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
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Abstract |
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Abbreviations: AK, actinic keratoses; BCC, basal cell carcinoma; CPD, cyclobutane pyrimidine dimers; PBS, phosphate-buffered saline; SCC, squamous cell carcinoma
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Introduction |
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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 (280315 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: CT 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 (5490%), and their precursor lesions actinic keratoses (AKs) (6070%) (58). Certain mutation hotspot regions are found in p53: in codons 173179, 235250 and 273278 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:
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.
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Materials and methods |
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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 (280315 nm) and 46% in UVA (315400 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 avidinebiotin-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 haematoxylineosin 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 1001000 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 58 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.
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Results |
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A randomly selected subset of 40 of these distinct p53 patches was micro-dissected and from 26 the DNA was isolated successfully. Sequencing exons 58 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 CT 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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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, 186355) 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, 279681) 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, 164333) 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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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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Discussion |
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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 CT 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 24 and 911 is small, since sequencing of all exons of p53 patches revealed that the vast majority of p53 mutations are indeed found in exons 58 [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 6588 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 830040 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 830040 000 patches per individual are present in the entire area of UV-exposed skin.
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Acknowledgments |
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Conflict of Interest Statement: None declared.
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References |
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