cellulaire, Toulouse, France

a r t i c l e

i n f o

a b s t r a c t

Article history:

Cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-

Received 7 November 2007

4PPs) are the two main classes of mutagenic DNA damages induced by UVB radiation.

Received in revised form

Numerous studies have been devoted so far to their formation and repair in human cells

11 January 2008

and skin. However, the biochemical methods used often lack the specificity that would allow

Accepted 15 January 2008

the individual study of each of the four CPDs and 6-4PPs produced at TT, TC, CT and CC din-

Published on line 4 March 2008

ucleotides. In the present work, we applied an HPLC-mass spectrometry assay to study the formation and repair of CPDs and 6-4PPs photoproducts in primary cultures of human ker-

Keywords:

atinocytes and fibroblasts as well as in whole human skin. We first observed that the yield of

DNA damage

dimeric lesions was slightly higher in fibroblasts than in keratinocytes. In contrast, the rate

DNA repair

of global repair was higher in the last cell type. Moreover, removal of DNA photoproducts

UV mutagenesis

in skin biopsies was found to be slower than in both cultured skin cells. In agreement with previous works, the repair of 6-4PPs was found to be more efficient than that of CPDs in the three types of samples, with no observed difference between the removal of the TT and TC derivatives. In contrast, a significant influence of the nature of the two modified pyrimidines was observed on the repair rate of CPDs. The decreasing order of removal efficiency was the following: C<>T > C<>C > T<>C > T<>T. These data, together with the known intrinsic mutational properties of the lesions, would support the reported UV mutation spectra. A noticeable exception concerns CC dinucleotides that are mutational hotspots with an UV-specific CC to TT tandem mutation, although related bipyrimidine photoproducts are produced in low yields and efficiently repaired. © 2008 Elsevier B.V. All rights reserved.

1.

Introduction

The incidence of skin cancer has considerably increased worldwide during the last few decades. Exposure to solar ultraviolet (UV) radiation has been unambiguously shown to be involved in the pathogenesis of most skin cancers. The harm-

∗

Corresponding author. Tel.: +33 4 38 78 31 91; fax: +33 4 38 78 50 90. E-mail address: [email protected] (T. Douki). 1568-7864/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.dnarep.2008.01.005

ful effects of UV radiation are mostly associated with both direct and indirect DNA damage [1] which can lead to the formation of mutations. UVB radiation, the most energetic part of the solar spectrum, is the most mutagenic and carcinogenic component. Absorption of UVB photons by DNA induces dimerization of adjacent pyrimidine bases that gives rise to

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two types of DNA damage, namely cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) [1]. Since the photoreactions may take place at each of the bipyrimidine dinucleotides, eight different dimeric photoproducts are expected to be generated upon UVB irradiation. However, their yields of formation strongly depend on the primary sequence of the DNA chain. Bipyrimidine photoproducts are preferentially produced at TT and TC sites whereas lower amounts are formed at CT and CC sites [2]. Furthermore, the ratio between the yield of CPDs and 6-4PPs strongly depends on the two adjacent bases involved in the lesions [2]. The role of the dimeric lesions in photocarcinogenesis may be inferred from the high proportion of p53 mutations, mostly C to T and CC to TT transitions, detected at bipyrimidine sites in skin tumors [3–5]. In addition, a link between the formation of CPDs and the mutagenesis induced by UVB radiation in mammalian cells has been clearly established [6]. Indeed, mutational hotspots in p53 gene are also frequent sites of UVB-induced formation of CPDs [7,8]. To counteract the effects of UV-induced DNA damage and to restore the genomic integrity, cells possess a series of repair systems. Thus, in mammalian cells, bipyrimidine photoproducts are removed by the nucleotide excision repair (NER) pathway. A defect in NER is at the origin of high UV-induced skin cancer incidence shown by most complementation groups of xeroderma pigmentosum patients [9]. Two distinct NER sub-pathways have been identified: the global genomic repair which deals with damage removal in the entire genome with usually a moderate efficiency, and the transcription coupled repair which selectively and very efficiently takes care of the damage in the transcribed strand of active genes [10]. Therefore, information is needed on both the formation frequency of a specific lesion and its rate of repair, in order to determine its biological impact. Interestingly, the repair efficiency of dimeric DNA damage depends on the type of bipyrimidine photoproducts. It is now well established in mammalian cells that 6-4PPs are more efficiently removed than CPDs [11–13]. The preferential repair of 6-4PPs with respect to CPDs has also been observed in whole human skin [14,15]. However, most of these conclusions have been inferred from immunological assays and no data are available so far on the repair rate of each of the eight possible bipyrimidine photoproducts. For instance, little is known on the possible different efficiency of the repair machinery towards the four different CPDs. These specific features could be partly inferred by sequencing techniques along a specific gene [16–18]. However, although extremely powerful, this approach yields data that also include local sequence effects. Therefore, the aim of the present study was to determine the individual rate of removal of CPDs and 6-4PPs from the DNA of human skin and primary cultures of cutaneous cells upon exposure to UVB radiation. For this purpose, lesions were quantified by an accurate high performance liquid chromatography method associated with tandem mass spectrometry (HPLC-MS/MS) detection. The assay, that allows the detection of the two classes of photoproducts for any of the four bipyrimidine dinucleotides [2] has been successfully applied to the determination of the distribution of dimeric photoproducts in cutaneous cells [19,20] and in human skin [21].

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Using this approach, we show that the nature of the two modified pyrimidines bases drastically affects the rate of removal of CPDs in cultured skin cells and whole human skin. These results thus raise questions in terms of impact of DNA repair on mutagenesis and molecular recognition by the repair proteins of lesions exhibiting very similar structures.

2.

Materials and methods

2.1.

Human skin biopsy and cell culture

Normal human skins were obtained immediately after breast plastic surgery from healthy Caucasian donors with their inform consent (Department of “Chirurgie Plastique et Maxillo-faciale”, CHU Grenoble, France). The skin phototype of all selected donors was either II or III according to Fitzpatrick classification [22]. Normal fibroblasts and keratinocytes were isolated from the same donor as previously described [19,21]. All experiments were performed using keratinocytes at passages 2 or 3 and fibroblasts at passages 5 or 6. The whole human skin was prepared as previously described [21]. Briefly, after four rinsing steps in PBS, another short one in ethanol 70% and five others in PBS, 6-mm punch biopsies were made. Biopsies were then put in 35-mm Petri dish and stored in dark in the presence of PBS until irradiation.

2.2.

UVB irradiation

The UVB light source used was a VL 215 G irradiator (Bioblock Scientific, Illkirch, France) equipped with two 15 W tubes with a spectrum distribution mostly emitting at 312 nm. The irradiance was measured by a VLX 3 W radiometer (Vilbert Lourmat, ´ France) fitted with a 312 nm probe. The irradiMarne la Vallee, ance received by the samples was 0.3 mW/cm2 with the lamp placed 80 cm above the targets. Petri dishes containing skin biopsies were exposed immediately, the lid being removed, to UVB radiation. Just after irradiation, PBS was replaced by fresh medium (DMEM/F12), and the skin biopsies were incubated at 37 ◦ C for increasing periods of time in order to assess the repair kinetic of DNA photoproducts. For each time point, three biopsies were pooled. Typically, the following conditions were used for the irradiation of cultured cells. Keratinocytes were seeded at 5 × 105 cells and fibroblasts at 6.5 × 105 cells per 100-mm Petri dish and grown to sub-confluence during 5 days. Just before irradiation, culture medium was removed and stored. Cells were rinsed twice with PBS and then UVB irradiation was performed in PBS with the lid removed. After irradiation, the cells were put back into their initial medium and incubated at 37 ◦ C for an appropriate period of time. In all cases, irradiations of whole human skin or primary keratinocytes and fibroblasts were performed in triplicate for each donor.

2.3.

DNA extraction and digestion

The protocol for DNA extraction from human skin involved first a grinding step in liquid nitrogen. Then, DNA was

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extracted by using the DNEasy Tissue Kit obtained from Qiagen (Courtabœuf, France). After the cold grinding step of the three pooled biopsies, the resulting powder was recovered with 360 ␮l of the first lysis buffer ATL prior to be incubated overnight at 55 ◦ C with proteinase K for a complete lysis of the tissue. A RNase A treatment and a second lysis step (buffer AL) were performed before loading the samples onto the DNEasy mini spin column. DNA was then eluted in two successive steps by using 200 ␮l of a 0.1 mM deferroxiamine mesylate solution each time. The sample was freeze-dried overnight and the resulting DNA residue was dissolved in 50 ␮l of 0.1 mM deferroxiamine mesylate solution. DNA extraction of keratinocytes and fibroblasts was performed by using a chaotropic method. Briefly, in the first step, the cell pellet was homogenized by pipeting the suspension in the presence of Triton-X100 to remove the plasma membrane. Nuclei were isolated by centrifugation and made soluble by addition of SDS. After successive treatments by RNases and proteinase, DNA was precipitated by addition of sodium iodide and 2-propanol. The resulting DNA pellet was finally dissolved in 50 ␮l of a 0.1 mM deferroxiamine mesylate solution. In all cases, extracted DNA was digested by incubation with phosphodiesterase II, DNAse II and nuclease P1 at pH 6 for 2 h at 37 ◦ C followed by an additional digestion step involving phosphodiesterase I and alkaline phosphatase at pH 8. After 0.1 M HCl was added, the samples were vortexed and centrifuged prior to be transferred into HPLC vials and then freeze-dried overnight. The resulting residues were made soluble in 40 ␮l of a 20 mM triethylammonium acetate solution (pH 6.5) before injecting onto the HPLC-MS/MS system.

2.4.

HPLC-MS/MS detection of DNA photoproducts

The samples were injected onto a model 1100 micro-HPLC system (Agilent technologies, Massy, France) coupled to a API 3000 triple quadrupolar mass spectrometer (PerkinElmer/SCIEX, Thornhill, Canada). The separation was achieved on an Uptisphere ODB octadecylsilyl silica gel column (150 × 2 mm, 3 ␮m particle size) from Interchim (Monluc¸on, France) with a gradient of acetonitrile in a 2 mM triethylammonium acetate

solution (pH 6.5). Chromatographic conditions and mass spectrometry features were as previously described [2]. The transitions used for the detection of the different bipyrimidine photoproducts and their retention times (Rts) were the following: 545 → 447 for the TT cyclobutane dimer (T<>T, Rt 14.6 min), 545 → 432 for the TT (6-4) photoproduct (TT 6-4PP, Rt 17.7 min), 531 → 195 for the TC and the CT cyclobutane dimers (T<>C and C<>T, respectively, Rt 7.8 and 9.9 min), 530 → 195 for the TC (6-4) photoproduct (TC 6-4PP, Rt 17.8 min) and 517 → 195 for the CC cyclobutane dimer (C<>C, Rt 4.2 min). The amount of each lesion was determined by using an external calibration obtained by the injection of known amounts of the authentic compound. Normal nucleosides were quantified by HPLC-UV with a detector set at 270 nm placed at the outlet of the column.

3.

Results

3.1. Larger yield of bipyrimidine photoproducts in fibroblasts than in keratinocytes Firstly, we determined the formation yields of the bipyrimidine photoproducts in fibroblasts and keratinocytes originating from the same donors. Upon UVB irradiation, bipyrimidine photoproducts were produced in the two cell types in the following decreasing order of frequency: T<>T > T<>C ≥ 6-4 TC > C<>T > C<>C > 6-4 TT. It may be added that none of other photoproducts, including 6-4PPs at CT and CC sequences and all Dewar valence isomers, was detected. Although, the relative distribution of photoproducts was found to be approximately similar for the two skin cell types, the combined yield of damage was significantly 1.6-fold higher (p < 0.02) in fibroblasts than in keratinocytes with 276 and 170 lesions/106 bases, respectively, following exposure to 200 J/m2 (Table 1). Significant differences were also observed in the formation for most of the lesions. Thus, we note a larger formation of T<>T and T<>C in fibroblasts than in keratinocytes (Table 1; p < 0.003). The UVB-induced generation of 6-4 TT was slightly larger in fibroblasts (p < 0.04). These observations emphasize that a similar relative photoproduct distribution was observed in both cell types, although the proportion of T<>C was slightly higher in fibroblasts.

Table 1 – Larger formation yield of photoproducts in fibroblasts than in keratinocytes after UVB irradiation (200 J/m2 )

3.2. cells

Lesions

Fibroblasts

T<>T 6-4 TT T<>C 6-4 TC C<>T C<>C

104.2 6.7 85.6 38.9 30.7 10.8

Sum

276.9 ± 13.0

Then, the repair kinetics of each photoproduct was determined in fibroblasts (Fig. 1) and keratinocytes (Fig. 2) following exposure to 200 J/m2 of UVB radiation. As expected, the two main 6-4PPs were efficiently removed at a similar rate from the DNA of human cells. In contrast, the repair rate of CPDs was found to strongly depend on their chemical structure for the two cell types. Indeed, as shown in Figs. 1 and 2, T<>T was much more slowly repaired than C<>T. T<>C and C<>C exhibited an intermediate repair rate which differed according to the cell type. Indeed, T<>C and C<>C were equally repaired in fibroblasts while C<>C was removed slightly faster than T<>C in keratinocytes. Although, the repair kinetics profiles looked similar in the two cell types, a more quantitative analysis was carried out

± ± ± ± ± ±

3.8 0.2 5.7 2.5 1.8 1.6

Keratinocytes 58.1 5.3 33.1 36.5 26.0 11.0

± ± ± ± ± ±

5.6* 0.6** 8.3* 4.2 6.4 7.0

170.0 ± 31.9***

Results are expressed in number of lesions per 106 normal bases and are the average ± standard deviation of three different donors. Difference between fibroblasts and keratinocytes was found to be statistically significant using the Student t test with *p < 0.003, **p < 0.04, ***p < 0.02.

Differences in the repair rate of CPDs in human

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Fig. 1 – Kinetics of repair of bipyrimidine photoproducts in fibroblasts exposed to UVB radiation (200 J/m2 ). Results are expressed in percentage of unrepaired lesions and are the average ± standard deviation of three different donors. The experiment for each donor was done in triplicate.

atinocytes with respect to fibroblasts (Table 2). Indeed, 8 h after UVB radiation, the level of remaining lesions was 1.2–2.8-fold lower in keratinocytes than in fibroblasts contingent on the photoproduct. Similarly, 24 h after UVB, the removal of 6-4PP was almost complete in the two cell types and the levels of remaining T<>T, T<>C, C<>T and C<>C were respectively 1.3, 1.7, 2.4 and 2.5-fold lower in keratinocytes than in fibroblasts (Table 2). Even 48 h after irradiation, this more efficient repair rate remained since the residual level of T<>T, T<>C, C<>T and C<>C were still 1.2, 1.6, 1.3 and 2.6-fold lower in keratinocytes, respectively. Moreover, the sum of remaining lesions for each class of photoproducts (CPDs or 6-4PPs) was also calculated in both cell types. Thus, the level of remaining CPDs was respectively 1.3 and 1.6-fold lower in keratinocytes than in fibroblasts 8 and 24 h after UVB radiation. Thereby 24 h after irradiation, around 60% of all CPD were still present in fibroblasts while this value fell to 37.5% in keratinocytes. For 6-4PPs, considering that their repair was almost complete 24 h after irradiation, the level of remaining 6-4PPs was estimated at 4 and 8 h; it was respectively 1.9 and 1.8-fold lower in keratinocytes than fibroblasts.

3.3. Differences in the repair rate of CPDs in whole human skin

Fig. 2 – Kinetics of repair of bipyrimidine photoproducts in keratinocytes exposed to UVB radiation (200 J/m2 ). Results are expressed in percentage of residual lesions and are the average ± standard deviation of three different donors. The experiment for each donor was done in triplicate.

in order to compare the rates of photoproduct removal in fibroblasts and keratinocytes. For this purpose, the proportion of remaining lesions at 8 and 24 h was determined for all photoproducts and in both cell types. A significantly faster removal of CPDs and 6-4PPs (p < 0.05) was observed in ker-

In a final series of experiments, we determined the repair kinetics of bipyrimidine photoproducts in whole human skin exposed to 1000 J/m2 of UVB radiation. This dose may seem like rather large but it should be reminded that a 26-fold protection is afforded by skin with respect to cultured keratinocytes under our experimental conditions, in agreement with recently published data [21]. Interestingly, the repair rate of CPDs in the DNA of human skin was also affected by the nature of the two modified pyrimidines. Like in fibroblasts and keratinocytes, T<>T was the most slowly repaired CPD in human skin while C<>T was the fastest one (Fig. 3). However, in contrast to cultured cells, C<>C was also quickly removed from skin DNA since it was excised at a rate similar to that of C<>T. T<>C exhibited an intermediate repair rate with respect to other CPDs. Altogether, 48 h after the irradiation, 54.3% of T<>T, 27.8% of T<>C, 12.7% of C<>C and 9.7% of C<>T were still present in the DNA. In contrast to CPDs, 6-4PPs were rapidly repaired and no difference was found between the rate of removal of the 6-4PPs at TT and TC sequences. However, the repair rate of 6-4PPs was lower

Table 2 – Efficiency of repair of bipyrimidine photoproducts in fibroblasts and keratinocytes 8 and 24 h after UVB irradiation (200 J/m2 )

Fibroblasts 8h 24 h Keratinocytes 8h 24 h

T<>T

6-4 TT

T<>C

6-4 TC

C<>T

C<>C

91.5 ± 6.0* 72.8 ± 1.3

10.1 ± 1.0* 2.0 ± 0.6

84.9 ± 5.1* 57.1 ± 8.9*

6.8 ± 0.7 0.7 ± 0.8

49.8 ± 0.1* 22.1 ± 3.3*

88.4 ± 5.6* 60.7 ± 2.9*

78.0 ± 1.8 55.5 ± 7.3

3.6 ± 1.4 1.5 ± 0.7

59.5 ± 5.6 33.5 ± 2.7

3.7 ± 2.3 1.5 ± 1.3

28.2 ± 2.9 9.1 ± 0.7

56.6 ± 6.5 24.2 ± 9.0

Results are expressed in percentage of unrepaired lesions and are the average ± standard deviation of three different donors (experiment for each donor was done in triplicate). Difference between fibroblasts and keratinocytes was statistically significant with *p < 0.05.

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Fig. 3 – Kinetics of repair of bipyrimidine photoproducts in human skin exposed to UVB radiation (1000 J/m2 ). Results are expressed in percentage of residual lesions and are the average ± standard deviation of four different donors.

in human skin than in culture of fibroblasts or keratinocytes (Figs. 1–3).

4.

Discussion

Although, repair of UVB-induced bipyrimidine photoproducts is extensively studied for many decades, little is known on the effect of the nature of the involved pyrimidine bases involved on the repair efficiency of a given type of photoproduct. Indeed, the available results have mostly been obtained with biochemical techniques based on the use of either antibodies or repair enzymes that are not able to discriminate between the four different lesions for a given class of photoproducts. Therefore, we undertook a more specific study by using a dedicated HPLC-MS/MS assay that allows individual quantification of all possible CPDs and 6-4PPs. We have previously reported some data on DNA repair in human cells using this analytical approach [19,20]. However, information was only provided on the removal of TT and TC lesions from the DNA of cultured cells. In the present study, repair rates were determined for all CPDs and the detectable 6-4PPs at only TT and TC sites. Cultured keratinocytes and fibroblasts, as well as skin, were studied. In addition, comparison could be made between the different types of human samples since they originated from the same skin explants. This was not possible in our previous studies where fibroblasts and keratinocytes were from breast skin and foreskin, respectively. The use of the HPLC-MS/MS method allowed us to obtain accurate quantitative data on the formation yields of the photoproducts in the two cultured cell types. In agreement with previous studies [23–25], the overall level of photoproducts was 1.6-fold higher in fibroblasts than in keratinocytes. These differences in the formation efficiency between the two cell types could be explained by the presence of keratin that might weaken the susceptibility of DNA to UVB radiation in keratinocytes. Interestingly, we previously observed that the yields of CPDs and 8-oxo-7,8-dihydro-2’-deoxyguanosine were 3.3 and 1.7 times larger in fibroblasts than in keratinocytes [26], respectively. These observations show that keratinocytes are protected over a wide range of wavelengths, which can eas-

ily be explained by the upper position of this cell type in skin and its larger exposure to harmful UV radiation. It may be also added that in agreement with our recently published data, the yield of photoproducts was 26 times lower in skin than in keratinocytes, showing the additional protection afforded by the stratum corneum. Emphasis was then placed on the comparison of the repair efficiency of the photoproducts in the two cell types. A small but significantly larger rate of removal was observed in keratinocytes with respect to fibroblasts (1.2–2.8 depending on the lesion 8 h after irradiation). Similar observations, based on the use of either specific antibodies [24] or unscheduled DNA synthesis assay [23,25], have been reported. A first explanation of this observation could be a difference in the response of the two cell types to UVB-induced damage. Indeed, evidence has been accumulating showing that p53 is an important determinant for normal NER activity in fibroblasts [27,28]. However, p53 does not seem to be required to maintain the global NER activity in either primary [29] or differentiated keratinocytes [30]. A second explanation for the lower rate of repair in fibroblasts could be the larger initial amount of damage in these cells. Indeed, we and others [19,20,31] have previously shown that the rate of removal of UVB-induced photoproducts, expressed in relative values, strongly decreases when the applied dose increases, and thereby when the initial level of damage increases. This may be accounted for by a saturation of the repair capacities of the cells which therefore can handle only a limited amount of lesions over a given period of time. As a result, the proportion of repaired lesions is lower when the initial level of damage is high. As far as the repair of the individual lesions is concerned, we first observed, in agreement with previous observations made in mammalian cells [11–13] and even in whole skin [14,15], that 6-4PPs are much more rapidly repaired than CPDs. We also confirm our previous observations of equal repair efficiency for both 6-4PP at TT and TC dinucleotides [19,20] in cultured cells. We could extend this observation to skin. It is worth mentioning that a much lower rate of removal bipyrimidine photoproducts was observed in skin than in cultured cells. Moreover, this difference might be even greater than it appears here as a higher dose of UVB would have been necessary to obtain the same amount of DNA damage in human skin as in cultured keratinocytes. Interestingly, the contribution of 6-4PPs to UV mutagenesis is often ruled out on the basis of their high repair efficiency in cultured cells. Accordingly, CPDs have been suggested to be responsible for the vast majority of the mutational events in cells [6]. The present observations suggest that 6-4PPs, which are highly mutagenic [32–34], could persist longer in skin than in isolated cells and therefore might be able to contribute to some extent to UV mutagenesis. This observation of a slower photoproducts removal in skin may reflect differences in repair capacities between actively growing and non-dividing cells. It may also be explained by lower DNA repair efficiency in keratinocytes in the process of differentiation in the upper layers of epidermis. Another explanation could be a decrease in repair capacities of skin maintained in survival medium. However, the presently reported results were obtained in explants treated within a few hours after surgery. In preliminary experiments, some skin samples were left overnight in survival medium prior to irradi-

d n a r e p a i r 7 ( 2 0 0 8 ) 704–712

ation and DNA repair analysis. The obtained data were similar to those shown in Fig. 3. These observations strongly suggest that our results are not extensively affected by a loss of repair capacities of the skin ex vivo. The validity of our results is also shown by a comparison with published in vivo data based on immunological approach [14]. Indeed, although the data are not directly comparable with our results because the authors used solar simulated sunlight, they noted that CPDs exhibit a half-life of 42.5 h for skin phototype II. Similarly, using another more specific method, the HPLC 32 P-postlabeling assay, the authors noted that approximately 50% of T<>T and 25% of T<>C remained in human skin in situ 48 h after solar simulated sunlight exposure [15,35]. The most striking observation of the present work was the effect of the chemical structure of CPDs on their repair rate. In previous studies, a slightly higher repair rate of T<>C with respect to T<>T was observed in both fibroblasts and keratinocytes [19,20]. Similar observations have also been made on the basis of semi-quantitative HPLC 32 P-labeling measurements [15,35,36]. The presently additional information on C<>T and C<>C as well as assessment of repair in skin unambiguously confirmed that the nature of the two pyrimidine bases involved in a CPD strongly affects its rate of removal from DNA. Indeed, in whole skin and cultured skin cells exposed to UVB radiation, CPDs were removed in the following decreasing order of efficiency: C<>T > C<>C > T<>C > T<>T. As a general observation it was found that T<>T and C<>T were found to be the lowest and fastest repaired CPDs, respectively, irrespective of the sample type. T<>C and C<>C were removed with intermediate efficiency, C<>C being repaired more efficiently than T<>C. The half time for repair of each CPD was estimated for the three types of samples. Ratio between these values for T<>T and C<>T were found to be 4.5, 8 and 5.2 in skin, keratinocytes and fibroblasts, respectively. It is interesting to look at these data in the light of known mutagenic properties of UV radiation on cells [37,38] and sunlight on skin as observed in tumors [3–5]. First, it may seem surprising that hardly any mutations occur at TT bipyrimidine sites although the corresponding CPD is repaired with a very poor efficiency. However, studies involving replication of synthetic probes carrying T<>T have unambiguously shown that this lesion was not mutagenic because the thymine residues involved in the dimeric lesion still code for adenine upon replication [33,39,40]. Second in the decreasing order of repair rate is T<>C which has been suspected to be responsible for the C to T transitions observed at TC sites [3–5,37,38]. Its relative long lifetime in cells is thus likely to account for this major mutational event. Our results also explain why C<>T, the photoproduct of the reverse sequence with respect to TC, does not influence the final UV mutation spectrum. The lack of mutations at CT dinucleotides cannot be explained by the mutational properties of the photoproduct that is expected to be present as U<>T upon deamination like T<>C is converted into T<>U, and which is likely to lead to incorporation of A in front of the uracil moiety [41,42]. The observation that C<>T is very efficiently repaired provides a clear explanation of the absence of mutation events at CT sites. Strikingly, C<>C was also found to be efficiently repaired, at least in skin and to a lesser extent in keratinocytes. This is

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surprising because CC to TT tandem mutations are regarded as the hallmark of UV mutagenesis, with this type of event representing 20% of the mutations in normal and 80% in XP cells [4,5]. It may also be reminded that CC dinucleotide is the less photoreactive among the four bipyrimidine sites [2,21]. These observations suggest that CC dimeric photoproducts exhibit strong mutagenic properties that allow them to appear in UV mutation spectra. It has been proposed that the main explanation for C<>C induced mutagenesis is linked to its deamination into U<>U [43]. This last reaction is also expected to be favored in XP patient because the slower repair of dimeric photoproducts allows deamination to be completed [44]. However, this explanation should apply to all cytosine-containing CPDs that were shown to exhibit similar deamination rates in model systems [45–47] and in DNA [44]. Our observations on the differential rate of repair of CPDs allow us to refine the model. Indeed, the repair of T<>C is rather slow even in normal skin and this explains why its contribution to UV mutation spectrum does not increase in XP cells. In contrast, C<>C is likely to be rapidly removed from normal tissue before occurrence of deamination and induction of mutations. In XP cells, the half-life of C<>C in DNA is likely to be much longer and its mutational consequences are more frequent. It should however be emphasized that this model does not explain why mutational events at CT dinucleotides are rare events in XP cells. A first explanation could be a poorer mutational potential of the uracil moiety in deaminated C<>T than in T<>U and U<>U. However, the former lesion has never been specifically studied, in contrast to other CPDs. Information is also needed on the individual repair rate of the four CPDs in XP cells and also on deamination of cytosine containing dimeric lesions in a cellular context. A first possible explanation to the clear difference in repair efficiency of the four different CPDs has to be searched in their structural effect on DNA. Indeed, such consideration accounts for the faster removal of 6-4PPs with respect to CPDs because the former lesions induce a much larger distortion of the DNA structure than the latter [48,49]. Unfortunately, data on the structural effect of CPDs on DNA are limited to T<>T [48,50,51]. Nevertheless, our results allow us to anticipate a sequence effect. Indeed, in fibroblasts, keratinocytes and skin, the fastest removed CPDs were those with a 5 -end cytosine moiety (CT and CC). In contrast, 5 -end thymine-containing CPDs were more slowly repaired. In addition to structural effects, these observations could be explained by the binding properties of the DNA damage sensor proteins such as DDB2 or XPC [52–54]. A role for the damaged DNA binding protein could also explain the observed differences between cultured cells in terms of lesion specificity and repair rates as the concentration of DDB2 influences the repair kinetics of both CPDs and 6-4PPs in mouse fibroblasts [55]. Indeed, it has been recently shown that in response to UVB radiation, the gene expression profile of human epidermis differs from that of cultured keratinocytes since it is mainly associated with DNA repair in epidermis while it is predominantly related to cell cycle arrest and apoptosis in cultured keratinocytes [56]. It can thus be proposed that the amount and the nature of the proteins that first detect DNA photoproducts are different in skin and cultured cells.

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5.

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Conclusion

The bipyrimidine photoproducts induced by the UV component of the solar spectrum have been identified almost half a century ago. Since then, a number of chemical and biological properties of these lesions have been gathered. However, the determination of the individual repair rate for each of the photoproducts arising from the different bipyrimidine dinucleotides was not unavailable in cultured cells and skin. These data are reported in the present work. No dinucleotide effect was observed for 6-4PP that is the most efficiently removed lesion. In contrast, the nature of the pyrimidine bases involved in the dimeric lesions strongly affects the repair rate of CPD. C<>T is the fastest eliminated photoproduct while T<>T is the slowest. T<>C and C<>C show intermediate kinetics of removal. The data fit with the known mutation spectrum of UVB light for T<>T, T<>C and C<>T but this does not explain the significant fraction of CC to TT tandem mutations observed in cells and tumors. Indeed, CC site is the least photoreactive dinucleotide and C<>C is quite efficiently repaired. Both structural studies aimed at understanding the mutational properties of this photoproduct and biochemical works on the recognition of individual CPDs are required to unravel the mechanism behind mutations at CC dinucleotides. Such studies could also explain the difference in repair profile and efficiency observed between cell types and whole skin.

Acknowledgments The authors wish to thank Pr. J Lebeau and Mrs MJ Belin (Department of “Chirurgie Plastique et Maxillo-faciale”, CHU Grenoble, France) for providing normal human skin from breast plastic surgery. We acknowledge Pr. MT Leccia (Department of Dermatology, CHU Grenoble, France) for her helpful assistance in sample collection from the Department of “Chirurgie Plastique et Maxillo-faciale” and for writing the inform consent and the skin phototype questionnaire.

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