Differential Role of Basal Keratinocytes in UV-Induced Immunosuppression and Skin Cancer

MOLECULAR AND CELLULAR BIOLOGY, Nov. 2006, p. 8515–8526 Vol. 26, No. 220270-7306/06/$08.00�0 doi:10.1128/MCB.00807-06Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Differential Role of Basal Keratinocytes in UV-InducedImmunosuppression and Skin Cancer�

Judith Jans,1†‡ George A. Garinis,1‡ Wouter Schul,1§ Adri van Oudenaren,2 Michael Moorhouse,3Marcel Smid,4 Yurda-Gul Sert,1 Albertina van der Velde,1 Yvonne Rijksen,1 Frank R. de Gruijl,5

Peter J. van der Spek,3 Akira Yasui,6 Jan H. J. Hoeijmakers,1 Pieter J. M. Leenen,2and Gijsbertus T. J. van der Horst1*

MGC, Department of Cell Biology and Genetics, Center for Biomedical Genetics, Erasmus University Medical Center, P.O. Box 1738,3000 DR Rotterdam, The Netherlands1; Department of Immunology, Erasmus University Medical Center, P.O. Box 1738,

3000 DR Rotterdam, The Netherlands2; Department of Bioinformatics, Erasmus University Medical Center, P.O. Box 1738,3000 DR Rotterdam, The Netherlands3; Department of Medical Oncology, Josephine Nefkens Institute,

Erasmus University Medical Center, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands4;Department of Dermatology, Leiden University Medical Center, Sylvius Laboratory,

2300 RA Leiden, The Netherlands5; Department of Molecular Genetics, Institute ofDevelopment, Aging and Cancer, Tohoku University, Sendai, 980-8575, Japan6

Received 8 May 2006/Returned for modification 12 June 2006/Accepted 30 August 2006

Cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs) comprise major UV-induced pho-tolesions. If left unrepaired, these lesions can induce mutations and skin cancer, which is facilitated byUV-induced immunosuppression. Yet the contribution of lesion and cell type specificity to the harmfulbiological effects of UV exposure remains currently unclear. Using a series of photolyase-transgenic mice toubiquitously remove either CPDs or 6-4PPs from all cells in the mouse skin or selectively from basalkeratinocytes, we show that the majority of UV-induced acute effects to require the presence of CPDs in basalkeratinocytes in the mouse skin. At the fundamental level of gene expression, CPDs induce the expression ofgenes associated with repair and recombinational processing of DNA damage, as well as apoptosis and aresponse to stress. At the organismal level, photolyase-mediated removal of CPDs, but not 6-4PPs, from thegenome of only basal keratinocytes substantially diminishes the incidence of skin tumors; however, it does notaffect the UVB-mediated immunosuppression. Taken together, these findings reveal a differential role of basalkeratinocytes in these processes, providing novel insights into the skin’s acute and chronic responses to UV ina lesion- and cell-type-specific manner.

Exposure to UV light has undesired health consequenceswith increasing impact; apart from acute effects (e.g., sunburn),skin tumors are considered a major threat, demonstrated bytheir increasing incidence in white populations, due to alteredlife style and the erosion of the protecting ozone layer (1, 43).Besides the ability of RNA (20, 21) and proteins (5) to absorblight at the UV wavelength, our recent findings have unequiv-ocally pointed to DNA as the biologically most relevant targetof UV radiation (12, 22, 35). UV induces the formation ofmajor dimer configurations, namely the cyclobutane pyrimi-dine dimers (CPDs) and 6-4 photoproducts (6-4PPs), gener-ated by covalent bonds between two adjacent pyrimidines, thatinterfere with biological processes (e.g., transcription and rep-lication) critical for cell viability (28).

To recognize and remove effectively the wide range of

hazardous DNA lesions, mammalian cells employ a resource-ful battery of DNA repair systems (10, 11, 15). For instance,nucleotide excision repair (NER) removes UV-induced DNAdamage as well as numerous other helix-distorting lesions (15).NER is divided in two subpathways: global genome NER (GG-NER) and transcription-coupled NER (TC-NER) that differprimarily in the way damage is recognized (2, 27, 42). In GG-NER, the protein complex XPC/hHR23B probes the completegenome for deformation of the DNA double helix (39). How-ever, whereas this complex readily recognizes the highly helix-distorting 6-4PPs, thereby allowing their fast removal, it poorlyrecognizes and removes the mildly distorting CPDs (2, 28). Inhumans, CPD recognition is enhanced in potentially genotoxicconditions due to p53-dependent upregulation of the p48 sub-unit of the DNA damage binding protein DDB (18). Rodents,however, lack the p53-responsive element in the p48 gene andare, therefore, unable to repair CPDs by GG-NER (18, 19). InTC-NER, damage recognition is initiated when an elongatingRNA polymerase II is stalled upon transcription-blocking le-sions (e.g., CPDs and 6-4PPs) on the template strand of activegenes. This initial step in TC-NER requires the action of CSBand CSA proteins. Subsequently, the XPB and XPD helicasesof the 10-subunit transcription factor TFIIH unwind the helixsurrounding the lesion. XPA verifies the damage whereas RPAstabilizes the complex. Next, a single-strand DNA fragment of�30 nucleotides long flanking the damage is excised by XPG

* Corresponding author. Mailing address: MGC, Department ofCell Biology and Genetics, Center for Biomedical Genetics, ErasmusUniversity Medical Center, P.O. Box 1738, 3000 DR Rotterdam, TheNetherlands. Phone: 31 10 4087455. Fax: 31 10 4089468. E-mail: [email protected].

† J.J. and G.A.G. contributed equally to this work.‡ Present address: Department of Molecular and Cell Biology, Uni-

versity of California at Berkeley, 125 Koshland Hall, Berkeley, Calif.§ Present address: Novartis, Institute of Tropical Disease, Singa-

pore.� Published ahead of print on 11 September 2006.

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and XPF/ERCC1 endonucleases. Finally, DNA synthesis ofthe excised strand resumes to fill the gap followed by ligationof the nick (8, 10, 15, 44).

The indispensable task of NER in removing (UV-induced)DNA lesions is highlighted by three clinically and geneticallyheterogeneous human syndromes that carry defects in NER-associated genes and that are all sensitive to UV exposure:xeroderma pigmentosum (XP), Cockayne syndrome (CS), andtrichothiodystrophy (7). Particularly, xeroderma pigmentosumpatients are characterized by a �1,000-fold increased suscep-tibility to sunlight-induced skin cancer (4).

Even so, many organisms are equipped with yet anothermechanism for repair of UV-induced DNA lesions, namedphotoreactivation (PR) (46). Unlike NER, PR is carried outby photolyases, monomeric enzymes that specifically recog-nize and repair either CPDs or 6-4PPs by damage reversal,thereby obviating the need for excision and DNA resynthe-sis. Photolyases require visible light as a source of energy tosplit the pyrimidine dimer and revert distorted base confor-mations back to their original state. However, despite theirstrong evolutionary conservation in many organisms (rang-ing from bacteria to marsupials), photolyase enzymes areabsent in placental mammals. Therefore, rodents and hu-mans rely solely on the complex and, for CPDs, the lessefficient NER system (46).

To unravel the contribution of the individual classes of pho-tolesions (i.e., CPDs versus 6-4PPs) to the deleterious outcomeof UV exposure in the skin, we have previously generatedtransgenic mice that ubiquitously express either the Potoroustridactylus CPD photolyase (�-act-CPD-PL) or the Arabidopsisthaliana 6-4PP photolyase (�-act-6-4PP-PL) from the �-actinpromoter (35). Light-dependent removal of CPDs and/or6-4PPs from the skin provided ample evidence that CPDs,rather than 6-4PPs, are mostly responsible for the majority ofthe adverse effects upon UV exposure, including sunburn,mutagenesis, and skin cancer (21, 33). However, the �-act-CPD-PL and �-act-6-4PP-PL mouse models provided littleinformation regarding the contribution of distinct cell types tothe biological processes associated UV exposure.

In this regard, the basal keratinocyte is particularly prone tooncogenic transformation, as it proliferates vigorously while itis burdened with the task to respond continuously to environ-mental triggers. Thus, we generated mice expressing the P.tridactylus CPD photolyase enzyme from the basal keratino-cyte-specific promoter keratin-14 (K14) (33). The K14-CPD-photolyase (K14-CPD-PL) transgenic mice allowed rapid,light-dependent removal of CPDs from basal keratinocytesonly, whereas DNA lesions in all other cell types (e.g., fibro-blasts and more differentiated keratinocytes) could only beremoved by the substantially slower NER. Besides the UV-mediated mutagenic effects, the suppression of the immunesystem subsequent to UV exposure contributes substantiallyto the development of skin cancer (41). Here, the K14-6-4PP-PL mice complete the set of available tools, allowing usto address lesion and cell type specificity in relation tosystemic immunosuppression and skin cancer upon exposureto UVB irradiation.

MATERIALS AND METHODS

Generation of K14-photolyase transgenic mice. The construct for the gener-ation of K14-6-4PP-PL transgenic mice was cloned in the vector pSP72 (Pro-mega) and contained the human keratin-14 promoter (2.3-kb PCR fragment,generated using forward primer 5�-AAGCTTATATTCCATGCTAG-3� and re-verse primer 5�-GGATCCTGAGTGAAGAGAAGG-3�) followed by the A.thaliana 6-4PP-PL cDNA. At the 3� end, exon 2 (the last 22 bp), intron 2, exon3, and the 3� untranslated region (including the polyadenylation signal) of thehuman �-globin gene were inserted. The expression constructs were excised fromthe plasmid using SalI, separated from vector DNA by agarose gel electrophore-sis, isolated from the gel with a GeneClean II kit (Bio 101), and further purifiedusing Elutip-D minicolumns (Schleicher and Schuell, Germany). The fragmentwas dissolved in injection buffer (10 mM Tris-HCl, pH 7.5, 0.08 mM EDTA) andinjected into the pronucleus of fertilized eggs derived from FVB/N intercrossesas described previously (16).

Transgenic animals were identified by Southern blot analysis of genomic tailDNA, using the 6-4PP-PL cDNA as a probe. To estimate the number of inte-grated copies, equal amounts of genomic DNA from transgenic mice weresubjected to Southern blot analysis. As a standard, we used equal amounts ofgenomic tail DNA supplemented with 0, 10, 30, or 100 pg of the corresponding6-4PP-PL expression construct. The hybridization signal obtained with the 6-4PP-PLcDNA probe was quantified using a Molecular Dynamics PhosphorImager andImageQuant software. After comparison of signal intensities, the transgene copynumber was estimated using the supplemented reference samples. Routine geno-typing of mice was performed by PCR analysis. The primer set 5�-GCACGATTCAGCAAGCAAGG-3� (forward primer) and 5�-CGGTACCTCTACCTATTTGAGTTTTG-3� (reverse primer) was used to amplify a 200-bp fragment of the6-4PP-PL coding region. Experiments were performed on mice in a mixed FVB/C57BL/6J background, except for the carcinogenesis and microarray experiments, inwhich animals were further crossed with hairless HRA/SKH mice. The generation of�-act-CPD-PL and �-act-6-4PP-PL transgenic mice, as well as genotyping proce-dures, has been described elsewhere (22, 35).

As required by Dutch law, the Dutch Ministry of Agriculture, Nature andFood Quality approved the generation of genetically modified mice. An inde-pendent Animal Ethical Committee (Dutch equivalent of the IACUC) approvedall animal studies.

RNA isolation and reverse transcriptase PCR (RT-PCR). Mouse skin RNAwas isolated, and cDNA synthesis was performed using Superscript II RNase Hreverse transcriptase (Life Technologies) according to the protocol of the sup-plier. A PCR was performed on the cDNA using a forward primer in thephotolyase transgene (5�-GCACGATTCAGCAAGCAAGG-3�) and a reverseprimer in exon 3 of the �-globin gene (5�-TGGACAGCAAGAAAGCGAG-3�).The presence of introns in the �-globin moiety of the photolyase transgenesallows discrimination between the cDNA-derived PCR product and possiblegenomic DNA contamination.

Photoreactivation in mouse cells and skin. Cells were grown on coverslips andwashed with phosphate-buffered saline, exposed to 20 J/m2 UV-C (Philips TUVgermicidal lamp), and subsequently kept in Hank’s buffer (137 mM NaCl, 5.4mM KCl, 4.4 mM KH2PO4, 0.33 mM Na2HPO4, 1.3 mM CaCl2, 0.81 mMMgSO4, 4.2 mM NaHCO3, 1 g/liter glucose, pH 7.4). Photoreactivation wasperformed by exposing cells for 1 h to light from four white fluorescent tubes(Philips TLD 18W/54) at a distance of 15 cm and shielded by a 5-mm glassfilter. Nonphotoreactivated cells were given the same treatment except thatdishes were covered with two layers of aluminum foil and put under the samefluorescent lamps. Immunocytochemical staining of CPDs and 6-4PPs usingthe antibodies TDM2 and 64M2, respectively, was performed as describedpreviously (35).

Mice were anesthetized, and hairs were removed from a small area on the backof the animal. One third of the hairless area was covered with black nonadhesivetape, and the remaining area was exposed to the light of two Philips TL-12 (40W) tubes emitting UVB light. Typically, 1 minimal erythemal dose (MED) wasobtained with an exposure of 2 min. Subsequently, half of the UV-exposed areawas covered with tape, and mice were exposed for 3 h to the light of 4 whitefluorescent tubes (GE Lightning Polylux XL F36W/840) filtered through 5 mm ofglass. Then, mice were sacrificed, and skin samples were taken from the unex-posed area, the UV-irradiated area that was covered, and the UV-irradiated areathat was exposed to PR light. Skin sections were stained as described previouslywith antibodies TDM2 or 64M2 recognizing CPDs or 6-4PPs, respectively (35).

Microarray experimental design, total RNA isolation, cDNA labeling, hybrid-ization, and data extraction. Two groups of hairless mice (three mice per group)were irradiated with 1 MED of UVB and PR for 3 h (group 1) or kept in the dark(group 2). Skin samples were isolated at 8 h after UV irradiation as described

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above. Total RNA was isolated as previously described (12), and RNA concen-tration and quality were assessed by spectrophotometry and by the use of anAgilent 2100 Bioanalyzer. Differentially labeled cDNA was prepared from RNAfrom either the UV-irradiated, PR-light-treated skin or the UV-irradiated, non-light-treated skin; it was mixed (per time point) and hybridized to cDNA mi-croarrays representing �15,000 mouse genes (obtained from The NetherlandsCancer Institute). Labeling and hybridization protocols were performed as pre-viously described. Dye incorporation bias was avoided by reverse labeling allsamples during the total RNA reverse transcription. A pair of self-hybridizations(dye reversed) preceded each batch of six microarrays. cDNA microarrays werescanned using a laser confocal scanner (Scanarray Express HT; Perkin ElmerInc.). Data were extracted by means of the Imagene software package version 5.0(Biodiscovery Inc., California). Detailed information on labeling and hybridiza-tion protocols and the microarray platform can be found at http://microarrays.nki.nl/download/index.html.

Data processing and analysis. Hierarchical clustering, principal componentanalysis, self-organizing maps, K-clustering, analysis of variance, gene ontologyclassification, and network analysis were performed by the Spotfire Decision Sitesoftware package 7.2, version 10.0 (Spotfire Inc., Massachusetts), the GeneOntology (GO) Mapper application (38), and Ingenuity Pathways Analysis soft-ware (www.ingenuity.com) as previously described (12). Significant overrepre-sentation of GO-classified biological processes was assessed by comparing thenumber of pertinent genes in a given biological process to the total number of therelevant genes printed on the NIA 15K cDNA microarray for that biologicalprocess (Fisher exact test, P � 0.05; false detection rate of �0.1) using thepublicly accessible software Ease (17).

Quantitative real-time PCR evaluation. Quantitative real-time PCR was per-formed with the DNA engine Opticon according to the instructions of themanufacturer (MJ Research). For quantitation of cDNA, primer pairs for Rad51(forward, 5�-TAC ATT GAC ACC GAG GGC AC-3�; reverse, 5�-CTG ACGCTT GGT AAA GGA GC-3�), Mcl-1 (forward, 5�-GAT GGC GTA ACA AACTGG G-3�; reverse, 5�-GGA AGA ACT CCA CAA ACC C-3�), Sumo (forward,5�-GCC AGT GAT GTG AAG AGA CC-3�; reverse, 5�-GGT GGG TTC TGAAAG TGG AG-3�), Hspcb (forward, 5�-AGA GCC TCA CCA ATG ACTGG-3�; reverse, 5�-ATG ATG AAC ACA CGG CGG A-3�), Maged (forward,5�-AGA ATG CCA CCA CAA AGG G-3�; reverse, 5�-ACT GGG AGA CTGAGG GAA AT-3�), Trp53 (forward, 5�-AAC TAT GGC TTC CAC CTG G-3�;reverse, 5�-GCT GTG ACT TCT TGT AGA TGG-3�), Gpx3 (forward, 5�-TCTACG AGT ATG GAG CCC TCA-3�; reverse, 5�-GCC CAG AAT GAC CAAGCC AA-3�), Ddb1 (forward, 5�-GCC AGT CAA AGA GGT GGG AA-3�;reverse, 5�-AAT GAT GCC AGT CTC CGA GG-3�), Sod1 (forward, 5�-GGGACA ATA CAC AAG GCT GT-3�; reverse, 5�-GCC AAT GAT GGA ATGCTC TC-3�), and Hprt-1 (forward, 5�-GGC AAC ATC AAC AGG ACTCC-3�; reverse, 5�-CGA AGT GTT GGA TAC AGG CC-3�) were designed togenerate intron-spanning products of 180 to 210 bp. Hypoxanthine guaninephosphoribosyltransferase 1 (Hprt-1) mRNA was used as an external stan-dard. For data analysis, the second derivative maximum method was applied:E1gene of interest

�CP (control � sample)/Ehprt-1�CP (control � sample), where E is

efficiency.Apoptosis. For detection of apoptotic cells in the skin, we used a terminal

deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL)assay (Fluorescein Apoptosis Detection System; Promega). Depilated areas onthe back of mice were exposed to UV and PR light as described above andsubsequently kept in the dark. Skin samples, taken 40 h after UV exposure, werefixed overnight in 4% paraformaldehyde, washed in phosphate-buffered saline,and embedded in paraffin. Skin sections (5 �m) were deparafinized and incu-bated as described by the manufacturer.

Hyperplasia. Mice were anesthetized and an area on the back was depilated byplucking. Mice were exposed to 1 MED UVB and PR light for four consecutivedays. One week after the start of the experiment, mice were sacrificed, and 8-�mskin sections were obtained. Sections were further processed and stained withhematoxylin and eosin.

Immunosuppression. Systemic immunosuppression was determined as before(3) with slight modifications. The animals were shaven on the back 1 day prior toUV treatment. For irradiation, a Bluepoint 2 source (Honle, Munchen, Ger-many) was used. Two circular areas (6.3 cm2 in total) were irradiated with 0.5MED (2,000 J/m2) each day for five consecutive days. The mice were skinsensitized 4 days after the last UV exposure by topical application of 5% picrylchloride (PCl) to nonirradiated shaved abdomen, chest, and feet. Four days aftersensitization, both ears of mice were challenged with 0.8% PCl in olive oil. At24 h after challenge, duplicate ear measurements were performed with an engi-neer’s micrometer (Mitutoyo Digimatic 293561; Veenendaal, The Netherlands).The responses were statistically evaluated using a two-tailed Student’s t test.

Skin carcinogenesis. Hairless photolyase mice and their wild-type (wt) litter-mates aged 8 to 12 weeks were exposed daily to 500 J/m2 UVB (Philips TL-12tubes) followed by 3 h of PR light (GE Lightning Polylux XL F36W/840 lamps).Mice were followed in time and thoroughly screened weekly for the occurrenceof skin abnormalities. Typically, carcinomas in wt animals were expected to occurafter 3 months of treatment. Mice were sacrificed when tumors of �4 mmoccurred. Biopsies of tumors were taken and processed for routine hematoxylin-eosin staining. Graphical representation of the prevalence versus time is basedon an actuarial method described by Kaplan and Meier (reviewed in reference45) and adapted to carcinogenesis by Peto et al. (33).

RESULTS

Generation of keratin-14-photolyase transgenic mouselines. The generation of the K14-CPD-photolyase mouse lineswas described previously (35). To obtain mice expressing the6-4PP-PL transgene in basal keratinocytes, a construct wasgenerated containing the A. thaliana 6-4PP-PL cDNA, pre-ceded by the human K14 promoter. To enhance mRNA sta-bility, a part of the human �-globin gene (i.e., exons 2 and 3,intron 2, the 3� untranslated region, and the polyadenylationsignal) was cloned behind the photolyase cDNA (Fig. 1A). Thephotolyase encoded by the A. thaliana 6-4PP-PL cDNA spe-cifically repairs 6-4PPs, leaving the CPDs unrepaired, as shownby PR experiments in UV-exposed dermal fibroblasts from�-actin-6-4PP-PL mice (Fig. 1B).

Oocyte injections resulted in several independent K14-6-4PP-PL mouse lines. The selected mouse line contained �25copies of the transgene, as determined by Southern blot anal-ysis (data not shown). RT-PCR analysis of total skin RNAshowed the presence of a 300-bp fragment, indicative of properexpression and splicing of the transgene (Fig. 1C).

Photoreactivation of the mouse skin. The transgenically ex-pressed photolyase enzyme is expected to allow light-depen-dent removal of DNA lesions in a subset of epidermal kerati-nocytes only. Previously, we showed that PR in K14-CPD-PLmice indeed resulted in CPD removal in basal keratinocytesspecifically. To investigate light-dependent removal of 6-4PPs,we applied an immunohistochemical assay on K14-6-4PP-PLmice, using an antibody specifically recognizing 6-4PPs (64M2).One-third of a depilated area on the back of mice was covered,while the remaining part was exposed to 1 MED of UVB. Next,half of the UV-exposed area was covered, while the remainingpart of the skin was exposed to PR light for 3 h. Skin biopsieswere taken, and sections were further processed for immuno-histochemical staining. As expected, the non-UV-irradiatedskin did not show any DNA lesions (Fig. 1D, No UV). Similarto our previous experiments, upon UV exposure, both CPDs(see reference 35) and 6-4PPs (this work) are induced in epi-dermis and upper dermis (Fig. 1D, UV 3 h dark). Importantly,upon exposure to PR light for 3 h, 6-4PPs are rapidly removedfrom a subset of cells in the epidermis only, indicating that theenzyme is indeed functional in basal keratinocytes (Fig. 1D, 3h PR light). Together, these findings validate the K14-6-4PP-PL mice for the investigation of the role of basal kerati-nocytes in the response of the skin to UV.

Acute effects upon exposure to UVB-light. (i) Apoptosis ofbasal keratinocytes requires the presence of CPDs. Exposureof the skin to UV light leads to apoptosis of keratinocytes and,upon chronic treatment, to epidermal hyperplasia, detected asthickening of the epidermis. Recently, we have shown thatremoval of CPDs, rather than 6-4PPs, from the entire skin is

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sufficient to prevent initiation of the apoptotic response (22),an effect that was also observed when CPDs were removedfrom basal keratinocytes only (35). We now also investigatedthe effect of enhanced removal of 6-4PPs in basal keratinocyteson apoptotic events; K14-6-4PP-PL mice and K14-CPD-PLmice (used as a control) were exposed to a single dose of UVB

(1 MED), followed by PR light for 3 h, and compared toUV-irradiated K14-photolyase mice that were kept in the dark.Forty hours after UVB-exposure, skin biopsies were taken, andskin sections were further processed for the TUNEL assay,thereby allowing the detection of apoptotic cells. As shown inFig. 2A, preferential elimination of 6-4PPs from basal kerati-

FIG. 1. Generation of K14-6-4PP photolyase transgenic mice. (A) Expression construct for the generation of K14-6-4PP-photolyase transgenicmice, containing the human K14 promoter, the A. thaliana 6-4PP-photolyase cDNA, and human genomic �-globin sequences, including exons 2and 3, intron 2, the 3� untranslated region, and the polyadenylation signal. Arrows indicate the position of the primers used for the RT-PCRexperiment. (B) Photoreactivation of 6-4PPs in cultured transgenic fibroblasts. Induction of CPD and 6-4PP lesions in cultured MDFs from6-4PP-PL transgenic mice by 20 J/m2 of UVC light and subsequent exposure to photoreactivating light for 1 h. Photolesions were detected byimmunofluorescent labeling using CPD- or 6-4PP-specific antibodies and fluorescein isothiocyanate-conjugated secondary antibodies. (C) RT-PCRanalysis of RNA from skin extracts of K14-6-4PP photolyase transgenic mice results in a 300-bp band. (D) 6-4PP lesions in the depilated dorsalskin of K14-6-4PP photolyase mice following exposure to 1 MED of UVB light and without (middle panel) or with (bottom panel) subsequentexposure to PR light. Photolesions were detected using 6-4PP-specific antibodies and horseradish peroxidase-conjugated secondary antibodies.Diaminobenzidine was used as substrate. Nuclei are visualized by methyl green staining. Note the uniform high density of nuclear labeling in thenon-PR tissue, in contrast to the heterogeneous and lighter nuclear labeling of the PR tissue.


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nocytes did not have a significant impact on the apoptoticresponse (Fig. 2A). This is fully consistent with previous ob-servations where the levels of apoptosis were not significantlyaltered upon PR of 6-4PPs in the ubiquitously expressing�-act-6-4PP-PL (22).

(ii) CPDs in basal keratinocytes are responsible for UV-induced hyperplasia. To study the effect of distinct DNA lesionsin basal keratinocytes on epidermal hyperplasia, K14-CPD-PLand K14-6-4PP-PL mice were exposed to UVB (1 MED) for fourconsecutive days. Every UV treatment was followed by exposureto PR light for 3 h, while UV-irradiated, non-PR light-treatedtransgenic mice were kept in the dark. Three days after the lastUV exposure, mice were sacrificed, and skin samples were stainedwith hematoxylin and eosin. As expected, a clear induction ofepidermal hyperplasia was observed in mice that were only ex-

posed to UVB (Fig. 2B). However, after exposure of K14-CPDphotolyase mice to PR light, i.e., upon removal of CPDs frombasal keratinocytes only, only a minor thickening of the epidermiscould be observed, indicating that epidermal hyperplasia wasnearly absent. In marked contrast, light-dependent enhanced re-moval of 6-4PPs from the basal keratinocytes of K14-6-4PP-PLmice did not significantly alter the skin hyperplasia observed inmouse skin after UVB. These findings identify the basal kerati-nocyte as the major cell type responsible for UV-induced hyper-plasia, and, in line with previous studies in �-act photolyasemouse models (21), point towards CPDs as the critical lesioninvolved in this process.

(iii) Photoreactivation of CPDs in basal keratinocytes pro-foundly alters the transcriptional response to UV in wholeskin. The above findings stress the importance of CPD lesions,

FIG. 2. Effect of photoreactivation of photoproducts from basal keratinocytes on UVB-induced acute responses. (A) Apoptotic response inphotolyase mice. Photolyase mice were exposed to 1 MED UVB, followed by exposure to PR light for 3 h or darkness. Apoptosis was measured40 h after UV exposure by a TUNEL assay. (B) Hyperplasia in photolyase mice. Mice were exposed to 1 MED UVB for four subsequent days,followed by exposure to PR light (3 h) or darkness. Four days after the last exposure, mice were sacrificed, and skin sections were stained withhematoxylin and eosin.


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rather than 6-4PPs, for a wide range of biological processes.Previously, we showed that genome-wide changes in transcrip-tion required the continuous presence of UV-induced CPDphotolesions in in vitro cultured transgenic CPD-PL mousedermal fibroblasts (MDFs) in a time-, dose-, and light-depen-dent manner (12). However, cultured cells are continuously un-der physiological stress, as they are in a state of continuous pro-liferation and have adapted to culture conditions. To assess thephysiological impact of persisting CPD photolesions in basal ke-ratinocytes on the transcriptional response, we used a functionalgenomics approach in K14-CPD-PL mice following exposure ofmouse skin to irradiation. Total RNA was isolated from wholeskin samples of UVB-irradiated (1 MED) K14-CPD-PL hairlessmice (n 3) either photoreactivated or not. Animals were sac-rificed 8 h post-UV exposure, a relevant time point that allowsthe expression differences between the photoreactivated and non-photoreactivated mice to evolve (12). In a dye-swap approach,differentially labeled cDNA prepared from RNA from either theUV-irradiated, PR light-treated skin or the UV-irradiated, non-light-treated skin was mixed and hybridized to cDNA microarraysrepresenting �15,000 mouse genes.

An unsupervised hierarchical clustering of the genes thatvaried significantly (P � 0.05 and �1.5-fold change) betweenthe UV-irradiated, PR- and non-PR-treated skin samples dem-onstrated the profound effect of PR (and by inference, ofremoval of CPDs from basal keratinocytes) on the transcrip-tional response to UVB in the skin. Of note, all samples clus-tered primarily into two main groups. This clustering was de-termined predominantly by PR status, suggesting that PR ofCPDs in basal keratinocytes comprises a dominant determi-nant of gene expression in the total skin (Fig. 3A).

To confirm the results of the hierarchical clustering, as wellas to reduce the dimensionality of the data set and improve thevisualization of meaningful variables between samples, we em-ployed a principle component analysis, as previously described(12). Herein, each sphere represents one sample that is posi-tioned in a reconstructed three-dimensional “gene space” sothat proximity between spheres demonstrates the degree ofsimilarity of expression profiles between samples (Fig. 3B). Inagreement with the previously demonstrated tree graph, allspheres representing the PR, non-PR, and untreated controlsamples segregated into distinct groups on the basis of their PRstatus, thus establishing the prime significance of PR and ourability to measure it on the transcriptional level using an in vivoexperimental model system.

(iv) The presence of a single type of UV-induced photole-sions (i.e., CPDs) in basal keratinocytes impinges on a widerange of biological processes in vivo. To avoid data preselec-tion and potential introduction of bias, we employed an unbi-ased approach to unveil the significantly overrepresented bio-logical processes and underlying networks employed by basalkeratinocytes to sustain key cellular functions upon UVB irra-diation and subsequent PR (or not). For this, all significantgenes responsible for clustering the PR and non-PR samplesinto separate groups were assembled according to the GOclassification system and integrated in the complete tree of“Physiological Processes (GO:0007582)” using a previously de-veloped three-dimensional interactive visualization system(http://www2.eur.nl/fgg/ch1/k14network/). Each illustrated GOterm was then scored for its relative overrepresentation by

examining separately the number of up- and downregulatedgenes with respect to the total population of genes printed onour cDNA platform for that GO term. Further insight wasgained by employing the commercially available Ingenuity da-tabase (www.ingenuity.com) to identify all the significantlytranscribed genes whose gene products participated in over-lapping and distinct networks. These networks were then ex-amined for their statistical significance and explored further bylisting the most significantly relevant functions associated withcombinations of genes that participated in each of these net-works (see “network analysis” in interactive visualizations athttp://www2.eur.nl/fgg/ch1/k14network/).

With exposure to 1 MED of UVB in the absence of PR, theresponse to oxidative stress and DNA damage along with theprotein and DNA metabolism and biosynthesis was promi-nently present among the cellular pathways that were signifi-cantly overrepresented (Fig. 3C) (a detailed list is available athttp://www2.eur.nl/fgg/ch1/k14network/). Subsequent analysisrevealed a number of gene networks involved in numerousvital cellular processes, ranging from cell growth and mainte-nance to immune responses. This suggests that the presence ofCPDs (as the most critical type of UV-induced photolesion) ina subset of keratinocytes is sufficient to trigger a broad spec-trum of transcriptional responses underlying a plethora of bi-ological processes in the mouse skin.

In keratinocytes, various genotoxic stresses that damage theDNA have previously been shown to result in the activation ofgenes involved in cell cycle checkpoints, leading to diversecellular responses including cell cycle arrest, repair of DNAdamage, and programmed cell death (25). Consistent with thetime frame (8 h post-UV) and dose employed in this study, anumber of genes associated with DNA repair (e.g., Rad511,Ubl12, Ddb11, and Fen11, where1 and2 indicate up- anddowregulation, respectively) were significantly modulatedupon the presence of CPDs in basal keratinocytes, suggestingthe essential role of these cells in provoking the DNA damageresponse in the UV-irradiated mouse skin. Interestingly, de-spite the fact that the majority of nondividing cells are ex-pected to mask this response in the mouse skin compared tothe cultured cells, the transcriptional regulation of the humanrecA homologue Rad51 is in agreement with our previous re-sults suggesting that the presence of unrepaired CPDs duringDNA replication may induce the formation of DNA breaksand/or homologous recombination (12).

Coupled to this response, a number of genes involved inhistone metabolism and chromatin modification were signifi-cantly upregulated in the absence of PR (e.g., Hmgn11,Hdac21, H1f01, Hira1, and Smarcb11), supporting previ-ous findings that showed human keratinocytes to induce theexpression of genes coding for specific histones and histonemodification proteins upon UVB irradiation (25). Interest-ingly, the latter could also be associated with an enhancedproliferative response for a subset of epidermal cells that couldeventually result in the epidermal hyperplasia, known to occurat later stages upon UVB irradiation.

Studies in cultured human keratinocytes in vitro (26, 34) andin the hairless murine skin in vivo (32, 37) have providedcompelling evidence that exposure of the skin to UV irradia-tion stimulates the skin’s enzymatic antioxidant defense sys-tem. In agreement, the presence of CPD lesions provoked the


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FIG. 3. Transcriptome analysis in K14-CPD-PL mice. (A) Tree graph representation of the similarity between significant gene expressionprofiles (analysis of variance, P � 0.05; �1.5-fold change) of irradiated non-PR (M1 to M3) and PR (M4 to M6) skin samples compared tonon-UV-irradiated control skin samples (C1 and C2). Note the clustering of all samples into two main groups correlating to exposure to PR (light)or not (dark) subsequent to UV irradiation. (B) Principle component analysis of irradiated PR and non-PR skin samples. Each sphere, coloredaccording to PR status, represents a skin tissue sample that is positioned in a reconstructed three-dimensional gene space so that proximity betweenspheres represents similarity between gene expression profiles of the corresponding matrix points. Note that mice are segregated according to theUV irradiation treatment and PR status. (C) Heat map representation of PR-dependent, significant gene expression profiles of genes involved instress-related responses including DNA damage repair, apoptosis, and oxidative stress. Changes in relative expression are represented by red(upregulated) and green (downregulated); black indicates no change compared to nonirradiated controls. (D) Verification of microarray data byquantitative real-time PCR. Relative changes in expression (n-fold) indicate the relative average expression levels of indicated genes in irradiatedK14-CPD-PL mouse skin (n 3) treated with PR (or not) compared to those from nonirradiated, non-PR-treated k14-CPD-PL mouse skinsamples.


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transcriptional upregulation of two major enzymatic antioxi-dants (e.g., Sod11 and Gpx31) known to confer cellular re-sistance to oxidative stress (34, 36), suggesting a tissue-specificantioxidant response to UVB-mediated phototoxicity medi-ated by CPD lesions. The PR-dependent nature of this re-sponse indicates that the presence of CPD lesions in basalkeratinocytes is sufficient for activation of this defense system.Of note, the significant overrepresentation of the response tooxidative stress was only part of the identified, broader re-sponse to stress, including genes associated with the responseto heat (e.g., Hspa81 and Hspcb1), inflammation, and stress-induced signaling (e.g., Nfatc41, Csf3r2, and Map2k32).

Even so, irreparable DNA damage, or insufficient repair intime, is expected to trigger the death of cells carrying excessDNA damage or in specific stages of differentiation throughthe activation of apoptotic responses. In this study, severalproapoptotic genes exhibited significant changes in expressionat 8 h post-UV treatment, though not unidirectionally, pre-sumably due to the heterogeneity in the response between cells(e.g., Elmo22, Ddx4L2, Pdcd51, Pdcd81, Pdcd112,Casp62, Trim351, Cfdp11, App11, Mcl11, and Tpt11).Since increased apoptosis was demonstrated in basal keratino-cytes 40 h after the exposure of the skin to UVB irradiation(Fig. 2), these data suggest the early presence of both pro- andantiapoptotic responses, whose final outcome may be largelydependent on the extent of DNA damage, the efficiency ofDNA repair itself, the stage of differentiation, and cell cyclephase.

Finally, in order for the cell to respond rapidly to growthconditions and insults induced by UVB irradiation, it needs tocover its metabolic needs through the tight regulation of sev-eral biosynthetic pathways. In agreement with this, the distin-guishable overrepresentation of metabolic genes (evidenced atvarious GO levels in this data set) was for the most partattributable to the upregulation of several ribosomal proteingenes (e.g., L31, L41, L51, L61, L101, S3a1, S41, S51,S71, and S81) along with genes involved in translationalelongation (e.g., Eef1g1, Eef21, Eef1a11, and Elf2s3y1),highlighting the role of ribosomal biogenesis and protein bio-synthesis in the economy of the cell.

Examining the mRNA levels of several genes associated withthe DNA damage response and other biological processes(Mcl1, Rad51, Sumo, Hpsb, Maged, Trp53, Gpx3, Ddb1, andSod1) in the skin of UV-irradiated, PR-treated (or not) K14-CPD-PL mice validated the accuracy of the microarray data(Fig. 3D).

Chronic effects upon exposure to UVB-light. (i)Removal ofCPDs from basal keratinocytes results in substantial protec-tion from skin carcinomas. CPD lesions are an importanttrigger for UV-induced skin cancer, as mice ubiquitously ex-pressing a CPD-PL transgene are protected substantially fromthe formation of these tumors, in strong contrast to mice ubiq-uitously expressing a 6-4PP-PL transgene (22). Whereas basalkeratinocytes are considered a crucial cell type involved in theformation of UV-induced skin tumors, it is still unknownwhether systemic effects may also play a significant role in skinneoplasia. To address directly whether distinct types of DNAlesions in keratinocytes specifically play an important role inskin carcinogenesis, we subjected hairless K14-CPD-PL andK14-6-4PP-PL animals to a single daily dose of UVB (1 MED),

immediately followed by exposure to PR light for 3 h, to spe-cifically remove either CPD or 6-4PPs from the basal kerati-nocytes, using hairless wt mice as a control. The fraction oftumor-free wt control animals declined shortly after the initi-ation of UV treatment (Fig. 4A). Correspondingly, the averagenumber of tumors (mostly squamous cell carcinomas and oc-casionally papillomas) per mouse increased considerably (Fig.4B). Preferential removal of 6-4PPs from basal keratinocytesdid not significantly reduce cancer incidence, while removal ofCPDs or both CPDs and 6-4PPs from basal keratinocytesyielded a very strong cancer protection (Kaplan-Meier analy-sis, P 0.01). Whereas by week 16 all 11 wt animals had oneor more tumors, by that time only 1/21 K14-CPD-PL or K14-CPD/6-4PP-PL transgenic mice had acquired the first tumor.From these data we conclude that apparently rapid removal ofCPDs, rather than 6-4PPs from basal keratinocytes only, issufficient to protect against carcinogenic events, thus providingcompelling evidence for both the relevance of basal keratino-cytes as well as of UV-induced CPD lesions in skin carcino-genesis.

(ii) Removal of CPDs from the total mouse skin, but notfrom basal keratinocytes alone, abrogates UV-mediated immu-nosuppression. Photocarcinogenesis does not merely dependon the induction of mutations; failing surveillance by the im-mune system plays a pivotal role in this process as well. It haslong been demonstrated that UV-induced skin tumors arehighly immunogenic and are immediately rejected when trans-

FIG. 4. Effect of photoreactivation of photoproducts from basalkeratinocytes on skin carcinomas. Photolyase mice (for K14-CPD-PL,n 10; K14-6-4PP-PL, n 9; K14-CPD/6-4PP-PL, n 11) and theirwt littermates (n 11) received daily UVB treatments (500 J/m2)followed by 3 h of PR light. (A) Kaplan-Meier plot showing thefraction of tumor-bearing mice in time after the first UV treatment.K14-CPD-PL and K14-CPD/6-4PP-PL animals remain tumor-freelonger than wt littermates (P 0.01). (B) The average number ofsquamous cell carcinomas per mouse in time after the first UV treat-ment.


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planted onto syngeneic mice, unless recipient animals are ex-posed to subcarcinogenic doses of UVB light prior to trans-plantation (9). Thus, UV radiation exerts immunosuppressiveeffects, allowing skin tumors to persist. Among the potentialchromophores that are thought to be responsible for eliciting asuppressive response upon UV are urocanic acid (UCA) andDNA. UCA is formed in the stratum corneum by deaminationof histidine. Upon absorption of UV light, the naturally occur-ring trans form isomerizes to into cis UCA that is known to actas a mediator of the UV-induced immunosuppression (14, 31).The dramatic skin cancer protection observed upon fast re-moval of CPDs may, therefore, be caused by both a reductionin the mutation load and an alteration of the immune response(22). Of note, this comprehensive set of transgenic photolyasemice now enables a thorough investigation of the role of DNAlesions in the UV-mediated immunosuppressive response.

First, we studied the systemic immune response in the ubiq-uitously expressing �-act-CPD-PL and �-act-6-4PP-PL mice.Mice were exposed to five daily UVB treatments, applied attwo spots on the shaved back. After every UV exposure, micewere exposed to PR light for 3 h or else kept in the dark. Fourdays after the last UVB exposure, all mice were sensitized byapplication of picryl chloride (PCl) on their shaved bellies.Four days later, the ear thickness was measured (time zero),and ears were challenged with PCl. Ear thickness was mea-sured again 24 h after the challenge, and the immune responseof each mouse was expressed as a percentage of ear swelling.To exclude that exposure to PR light might affect the immuneresponse, we first studied the suppression of the immune re-sponse in mice that did not express the photolyase transgene(Fig. 5A). As expected, exposure to UVB, but importantly notto PR light alone, resulted in a reduced immune response,confirming the immunosuppressive effects of UVB irradiationand the use of PR as a valid tool to study the contributionof DNA lesions to immunosuppression. Exposure of �-act-CPD-PL mice to UVB irradiation resulted in a clear suppres-sion of the immune system (Fig. 5B). Strikingly, ubiquitousphotolyase-mediated removal of CPDs abolished completelythe immunosuppressive effects of UV light, revealing a criticalrole for these lesions in the response. In contrast, exposingirradiated �-act-6-4PP-PL mice to PR light did not alter theimmunosuppression (Fig. 5C).

Keratinocytes are thought to play an important role in mod-ulation of the immune response by releasing various cytokinesupon exposure to UV. To examine whether the presence ofDNA damage in other cells than the basal keratinocytes playsa similarly critical role in induction of immune suppression asit does in acute sunburn, hyperplasia, and carcinogenesis, westudied the systemic immunosuppression upon UV exposure inK14 photolyase mice. Figure 6 shows that UV exposure of bothK14-6-4PP or K14-CPD PL mice resulted in suppression of thesystemic immune response, comparable to the suppression inwt mice. As such, K14-CPD PL mice remarkably contrast with�-act-CPD-PL mice, in which ubiquitous removal of CPDseliminates immunosuppression. These findings suggest that,whereas CPDs in basal keratinocytes comprise a major triggerfor UV-induced sunburn and carcinogenesis, DNA lesions(and in particular CPDs) in other cell types are primarilyresponsible for immunosuppression.

DISCUSSION

Photolyase transgenic mice. The generation of ubiquitouslyexpressing �-act-CPD and �-act-6-4PP photolyase transgenicmice, along with basal keratinocyte-specific K14-CPD photol-yase transgenic mice has been of great value to delineate therole of distinct DNA lesions in acute UV effects (i.e., sunburn)and skin cancer (22, 35). To further improve our knowledge onlesion and cell type specificity in relation to photocarcinogen-esis, we now have generated mice expressing the 6-4PP-pho-tolyase from the basal keratinocyte-specific promoter K14. Ex-posure of UV-irradiated K14-6-4PP-PL mice to visible lightconfirmed the functionality and specificity of the transgene, as

FIG. 5. Systemic immunosuppression in wt and �-act-photolyasemice. wt (A), �-act-CPD-PL (B), and �-act-6-4PP-PL (C) mice wereirradiated with 0.5 MED UVB for five consecutive days. The micewere skin-sensitized 4 days after the last UV exposure by topicalapplication of PCl to nonirradiated shaved abdomen, chest, and feet.Four days after sensitization, both ears of the mice were challengedwith 0.8% PCl in olive oil. At 24 h after the challenge, duplicate earmeasures were performed with an engineer’s micrometer. Thirty-fouranimals per genotype were used. Error bars indicate the standarderrors of the means.


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it allowed efficient repair of the 6-4 PP lesions in the basalkeratinocytes only, whereas such lesions persisted in all othercell types (Fig. 1D).

Role of basal keratinocytes in apoptosis and hyperplasia.Using the K14-6-4PP-PL mouse model, we have shown thatremoval of 6-4PPs from basal keratinocytes had no effect oneither apoptosis (sunburn) or hyperplasia, consistent with ourprevious findings with ubiquitously expressing 6-4PP-PL trans-genic mice (21). In contrast, removal of CPDs from basalkeratinocytes only substantially decreased the incidence of celldeath (33) (Fig. 2A), as well as the proliferative responseresulting in hyperplasia (this study). A similar observation wasmade after ubiquitous removal of CPDs from the mouse skin.Taken together, these findings indicate a central role of basalkeratinocytes and CPD lesions in the onset of these UV-in-duced acute skin effects. These findings also corroborate theobservation that in rodent cells CPDs—in contrast to6-4PPs—are hardly recognized and virtually not repaired byGG-NER. Thus, whereas an additional mechanism for therepair of 6-4PPs does not significantly improve their UVresistance, providing mice with the CPD-photolyase enzymeproves beneficial.

Photoreactivation-dependent transcriptional responses ofthe UVB-exposed K14-CPD-PL mouse skin. Using culturedMDFs, PR was previously shown to impact, at the transcrip-

tional level, biologically significant cellular UV-induced re-sponses (12) and, thus, was expected to affect gene expressionconsiderably in our in vivo experimental model system. Impor-tantly, hierarchical clustering as well as principal componentanalysis grouped the significantly transcribed genes primarilyby PR status (Fig. 3A and B), thereby establishing CPDs inbasal keratinocytes as a major determinant of gene expressionchanges in the UV-exposed mouse skin. The fact that thetranscriptional response to CPDs is time dependent, with dif-ferent kinetics depending on the UV dose, thoroughly explainswhy, in this study, the PR samples were still remote from thecontrols in the three-dimensional space at this relatively earlytime point. In this light, the fact that the systemic immunosup-pression is maintained in PR UV-treated CPD-PL mice, and assuch dissociates from the acute inflammation and carcinogen-esis, is highly interesting.

Exposure of the K14-CPD-PL mouse skin to UV revealedseveral PR-dependent, and thus CPD-provoked biological pro-cesses (Fig. 3C). In cultured cells, DNA photoproducts canstall replication, thereby generating a more toxic lesion, thedouble-strand break (12). In this study, we identified genesinvolved in DNA recombination and the repair of DNA breaks(e.g., Rad51 and Ubl1), which suggests that, rather than CPDsthemselves, CPD-dependent replication products (e.g., stalledreplication forks and DNA breaks) in basal keratinocytes arelikely to contribute to UV effects in the skin, which is in perfectagreement with our in vitro data (12). There has been a long-standing question whether damage to DNA can affect nucleo-somal stability, thereby altering lesion accessibility (40). Forinstance, exposure of human epidermal keratinocytes to UVBinduces the expression of genes involved in histone synthesis(6). We now have shown that CPDs are a major determinant inupregulating the expression of genes involved in the modifica-tion and maintenance of nucleosomal structures. Additionally,the upregulation of genes associated with ribosomal biosynthe-sis, translational elongation, and histone synthesis could allreflect the onset of a proliferative response, underlying theobserved hyperplasia and/or counteracting cell loss due to apop-tosis. None of these genes was upregulated upon photoreacti-vation of CPDs in the mouse skin, signifying the autonomousand predominant nature of the basal UV-induced cell responseto CPDs. Both the proapoptotic response at the transcriptionallevel and the subsequent induction of cell death (40 h after UVirradiation) in irradiated non-PR mouse skin underscore thedestructive potential of CPDs (or CPD-dependent replicationproducts), revealing that their sole presence in basal keratino-cytes suffices to trigger apoptosis. In addition to the data pre-sented here, a number of additional genes were shown to dem-onstrate PR-dependent expression. These annotated data sets areavailable for interactive querying and hypothesis-driven analysesat http://www2.eur.nl/fgg/ch1/k14network/.

Role of basal keratinocytes in skin cancer. Previously, wehave shown that ubiquitous removal of CPDs from the skinprovides a high level of protection from cancer initiation,pointing to this lesion as the major trigger for photocarcino-genesis (21) To examine whether the presence of UV-inducedphotolesions in basal keratinocytes constitutes the primarycause of skin cancer, we investigated the incidence of skintumors in K14-CPD and K14-6-4PP photolyase mice uponexposure to UVB and subsequent specific removal of lesions

FIG. 6. Systemic immunosuppression in K14-photolyase mice.K14-CPD-PL (A) and K14-6-4PP-PL (B) mice were irradiated withUVB for five consecutive days. The mice were skin sensitized 4 daysafter the last UV exposure by topical application of PCl to the nonir-radiated shaved abdomen, chest, and feet. Four days after sensitiza-tion, both ears of the mice were challenged with 0.8% PCl in olive oil.At 24 h after the challenge, duplicate ear measures were performedwith an engineer’s micrometer. Thirty-four animals per genotype wereused. Error bars indicate the standard errors of the means.


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by PR. Strikingly, fast removal of CPDs from basal keratino-cytes dramatically decreased the incidence of skin cancer inK14-CPD-PL mice (Fig. 4B) to a level similar to that observedwhen CPDs were ubiquitously removed from the skin. Thisfinding points to basal keratinocytes as the primary cell type forthe tumorigenesis observed in the irradiated mouse skin,thereby limiting the impact of systemic mechanisms. Evenmore, since PR in keratinocytes can prevent apoptosis and kera-tinocyte-associated carcinogenesis, an interesting follow-up ex-periment would be to expose K14-CPD-PL/K14-6-4PP-PL dou-ble transgenic mice to the maximum tolerable dose of UV (whichin the absence of photoreactivation likely would be lethal) andfind out whether melanomas develop.

DNA damage and immunosuppression. The relevance ofUV-mediated immunosuppressive effects in carcinogenesiswas first demonstrated by Fisher and Kripke, who showed thattransplantation of UV-induced skin tumors to syngeneic micecan result in tumor rejection (9). If, however, recipient micehad received subcarcinogenic doses of UV light, rejection didnot take place, indicating that UV light allows persistence ofskin tumors by suppressing the immune system. However, themechanism underlying this process is not yet clear. Both UCAand DNA damage can act as chromophores responsible forimmunosuppression (24, 31, 41). Importantly, treating micewith HindIII-containing liposomes was previously shown toinduce immune suppression (30). These data in combinationwith the earlier data published by Kripke and coworkers (24)indicate that DNA damage, either as double-strand breaks(HindIII) or pyrimidine dimer formation, is sufficient to acti-vate immune suppression. Utilizing the ubiquitously expressing�-act-photolyase mice, it is now possible to investigate to whatextent specific UV-induced DNA lesions affect the immunesystem by removing either CPDs or 6-4PPs in a light-depen-dent manner. In contrast to 6-4PPs, ubiquitous removal ofCPDs abolished UV-induced immunosuppression, suggestingthat photoisomerization of trans UCA alone is not sufficient toinduce the suppressive effects; CPD lesions are a prerequisite.In contrast to CPDs, repair of 6-4PPs by NER is fast andadequate, providing mice with a system that further enhancesremoval of 6-4PPs and does not significantly affect the biolog-ical outcome. However, providing mice with CPD-photolyase,thereby allowing fast removal of the most abundant UV lesion,does affect the biological outcome and appears beneficial tothe animal. Thus, these results confirm and expand on theearlier findings (24, 30) suggesting that DNA damage (strandbreaks, pyrimidine dimers, or 6-4 photoproducts) induces im-mune suppression but that only pyrimidine dimer formation isinvolved in photocarcinogenesis.

To investigate to what extent 6-4PPs are capable of elicitingimmunosuppressive effects, one could breed photolyase micewith available NER-deficient mice. For instance, completeNER abrogation, (e.g., Xpa knockout mice), strongly increasesthe susceptibility to UV-induced systemic immunosuppression(13, 29). However, whereas in mice lacking only TC-NER (e.g.,Csb mutant mice) or GG-NER (e.g., XPC mice) systemic im-munosuppression is substantially reduced, local immunosup-pression correlates with local Langerhans cell depletion andoccurs at lower UV doses in TC-NER-deficient mice (Csb andXpa mice) (23). In contrast, the UV dose required to suppressthe immune system in XPC mice is similar to the dose required

in wt mice. Thus, TC-NER deficiency likely enhances localimmunosuppression. Furthermore, only a total NER defectresults in increased systemic immunosuppression. Crossing ourphotolyase transgenic mice to NER-deficient mice will eluci-date the role of 6-4PPs in this process and indicate whethertheir presence on transcribed versus nontranscribed regionsleads to differential effects.

Role of basal keratinocytes in immunosuppression. Next, wesought to examine the relevant cell types involved in the im-mune suppression induced by UV. For example, Langerhanscells migrate towards the draining lymph nodes in irradiatedskin, shifting the Th1/Th2 balance towards a Th2 response(41). Using the K14-driven photolyase mice, it was possible toremove DNA lesions from basal keratinocytes, whereas dam-age remained in, e.g., Langerhans cells and differentiated ke-ratinocytes. Removal of DNA lesions from basal keratinocytesdid not abolish systemic immunosuppression, suggesting thatDNA lesions in other cell types are sufficient to induce thisresponse. This discrepancy between systemic immune suppres-sion and the acute UV effects related to DNA damage in basalkeratinocytes (i.e., sunburn and hyperplasia) is in line withprevious findings in Csb mice (12). These mice demonstrateUV sensitivity with acute epithelial effects, yet their systemicimmune suppression profile is comparable to that of wt mice.To unravel the cell type(s) responsible for UV-induced immu-nosuppression, transgenic mice have been generated in whichthe photolyase transgene is expressed specifically in other skincell types, e.g., Langerhans cells.

ACKNOWLEDGMENTS

We thank O. Nikaido for providing us with the TDM2 and 64M2antibodies. Also, we thank Jun-Ichi Miyazaki (Osaka University Med-ical School, Osaka, Japan) for providing us with the pCY4B vector andJ. Garssen for the supply of picryl chloride.

This work was supported by the Dutch Cancer Foundation (EUR98-1774 and EMCR 2002-2701), the Interuniversitary Research Insti-tute for Radiopathology and Radioprotection (IRS grant 7.3.5), theAssociation for International Cancer Research (AICR 98-259 andAICR 03-128), and the Japanese Ministry of Education, Science andCulture (MONBUSHO grant 10044231).

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Differential Role of Basal Keratinocytes in UV-Induced Immunosuppression and Skin Cancer