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[Cancer Research 66, 2962-2969, March 15, 2006]
© 2006 American Association for Cancer Research

Molecular Biology, Pathobiology, and Genetics

Enhanced Photocarcinogenesis in Interleukin-12–Deficient Mice

Akira Maeda1, Stefan W. Schneider3, Motohiro Kojima2, Stefan Beissert3, Thomas Schwarz1 and Agatha Schwarz1

Departments of 1 Dermatology and 2 Pathology, University Kiel, Kiel, Germany and 3 Department of Dermatology, University Münster, Münster, Germany

Requests for reprints: Thomas Schwarz, Department of Dermatology, University of Kiel, Schittenhelmstrasse 7, D-24105 Kiel, Germany. Phone: 49-431-5971500; Fax: 49-431-5971503; E-mail: tschwarz{at}dermatology.uni-kiel.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
UV-induced DNA damage is the basis for the development of UV-mediated skin cancer because reduction of DNA damage lowers the risk for photocarcinogenesis. The cytokine interleukin (IL)-12 was shown to exhibit the capacity to reduce UV-induced DNA damage presumably via induction of nucleotide excision repair. Because IL-12 is also produced in the skin, we wondered whether endogenous IL-12 protects from photocarcinogenesis. Therefore, we used knockout mice that lack the IL-12p40 chain and thus do not secrete biologically active IL-12. IL-12p40 knockout (IL-12p40–/–) and wild-type (wt) mice were exposed thrice weekly to UV. Skin biopsies obtained after 6 weeks revealed significantly increased numbers of sunburn cells in IL-12p40–/– mice. Additionally, a higher load of UV-induced pyrimidine dimers could be detected in the skin of UV-exposed IL-12p40–/– mice. Staining of epidermal sheets with an antibody against the tumor suppressor gene p53 revealed a higher number of p53 patches in the skin of IL-12p40–/– mice. After ~200 days, first skin tumors developed. Kaplan-Meier analysis indicated a significantly increased probability of tumor development in the IL-12p40–/– mice. In addition, the number of tumors developing in the individual mice was significantly higher in IL-12p40–/– mice than in wt mice. Tumors obtained in IL-12p40–/– mice grew faster than those obtained from wt mice on inoculation into nu/nu mice. This was confirmed in an electrophysiologic assay evaluating the intrinsic invasive potency of tumor cells. Together, these data indicate that IL-12 deficiency is associated with an increased risk to develop UV-induced skin cancer, implying that endogenous IL-12 may protect from photocarcinogenesis. (Cancer Res 2006; 66(6): 2962-9)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The incidence of UV-induced skin cancer is rapidly rising, accounting for >40% of all human cancers in the United States (1). Solar/UV radiation is certainly the predominant causal factor. Exposure to the midwave range (UVB, 290-320 nm) plays the major role in the induction of nonmelanoma skin cancer; preponderance of evidence indicates that UV radiation, including the long wavelength range (UVA, 320-400 nm), is also involved in the etiology of melanoma, although the situation is much less clear than in nonmelanoma skin cancer. The major event in photocarcinogenesis is the induction of DNA damage by UVB. UVB primarily induces two types of DNA lesions: cyclobutane pyrimidine dimers and (6-4) photoproducts (2). DNA damage is induced by rather low even suberythemogenic UVB doses. The vast majority of UVB-induced DNA lesions is removed by the nucleotide excision repair (NER), the essential endogenous DNA repair system (3). The importance and efficacy of the NER is best illustrated by the disease xeroderma pigmentosum (XP). Due to genetic mutations in the various NER genes, these unfortunate patients do not exhibit a functional NER and suffer from a dramatically increased risk to develop UV-induced skin cancer at early ages (4). Accordingly, mice, in which the different genes of the NER complex have been knocked out, develop earlier and have a higher number of skin tumors on chronic UVB exposure when compared with wild-type (wt) mice (5). In turn, accelerated or enhanced removal of UVB-induced DNA damage (e.g., via the topical application of the bacterial DNA repair enzyme T4 endonuclease V) reduced the development of skin tumors in chronically UVB-exposed mice (6). Likewise, topical T4 endonuclease V was shown to reduce the risk to develop actinic keratoses, the pre-stage of skin cancer, in XP patients (7).

The immunomodulatory cytokine interleukin (IL)-12 was recently shown to reduce the amount of DNA damage both in vitro and in vivo (8). Intracutaneous injection of IL-12 into murine skin before UVB exposure reduced the amount of DNA damage significantly in skin samples taken 16 hours after UV exposure. In contrast, no difference was observed between IL-12- and sham-treated mice when skin biopsies were taken immediately after UV exposure. This excluded the possibility that IL-12 acted as a UVB filter. The fact that no differences in the amounts of DNA lesions were present immediately after UVB irradiation but occurred at later time points could only be explained by enhanced removal of photoproducts. This unique effect of IL-12, however, was not observed in Xpa knockout mice, which, due to a mutation in the XPA gene, do not exert a functional NER (5). Hence, it was concluded that IL-12 might induce NER (8). Because IL-12 can be produced locally in the skin either by keratinocytes or by macrophages (9–11), it may be speculated that endogenous production of IL-12 might protect from photocarcinogenesis. To address this issue, we used knockout mice that lack the IL-12p40 chain and thus do not secrete biologically active IL-12, which is a heterodimeric cytokine consisting of a p35 and a p40 chain (12). Here, we show that IL-12p40 knockout (IL-12p40–/–) mice, on chronic UVB exposure, develop skin tumors earlier and at a higher frequency compared with wt mice. In addition, tumors generated in IL-12p40–/– mice exerted a more aggressive growth behavior. Because lack of the production of IL-12 results in enhanced photocarcinogenesis, one can, in turn, conclude that endogenously produced IL-12 might protect from the development of UV-induced skin cancer and thus represents an additional natural protection mechanism.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice. C57BL/6 mice and immunodeficient nu/nu mice (HsdCpb: NMRI-Foxn1nu) were purchased from Harlan-Winkelmann (Borchen, Germany). IL-12p40–/– mice were kindly provided by I. Förster (Institute for Medical Microbiology, Immunology and Hygiene, Technical University Munich, Munich, Germany).

UV irradiation and tumor induction. Within the solar spectrum, the UVB range (290-320 nm) is responsible for carcinogenesis. Therefore, a bank of four Philips UVB TL40W/12 sunlamps (Philips, Hamburg, Germany) with an emission spectrum ranging from 280 to 350 nm with a peak at 313 nm was used for irradiation. The average output measured with an UVB meter (Waldmann, Schwenningen, Germany) was 10 W/m2. The mice were placed on a shelf 20 cm below the light bulbs for irradiation. The cage order was systematically rotated before each treatment to compensate for uneven illumination along the shelf as described previously (13). The mice (25 for each group) were shaved with electric clippers on the entire dorsum once weekly. Beginning at 10 weeks of age, mice were irradiated thrice weekly with 5 kJ/m2 for 4 weeks, 10 kJ/m2 for 4 weeks, 15 kJ/m2 for 4 weeks, and 20 kJ/m2 for 5 months. Tumor development was monitored twice weekly during the irradiation period and for additional 8 months thereafter. The location and growth of each tumor exceeding 2 mm in diameter were recorded. Representative excision biopsies from all tumors were taken, fixed in paraformaldehyde, and embedded in paraffin. Sections were stained with H&E and documented by a video computer-assisted digital image processing technique (DISKUS version 3.99 for Windows 95, C.H. Hilgers, Königswinter, Germany). Tumors were analyzed in a blinded fashion and grouped as highly, moderately, or poorly differentiated according to the following criteria: high (predominantly mature squamous cells, numerous horn pearls, slight atypicality), moderate (less keratinization, few horn pearls with incompletely keratinized centers), and poor (no horn pearls, almost no keratinization, many cells atypical).

For evaluation of the degree of proliferative activity, sections were stained with an antibody directed against Ki-67 (Vector Laboratories, Burlingame, CA) using immunoperoxidase technique. Sections were also stained with a polyclonal antibody directed against keratin 10 (Covance, Berkeley, CA) as a differentiation marker.

Evaluation of sunburn cells. Biopsies were taken from dorsal skin of mice that were exposed to 5 kJ/m2 UVB thrice weekly for 4 weeks, fixed in formaldehyde, and embedded in paraffin. Tissue sections (5 µm) were stained with H&E, and the number of sunburn cells, defined as apoptotic cells within the epidermis exhibiting a shrunken eosinophilic cytoplasm and a condensed nucleus, was counted throughout the epidermis of sections per sample using a grid measuring 1 x 1 cm inserted in a conventional microscope. Sunburn cells per 10-mm-long epidermis were counted. At least five fields of each sample were evaluated in a blinded fashion, and the number of sunburn cells (mean ± SD) was calculated.

Immunohistochemical staining for UV-induced DNA damage. Skin biopsies were fixed in 7% buffered paraformaldehyde, dehydrated, and embedded in paraffin. After deparaffinization with xylol and ethanol at decreasing concentrations, sections were immersed in an aqueous solution containing 0.1 mol/L citric acid and 0.1 mol/L sodium citrate and subsequently microwave treated for 15 minutes to unmask antigenic epitopes. Endogenous peroxidase activity was blocked by incubation with 3% H2O2 in PBS for 20 minutes. After rinsing with PBS, nonspecific binding sites were blocked with 2% bovine serum albumin (BSA) for 30 minutes at room temperature followed by incubation with the primary antibody diluted in 1% BSA overnight. A monoclonal antibody directed against pyrimidine dimers (Kamiya Biomedical Co., Seattle, WA) was used at a dilution of 1:2,000. Staining was done by an indirect immunoperoxidase technique using the following reagents: biotinylated universal antibody, streptavidin-horseradish peroxidase (HRP), and NovaRED substrate. All reagents were purchased from Vector Laboratories. Negative controls comprised omission of the first antibody.

Detection of p53 patches. Ears were obtained from mice that were exposed to 5 kJ/m2 UVB thrice weekly for 4 and 8 weeks, respectively and mechanically split into dorsal and ventral sides. Sheets were floated on PBS containing 20 mmol/L EDTA (pH 7.4) at 37°C for 2.5 hours. After washing with PBS, the dermis was removed by using two forceps. The epidermal sheets were spread floating on PBS in a Petri dish and subsequently fixed in PBS-buffered 4% formaldehyde solution for 15 minutes at room temperature. Subsequently, endogenous peroxidase was blocked in methanol containing 1.5% H2O2 during 20-minute incubation in a shaker. Sheets were washed thrice for 5 minutes in PBS containing 0.5% Tween 20 (Sigma, St. Louis, MO). Nonspecific binding was blocked with 10% normal rabbit serum and 0.2% BSA in PBS containing 0.5% saponin (Merck, Darmstadt, Germany) for permeabilization. The monoclonal mouse anti-p53 antibody Pab240 (Novacastra Laboratories Ltd., Newcastle, United Kingdom) was applied at 1:2,500 dilution overnight at 4°C. The Pab240 antibody recognizes only mutant but not wt p53. Then, the sheets were incubated with biotinylated goat anti-rabbit secondary antibody (1:200) for 30 minutes at 37°C, streptavidin-HRP, and substrate sequentially. The sheets were mounted with Kaiser's glycerol gelatin (Merck) without counterstaining.

The number and size of p53 clones in the epidermal sheets were evaluated in a blinded fashion by light microscopy using x200 magnification. The epidermis was investigated systematically to precisely cover the slide area without double reading or missing sectors. The number of p53-positive cell groups (clones) and the number of cells per clone were recorded. To determine the sheet area, each epidermal sheet was documented by a video computer-assisted digital image processing technique (DISKUS version 3.99 for Windows 95).

Establishment of tumor cell lines. Tumor specimens were surgically removed from the tumor-bearing IL-12p40–/– or wt mice and rinsed twice with PBS supplemented with 1% antibiotic/antimycotic solution (PAA Laboratories, Linz, Austria). Small pieces of <2 mm3 were seeded in six-well culture plates containing RPMI 1640 supplemented with 20% FCS (Life Technologies, Breda, the Netherlands), 1% L-glutamine (PAA Laboratories), 1% MEM nonessential amino acid (PAA Laboratories), 0.25% ciprofloxacine, and 0.5% antibiotic/antimycotic solution. All cells were maintained at 37°C in a humidified atmosphere containing 5% CO2. When the cultures had reached ~80% confluence, the adherent cells were detached with 0.1% trypsin/0.05% EDTA (Life Technologies) and used for subsequent passage.

Proliferation assay. The proliferative capacity of tumor cell lines obtained from both IL-12p40–/– and wt mice was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) proliferation assay. Cells were seeded in triplicate into 96-well plates at a density of 5 x 103 per well. At 0, 2, 3, or 7 days after seeding, MTT solution (20 µL; 5 mg/mL in PBS) was added to each well followed by incubation for 4 hours at 37°C. After stop buffer [10% SDS, 50% formamide (pH 4.7)] was added to each well, cells were incubated at 37°C overnight. Spectrophotometric absorbance of each sample was measured at 595 nm using a microplate reader (Bio-Rad, Hercules, CA).

To determine the colony-forming efficiency, the tumor cell lines obtained from IL-12p40–/– and wt mice were seeded in the culture dishes at a concentration of 1 x 104 cells/mL. After seeding for 7, 10, and 14 days, cells were stained with crystal violet to evaluate the number of colonies.

Measurement of cytokines. Tumor cells (IL-12p40–/– and wt mice) established as described above were seeded at a concentration of 5 x 105/mL. After seeding for 48 hours, supernatants were harvested and tested for IL-6, IL-10, and transforming growth factor-ß1 (TGF-ß1) using ELISA (Bender Medsystems, Vienna, Austria).

Transplantation of UV-induced tumors into nu/nu mice. To assess tumor growth of UV-induced tumors in vivo, tumors were transplanted into immunodeficient nu/nu mice. Five tumors dissected from each IL-12p40–/– or wt mice were washed twice with PBS/1% antibiotic/antimycotic solution and gently cut into small pieces measuring 3 mm. Subsequently, tumor pieces were transplanted to nude mice by s.c. inoculation into the back. Tumor size was monitored once weekly after inoculation by measuring in three dimensions with calipers. Mice with necrotic tumors or tumors ≥1 cm in diameter were immediately killed.

Transepithelial electrical resistance breakdown assay. For transepithelial electrical resistance (TEER) measurements, we used the subclone C7 isolated from Madin-Darby canine kidney cells (MDCK-C7 cells). C7 cells form an electrical tight cell monolayer and represent principle cells of the collecting duct. MDCK-C7 cells were grown on filter membranes (growth area, 4.2 cm2; pore diameter, 0.4 µm; thickness, 20 µm; Falcon, Heidelberg, Germany). MDCK-C7 cells (106) were seeded on each filter. Medium exchange and TEER measurement started 48 hours after seeding MDCK-C7 cells. After another 4 to 10 days, 106 tumor cells (originated in either IL-12p40–/– or wt mice) were added into the upper compartment of the filter cup. For control experiments, no tumor cells but MDCK-C7 cells were seeded in the upper chamber. For TEER measurements as published in detail previously (14, 15), we used commercially available STX-2 chopstick electrodes (EVOM; WPI, Sarasota, FL). Background electrical resistance built up by filter and medium only was constant and extremely low (25 {Omega} cm2). MDCK-C7 cell monolayers were used after exhibiting a resistance of ≥8 k{Omega} cm2. A resistance of ≥1 k{Omega} cm2 already implicates a tight MDCK-C7 monolayer. TEER was measured at least once daily in six-well dishes. TEER values were corrected for background resistance. All experiments were done in duplicates.

Statistical analysis. The method of Kaplan and Meier was used to describe the probability of tumor development in the carcinogenesis study. This is a life-table analysis and takes into account animals that die before developing a tumor. The differences in tumor latent periods were analyzed by the Mann-Whitney U test. The differences in the average numbers of tumors per mouse were analyzed by {chi}2 test. The other differences were analyzed by the Student's t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
UV-induced tumors develop earlier and more frequently in IL-12p40–/– mice. To address whether IL-12p40–/– mice are more susceptible to photocarcinogenesis, IL-12p40–/– and wt mice were exposed to a chronic UVB regimen, which is known to induce skin tumors after ~200 days. The period of time between the first UV exposure and the appearance of the first visible skin tumors was significantly different in the two groups. Although both IL-12p40–/– and wt mice were susceptible to photocarcinogenesis, tumor development was accelerated in IL-12p40–/– mice (Fig. 1A ). Furthermore, the incidence of UV-induced skin tumors was significantly enhanced in the IL-12p40–/– mice; more mice developed tumors, and the overall number of tumors observed was higher compared with wt mice (Fig. 1B). The preferential sites for tumor development were the back and the ears. In IL-12p40–/– mice also, tumors developed in the conjunctiva. Histopathologic examination revealed that all tumors induced by UV were squamous cell carcinomas. The vast majority of tumors developed in wt mice were highly differentiated, whereas almost one third of the tumors developed in IL-12p40–/– mice were poorly differentiated (Table 1 ). Poor differentiation was associated with a loss of the expression of keratin 10. The expression of the proliferation marker Ki-67 correlated with the differentiation stage (Fig. 2 ).


Figure 1
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  		Figure 1. Increased photocarcinogenesis in IL-12p40–/– mice. Mice were exposed to UV at their weekly shaved backs thrice weekly. UV (5 kJ/m2) was given per exposure for 4 weeks followed by 10 kJ/m2 UV per exposure for 4 weeks, 15 kJ/m2 UV per exposure 4 weeks, and 20 kJ/m2 UV per exposure for the remaining 5 months. A, probability of tumor development was analyzed according to Kaplan-Meier for IL-12p40–/– and wt-control mice. B, average numbers of tumors per mouse (n = 20 for both groups). *, P < 0.05 versus wt mice; **, P < 0.02 versus wt mice.

 

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  		Table 1. Site and histologic differentiation of tumors induced by UV irradiation in IL-12p40–/– and wt mice

 

Figure 2
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  		Figure 2. Differentiation stages and Ki-67 expression of UV-induced tumors. UV-induced tumors of both IL-12p40–/– and wt mice were excised, paraffin embedded, cut, and stained with H&E. Differentiation was graded as high, moderate, or poor. In addition, sections were subjected to immunohistochemical staining with antibodies directed against Ki-67 and keratin 10 (K10). Bar, 50 µm.

 
Increased number of sunburn cells, pyrimidine dimers, and p53 clusters in the epidermis of IL-12p40–/– mice. Because UV-induced DNA damage is the major molecular trigger for sunburn cell formation (16), a decrease in the DNA repair is associated with an increased number of apoptotic keratinocytes on UV exposure as shown in NER-deficient mice (17). Accordingly, an enhanced number of sunburn cells was described in IL-12p40–/– mice on acute UV exposure (8). This finding was compatible with the hypothesis that IL-12–deficient mice suffer from decreased DNA repair. To determine whether chronic UV exposure also induces an increased number of apoptotic keratinocytes, IL-12p40–/– and wt mice were exposed to 5 kJ/m2 UV thrice weekly for 4 weeks. Biopsies were taken, and the number of sunburn cells was counted. IL-12p40–/– mice revealed a significantly increased number of apoptotic keratinocytes in the epidermis (Fig. 3A ).


Figure 3
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  		Figure 3. Increased sunburn cells and pyrimidine dimers in IL-12p40–/– mice on chronic UV exposure. Mice were irradiated thrice weekly with 5 kJ/m2 for 4 weeks. Biopsies were taken and sections were stained with H&E. A, number of sunburn cells per 1-cm-long epidermis. Columns, mean; bars, SD. B, sections were stained with an antibody directed against UV-induced pyrimidine dimers using peroxidase method. Bar, 20 µm. *, P < 0.02 versus wt mice.

 
To finally prove whether the load of UV-induced DNA damage is higher in IL-12p40–/– mice, biopsies were taken and stained with an antibody that recognizes UV-specific pyrimidine dimers. The staining of the nuclei was more pronounced in skin of UV-exposed IL-12p40–/– mice in comparison with wt mice (Fig. 3B). This indicates that the amounts of UV-induced photolesions are higher in the absence of IL-12 presumably due to decreased DNA repair (8).

UV induces mutations in the p53 gene. Thus, in sun-exposed skin, numerous clones of p53-mutant keratinocytes can be detected (18–20). At least in mice, these clones correlate with the tumor risk (21). Because DNA repair-deficient mice develop p53 patches faster and more frequently (22), we were interested to study whether p53 patches are stronger expressed in IL-12p40–/– mice on chronic UV exposure. To address this issue, epidermal sheets of UV-exposed skin from both IL-12p40–/– and wt mice were obtained and stained with an anti-p53 antibody (Fig. 4A ). In IL-12p40–/– mice, a significantly higher frequency of p53 patches was detected (Fig. 4B). In addition, the individual size of the clones was significantly larger than that observed in wt mice exposed to same UV doses (Fig. 4C).


Figure 4
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  		Figure 4. Increased p53 clones in IL-12p40–/– mice. Mice were irradiated thrice weekly with 5 kJ/m2 for 4 and 8 weeks, respectively. After UVB irradiation, ears were excised and epidermal sheets were prepared. A, epidermal sheets were stained with mouse anti-p53 antibody. B, size of p53 clones in the epidermal sheets was evaluated by light microscopy. Columns, mean; bars, SD. C, number of p53 clones in the epidermal sheets was evaluated by light microscopy. Columns, mean; bars, SD. Bar, 50 µm. *, P < 0.0005 versus wt mice; **, P < 0.05 versus wt mice.

 
In vitro proliferative capacity of tumor cell lines. To study the growth behavior of the UV-induced tumors, tumor specimens were obtained from both IL-12p40–/– and wt mice. Pieces of <2 mm3 were seeded into six-well culture plates and cultured in RPMI 1640 supplemented with 20% FCS. When the cultures had reached ~80% confluence, the adherent cells were detached with 0.1% trypsin/0.05% EDTA and used for subsequent passage. To determine the capacity to proliferate, cell lines were subjected to a MTT proliferation assay, which was done immediately (0), 2, 3, or 7 days after seeding. In all samples tested, the proliferative capacity was higher in cell lines obtained from UV-exposed IL-12p40–/– mice when compared with cell lines developed in wt mice (Fig. 5A ).


Figure 5
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  		Figure 5. Proliferation rate by UV-induced skin tumors. Cells were obtained from tumors induced by UV in either IL-12p40–/– or wt mice and put into culture. A, proliferation measured using a MTT assay immediately (0 hour), 2 (48 hours), 3 (72 hours), or 7 days after onset of culture. Y axis, spectrophotometric absorbance of each sample measured at 595 nm using a microplate reader. Columns, mean; bars, SD. B, cells were seeded at low concentrations (1 x 104/mL), and the outgrowth of colonies was analyzed 7, 10, and 14 days after seeding by visualizing the colonies with crystal violet. *, P < 0.0001 versus wt mice; **, P < 0.001 versus wt mice; ***, P < 0.001 versus wt mice.

 
Although the MTT assay is frequently used to analyze cell proliferation, it actually measures mitochondrial respiration. To consolidate these findings, colony formation assay was done. Cells were seeded at low concentrations, and the outgrowth of colonies was analyzed 7, 10, and 14 days after seeding by visualizing the colonies with crystal violet. The number of colonies increased with time in cells obtained from both IL-12p40–/– and wt mice (Fig. 5B). However, the frequency of colonies was much higher in tumor cells obtained from IL-12p40–/– mice than from wt mice.

Cytokine release by tumor cells. Because proinflammatory cytokines have been suggested to support the growth of tumor cells in a paracrine and autocrine fashion (23), supernatants were harvested from tumor cell lines 48 hours after seeding and tested for IL-6 and TGF-ß. In addition, the concentrations of the immunosuppressive cytokine IL-10 were measured. Cell lines obtained from IL-12p40–/– mice secreted remarkably enhanced levels of IL-6 in comparison with cell lines obtained from wt mice, whereas no differences were observed for TGF-ß (Fig. 6 ). In addition, tumor cells obtained from IL-12p40–/– mice released slightly enhanced levels of IL-10.


Figure 6
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  		Figure 6. Cytokine release by UV-induced tumors. Supernatants were harvested from tumor cell lines obtained from IL-12p40–/– or wt mice 48 hours after seeding and tested for IL-6, IL-10, and TGF-ß (in pg/mL) using ELISA. Columns, mean of triplicate measurements; bars, SD. *, P < 0.05 versus wt mice; **, P < 0.02 versus wt mice.

 
Tumors generated in IL-12p40–/– mice grow faster in vivo. Because of the enhanced proliferative capacity and the poorer differentiation, we surmised that the tumors generated in the IL-12p40–/– mice might exert a more aggressive phenotype. To address this issue, tumor samples of ~3 mm in diameter were inoculated s.c. into nu/nu mice. Because nu/nu mice are immunodeficient, the tumors are not rejected and grown. The recipient mice were checked once weekly for the development of tumors. In addition, measuring in three dimensions with calipers monitored the tumor volume. In mice that were inoculated with tumors obtained from IL-12p40–/– mice, tumors developed earlier. First tumors in these mice were visible between days 10 and 20, whereas no tumors were observed at these time points in mice that had received tumor cells generated in wt mice (Fig. 7 ). In addition, the volumes increased much more rapidly in transplanted tumors obtained from IL-12p40–/– mice in comparison with tumors generated in wt mice (Fig. 7). This indicates that UV-induced tumors from IL-12p40–/– mice exert a more aggressive growth behavior in vivo.


Figure 7
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  		Figure 7. Skin tumors derived from IL-12p40–/– mice grow more aggressively in vivo. Tumors (3 mm in diameter) obtained from either UV-exposed IL-12p40–/– mice or UV-exposed wt mice were cut into small pieces. Tumor pieces were transplanted s.c. into nude mice. Tumor size was measured with a caliper and documented every week after transplantation. Y axis, increase of tumor volume. *, P < 0.0001 versus wt mice on day 18; **, P < 0.0001 versus wt mice on day 21; ***, P < 0.00001 versus wt mice on day 28; ****, P < 0.00001 versus wt mice on day 35.

 
Analysis of intrinsic invasive potency using an electrophysiologic invasion assay. To consolidate these data, the intrinsic invasive potency of the tumor cells was tested in an electrophysiologic invasion assay. In this assay, breakdown of the TEER across a renal cell monolayer indicates opening of tight junctions induced by tumor cell invasion. Hereby, reduction of TEER correlates with the invasive capacity of cocultured tumor cells. Thus, the ratio of the TEER in the presence (TEERcoculture) and absence (TEER0) of tumor cells can be used as a variable for the intrinsic invasive potency of tumor cells. TEER was measured 48 and 64 hours after coculture with tumor cells (Fig. 8 ). At both time points, the TEERcoculture/TEER0 ratio was significantly lower on coculture of tumor cells obtained from IL-12p40–/– mice, indicating a more aggressive growth behavior of tumor cells induced by UV in IL-12p40–/– mice than in wt mice.


Figure 8
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  		Figure 8. Measurement of invasive capacity of tumor cells in an electrophysiologic invasion assay. Tumor cells (106) obtained from IL-12p40–/– or wt mice were seeded onto monolayers of MDCK-C7 cells. After 48 and 64 hours, TEER was measured. Y axis, ratio between the TEER in the presence or absence of tumor cells. Columns, mean; bars, SD. *, P < 0.02 versus wt mice 48 hours; **, P < 0.05 versus wt mice 64 hours.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It was the aim of this study to clarify whether endogenous production of IL-12 reduces the risk to develop skin cancer on chronic UV exposure. The rationale for this question was based on the previous observation in both murine and human systems that IL-12 exhibits the capacity to reduce UV-induced DNA damage presumably via induction of NER (8). Induction of DNA damage by UVB is the major hallmark event in photocarcinogenesis. Therefore, it is assumed that reduction of the load of DNA damage via accelerated repair or via elimination of cells carrying DNA damage by induction of apoptosis lowers the risk for skin cancer. An efficient strategy to remove DNA damage is the application of exogenous DNA repair enzymes like T4 endonuclease V or photolyase (6, 24). In fact, topical administration of T4 endonuclease V in a liposome formulation reduced the frequency of skin tumors on chronic UV exposure in mice significantly (6). In addition, a randomized placebo-controlled study in XP patients showed that the number of actinic keratoses was reduced in the group that applied topical T4 endonuclease V in comparison with the placebo group (7). Until recently, it was thought that the NER, as an essential repair system, is constitutively expressed and not subjected to any regulation. However, Eller et al. (25) were the first to show that administration of DNA oligonucleotides induces DNA repair via a p53-dependent mechanism. Accordingly, a recent in vivo study showed that topical pretreatment with DNA oligonucleotides enhanced the rate of DNA photoproduct removal, decreased UV-induced mutations, and reduced photocarcinogenesis in UV-irradiated hairless wt and partially repair-deficient mice (Xpc+/–; ref. 26).

Because IL-12 was also found to reduce DNA damage, application of IL-12 should also protect from photocarcinogenesis. However, in contrast to DNA oligonucleotides, administration of IL-12 is problematic for various reasons. IL-12 has a molecular weight of ~70 kDa; thus, penetration into the skin might be impaired or even impossible. Because of its heterodimeric structure, production of IL-12 is an expensive procedure and thus not suitable for practical use. However, IL-12 can be produced in the skin by either keratinocytes or macrophages (9–11). Hence, it might be possible that endogenous production of IL-12 might protect from photocarcinogenesis. To address this issue, we used IL-12p40–/– mice that do not produce functional IL-12 (12).

IL-12p40–/– mice revealed an increased number of sunburn cells on chronic UV exposure, thus confirming previous data (8). Because UV-induced DNA damage is certainly not the only but one of the major molecular triggers for apoptosis (16), the increased number of apoptotic keratinocytes suggested enhanced amounts of DNA damage in the knockout mice compared with wt mice. This was confirmed by immunohistochemical detection of UV-induced pyrimidine dimers, which were found more pronounced in skin of IL-12p40–/– mice than of wt mice on UV exposure. In addition, significantly more and larger p53 patches were detected in the IL-12p40–/– mice. It was recently shown that the order in which NER-deficient mice developed these patches was predictive of the order in which they developed tumors (22). Accordingly, IL-12p40–/– mice developed skin tumors earlier and at a higher frequency in comparison with wt mice. In addition, the tumors that were induced by UVB in IL-12p40–/– mice revealed a more aggressive growth behavior as shown in vivo by transplantation into nu/nu mice.

Although the transplantation studies into nu/nu mice do not allow to address the full functions of endogenous IL-12 during carcinogenesis, they rule out the possibility that the tumors simply grow faster because of the absence of IL-12. If this would be the case, we should not have seen a difference in the tumor growth of inoculated tumors obtained from IL-12p40–/– and wt mice. The higher aggressiveness is also shown in vitro by using a recently described electrophysiologic assay (14, 15). Whether the higher aggressiveness represents variations in the differentiation stage or differences between IL-12p40–/– and wt mice is hard to dissect because the vast majority of tumors obtained in the knockout mice revealed a higher stage of dedifferentiation.

Human and murine squamous cell carcinomas have been reported to produce proinflammatory cytokines, including IL-1{alpha}, IL-6, granulocyte macrophage colony-stimulating factor, and IL-8. Production of several members of this cytokine family has been associated with increased tumor growth or metastasis in a variety of neoplasms (23). Cytokine analysis revealed that tumor cells generated from IL-12p40–/– mice release enhanced levels of IL-6. It remains to be determined whether this contributes to the enhanced proliferation in an autocrine or paracrine manner or to the enhanced invasion because IL-6 has been reported to induce matrix metalloproteinases (MMP), which by themselves promote angiogenesis and invasion by acting on extracellular matrix proteins (27). Preliminary PCR data revealed a higher expression of MMP-2, MMP-3, and MMP-9 in tumors obtained from IL-12p40–/– mice when compared with tumors obtained from wt mice. However, we could not detect a difference in the expression of the enzymes in skin from IL-12p40–/– and wt mice. It remains to be determined whether altered expression of MMP contributes to the more aggressive growth behavior of the tumors generated in IL-12p40–/– mice. Thus, this subject is an area of future investigations.

Immunosuppression plays an important role in photocarcinogenesis (28). This is not only based on experimental in vitro and in vivo data but also on clinical facts. Evidence exists for a strong correlation between the risk to develop skin cancer and immunosuppression. Chronically immunosuppressed individuals, like transplant patients, exhibit a significantly enhanced risk for skin cancer (29). Many factors may be involved in this process, including drugs like azathioprine, which, as recently shown, in concert with UVA radiation, may synergistically enhance mutagenicity (30). This risk certainly increases with the cumulative UV load. In turn, restoration or even enhancement of an immune response [e.g., by topical or systemic application of immunomodulators (IFNs, imiquimod)] has turned out as a successful therapeutic strategy for the treatment of skin cancer (31, 32). IL-12 is an immunomodulatory cytokine and one of the major players involved in orchestrating both innate and acquired immune responses (33). It is critical for the development of T helper 1 responses. In addition, IL-12 is able to prevent UV-induced immunosuppression. Injection of IL-12 before hapten application onto UV-exposed skin prevents the development of immunosuppression (34–36). Even more importantly, IL-12 was discovered to break established immunotolerance and to antagonize the suppressive activity of regulatory T cells. Although the mechanism for breaking tolerance and antagonizing the activity of regulatory T cells still remains to be determined, it was recently discovered that the prevention of UV-induced immunosuppression by IL-12 is mediated via its capacity to reduce DNA damage (37), the major molecular trigger for UV-mediated immunosuppression (38, 39). Because of the crucial role of IL-12 in generating immune reactions, IL-12p40–/– mice have been shown to be impaired in several immune responses (e.g., contact hypersensitivity; ref. 12). It remains to be determined whether the compromised immune system contributes to the enhanced photocarcinogenesis in IL-12p40–/– mice. To the best of our knowledge, IL-12p40–/– mice have not been shown thus far to exhibit an increased risk to develop malignant tumors.

Photocarcinogenesis is a highly complex process and thus can only be studied in in vivo models. In turn, it is difficult to pinpoint mechanisms especially when studying a cytokine that exhibits many activities, such as IL-12. This caveat applies also for the dissection of whether the enhanced carcinogenesis in the absence of IL-12 is due to the immunodeficient state or due to the decreased DNA repair. We initially considered crossing the IL-12p40–/– mice with IFN-{gamma}-deficient mice and to compare the frequency of UV-induced skin cancer with that of IFN-{gamma}-deficient mice. However, even in this case, one could argue for the involvement of another type of immunodeficiency unrelated to IFN-{gamma} in the IL-12p40–/– mice because the alteration of the immune system by IL-12 deficiency certainly cannot be explained with IFN-{gamma} deficiency alone. Because of these limitations, we, of course, cannot exclude with absolute certainty that the enhanced photocarcinogenesis in IL-12p40–/– mice is because of an impaired immune response. Nevertheless, we think that the effect is at least partially due to the effect of IL-12 on DNA damage. We previously could show that IL-12p40–/– mice exhibit a significantly enhanced number of apoptotic keratinocytes (sunburn cells) on an acute UV exposure (8). The data could be confirmed in this study. Here, it was shown that the number of sunburn cells in IL-12p40–/– mice is also significantly increased on chronic UV exposure. Although many factors are involved (16), the severity of UV-induced DNA damage seems to be one of the major determinants whether a cell undergoes apoptosis or not. Accordingly, DNA repair-deficient mice revealed enhanced frequency of sunburn cells (17). Thus, the number of sunburn cells can be used as a surrogate marker for the load of DNA damage. Because IL-12p40–/– mice revealed an increased number of apoptotic keratinocytes in comparison with wt mice, we concluded that, due to the constitutive expression of IL-12, the amounts of DNA damage in wt mice are lower. This was confirmed by immunohistochemistry, which showed a higher load of pyrimidine dimers in UV-exposed skin of IL-12p40–/– mice. The impaired DNA repair in IL-12p40–/– mice is also reflected in the higher number of p53 clones in the epidermis of these animals. UVB mutates the p53 gene and thus allows expansion of cell clones because the mutated cells are not eliminated by apoptosis because of the lack of functional p53 (40).

UVB-induced apoptosis has been recognized as a protective mechanism that eliminates cells carrying a certain amount of DNA damage. Hence, alterations in the apoptosis rate can have a significant effect on photocarcinogenesis. Indeed, inhibition of apoptosis (e.g., by deletion of p53) was shown to enhance the incidence of skin tumors on chronic UV exposure in mice (41). In turn, strategies exist to enhance UVB-induced apoptosis as recently shown for IL-1 (42, 43). However, it remains to be determined whether this is associated with reduced photocarcinogenesis in vivo. In contrast, the increased numbers of sunburn cells in IL-12p40–/– mice do not indicate a reduced risk for carcinogenesis because the increase of apoptosis in this case may be related to enhanced amounts of DNA damage that may reflect decreased DNA repair. All these findings support our conclusion that the enhanced susceptibility for UV-induced skin cancer in IL-12p40–/– mice is at least partially due to an impaired removal of DNA damage. Thus, endogenously produced IL-12 might protect from the development of UV-induced skin cancer and may represent an additional natural protection mechanism.

In turn, one can conclude that overexpression of IL-12 may support accelerated removal of DNA damage. Thus, it is tempting to speculate on the therapeutic potential of topically applied IL-12 to prevent UV-induced skin cancer. However, as mentioned above, application of IL-12 might not represent a practical approach for technical but also for cost reasons. On the other hand, as shown in this study, because endogenous production of IL-12 suffices to reduce the risk for skin cancer, it is tempting to speculate about approaches to induce endogenous IL-12. Cytokine inducers, like imiquimod or CpG, might represent such substances.


   Acknowledgments
 
Grant support: German Research Foundation grants SFB 415, A16 (T. Schwarz), SCHW1177/1-1 (A. Schwarz), and BE 1580/7-1 (S. Beissert), Federal Ministry of Environmental Protection grants St.Sch_4373 and St.Sch_4491 (T. Schwarz), and Centre de Recherches d'Investigations Epidermiques et Sensorielles Research Award 2004 (T. Schwarz).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Ehrhardt Proksch for help with the immunohistochemical analyses and Susanne Dentel and Martina Wedler for excellent technical assistance.

Received 10/ 6/05. Revised 12/27/05. Accepted 1/19/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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