Relationship between UV-induced mutant p53 patches and skin tumours, analysed by mutation spectra and by induction kinetics in various DNA-repair-deficient mice

Heggert Rebel, Nicolien Kram 1, Anja Westerman 1, Sander Banus 2, Henk J. van Kranen 1 and Frank R. de Gruijl *

Department of Dermatology, Leiden University Medical Centre, Sylvius Laboratory, PO Box 9503, 2300 RA, Leiden, The Netherlands, 1 Laboratory of Toxicology, Pathology and Genetics and 2 Laboratory of Vaccine-Preventable Diseases, National Institute of Public of Health Effects Research, PO Box 1, 3720 BA Bilthoven, The Netherlands

* To whom correspondence should be addressed. Tel: +31 71 5271902; Fax: +31 71 5271910; Email: F.R.de_Gruijl{at}lumc.nl


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Clusters of p53 immunopositive epidermal keratinocytes (so-called p53 patches, clones or foci) are found in sun or ultraviolet (UV) light-exposed skin. We investigated to what extent these p53 patches are genuine precursors of skin carcinomas in chronically irradiated hairless (SKH1) mice. The mutation spectra of exons 5–8 of the p53 gene of laser-micro-dissected mutant p53 patches and carcinomas were therefore compared. The mutations we found were mainly UV-signature mutations (C->T and CC->TT at dipyrimidine sites) located at known hotspots. No significant differences were found between both spectra, indicating that all p53 patches harbour mutations with which they could progress to carcinomas. To examine whether these p53 patches can be used as tumour risk indicators, we made an extensive comparison of the induction kinetics of these patches and carcinomas in genetically modified mice with various defects in nucleotide excision repair (NER), i.e. xeroderma pigmentosum A (Xpa), Xpc and Cockayne syndrome B (Csb) and wild-type mice. In this aforementioned order, the mouse strains developed both p53 patches and carcinomas in the course of daily exposure to 40 J/m2 UV. Hence, the order in which the NER-deficient mice developed patches was predictive of the order in which they developed tumours. The induction kinetics of the patches in Xpc-deficient mice differed notably from the others: there was a stationary phase (days 13–41) where the numbers were limited to 5–10 patches per mouse before an explosive increase which ran parallel to the other groups. The chance that a p53 patch progresses to carcinoma is relatively small (estimated at 1 out of 8300–40 000/individual when the first tumour appears), but our results are strongly indicative of a causal relationship between p53 patches and carcinomas.

Abbreviations: AK, actinic keratoses; BCC, basal cell carcinoma; CPD, cyclobutane pyrimidine dimers; PBS, phosphate-buffered saline; SCC, squamous cell carcinoma


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The most frequently diagnosed type of cancer in the fair-skinned Caucasian populations is non-melanoma skin cancer (NMSC): ~62 700 new registered cases in 2001 in the UK (www.cancerresearchuk.org). The main categories are basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), estimated to have ~0.05 and 0.7% case-fatality rates, respectively (1). Although the mortality rates decreased, the incidence of NMSCs increased rapidly during the last decades resulting in a growing public health burden (2,3).

UV radiation, either solar or artificial, exerts genotoxic stress that can cause SCC in the skin. The shortest wavelengths in the solar spectrum that reach the earth surface are in the UVB band (280–315 nm). UVB radiation induces a variety of damage in DNA, mainly cyclobutane pyrimidine dimers (CPDs) and (6,4)photoproducts (6-4PPs). If not adequately removed by the DNA-repair machinery, this damage gives rise to characteristic so-called ‘UV-signature mutations’: C->T and CC->TT transitions at dipyrimidine sites (4). These mutations are found, among others, in the tumour suppressor gene p53 in the majority of SCCs (54–90%), and their precursor lesions actinic keratoses (AKs) (60–70%) (58). Certain mutation hotspot regions are found in p53: in codons 173–179, 235–250 and 273–278 in humans (5,911) and codons 146, 173, 267 and 272 in mice (8) (murine codon numbering according to the latter reference; in other references often numbered +3).

DNA-repair-deficiency in XP patients results in persisting levels of UV lesions and accelerated cancer incidence. Xpa-deficient hairless mice, devoid of nucleotide excision repair (NER), develop UVB-induced carcinomas 4x faster than their wild-type littermates (12), as computed from the ratio of the median tumour induction times (t50s), 78 and 331 days, respectively. Xpc-(global genome repair) deficient mice and Csb-(transcription-coupled repair)-deficient mice develop their carcinomas three and two times faster, respectively (13). DNA lesions (CPDs and 6-4PPs) in non-transcribed strands appear to be a dominant determinant of susceptibility to UV-induced SCCs.

Carcinogenesis is a multi-step process in which an initial mutation is followed by additional genetic alterations eventually leading to growth deregulation and ultimately metastasis. In addition to early p53 point mutations, AK and SCCs show loss of heterozygosity (LOH) in the p53 gene. Progression of AK to SCC correlates with LOH in the P16INK4A gene (14) and pronounced decrease of retinoid receptor expression (15). In invasive SCCs P16INK4A and P14ARF are commonly inactivated (16). In human skin up to 46% (11/24) of the SCCs were found to carry mutations in H-RAS in codon 12 (17). In mouse models ras mutations were only detected at low incidence (20% of the skin tumours) in C3H mice (18).

Immunohistochemically detectable clusters of epidermal cells with accumulated nuclear p53 protein (‘p53 patches’, ‘p53 clones’ or ‘p53 foci’) are found in UV-irradiated skin long before tumours arise (19,20). Using laser micro-dissection and mutation analysis on single cells, p53 patches were proven to be clonal outgrowths of a keratinocyte, and therefore can be called p53 clones (21). In p53 patches UV-signature hotspot mutations are found that are also present in SCCs (20,22). The number of p53 patches increases with age in humans (23,24) and a higher incidence is found in skin adjacent to SCCs than in skin adjacent to BCCs (24). The body of data indicates that the induction of p53 patches represents a step prior to the development of AK and subsequent SCC. This is further confirmed in hairless mouse experiments with well-controlled UV dosages where mutant p53 patches were found to display the same dose-time dependency as SCCs (25). Moreover, increased densities of p53 patches in XPC patients (26) and in UV-exposed Xpa-deficient mice (25) provide further evidence for a causal relationship between p53 patches and SCCs, and therefore the density of mutant p53 patches in the skin may be an indicator of SCC risk.

On the other hand, some results do not seem to confirm such a direct relationship between the frequency of p53 patches and the incidence of SCCs. First of all, the mutant p53 patches can be numerous, while the incidence of SCCs is low: ~10 000 mutant p53 patches/mouse are expected at the median latency time of a first SCC in a SKH1 hairless mouse (25). This indicates that most mutant p53 patches do not progress to SCC, at least not during the lifetime of a mouse. Secondly, in a French study (27) no significantly higher p53-patch densities were found in patients with multiple carcinomas compared with age-matched patients with one carcinoma.

The central question that arises is whether (all) p53 patches are true precursors of SCCs. Here we divide this question into two sub-questions:

  1. Do all p53 patches harbour potential tumorigenic mutations or is there a selection towards particular mutations during carcinoma development?
    The majority of the p53 mutations in SCCs can be found in hotspots at codons 146, 173, 267 and 272 in SKH1 mice (8). If there were to be a mutation-based selection towards certain hotspots, one would expect to find a wider variety of mutations in the p53 patches. Therefore, we sequenced the p53 gene of UVB-induced SCCs and p53 patches in hairless SKH1 mice and compared the two mutation spectra.
  2. Do the induction kinetics of p53 patches consistently match those of carcinomas in various tumour-prone DNA-repair-deficient backgrounds?

Deficiency in DNA repair results in persisting UV-induced lesions and accelerated cancer prevalence. In agreement with this, we previously found accelerated p53-patch induction in chronic UVB-irradiated Xpa-KO mice (25). In the present expanded study, we systematically examined whether the induction times of UV-induced p53 patches in three different DNA-repair-deficient mouse strains (Xpa, Xpc and Csb-KO) correspond with the subsequent tumour induction times.


    Materials and methods
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 Abstract
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 Materials and methods
 Results
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Animals
Hairless mice (Crl:SKH1-HR, Charles River, Maastricht, The Netherlands) were entered into the experiments at ages ranging from 6 to 9 weeks and were housed individually in standard type I Macrolon cages. Standard mice chow (Hope Farms RMB-H) and tap water were available ad libitum. As legally required, approval for the experiments was obtained from the University's ethics commission on animal experiments.

Hairless Xpa-deficient mice (28) were kindly provided by Dr H.van Steeg, Bilthoven, The Netherlands. The Xpc-deficient mice (a kind gift of Dr E.C.Friedberg, Dallas, TX) and Csb-deficient mice (a kind gift of Dr G.T.J.van der Horst, Rotterdam, The Netherlands) used in this study have been described in detail previously (29,30). In short, Xpa-deficient mice were generated by replacing exons 3 and 4 of the Xpa gene with a neo marker cassette; Xpc-deficient mice were generated by deletion of exon 10 of the Xpc gene, and in Csb-deficient mice a premature stop codon (mimicking a mutation in a CSB patient) was introduced in exon 5 of the Csb gene. The strains were generated in Ola-129/C57BL/6 background, C57BL/6 background, and Ola-129/C57BL/6/FVB background, respectively. Hairless Xpa-, Xpc- and Csb-deficient mice were generated by cross breeding Xpa-, Xpc- and Csb-deficient mice with hairless SKH1 mice for 7, 3 and 3 generations, respectively. The progeny was genotyped by PCR assays on tail DNA. Of each strain both male and female mice were used in a 1:1 ratio in all the experiments, and responses of the homozygous knockouts were compared with those of their heterozygous and wild-type littermates.

UV irradiation
Hairless SKH1 mice were chronically irradiated with a daily dose of 40, 250 or 500 J/m2 UV from Philips TL-12/40W fluorescent tubes [54% output in UVB (280–315 nm) and 46% in UVA (315–400 nm)] in automatic time switched irradiation cabins (31). The dosages corresponded to 0.08, 0.5 and 1.0 MED in wild-type hairless mice, respectively, where an MED is the minimal erythemal/oedemal dose.

For mutation analysis of p53 patches, a group of eight SKH1 mice was irradiated with 250 J/m2/day for 59 and 75 days. Within 24 h after the last UV irradiation, four mice per time point were killed and epidermal sheets were isolated for immunohistochemistry, and subsequent laser micro-dissection and mutation analysis.

Hairless Xpa-, Xpc- and Csb-deficient mice and their proficient littermates were irradiated in a chronic experiment at low daily UV dosages of 0.08 MED (40 J/m2 from TL-12 lamps). The reason for low dosages is the fact that Xpa and Csb-deficient mice are very sensitive to UV, both having a roughly 10 times lower MED than their wild-type littermates (12,13). After several time points from 10 to 188 days four mice of the three different strains were killed by CO2 asphyxiation and epidermal sheets were isolated for immunohistochemistry.

Four hairless Xpc-deficient mice and four of their wild-type littermates were also irradiated at high exposure levels of 500 J/m2 UV from TL-12 lamps/day corresponding to 1.0 MED of these animals (32). After 15 days of irradiation the mice were killed by CO2 asphyxiation and epidermal sheets were isolated for immunohistochemistry.

For the induction of SCCs in control mice 15 wild-type SKH1 mice received 40 J/m2/day. When tumours reached >4 mm diameters, animals were killed. The dissected tumours were split in two: one part was snap frozen in liquid nitrogen for p53-mutation analysis, while the other part was fixed in 4% buffered formaldehyde at 4°C for 24 h for immunohistochemistry.

The UV induction of SCCs in Xpa, Xpc and Csb-deficient mice has been studied before (12,13), where 0.08 MED from F40 sunlamps (80 J/m2) was delivered to the animals. The formerly produced F40 lamps are similar to TL-12 lamps, but two times less efficient in biological effects like oedema, erythema and carcinogenesis due to a slight shift in the spectrum towards longer wavelengths (25). In the present study additional UV-carcinogenesis experiments were performed with 14 hairless Xpc-deficient mice and 13 of their wild-type and heterozygous littermates that were daily exposed to 1000 J/m2 UV from F40 lamps (1 MED for wild type and Xpc–/– mice), and 12 hairless Xpc-deficient and 12 wild-type littermates that were exposed to 32 J/m2 from F40 lamps. Weekly, the diameters and numbers of skin tumours were scored on standard forms. When the tumours reached diameters >4 mm, the mice were killed.

Mutant p53 (Pab240) immunostaining in epidermal sheets
Within 24 h after the last irradiation mice were killed and pieces of dorsal skin of 21 x 34 mm were excised and incubated overnight at 4°C floating on phosphate-buffered saline (PBS) containing 200 µg/ml thermolysin (P-1512; Sigma Chemical, St Louis, MO) and 2 mM CaCl2, pH 7.8. Epidermal sheets were obtained by rolling the complete epidermis with the corneal site onto a polystyrene tube while holding and detaching the dermis with forceps. Epidermal sheets were spread floating on PBS in a petri dish, and subsequently fixed in PBS buffered 4% formaldehyde solution (Merck, 1.04003, 37%, Z.A., Darmstadt, Germany), for 10 min at room temperature. After a brief PBS wash, antigen retrieval was performed by 5 min boiling in 10 mM citrate buffer (pH 6.0). The epidermal sheets were washed in 4 ml polystyrene tubes filled with PBS. Subsequently endogenous peroxidase was blocked in methanol, containing 1.5% H2O2, during 20 min incubation in an end-to-end rotor. Sheets were washed three times by 5 min incubation in PBS, containing 0.5% Tween-20 (polyoxyethylene sorbitan monolaurate, Sigma, P-1379, St Louis, MO). Non-specific binding was blocked with 5% normal rabbit serum and 0.2% BSA in PBS to which 0.1% saponine was added for permeabilization.

The mutant-specific anti-p53 antibody Pab240 (Novocastra, NCL-p53-240, Newcastle, UK) (19) was diluted 1:25 in PBS, containing 5% normal rabbit serum, 0.2% BSA and 0.1% saponine, in which the epidermal sheets were incubated overnight at 4°C. Unbound antibody was removed in a triple wash of 5 min in PBS/Tween 0.5%. The sheets were incubated for 1 h, at room temperature in the 1:50 diluted secondary antibody, rabbit anti mouse (IgG1)-biotin (Zymed, 61-0140, San Francisco, CA), in PBS containing 0.2% BSA and 0.1% saponine. Excess of this antibody was removed in a triple wash of 5 min in PBS. The sheets were then incubated for 45 min in avidine–biotin-peroxidase complex (DakoCytomation, ABC complex, K0355, Copenhagen, Denmark). After a triple wash of 5 min in PBS, the sheets were stained for 5 min in 50 ml substrate solution, containing 40 mg 3,3'-diaminobenzidine (DAB, Sigma, D-5905, St Louis, MO) and 100 µl 30% H2O2. Peroxidase reaction was stopped by a triple wash of 5 min in PBS. The sheets were mounted basal side up, in paragon (7.0% gelatine and 50% glycerol in distilled water).

A grid, placed on top of each epidermal sheet preparation, was used to score the number of p53 clones in 20 squares (total area 29.0 x 18.5 mm), using a light microscope (Pl x25/0.5 objective).

Staining of tumour sections
The tumour parts fixed in 4% buffered formaldehyde were subsequently sectioned in paraffin and stained with routine haematoxylin–eosin staining. Independent analysis on coded slides was performed by a veterinary pathologist, and histopathologically confirmed SCCs were used for mutation analysis.

Mutant p53 (Pab240) staining was performed on paraffin slides of SCCs according to the protocol for epidermal sheets as described above. Additionally the sections were counter stained with haematoxylin and mounted with Depex.

Laser micro-dissection
For micro-dissection eight preparations of epidermal sheets stained for Pab240-positive patches were incubated in 60°C distilled water, allowing the mounting medium (paragon) to melt to release the sheets. After three washes with distilled water, the free epidermal sheets were placed on object glasses. With a laser dissection microscope (P.A.L.M. GmbH, Bernried, Germany) equipped with a x40/1.3 NA, individual p53 patches of ~100–1000 cells were micro-dissected and shot with a laser pulse into a sterile tube for DNA isolation.

Detection of p53 gene mutations
For mutation analysis, DNA was isolated from protein kinase C digested frozen SCC parts and from micro-dissected p53 clones, using QIAamp spin columns (Qiagen Gmbd Hilden, Germany) according to the manufacturer's protocol. This DNA was amplified by PCR on exons 5–8 of the p53 gene and then subjected to a pre-screening with degenerated gel electrophoresis (DGGE). DNA samples of tumours with aberrant DGGE bands were subsequently sequenced and analysed for mutations. The methods and primers applied are essentially as described in Ref. (33). Instead of M13-extended primers, unextended primers and fluorescent dideoxy-nucleotide triphosphates from the Thermo Sequenase II Dye Terminator Cycle Sequencing kit (Amersham Biosciences, Buckinghamshire, UK) were used, according to the manufacturer's protocol.


    Results
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 Materials and methods
 Results
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 References
 
P53-mutation spectra of p53 patches and SCCs
P53 patches were induced in a group of eight hairless SKH1 mice during chronic irradiation with a daily dose of 0.5 MED UV (250 J/m2). After 59 or 75 days of irradiation, four mice were killed and the dorsal skins were used to prepare epidermal sheets. The sheets were stained with mutant-conformation-specific p53 antibody Pab240 (19). Clusters of cells with intense nuclear staining, so-called mutant p53 patches, p53 clones or p53 foci, were microscopically visible in all sheets. The epidermal sheets were with complete hair follicles attached, and the mutant p53 patches were exclusively found in the interfollicular epidermis. In epidermal sheets of hairless SKH1 mice that were irradiated for 59 and 75 days, we found a mean number of 508 [standard error of the mean (SEM): 107] and 774 (SEM: 25) clusters of ≥10 p53-positive cells, respectively.

A randomly selected subset of 40 of these distinct p53 patches was micro-dissected and from 26 the DNA was isolated successfully. Sequencing exons 5–8 of the p53 gene (summarized in Table I) showed that 17/19 (89%) of the mutations were associated with dipyrimidine target sites, and most of those (15/19 = 79%) were C->T and CC->TT transitions, characteristic of UVB. We found these mutations in 14/26 (=54%) of the mutant p53 patches (mutation frequencies are summarized in Table II); five mutant p53 patches contained double mutations. The dominant C->T hotspot mutation in codon 267 was found in 8/14 (=57%) of the p53 patches. UV-signature mutations at dipyrimidine sites were also found in the known hotspots codons 172 and 272; but not in codon 146.


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Table I. p53 mutation spectra of SCCs versus mutant p53 patches in SKH1 mice

 

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Table II. Numbers of p53 mutations in SCC versus p53 patches

 
For comparison, SCCs were taken from wild-type control mice in the experiment dealt with in the next section. Histopathologically diagnosed SCCs were stained with mutant-p53 antibody Pab240 and from the 19 Pab240-positive SCCs the DNA was isolated. Exons 5–8 of the p53 gene were sequenced and C->T transitions at dipyrimidine sites were found in 15 of the 19 SCCs (=79%), see Tables I and II. Of these mutations 8 (=53%) were found in codon 267, and 2 (=13%) in codon 272. The percentages at these two hotspots did not significantly differ ({chi}2-test) from the percentages found in the p53 patches (57% in codon 267 and 7% in codon 272).

Sensitivity to p53 patch induction corresponds with tumour risk in DNA-repair-deficient mice
We compared three DNA-repair-deficient strains and their wild-type littermates on their sensitivity to induction of p53 patches. Xpa and Csb-deficient mice are very sensitive to UVB exposure and have an MED which is only 10% of that of wild-type mice. To be able to compare Xpa, Csb, Xpc-deficient and wild-type mice all strains were therefore irradiated with sub-acute low doses (0.08 wild-type MED/day, 40 J/m2 UV from TL-12 lamps). During the first days of irradiation some mild acute effects like oedema and erythema appeared only in the dorsal skin of Xpa and Csb-deficient mice. After the first week of chronic irradiation, the acute effects disappeared. After several time intervals, dorsal skin was isolated and epidermal sheets (size 21 x 34 mm) were immunostained with mutant-specific anti-p53 antibodies. The average numbers of p53 patches over the entire sheets of four animals were scored and plotted against time, see Figure 1. The p53 patches appeared first in Xpa–/– and Xpc–/– mice, already at day 13. High numbers (257, 95% CI, 186–355) of p53 patches were found in Xpa–/– mice at 27 days while the numbers of p53 patches in Xpc–/– mice remained on average smaller than 10 during the initial period (up to day 41). After this period a strong increase was seen in the Xpc–/– mice, reaching 436 patches (95% CI, 279–681) at day 62. In Csb–/– mice the first p53 patches were found at day 30, while high numbers were found after day 60, reaching 233 patches (95% CI, 164–333) at day 69. At several time points significant differences were found between the DNA-repair-deficient strains at 27, 30 and 48 days (P-value ranging from 0.041 to <0.001, Student's t-test on log values).



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Fig. 1. Median numbers of mutant p53 patches in Xpa (open circles), Xpc (open diamonds) and Csb-deficient hairless mice (open triangles) and wild-type littermates (in solid symbols) (n = 4) at 0.08 of a wild-type MED/day.

 
Patches of p53 were found in the DNA-repair-deficient mice long before patches could be detected in wild-type mice. The first p53 patches in all heterozygous NER-deficient and homozygous wild-type mice at this low dose of 0.08 MED were found only at day 118, while high numbers were found at 180 days (116 patches, 95% CI 50–273). No significant differences were found between the wild-type littermates of the three different mouse strains.

In the following order: Xpa–/– mice, Xpc–/– mice and Csb–/– mice, developed p53 patches well before wild-type mice, and this order corresponds to the order in which the mice develop SCCs (12,13).

Elevated numbers of p53 patches in hairless Xpc-deficient mice at 1 MED/day
Since Xpc-deficient mice have an MED comparable with wild-type animals, we examined whether at this much higher level of photoproducts the p53 patch density would still be indicative of the increased tumour growth rate. Therefore, we scored the numbers of Pab240-positive patches in Xpc-deficient mice and compared them with their homozygous and heterozygous wild-type littermates after 15 days of chronic UV irradiation at 1 MED (500 J/m2 UV from TL-12 lamps)/day. The numbers of p53 patches in Xpc+/+ and +/– mice did not significantly differ and averaged 7.3 ± 2.8 (SEM) while in Xpc–/– mice 107 ± 11 (SEM) patches were found (see Figure 2), i.e. a 14-fold elevation. The yield of 107 p53 patches found at 15 days at 1 MED/day in Xpc–/– is comparable with that in wild-type SKH1 mice at 23 days [data obtained from Ref. (25)], and is substantially (also 14-fold) higher than that observed with 15 days of 0.08 MED/day in Xpc-deficient mice (see Figure 1).



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Fig. 2. Average numbers of mutant p53 patches in Xpc-deficient mice (open diamonds) and wild-type littermates (closed diamonds) (n = 4) at 1.0 of a wild-type MED/day. Wild-type SKH1 data at 1.0 MED (closed circles and regression line) are derived from (25).

 
Elevated numbers of tumours in hairless Xpc-deficient mice
Previously, we reported a 2.8 times accelerated UV-induced carcinogenesis in Xpc-deficient mice at low dose (0.08 wild-type MED/day) chronic UV irradiations (13). To be able to compare our p53 patch results at 1 MED/day with tumour data of Xpc-deficient mice, we subjected hairless Xpc-deficient mice and their wild-type littermates to a UV-carcinogenesis experiment at 1 MED (1000 J/m2 UV from F40 lamps)/day. At this dose, Xpc–/– mice were highly significantly tumour prone (P < 0.0001, Mann–Whitney U-test, see Figure 3), and showed a 1.4 times acceleration of UV carcinogenesis (computed as the ratio of the median induction times of tumours ≥1 mm in controls and Xpc-deficient mice, 102 and 75 days, respectively). No significant difference was found between Xpc+/– and Xpc+/+ mice.



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Fig. 3. Kaplan–Mayer plots of UV-induced carcinogenesis (SCCs ≥ 1 mm) in Xpc-deficient mice (open diamonds) and wild-type littermates (closed diamonds).

 
In agreement with this accelerated carcinogenesis at 1 MED UV/day, increased numbers of tumours in Xpc–/– were found at certain time points: 18 times higher at day 77, and 40 times at day 84, and 16 times at day 91 (see Figure 4).



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Fig. 4. Average tumour yield (diameter ≥ 1 mm) of Xpc-deficient mice (open diamonds) and wild-type littermates (closed diamonds) during chronic UV irradiation of 1.0 MED/day (1000 J/m2 of F40 lamps).

 
Remarkably, the median induction time of tumours (t50) at 0.08 MED/day in Xpc–/– mice differed very little from the present one at 1 MED/day (84 versus 75 days, see Figure 5), in contrast to wild-type littermates (238 versus 102 days). Although the induction of mutant p53 patches in Xpc-deficient mice differed strongly (14x) between these two exposure regimens (on average 7.8 p53 patches at 0.08 MED/day and 107 p53 patches at 1 MED/day after 15 days of irradiation), the tumour induction did not. At a lower level of 0.032 MED UV/day the t50 is 117 days. The differences in t50s between and Xpc–/– and their wild-type littermates at 0.032 and 0.08 MED are comparable: factor 2.9 versus 2.8.



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Fig. 5. UV-dose dependency of carcinogenesis in Xpc-deficient hairless mice. 0.032, 0.08 and 1 MED correspond to 32, 80 and 1000 J/m2 of F40 lamps, respectively. The tumour induction times at 0.08 MED/day originate from (13). The t50s of Xpc-deficient mice are shown in open diamonds and wild-type littermates in closed diamonds.

 
Histological analysis of a randomly selected subset of the tumours (from Xpc–/– and their wild-type littermates) confirmed that these tumours were SCCs and their precursors, AKs.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this study we investigated to what extent mutant p53 patches are true precursors of SCCs induced by chronic UV exposure. We determined mutations in the p53 gene of UV-induced p53 patches and SCCs in order to ascertain whether any selection occurred of mutations in progression to SCC. Abundant UV-signature mutations (C->T and CC->TT transitions at dipyrimidine sites) reflected that UVB radiation was the cause in both mutant p53 patches and SCCs. No significant differences between the mutant p53 patches and SCCs were found in the frequencies of C->T hotspot mutations at codons 267 and 272 of the p53 gene, indicating that no pronounced selection based on mutation spectrum occurs during UV-induced carcinogenesis. Our data constitute an experimental confirmation of the finding by Ren et al. (34) who found ‘no discernible differences in humans among p53 patches, dysplasias, carcinomas in situ and SCCs with respect to the prevalence of UV-specific mutations’, as well as those more recently found for murine UV carcinogenesis (35).

Although the same hotspot mutations in murine SCCs were described in other studies, the percentages tended to be lower: we found that 53% of the SCCs carried C->T transitions in codon 267, whereas Dumaz et al. (8) found 39% and Queille (36) 46%. This slight difference may be attributable to the fact that we only included SCCs that are positive for Pab240 antibody in order to have a properly matched comparison with the Pab240-positive patches. Pab240 recognizes an epitope that is exposed in mutant p53 proteins due to a conformational exchange, while the epitope is cryptic in wild-type p53 proteins (37). Around 80% of UV-induced SCCs show positive immunostaining with Pab240 (19). Selection of our SCCs on Pab240 immunoreactivity, might have resulted in a slightly higher percentage of 267-hotspot-mutation-bearing SCCs. The possibility that differences in mutation spectrum between p53 patches and SCCs would be present in exons 2–4 and 9–11 is small, since sequencing of all exons of p53 patches revealed that the vast majority of p53 mutations are indeed found in exons 5–8 [96% in human (38) and 92% in mice (35)]. Hence, we may safely conclude that p53 patches and SCCs carry identical p53-mutation spectra, which implies that all p53 patches have the potential to develop into SCCs.

To test whether mutant p53 patches are directly related to skin tumour risk, we systematically examined the time course of UV-induced p53 patches in three DNA-repair-deficient tumour-prone mouse strains and their heterozygous and wild-type littermates. The order of induction of the majority of p53 patches was first in Xpa-deficient, second in Xpc-deficient, third in Csb-deficient mice and last in wild-type littermates, i.e. corresponding with the order of tumour susceptibility (12,13). This confirms that mutant p53 patches are reliable indicators of tumour risk for UV carcinogenesis in mice with various tumour prone genetic backgrounds.

The induction kinetics of p53 patches in Xpc-deficient mice (and perhaps in Xpa-deficient mice) appear to differ from those of Csb-deficient and wild-type littermates: for a relatively long period (from day 13 till 41) the numbers of p53 patches in Xpc-deficient mice remain at ~10/mouse, while after 41 days a rapid increase takes place. In Xpa-deficient mice there is an indication of a similar plateau between 13 and 20 days. This suggests that there is an initial equilibrium between the generation and loss of mutant p53 patches, which later on shifts to a predominant generation of mutant p53 patches.

The plots (Figure 1) do not show a smooth, steady growth in the numbers of p53 patches during chronic UV irradiation due to a relatively large variation among the mice. The reason for this is not entirely clear, but acquisition of the data in successive cohorts of animals in the course of a year may have contributed to the variation. In our previous paper (25) we reported a more steady increase during chronic UV irradiation in wild-type mice.

With regard to the prevention of SCCs, GGR appears to have more impact than TCR, and this apparently also holds for p53 patch induction. Although a defect in both GGR and TCR (in Xpa-deficient mice) causes the highest sensitivity, a defect in GGR (Xpc) accelerates p53 patch and SCC development more than a defect in TCR (Csb). An explanation for the fact that the Csb-deficient mouse is less sensitive to p53 patch and SCC induction compared with the Xpc-deficient mouse may be apoptosis. Since apoptosis can be triggered by stalled RNA polymerase due to photoproducts in active genes, Csb-deficient mice are more sensitive to UV-induced apoptosis than Xpc-deficient mice (39,40). An adequate apoptotic response in Csb-deficient mice could thus eliminate heavily damaged epidermal keratinocytes thereby reducing mutations and subsequently suppressing p53 patch induction. Though Xpa-deficient mice are also sensitive to apoptosis induction, apoptosis is apparently not able to overcome the high mutation rate caused by persisting photoproducts due to a combined crippling of TCR and GGR.

Another mechanism to reduce mutations in epidermal keratinocytes in Csb-deficient mice might be parakeratosis. As our group reported earlier (13), of the three repair-deficient strains studied, only the Csb-deficient mouse strain exhibits parakeratosis: an abnormal keratinisation of the stratum corneum with persistent keratinocyte nuclei, macroscopically visible as scaling. This extended cornified layer retaining DNA could simply absorb more DNA-damaging UV thereby reducing the UV dose that reaches the target cells: the proliferating basal epidermal keratinocytes.

In contrast to CSB patients, Csb-deficient mice are tumour prone (13,30). This could be attributable to a lack of Xpe (Ddb2, damaged-DNA-binding gene) induction in mice after UV exposure (41), and a related lack of GGR of CPD. Hence, GGR of CPD cannot serve as a backup of failing TCR in (Csb-deficient) mice, but it can in humans (42) which may explain why CSB patients are not known to run an increased risk of skin cancer. Ectopic expression of Ddb2 in mice reduces UV carcinogenesis (S.Alexeev, H.Kool, H.Rebel, M.Fousteri, C.Backendorf, F.R.de Gruijl, H.Vricling and L.H.F.Mullenders, manuscript submitted) and experiments are in progress to ascertain to what extent it corrects the tumour-proneness of Csb-deficient mice.

At chronic high UV doses of 1 wild-type MED/day, the numbers of p53 patches were elevated in Xpc-deficient mice in comparison with wild-type control mice. The yield of 107 p53 patches/mouse found at 15 days in Xpc-deficient mice could be compared with 23 days (1.5 times later) in wild-type mice. This corresponds well with the 1.4 times accelerated tumour induction at this dose. The difference between Xpc-deficient and wild-type mice was bigger at 0.08 MED UV/day: a yield of ~100 patches was found at day 50 in Xpc-deficient mice and in wild-type mice at day 180 (3.6 times later), see Figure 1. This corresponds again well with tumour data in which the t50 was 2.8 times shorter than in wild-type mice at 1 MED/day (13). The numbers of mutant p53 patches in Xpc-deficient versus wild-type mice are thus indicative of tumour risk, independent of the UV dose.

The UV-carcinogenesis experiments with three different UV doses revealed that the t50s of tumour induction in Xpc-deficient mice were 2.9, 2.8 and 1.4 times shorter than in wild-type littermates, indicating a rate-limiting effect in tumour development at daily dosages over 0.08 MED.

In all epidermal sheets, the p53 patches were exclusively located in the interfollicular epidermis. It has been hypothesized that BCCs would originate from basal stem cells in the hair follicle and SCCs from interfollicular stem cells. In our hairless SKH1 mouse model several skin tumours and pre-cancerous stages can be induced upon chronic UV irradiation, including SCC, AK, kerato-acanthoma, seborrheic keratosis, bowenoid tumour and papilloma (8,43), but BCCs were never found. Thus our findings very much support that the precursors of SCCs are derived from interfollicular stem cells and not from stem cells in the hair follicle. The density of persistent p53 patches should then be limited by the number of interfollicular stem cells (44). In the heterozygous Ptch knockout mouse model UV enhances the growth of BCCs and trichoblastomas (45). It would be interesting to examine whether in this model UV-induced p53 patches are located in the hair follicles.

Having established p53 patches as the most likely precursors of SCCs, it would be interesting to estimate the SCC risk from the density of p53 patches in the skin. The risk of transformation from AK to SCC within 1 year has been estimated to be 1/1000 (46). Since the SCC risk increases with age, the problem could be approached by looking at the density of p53 patches at the time the first SCC arises. At the median time the first SCC arises in the SKH1 hairless mouse ~10 000 mutant p53 patches/mouse can be expected, based on our previous data (25). Backvall (24) reported human data in which on average 0.15 p53 patches/mm with an averaged size of 1.4 mm were estimated in paraffin sections of SCC patients. Based on the assumption that (circular) p53 patches had been cut randomly, the expected real diameter of a p53 patch would then be 1.8 mm and the density of 8.3/cm2 is easily calculated as a rough estimate. Assuming that the sun-exposed skin is 1000 cm2, the total number would come out as 8300 patches per individual. Ren (34) reported about patients of 65–88 years old with dysplasia, CIS (carcinoma in situ) or SCC with an average of 40 p53 patches/cm2, which for 1000 cm2 of sun-exposed skin would result in 40 000 patches per patient. This suggests that the first SCC appears to arise on average at the moment 8300–40 000 p53 patches per individual are present. This range of number of patches corresponds well with our murine data in which 10 000 mutant p53 patches were estimated at the time the first SCC appears. We can conclude that the chance for a p53 patch to develop into an SCC is relatively small, but a strong relationship exists between the numbers of p53 patches and SCCs.

However, in a human study (27) no significant difference was found in the p53 patch density between a group of patients with one carcinoma and an age-matched group of patients with multiple carcinomas. One of the reasons for this might be regression. It is known that p53 patches regress when UV is discontinued (19,22). The density of p53 patches in patients is thus dependent on the season and on behaviour. This variability could limit the reliability of mutant p53 patches as a surrogate for human SCC development.

Moreover, failing immunity in Rag1 mice (47) or in mice that received an immunosuppressive agent [(48); Y.G.L.de Graaf, H.Rebel, R.Willemze, F.R.de Gruijl and J.N.Bouwes Bavinck, unpublished data] did not accelerate p53 patch induction, whereas this immune deficiency is known to increase the rate of SCC development. Hence, there appears to be a good correlation between p53 patch density and SCC risk, which may solely be modified by immunomodulatory effects.

In summary, we can state that UV-induced p53 patches are, independent of the kind of p53 mutation, potential precursors for SCCs. P53 patches are reliable indicators of tumour risk in various DNA-repair-deficient backgrounds, and in the chronic UV dose range we used. On average, the first SCC arises when 8300–40 000 patches per individual are present in the entire area of UV-exposed skin.


    Acknowledgments
 
We thank H.Sturkenboom for loyal animal caretaking, H.Dullens for access to the laser dissection microscope, R.Beems for HE sections and diagnosis of the tumours and H.van Steeg, G.T.J.van der Horst, and E.C.Friedberg for providing the Xpa, Csb and Xpc-deficient mice. We also thank J.Nijhof for critical reading of the manuscript. This study was financed mainly by grant UU97-1531 from the Dutch Cancer Society (KWF).

Conflict of Interest Statement: None declared.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received May 25, 2005; revised July 12, 2005; accepted July 22, 2005.