Chronic exposure to UV radiation (UVR), especially in the UVA (315–400 nm) and UVB (280–315 nm) spectrum of sunlight, is the major risk factor for the development of nonmelanoma skin cancer. UVR is a complete carcinogen, which both initiates and promotes carcinogenesis. We found that protein kinase C ε (PKCε), a member of the phospholipid-dependent threonine/serine kinase family, is an endogenous photosensitizer, the overexpression of which in the epidermis increases the susceptibility of mice to UVR-induced cutaneous damage and development of squamous cell carcinoma. The PKCε transgenic mouse (FVB/N) lines 224 and 215 overexpressed 8- and 18-fold PKCε protein, respectively, over endogenous levels in basal epidermal cells. UVR exposure (1 kJ/m2 three times weekly) induced irreparable skin damage in high PKCε-overexpressing mouse line 215. However, the PKCε transgenic mouse line 224, when exposed to UVR (2 kJ/m2 three times weekly), exhibited minimum cutaneous damage but increased squamous cell carcinoma multiplicity by 3-fold and decreased tumor latency by 12 weeks. UVR exposure of PKCε transgenic mice compared with wild-type littermates (1) elevated the levels of neither cyclobutane pyrimidine dimer nor pyrimidine (6-4) pyrimidone dimer, (2) reduced the appearance of sunburn cells, (3) induced extensive hyperplasia and increased the levels of mouse skin tumor promoter marker ornithine decarboxylase, and (4) elevated the levels of tumor necrosis factor α (TNFα) and other growth stimulatory cytokines, granulocyte colony–stimulating factor, and granulocyte macrophage colony–stimulating factor. The role of TNFα in UVR-induced cutaneous damage was evaluated using PKCε transgenic mice deficient in TNFα. UVR treatment three times weekly for 13 weeks at 2 kJ/m2 induced severe cutaneous damage in PKCε transgenic mice (line 215), which was partially prevented in PKCε-transgenic TNFα-knockout mice. Taken together, the results indicate that PKCε signals UVR-induced TNFα release that is linked, at least in part, to the photosensitivity of PKCε transgenic mice.
Damage of the skin by sunlight is the most common recurrent injury (e.g., sunburn, photoaging, and skin cancer) in humans (1, 2). UVA (315–400 nm), UVB (280–315 nm), and UVC (190–280 nm) are the three components of the UV spectrum (2, 3). Because stratospheric ozone absorbs most of the radiation below 310 nm (UVC), the UV radiation (UVR) that reaches us on earth comprises mostly UVA (90–99%) and UVB (1–10%). UVA and UVB are the most prominent and ubiquitous carcinogenic wavelengths in our natural environment (1, 2). Chronic exposure to UVR is the most common etiologic factor linked to the development of squamous cell carcinomas and basal cell carcinomas, the most common nonmelanoma forms of human skin cancer (4). Squamous cell carcinoma, unlike basal cell carcinoma, invades the nearby tissues (4). The first site of metastasis usually is a regional lymph node before metastatic growth in distant sites such as the lung and brain. Although mortality due to squamous cell carcinoma and basal cell carcinoma is low, it still poses a significant societal risk (4).
UVR is a complete carcinogen, which both initiates and promotes carcinogenesis. UVR initiates photocarcinogenesis by directly damaging DNA (5, 6, 7). UVR-induced photoproducts include cyclobutane pyrimidine dimer, pyrimidine (6-4) pyrimidone dimer, and Dewar photoisomer of the pyrimidine (6-4) pyrimidone dimer (5, 6). The cyclobutane pyrimidine dimer is the predominant photoproduct, accounting for 85% of the primary DNA lesions in UV-irradiated DNA (5). The majority of the DNA lesions are removed by the nucleotide excision repair (5, 6, 7). However, upon DNA replication, some cells acquire transition mutations (C→T) and tandem double mutations (CC→TT) arising at dipyrimidine sites (6, 7). These mutations are frequently observed in UV-induced squamous cell carcinoma in mice and humans (5). Among a series of gene mutations (TP53, PITCH, and oncogenes) that are associated with UV-induced skin cancer, C→T and CC→TT point mutations in the p53 gene are most frequent (8, 9). UVR can induce several types of epidermal injury including sunburn cell (apoptotic cell) formation (8, 10). The sunburn cells can be initiated by UV-induced DNA damage and subsequent induction of p53 protein. The p53-dependent apoptosis of UV-damaged normal cells (sunburn cells) is prevented due to p53 mutation. Thus, these mutated cells can clonally expand to form squamous cell carcinoma after subsequent UVR exposures.
The tumor promotion component of UVR carcinogenesis, which involves clonal expansion of the initiated cells, is probably mediated by aberrant expression of genes altered during tumor initiation. UVR has been reported to alter the expression of genes regulating inflammation, cell growth and differentiation, and oncogenesis. Specific examples include up-regulation of the expression of p21 (WAF1/C1P1; ref. 10), p53 (8), AP-1 activation (11), ornithine decarboxylase (ODC; ref. 12), COX2 (13), tumor necrosis factor α (TNFα), and a wide variety of cytokines and growth factors (14). UVR-induced initial signals linked to the development of skin cancer are not defined. We found that PKCε overexpression in epidermal cells of FVB/N mice sensitizes the skin to UVR-induced cutaneous damage and development of squamous cell carcinoma.
PKC, a family of phospholipid-dependent serine/threonine kinases, is not only the major intracellular receptor for the mouse skin tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA; refs. 15 and 16) but also is activated by a variety of stress factors including UVR (15, 17). PKCε is among six isoforms (α, δ, ε, η, μ, and ζ) expressed in the mouse skin (18). To determine the in vivo functional specificity of PKCε in mouse skin carcinogenesis, we generated PKCε transgenic mouse (FVB/N) lines 224 and 215 that overexpress approximately 8- and 18-fold, respectively, PKCε protein over endogenous levels in basal epidermal cells (19, 20). PKCε transgenic mice were observed to be highly sensitive to the development of squamous cell carcinoma elicited by the 7,12-dimethylbenz(a)anthracene (100 nmol)–TPA (5 nmol) tumor promotion protocol (19, 20). We now summarize in this communication the data indicating that PKCε overexpression sensitizes skin to UVR-induced cutaneous damage and development of squamous cell carcinoma possibly at the promotion step of carcinogenesis, and this is probably accomplished by promoting the enhanced induction and release of specific cytokines such as TNFα.
MATERIALS AND METHODS
PKCε transgenic mice were generated as described previously (19, 20). Transgenic mice were maintained by mating hemizygous transgenic mice with wild-type FVB/N mice. TNFα knockout mice were obtained from the commercial supplier (The Jackson Laboratory, Bar Harbor, ME). The chimeric TNFα-deficient mice were bred for 10 generations for mutant transmission to FVB/N mice for unified genetic background. Each generation was genotyped, and only +/− mice were bred. Once the line was predominantly FVB/N, brother–sister matings were the most rapid way to generate homozygous mice, which were routinely checked for lack of expression of TNFα protein. TNFα knockout–FVB/N were cross-bred with PKCε transgenic (line 215) mice to generate TNFα knockout–PKCε transgenic mice.
Polymerase Chain Reaction Genotyping of Protein Kinase C ε Transgenic and Tumor Necrosis Factor α Knockout Mice.
Tail clips from PKCε transgenic or TNFα knockout mice were obtained and digested overnight using 600 μL of genomic lysis buffer [20 mmol/L Tris-HCl (pH 8), 150 mmol/L NaCl, 100 mmol/L EDTA, (pH 8), and 1% (w/v) SDS] and 3 μL of proteinase K (20 mg/mL; Gibco-BRL, Gaithersburg, MD) at 55°C. Three μL of 4 mg/mL RNase A [in 10 mmol/L Tris-HCl (pH 7.5) and 15 mmol/L NaCl] were added, mixed, and incubated at 37°C for 1 hour. Two hundred μL of protein precipitation solution (Gentra Systems Inc., Minneapolis, MN) were added to each sample, mixed, and placed on ice for 5 minutes. The samples were microcentrifuged at 14,000 rpm for 10 minutes at 4°C. The supernatant was decanted into a tube containing 600 μL of 100% isopropanol. The tubes were mixed and centrifuged for 5 minutes at 4°C. The pellets were washed with 70% ethanol, centrifuged, and air-dried. The pellets were resuspended in 300 to 500 μL of double distilled water and quantitated.
One hundred ng of genomic DNA were used for subsequent PCR genotyping of PKCε transgenic or TNFα knockout mice. All reactions were carried out in 50 μL of total volume and consist of reaction buffer [10 mmol/L Tris-HCl (pH 8), 2.5 mmol/L MgCl 2, and 50 mmol/L KCl], 200 mmol/L dNTPs (dATP, dCTP, dGTP, and dTTP), and 1 unit of HotStartTaq (Qiagen, Valencia, CA).
For analysis of the PKCε transgene, a 1-kb fragment between the T7 tag and the rabbit β-intron was amplified. Amplification conditions consisted of an initial hold at 95°C for 15 minutes, followed by 35 successive cycles of 30 seconds at 94°C (denaturing), 30 seconds at 60°C (annealing), and 30 seconds at 72°C (extension), with a final extension step of 72°C for 7 minutes. The PCR product was then run on a 1% agarose gel in 1× Tris-Acetate-EDTA at 70 volts for 1 hour.
For analysis of the TNFα knockout mice, the PCR strategy designed by The Jackson Laboratory was used. In brief, primers for the neomycin selectable marker were used to identify the knockout allele, whereas primers specific to the TNFα gene were used to identify the wild-type allele. Primers were mixed, and multiplexing PCR was performed. Amplification conditions consist of an initial hold at 95°C for 15 minutes; followed by 12 successive cycles of 20 seconds at 94°C (denaturing), 30 seconds at 64°C (annealing), and then 30 seconds at 72°C (extension); followed by 25 cycles at 20 seconds at 94°C (denaturing), 30 seconds at 58°C (annealing), and then 30 seconds at 72°C (extension) with a final extension step of 72°C for 7 minutes. The PCR products were then run on a 1.5% agarose gel in 1× Tris-Acetate-EDTA at 70 volts for 1 hour.
The mice were housed in groups of two to three in plastic bottom cages in light-, humidity-, and temperature-controlled rooms; food and water were available ad libitum. The animals were kept in a normal rhythm of 12-h-light and 12-h-dark periods. The UVR source was Kodacel-filtered FS-40 sun lamps (approximately 60% UVB and 40% UVA). UVR dose was routinely measured using UVX-radiometer. The dorsal skin of the mice was shaved 3 to 4 days before experimentation. Mice were used for experimentation at 7 to 9 weeks of age. Mice were exposed to UVR three times weekly (Monday, Wednesday, and Friday). If needed, mice were also shaved during the course of the tumor induction experiment. Tumor multiplicity was observed every other week. Carcinomas were recorded grossly as downward-invading lesions, which were confirmed histologically.
Quantitation of Deoxyribonucleic Acid Photodamage.
At appropriate times after UVR exposure, epidermal DNA was isolated and quantitated as described previously (6). The levels of cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidone dimers in the epidermal DNA were determined by radioimmunoassay in Dr. Mitchell’s laboratory (M. D. Anderson Cancer Center, Department of Carcinogenesis, Smithville, TX) as described previously (6).
Analyses of cytokines were performed by Linco-diagnostic by Multiplex Biomarker Assay using Luminex xMAP Technology (Linco Research, St. Charles, MO).
Assay of Ornithine Decarboxylase Activity.
For the assay of ODC activity from mouse epidermis, mice were sacrificed by cervical dislocation at the appropriate time after treatment, and the epidermis from individual mice was separated from the dermis by a brief heat treatment (57°C for 30 seconds). Epidermal preparations were homogenized in 50 mmol/L Tris-HCl buffer (pH 7.2) containing 0.1 mmol/L pyridoxal phosphate, 1 mmol/L dithiothreitol, and 0.1 mmol/L EDTA. The epidermal extracts were centrifuged at 30,000 × g for 15 minutes to give a soluble supernatant. Soluble epidermal ODC activity was determined by measuring the release of 14CO2 from dl-[1-14C]ornithine (21).
Real-Time Quantitative Polymerase Chain Reaction.
Total RNA was isolated using the RNeasy RNA isolation kit (Qiagen) and DNase treated, and 1 μg was used to prepare cDNA using Ready-to-Go reverse transcription-PCR beads (Amersham Biosciences, Arlington Heights, IL). Quantitative reverse transcription-PCR was performed by monitoring in real time the increase in fluorescence of the SYBR Green dye as described using the iCycler detection system (Bio-Rad, Cambridge, MA). We also quantified transcripts of the 18 s RNA as an endogenous RNA control, and each sample was normalized on the basis of its 18 s content.
The tissue to be examined was excised promptly after euthanasia and immediately placed in 10% neutral-buffered formalin (20). The tissue was fixed for at least 1 hour in formalin and then embedded in paraffin. Four-μm sections were cut for hematoxylin and eosin staining. Skin sections were analyzed by a board-certified anatomic pathologist.
Analysis of Proliferating Cell Nuclear Antigen-Positive Cells and Epidermal Thickness.
PKCε transgenic mice and their wild-type littermates were exposed to UVR (2 kJ/m2) four times (Monday, Wednesday, Friday, and Monday), and mice were sacrificed at 0.5, 1, 3, 6, 12, 24, and 48 hours after the fourth treatment. There were two mice per treatment group. Skin specimens were fixed in 10% neutral-buffered formalin for 24 hours and embedded in paraffin. Four-μm-thick sections were cut for proliferating cell nuclear antigen (PCNA) staining, as described below.
The slides were incubated overnight at 4°C with primary antibodies. The primary antibodies used required antigen retrieval pretreatment by incubating samples in 95°C Tris-urea solution for 35 minutes. Subsequent incubation steps were performed in a moist chamber at room temperature. After intermediate washing steps in Tris-buffered saline (pH 7.4), the sections were incubated with biotin-labeled rabbit antimouse immunoglobulin G for 15 minutes at room temperature and then with streptavidin-peroxidase complexes for 15 minutes at room temperature. Visualization was performed using diaminobenzidine as a substrate for the peroxidase reaction. Slides were transferred into tap water and counterstained with hematoxylin for 4 minutes. Negative controls were included for each study and used normal mouse serum. No immunoreactivity was observed in these control sections. Specimens were analyzed using an Olympus BX 51 microscope.
For the quantitation of PCNA-positive staining cells, ten random areas were selected for each mouse at each time point. The number of cells demonstrating positive labeling and the total number of cells counted (1000) were recorded. An average percentage was then calculated based on the total number of cells and the number of positive staining cells from each set of 10 fields counted. Results are expressed as mean of percentages ± SEM.
For measurement of epidermal thickness, two random areas were selected for each mouse at each time point. Pictures were taken with a Nikon 35-mm camera. Microsoft Photo Editor software was used to measure skin thickness. The unit for skin thickness was pixel number. Each value represents the average of 10 measurements for each mouse. Results are expressed as mean of pixel number ± SEM.
Sensitivity of Protein Kinase C ε Transgenic Mice to the Development of Squamous Cell Carcinoma Elicited by Repeated Exposures to Ultraviolet Radiation.
PKCε transgenic line 215, which overexpresses about 18-fold PKCε protein more than wild-type littermates in the epidermis, elicited severe cutaneous damage after exposure to UVR (Fig. 1). The UVR source was Kodacel-filtered FS-40 sunlamps, which emit approximately 60% UVB and 40% UVA. UVR-induced skin damage in high PKCε-overexpressing mice (line 215) after exposure to UVR dose (either 1 or 2 kJ/m2) was extensive and irreparable, and the experiment could not be continued until the appearance of carcinomas (Fig. 1). However, the low PKCε-overexpressing mice (line 224) tolerated three times weekly UVR exposures (2 kJ/m2) for 38 weeks and, compared with their wild-type littermates, elicited increased squamous cell carcinoma multiplicity by 3-fold and decreased tumor latency by 12 weeks (Fig. 2).
A Comparison of Ultraviolet Radiation-Induced Deoxyribonucleic Acid Photodamage of Protein Kinase C ε Transgenic Mice and Their Wild-Type Littermates.
UVR-induced DNA damage in PKCε transgenic mice (lines 215 and 224) was compared with their wild-type littermates. In this experiment (Fig. 3), the mice were exposed to UVR (2 kJ/m2) only once and were sacrificed at the indicated times after UVR treatment. The levels of cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidone dimers in the epidermal DNA were determined by the radioimmunoassay as described previously (6). UVR-induced levels of both cyclobutane pyrimidine dimers (Fig. 3,A) and pyrimidine (6-4) pyrimidone dimers (Fig. 3,B) in PKCε-transgenic mice (line 215) were significantly higher (P < 0.01) as early as 0.5 hour after treatment. UVR-induced levels of both cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidone dimers in wild-type and PKCε-transgenic mice (line 215) were not significantly different (P = 0.925) at 1 hour post UVR exposure. At 24 hours post UVR treatment, PKCε transgenic mice (line 215) had significantly lower levels of cyclobutane pyrimidine dimers (Fig. 3,A) and pyrimidine (6-4) pyrimidone dimers (Fig. 3,B; P < 0.01). In a separate experiment (Fig. 3,C and D), PKCε mice (line 224) were exposed only once to UVR (2 kJ/m2). Mice were sacrificed at 1, 3, 6, 16, and 24 hours post UVR treatment. The levels of cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidone dimers in the epidermal DNA were analyzed. At all time points post UVR treatment, UVR-induced levels of cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidone dimers in PKCε transgenic mice and wild-type littermates were similar (Fig. 3 C and D).
UVR-induced DNA damage (cyclobutane pyrimidine dimers) in PKCε transgenic mice (line 215) was also compared with their wild-type littermates after chronic UVR exposure. In this experiment (Fig. 3,E and F), the mice were exposed to UVR (2 kJ/m2) four times (Monday, Wednesday, Friday, and Monday). Mice were sacrificed at the indicated times after the 4th UVR treatment. The levels of cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidone dimers in the epidermal DNA were determined by the radioimmunoassay as described previously (6). UVR-induced levels of cyclobutane pyrimidine dimers (Fig. 3,E) and pyrimidine (6-4) pyrimidone dimers (Fig. 3,F) in PKCε-transgenic mice were significantly lower (P < 0.01) at all time points (0.5, 1, 3, 6, 12, 24, and 48 hours) after treatment. UVR-induced cyclobutane pyrimidine dimers in wild-type and PKCε-transgenic mice reached a peak level (18-fold) at 0.5 and 3 hours, respectively, after treatment (Fig. 3,A). At 48 hours post UVR treatment, PKCε transgenic mice and their wild-type littermates have about 80% cyclobutane pyrimidine dimers repaired (Fig. 3,E). Similar results were observed with pyrimidine (6-4) pyrimidone dimers (Fig. 3 F).
Protein Kinase C ε Overexpression Induces Epidermal Proliferative Markers Proliferating Cell Nuclear Antigen and Ornithine Decarboxylase.
We explored the possibility that UVR sensitivity of PKCε transgenic mice may be the result of imbalance between cell proliferation and cell death. In this experiment (Figs. 4 and 5), PKCε transgenic mice and wild-type littermates were exposed to UVR (2 kJ/m2) four times. At the indicated times after the fourth UVR exposure, dorsal skin was removed and fixed in 10% formalin for the analysis of PCNA-positive cells and epidermal thickness (Figs. 4,A–C) or processed for assays of ODC (Fig. 4,D) and ODC mRNA (Fig. 4,D, inset). It is noteworthy that the zero-hour time point in these experiments (Figs. 4 and 5) is in fact 72 hours after the third UVR exposure. The percentage of PCNA-positive cells at zero time in PKCε transgenic mice was significantly higher (P < 0.01) than their wild-type littermates. The percentage of PCNA-positive cells in PKCε transgenic mice at 12, 24, and 48 hours after the fourth UVR treatment was not significantly different (P > 0.1) compared with the zero time point. In contrast, in wild-type mice, the percentage of PCNA-positive cells at 48 hours post UVR exposure was significantly higher (P < 0.001) compared with 0, 12, and 24 hours post UVR treatment (Fig. 4,B). Similarly, UVR-induced skin thickness at zero time point in PKCε transgenic mice was significantly greater than their wild-type littermates. The epidermal thickness in PKCε transgenic mice at 12, 24, and 48 hours after the fourth UVR treatment was not significantly different (P > 0.1) compared with the zero time point. In contrast, in wild-type mice, the epidermal thickness at 48 hours post UVR exposure was significantly greater (P < 0.001) compared with 0, 12, and 24 hours post UVR treatment (Fig. 4 C).
ODC, which decarboxylates ornithine to form putrescine, is the key enzyme in mammalian polyamine biosynthesis (22, 23). UVR-induced ODC activity has been well documented to be the essential component of the mechanisms of UVR carcinogenesis (21, 22, 23). This prompted us to compare the response of PKCε transgenic mice to their wild-type littermates for UVR-induced ODC activity. These results are illustrated in Fig. 4,D. UVR-induced levels of epidermal ODC mRNA correlated with the level of expression of PKCε protein in PKCε transgenic mouse lines (Fig. 4,D, inset). Maximum ODC activity, about an 11-fold increase over wild-type levels, in PKCε transgenic mice (215) was observed between 6 and 24 hours after the fourth UVR exposure. A significant increase in ODC activity in PKCε transgenic mice was still observed 48 hours post UVR exposure (Fig. 4 D).
Protein Kinase C ε Overexpression Suppresses Ultraviolet Radiation-Induced Formation of Sunburn Cells.
We determined the effects of chronic UVR exposures on the appearance of sunburn cells in the epidermis (Fig. 5). Sunburn cells, which appear in the epidermis after UVR exposures, are the keratinocytes undergoing apoptosis (8). Sunburn cells were identified in hematoxylin and eosin–stained histologic sections of the skin by their intensely eosinophilic cytoplasm and small and dense nuclei (Fig. 5,A and B). The UVR-induced percentage of sunburn cells in PKCε transgenic mice was significantly lower than their wild-type littermates (Fig. 5 C).
A Comparison of Ultraviolet Radiation-Induced Levels of Cytokines in Protein Kinase C ε Transgenic Mice and Their Wild-Type Littermates.
UVR has been shown to induce epidermal keratinocytes to release proinflammatory cytokines [interleukin (IL)-1 and TNFα], chemotactic cytokines [IL-6, IL-7, IL-15, granulocyte macrophage colony-stimulating factor (GM-CSF), and TNFα], and cytokines regulating immunity (IL-10, IL-12, and IL-18). As shown in Fig. 6, PKCε transgenic mice were more sensitive than their wild-type littermates to induce TNFα, granulocyte colony-stimulating factor (G-CSF), and GM-CSF levels after UVR exposure. The UVR-induced levels of epidermal TNFα mRNA and the TNFα protein correlated with the level of expression of PKCε protein in the transgenic mouse lines (Fig. 6). We also found that PKCε transgenic mice were more sensitive than their wild-type littermates to induce the release of serum levels of cytokines (TNFα, G-CSF, GM-CSF, IL-5, IL-6, and IL-10) after UVR exposure (data not shown).
The Link of Tumor Necrosis Factor α to Ultraviolet Radiation Cutaneous Damage of Protein Kinase C ε Transgenic Mice (Line 215).
To directly determine the role of TNFα in UVR-induced cutaneous damage in PKCε transgenic mice, we generated TNFα-deficient-PKCε transgenic mice by cross-breeding TNFα knock-out mice with PKCε transgenic mice (Fig. 7,B). UVR induced TNFα levels in wild-type mice but not in TNFα-deficient-PKCε transgenic mice (Fig. 7,A). UVR treatment three times weekly for 13 weeks at 2 kJ/m2 induced severe cutaneous damage in PKCε transgenic mice (Fig. 7,C), which was decreased in TNFα-HT and TNFα knockout-PKCε transgenic mice (Fig. 7 C).
The dorsal skin of UVR-exposed PKCε transgenic mice (Fig. 7,D) exhibited severely hyperplastic interfollicular epidermis with alternating regions of ulceration associated with severe scaring. The scar tissue also contained remarkable amounts of inflammatory infiltrate. In addition, the skin histopathology exhibited a disorganization of the hair follicle and hyperplasia of the bulb region (Fig. 7,D). Skin samples collected from PKCε transgenic-TNFα heterozygous (PKCε TNFαHT) mice had intact epidermis, with associated hyperplasia in both the follicular and interfollicular epidermis. The PKCε transgenic-TNFα knockout (PKCε TNFαKO) skin was intact and showed significant reduction in both the interfollicular and follicular hyperplasia, relative to the PKCε transgenic mice. In all three mouse lines, the sebaceous glands seemed to be hyperplastic (Fig. 7 D).
PKC represents a large family of phosphatidylserine-dependent serine/threonine kinases (15, 17, 18). Based on structural similarities and cofactor dependence, 11 PKC isoforms have been classified into three subfamilies: the classical, the novel, and the atypical (18). The classical PKCs (α, βI, βII, and γ) are dependent on phosphatidylserine, diacylglycerol, and Ca2+. The novel PKCs (δ, ε, η, and θ) retain responsiveness to diacylglycerol and phosphatidylserine but do not require Ca2+ for full activation. The atypical PKCs (λ and σ) only require phosphatidylserine for their activation (18). PKC isoforms exhibit functional specificity in their signals to oncogenesis (19, 20, 21, 24). PKCε participates in the regulation of diverse cellular functions including gene expression (25, 26, 27), cell adhesion (28), mitogenicity (29, 30), and cellular motility (31). PKCε is among the six (18) isoforms expressed in mouse epidermis. PKCε has been shown to promote malignant transformation (20). For example, overexpression of PKCε in Rat-6 NIH-3T3 fibroblasts led to increases in growth rates, anchorage independence, and tumor formation in nude mice (32, 33). Additionally, PKCε overexpression transformed nontumorigenic rat colonic epithelial cells (34). Overexpression of PKCε results in transformed androgen-dependent LNCaP tumor cells to androgen-independent cells (35). We previously reported that PKCε overexpression increases the susceptibility of FVB/N mice to develop squamous cell carcinoma (19, 20). We now present that PKCε is an endogenous photosensitizer that enhances UVR-induced cutaneous photodamage and development of squamous cell carcinomas.
The PKCε transgenic mouse line 215, which overexpressed 18-fold PKCε protein than wild-type littermates, elicited severe cutaneous damage with a UVR dose as low as 1 kJ/m2. UVR-induced skin wounds in high PKCε expressing transgenic line 215 were irreparable, and the experiment could not be continued further until the development of squamous cell carcinoma (Fig. 1). Cutaneous damage in low PKCε-expressing transgenic line 224 was less pronounced than line 215. Compared with wild-type littermates, the PKCε transgenic mice (line 224) elicited increased squamous cell carcinoma multiplicity by 3-fold and decreased tumor latency by 12 weeks (Fig. 2).
UVR-induced DNA photodamage did not correlate with the photosensitivity of PKCε transgenic mouse line 215. A single UVR exposure induced a significantly higher amount of both cyclobutane pyrimidine dimer (Fig. 3,A) and pyrimidine (6-4) pyrimidone dimer (Fig. 3 B) in PKCε transgenic mouse line 215. However, UVR-induced levels of both cyclobutane pyrimidine dimer and pyrimidine (6-4) pyrimidone dimer in PKCε mouse line 224 were similar to their wild-type littermates. Furthermore, chronic UVR exposure in PKCε transgenic mouse line 215, compared with the wild-type littermates, suppressed DNA photodamage. A simple explanation for lower amount of cyclobutane pyrimidine dimer and pyrimidine (6-4) pyrimidone dimer in PKCε transgenic mouse line 215 may be due to a significantly greater amount of hyperplasia causing a decrease in the amount of UVR to penetrate the epidermis. There is probably no difference in UVR-induced DNA damage between the PKCε transgenic and wild-type mice. Evidence indicates that UVR-induced cyclobutane pyrimidine dimer and pyrimidine (6-4) pyrimidone dimer are the predominant mutagenic lesions and are linked to the induction of skin cancer in both mice and human (5, 6, 7). It is also likely that DNA lesions other than cyclobutane pyrimidine dimer and pyrimidine (6-4) pyrimidone dimer may be involved in UVR-induced development of squamous cell carcinoma in PKCε transgenic mice. However, the present results do not provide evidence against the role of the initiation step of UVR carcinogenesis in determining the sensitivity of PKCε transgenic mice to UVR-induced cutaneous damage and induction of squamous cell carcinoma. The foregoing discussion emphasizes the role of promotion steps of UVR carcinogenesis in explaining the enhanced susceptibility of PKCε transgenic mice to UVR carcinogenesis.
Under similar UVR treatments, PKCε transgenic mice were more susceptible to UVR-induced hyperplasia than their wild-type littermates (Fig. 4). However the number of sunburn cells was decreased in UVR-treated skin of PKCε transgenic mice. Thus, PKCε overexpression in mouse epidermis seems to induce epidermal cell proliferation but suppresses apoptosis (Fig. 5). The ability to induce hyperplasia is one of the properties of skin tumor promoters (36). Consistent with these findings, the TPA-induced epidermal hyperplasia in PKCε transgenic mice has been shown to be increased by 50% compared with wild-type mice. Increased cell proliferation is an essential component of the mechanism of carcinogenesis (37). UVR-induced hyperplasia may be linked to the photosensitivity of PKCε transgenic mice.
PKCε transgenic mice were observed to be highly sensitive to UVR-induced increased levels of ODC and TNFα. TNFα release was proportional to the level of expression of the transgene PKCε. ODC and TNFα are well-documented mediators of skin tumor promotion by TPA and UVR treatments (37, 38, 39, 40, 41). In this context, it is noteworthy that DFMO, a suicide inhibitor of ODC, completely prevented the UVR-induced development of skin tumors in SKH mice (42). Also, TNFα knockout mice were resistant to skin carcinogenesis (37, 38, 39). It is likely that UVR-induced expression of TNFα is linked to the development of squamous cell carcinoma in PKCε transgenic mice.
PKCε transgenic mice were also observed to be more sensitive than their wild-type littermates to UVR-induced release of cytokines (IL-5, IL-6, G-CSF, GM-CSF, and IL-10) other than TNFα. It is likely that there is cross-talk among cytokines. It is notable that TNFα has been shown to regulate the production of several cytokines. In this context, the work of Marino et al. (43) with TNF-deficient mice is noteworthy. In the findings of Marino et al. (43), lipopolysaccharide-induced serum levels of cytokine IL-1β, IL-6, IL-10, IL-12, and interferon-γ were not altered in TNF-deficient mice. TNFα-deficient mice are resistant to the induction of skin cancer elicited by 7,12-dimethylbenz(a)anthracene-TPA (or okadaic acid) or by complete carcinogenesis protocol (37, 38, 39). Taken together, it seems that TNFα is a key proinflammatory cytokine linked to the induction of skin cancer. Recently, we have also reported that TNFα may play a role in TPA-promoted development of metastatic squamous cell carcinoma in PKCε transgenic mice (44). The results with the PKCε transgenic–TNFα knockout mice (Fig. 7) indicate that PKCε signals UVR-induced TNFα release that is linked at least in part to the photosensitivity of PKCε transgenic mice.
The mechanism by which PKCε activation transduces signals for TNFα release is unknown. The biological effects of UVR have been linked to the up-regulation of mitogen-activated protein kinases. In this context, the pioneering work of Dong et al. (45, 46, 47, 48) is noteworthy. They reported that UVR induces functional activation of mitogen-activated protein kinases (extracellular signal-regulated kinases and p38), which phosphorylate ribosomal kinases and p53 and activate PI3K (45, 46, 47, 48, 49, 50). UVR-induced downstream signaling components that are mediated by the mitogen-activated protein kinase family include activation of immediate early genes c-fos and c-jun and transcription factors AP-1 and nuclear factor κB (NFκB; ref. 11). UVR also up-regulates STAT-3 and NFAT transcription factors (51). There is now direct evidence that PKCε may mediate its oncogenic properties by directly activating the classic mitogenic signaling pathway involving Ras and Raf-1 kinase (29, 30, 52, 53, 54). Alternatively, TFGβ family members have been proposed to be, in part, responsible for the downstream effects of PKCε (27). Rat-6 fibroblasts, which overexpress PKCε, have been shown to secrete active forms of TGFβ2 and TGFβ3 in conjunction with an as yet unidentified mitogen, indicating that growth-stimulating autocrine/paracrine loops may be involved in the oncogenic activity of PKCε (27).
TNFα signal transduction pathways in UVR-induced cutaneous damage and development of squamous cell carcinoma are not known. TNFα mediates the activation of two transcriptional factors, AP-1 and NFκB, linked to the expression of TNFα-induced genes involved in immunity and inflammatory responses and control of cellular proliferation, differentiation, and apoptosis (55, 56). The role of AP-1 and NFκB activation in TNFα signal transduction pathway to the development of squamous cell carcinoma and PKCε transgenic mice is unknown and is important in view of the fact that NFκB activation is an oncogenic signal in systems other than skin (55, 56).
In summary, targeted overexpression of PKCε in epidermis sensitizes PKCε transgenic mice to UVR-induced cutaneous damage and development of squamous cell carcinoma. UVR is a complete carcinogen, and PKCε seems to impart photosensitivity at the promotion step of UVR carcinogenesis, probably involving interplay of several mechanisms including the role of specific cytokines such as TNFα. A major mechanism of UVR carcinogenesis also constitutes oxidative stress with generation of free radicals, leading to lipid and DNA damage and gene mutation (57). Inability to metabolize free radicals due to defects in detoxifying enzymes (e.g., glutathione S-transferases) may increase susceptibility to cutaneous carcinogenesis (58). Furthermore, impairment of immune responses may also predispose skin cancer (59). PKCε transgenic mice provide a useful model to investigate the molecular components of UVR carcinogenesis.
Grant support: NIH grants CA35368 and CA102431.
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