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[Cancer Research 66, 713-722, January 15, 2006]
© 2006 American Association for Cancer Research

Molecular Biology, Pathobiology, and Genetics

Protein Kinase C {delta} Overexpressing Transgenic Mice Are Resistant to Chemically but not to UV Radiation–Induced Development of Squamous Cell Carcinomas: A Possible Link to Specific Cytokines and Cyclooxygenase-2

Moammir H. Aziz1, Deric L. Wheeler2, Bhushan Bhamb1 and Ajit K. Verma1

1 Department of Human Oncology, Medical School, University of Wisconsin, Madison, Wisconsin and 2 Fred Hutchinson Cancer Research Center Division of Basic Sciences, Seattle, Washington

Requests for reprints: Ajit K. Verma, Department of Human Oncology, Medical School, University of Wisconsin, Madison, WI 53792. Phone: 608-263-9163; Fax: 608-262-6654; E-mail: akverma{at}facstaff.wisc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Protein kinase C {delta} (PKC{delta}), a Ca2+-independent, phospholipid-dependent serine/threonine kinase, is among the novel PKCs ({delta}, {varepsilon}, and {eta}) expressed in mouse epidermis. We reported that FVB/N transgenic mice that overexpress (~8-fold) PKC{delta} protein in basal epidermal cells and cells of the hair follicle are resistant to the development of both skin papillomas and squamous cell carcinoma (SCC) elicited by 7,12-dimethylbenz(a)anthracene initiation and 12-O-tetradecanoylphorbol-13-acetate (TPA) promotion protocol. We now present that PKC{delta} overexpression in transgenic mice failed to suppress the induction of SCC developed by repeated exposures to UV radiation (UVR), the environmental carcinogen linked to the development of human SCC. Both TPA and UVR treatment of wild-type mice (a) increased the expression of proliferating cell nuclear antigen (PCNA) and apoptosis; (b) stimulated the expression of cytokines tumor necrosis factor-{alpha} (TNF-{alpha}), granulocyte macrophage colony-stimulating factor (GM-CSF), and granulocyte CSF (G-CSF); and (c) increased cyclooxygenase-2 (COX-2) expression and expression of phosphorylated Akt (p-Akt), p38, extracellular signal-regulated kinase-1 (ERK1), and ERK2. PKC{delta} overexpression in transgenic mice enhanced TPA-induced but not UVR-induced apoptosis and suppressed TPA-stimulated but not UVR-stimulated levels of cell PCNA, cytokines (TNF-{alpha}, G-CSF, and GM-CSF), and the expression of COX-2, p-Akt, and p38. The results indicate that UVR-mediated signal transduction pathway to the induction of SCC does not seem to be sensitive to PKC{delta} overexpression. The proapoptotic activity of PKC{delta} coupled with its ability to suppress TPA-induced expression of proinflammatory cytokines, COX-2 expression, and the phosphorylation of Akt and p38 may play roles in the suppression of TPA-promoted development of SCC. (Cancer Res 2006; 66(2): 713-22)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Protein kinase C (PKC) represents a large family of phosphatidylserine-dependent serine/threonine kinases (1–4). Based on structural similarities and cofactor dependence, at least 11 PKC isoforms have been classified into three subfamilies: the classic (cPKC), the novel (nPKC), and the atypical (aPKC). The cPKCs ({alpha}, ßI, ßII, and {gamma}) are dependent on phosphatidylserine, diacylglycerol (DAG), and Ca2+. The nPKCs ({delta}, {varepsilon}, {eta}, and {theta}) retain responsiveness to DAG and phosphatidylserine but do not require Ca2+ for full activation. The aPKCs ({lambda} and {varsigma}) only require phosphatidylserine for their activation. At least five PKC isoforms ({alpha}, {delta}, {varepsilon}, {eta}, and {zeta}) are expressed in epidermal keratinocytes (5). PKC isoforms are differentially expressed in proliferative (basal layer) and nonproliferative compartments (spinous, granular, cornified layers), which exhibit divergence in their roles in the regulation of epidermal cell proliferation, differentiation, and apoptosis. Immunocytochemical localization of PKC isoforms indicate that PKC{alpha} is found in the membranes of suprabasal cells in the spinous and granular layers. PKC{varepsilon} is mostly localized in the proliferative basal layers. PKC{eta} is localized exclusively in the granular layer. PKC{delta} is detected throughout the epidermis (5).

To determine the in vivo functionality of PKC{alpha}, PKC{delta}, and PKC{varepsilon} in mouse skin carcinogenesis, we generated transgenic mice overexpressing an epitope-tagged PKC ({alpha}, {delta}, or {varepsilon}) under the control of human keratin 14 promoter (6–8). Transgenic mice overexpressing PKC{alpha} were also generated by other laboratories (9, 10). The susceptibility of PKC ({alpha}, {delta}, or {varepsilon}) overexpressing mice to skin carcinogenesis was determined by initiation with 7,12-dimethylbenz(a)anthracene (DMBA) and promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA; refs. 6–8). PKC{alpha} overexpression has no effect on skin tumor promotion susceptibility (8–10). PKC{delta} and PKC{varepsilon} transgenic mice exhibited different responses to skin tumor promotion by TPA. PKC{delta} suppressed both formation of skin papillomas and carcinomas (6). PKC{varepsilon} transgenic mice developed only squamous cell carcinomas (SCC), which could metastasize (7, 11). These results of the different roles of PKC{alpha}, PKC{delta}, and PKC{varepsilon} in the induction of skin cancer by DMBA-TPA protocol are not highly relevant to human skin cancer, which is mostly linked to chronic exposure to UV radiation (UVR; refs. 12, 13).

UVA (315-400 nm), UVB (280-315 nm), and UVC (190-280 nm) are the three components of the UV spectrum (12, 13). Because stratospheric ozone absorbs most of the radiation below 310 nm (UVC), the 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 (12, 13). Chronic exposure to UVR is the most common etiologic factor linked to the development of SCCs and basal cell carcinomas (BCC), the most common nonmelanoma forms of human skin cancer (14). SCC, unlike BCC, invades the nearby tissues (14). 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 SCC and BCC is low, it still poses a significant societal risk (14). We determined the sensitivity of PKC{delta} and PKC{varepsilon} overexpressing transgenic mice to the development of skin cancer by chronic exposure to UVR. We found that PKC{varepsilon} expression also sensitizes skin to UVR-induced cutaneous damage and development of SCC (15, 16). In contrast, PKC{delta} overexpression failed to affect UVR carcinogenesis, which forms the focus of this presentation. We present data in this communication, indicating that (a) PKC{delta} overexpressing transgenic mice are not resistant to UVR-induced mouse skin carcinogenesis, (b) both TPA and UVR tumor promotion involve common molecular targets [e.g., cytokines: TNF-{alpha}, granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage CSF (GM-CSF), cyclooxygenase-2 (COX-2), and phosphorylated Akt (p-Akt), p38, ERK1, and ERK2] possibly regulated by unique signal transduction pathways, and (c) PKC{delta} overexpression inhibits TPA-stimulated but not UVR-stimulated expression of these molecular targets.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
TPA was purchased from Alexis Corp. (San Diego, CA). DMBA was purchased from Aldrich Chemical Co., Inc. (Milwaukee, WI). The acrylamide, bisacrylamide, SDS, 0.45 µmol/L supported nitrocellulose membrane, Bio-Rad Protein Assay kit, and SDS-PAGE standards were purchased from Bio-Rad Laboratories (Hercules, CA). The source of antibodies were proliferating cell nuclear antigen (PCNA; DAKO Corp., Carpinteria, CA), COX-1, COX-2 (Cayman Chemical, Ann Arbor, MI); PKC{delta}, p38, p-p38, ERK1/2, p-ERK1/2, ß-actin (Santa Cruz Biotechnology, Santa Cruz, CA), and Akt and p-Akt (Ser473; Cell Signaling Technology, Beverly, MA). The enhanced chemiluminescence Western blotting reagents were purchased from Amersham Life Sciences, Inc. (Arlington Heights, IL). FS-40 sunlamps were purchased from National Biological/ETA Systems (Twinsburg, OH). Kodacel filter was purchased from Eastman Kodak Co. (Rochester, NY). FVB/N mice were purchased from Taconic (Germantown, NY).

PKC{delta} Transgenic Mice
PKC{delta} transgenic mice were generated as described previously (6–8). All animal protocols were approved by the institutional review board. Transgenic mice were maintained by mating hemizygous transgenic mice with wild-type FVB/N mice. The mice were housed in groups of two to three in plastic-bottomed cages in light-controlled, humidity-controlled, and temperature-controlled rooms; food and water were available ad libitum. The animals were kept in a normal rhythm of 12-hour light and 12-hour dark periods. The transgene was detected by PCR analysis using genomic DNA isolated from 1-cm tail clips (17).

Tumor Induction Experiments
Skin carcinogenesis by DMBA initiation and TPA promotion protocol. Mouse skin tumors were induced by the initiation-promotion regimen (6–8). For mouse skin tumor initiation, a single dose (100 nmol) of DMBA in 0.2 mL of acetone was applied topically to the shaved backs of mice. Two weeks after initiation, TPA (5 nmol) in 0.2 mL of acetone was applied twice weekly to the skin for the duration of the experiment.

Skin carcinogenesis by repeated UVR exposures. The UVR source was Kodacel-filtered FS-40 sun lamps (~60% UVB and 40% UVA). Mice were exposed to UVR (2 kJ/m2) from a bank of six Kodacel-filtered sunlamps. UVR dose was routinely measured using a UVX radiometer. Mice were used for experimentation at 7 to 9 weeks of age. The dorsal skin of the mice was shaved 3 to 4 days before experimentation. Mice were exposed to UVR thrice weekly (Monday, Wednesday, and Friday). If needed, mice were also shaved during the course of the tumor induction experiment. Tumor multiplicity was observed and recorded every other week. Carcinomas were recorded grossly as downward-invading lesions, which were confirmed histologically (16, 17).

Histology
The tissue to be examined was excised promptly after euthanasia and placed immediately in 10% neutral buffered formalin. The tissue was fixed for 24 hours in formalin and then embedded in paraffin. Sections of 4-µm thickness were cut for H&E staining. Specimens were analyzed using an Olympus BX 51 microscope. Sunburn cells (apoptotic cells) in the UVR-treated skin were identified as intensely eosinophilic cytoplasm and small and dense nuclei. Dead cells in the DMBA-TPA treated skin were scored as densely stained cells with condensed nuclei.

PKC{delta} Immunohistochemistry
The tissue to be examined was excised promptly after euthanasia and immediately placed in 10% neutral buffered formalin. The tissue was fixed for 1 hour in formalin and then embedded in paraffin. Four-micrometer sections were cut for PKC{delta} immunostaining. Deparaffinized slides were used for immunostaining with the DAKO immunoperoxidase LSAB1 kit. Endogenous peroxidase activity was blocked with 3% H2O2. Nonspecific binding was blocked with normal swine serum. The slides were incubated overnight with the PKC{delta} antibody and developed with biotinylated secondary antibody, streptavidin-conjugated horseradish peroxidase, and 3,3'-diaminobenzidine. Specimens were analyzed using an Olympus BX 51 microscope.

Analysis of PCNA-positive Cells by Immunohistochemistry
PKC{delta} transgenic mice and their wild-type littermates were exposed to UVR (2 kJ/m2) four times (Monday, Wednesday, Friday, and Monday) or treated topically with TPA (5 nmol in 0.2 mL acetone) thrice (Monday, Thursday and Monday). The mice were sacrificed at 1, 5, and 24 hours after the fourth UVR treatment as well as 1 and 5 hours after the third TPA treatment. Skin specimens were fixed in 10% neutral buffered formalin for 24 hours and embedded in paraffin. Four-micrometer-thick sections were cut for PCNA staining. Deparaffinized slides were used for immunostaining using DAKO immunoperoxidase LSAB1 Kit (DAKO) as described below.

Endogenous peroxidase activity was blocked with 3% H2O2. Nonspecific protein binding was blocked with normal swine serum. After antigen retrieval by incubating samples in 95°C Tris-urea solution for 35 minutes, the slides were incubated overnight with the appropriate primary antibody in moisture chamber. Subsequent incubation steps were done in a moist chamber at room temperature. After intermediate washing steps in TBS (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 done 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 using 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, 10 random areas were selected for each mouse at each time point. The number of cells showing positive labeling and the total number of cells counted (1,000) 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 ± SE.

Western Blot Analysis
Mice were shaved and depilated before experimentation. The mouse skin was excised and scraped to remove the s.c. fat. The epidermis was scraped off and homogenized in the lysis buffer [50 mmol/L HEPES, 150 mmol/L NaCl, 10% glycerol, 1% Triton X-100, 1.5 mmol/L MgCl2, 10 µg/mL aprotinin, 10 µg/mL leupeptin, 1 mmol/L phenylmethylsulfonyl fluoride, 200 µmol/L Na3VO4, 200 µmol/L NaF, and 1 mmol/L EGTA (final pH 7.5)]. The homogenate was centrifuged at 14,000 x g for 30 minutes at 4°C. Twenty micrograms of cell lysate were fractionated on 10% Criterion precast SDS-polyacrylamide gel (Bio-Rad Laboratories). The proteins were transferred to 0.45 µm Hybond-P polyvinylidene difluoride transfer membrane (Amersham Life Sciences). The membrane was then incubated with the indicated antibodies followed by a horseradish peroxidase secondary antibody (Santa Cruz Biotechnology), and the detection signal was developed with Amersham's enhanced chemiluminescence reagent and autoradiography using BioMax film obtained from Kodak. The quantitation of Western blots was done by densitometric analysis using Tatallab Nonlinear Dynamic Image analysis software (Nonlinear USA, Inc., Durham, NC).

Cytokine Analysis
At appropriate times after treatments, the PKC{delta} transgenic mice and their wild-type littermates were sacrificed. The mouse skin was excised and scraped to remove s.c. fat. The epidermis was scraped off and homogenized in the lysis buffer [50 mmol/L HEPES (pH 7.5), 150 mmol/L NaCl, 10% glycerol, 1% Triton X-100, 1.5 mmol/L MgCl2, 10 µg/mL leupeptin, 1 mmol/L phenylmethylsulfonyl fluoride, 200 µmol/L Na3VO4, 200 µmol/L NaF, and 1 mmol/L EGTA]. The homogenate was centrifuged at 14,000 x g for 30 minutes at 4°C. The analyses of cytokines from the epidermal extracts were preformed by Linco diagnostics using the Luminex Multi-Analyte Detection Assay (St. Charles, MO).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PKC{delta} overexpression in transgenic mice does not suppress development of SCC elicited by repeated UVR exposures. We compared the susceptibility of PKC{delta} overexpressing transgenic mice and their wild-type littermates to the induction of skin tumors developed by either DMBA-TPA protocol or by repeated UVR exposures. In these experiments, both male and female FVB/N transgenic mice that overexpress about 8-fold PKC{delta} protein and PKC{delta} activity above their wild-type littermates were either exposed to UVR (2 kJ/m2) thrice weekly or treated once with DMBA and then twice weekly with TPA (5 nmol) for 35 weeks. There were at least 20 mice per group. Kaplan-Meier analysis of carcinoma incidence is illustrated in Fig. 1A and B. At 35 weeks, the UVR-induced carcinoma incidence in male PKC{delta} transgenic mice and their wild-type littermates were 82% and 63%, respectively (P = 0.17). Similar results were observed in female mice. In contrast, both the papilloma multiplicity (data not shown) and the carcinoma incidence in PKC{delta} transgenic mice were decreased about 80% compared with wild-type mice in DMBA-TPA groups (Fig. 1A and B).


Figure 1
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  		Figure 1. Effects of PKC{delta} overexpression in FVB/N transgenic mice on the induction of SCC elicited either by DMBA-TPA protocol or by repeated exposure to UVR. Kaplan-Meier survival analyses of carcinoma data. PKC{delta} transgenic mice and their wild-type littermates were used for the induction of skin tumors. Mouse skin tumors in mice were elicited either by the DMBA-TPA (A) protocol or by repeated exposures to UVR (B) as described in Materials and Methods. There were 20 mice per treatment group. Carcinoma incidence data were statistically analyzed using Wilcoxon rank sum test. The number of UVR-induced SCC in PKC{delta} transgenic mice was not statistically different at all weeks compared with the wild-type littermates (P > 0.1). Expression of PKC{delta} protein in paraffin-embedded SCC specimens by immunocytochemistry (C and D).

 
PKC{delta} expression in carcinomas elicited by either DMBA-TPA protocol or repeated exposure to UVR. We have previously reported the analysis of PKC{delta} protein in skin tumors developed in PKC{delta} overexpressing mice and their wild-type littermates (6). The level of PKC{delta} remained constant in the epidermis of wild-type mice even after 25 weeks of biweekly treatment. Expression of endogenous PKC{delta} in papilloma from wild-type mice was reduced compared with the level of uninvolved epidermis of TPA-treated mice. Unlike papillomas examined in wild-type mice, total PKC{delta} protein was maintained in papillomas from PKC{delta} overexpressing mice (6). Consistent with these previously reported results, PKC{delta} was still overexpressed in the DMBA-TPA–induced tumors in PKC{delta} overexpressing transgenic mice (Fig. 1C). In contrast, PKC{delta} expression was considerably reduced in carcinomas developed either by the DMBA-TPA protocol in wild-type mice (Fig. 1C) or by repeated UVR exposures in both PKC{delta} overexpressing transgenic and wild-type mice (Fig. 1D). The results of loss of the PKC{delta} in mouse SCC are in accord with the recent results reported by D'Costa et al. in human SCC (18).

PKC{delta} overexpression enhanced TPA-induced but not UVR-induced apoptosis. PKC{delta} has been well documented as a proapoptotic protein through its association either as holoenzyme or catalytic fragment with mitochondria (19–24). A possibility was explored to determine whether PKC{delta} overexpression has differential effects on TPA-induced or UVR-induced levels of apoptosis. In this experiment (Fig. 2), the PKC{delta} transgenic mice and their wild-type littermates were treated in parallel with DMBA-TPA or UVR. At various times after treatment, the percent apoptotic cells in H&E-stained sections were analyzed (Fig. 2A and B). PKC{delta} overexpression did not significantly (P > 0.1) alter the number of sunburn cells (apoptotic cells) in UVR-treated skin (Fig. 2B and D). Sunburn cells, which appear in the epidermis after UVR exposures (Fig. 2B), are the keratinocytes undergoing apoptosis (25). Sunburn cells were identified in H&E-stained histologic sections of the skin by their intensely eosinophilic cytoplasm and small, dense nuclei (Fig. 2B). Treatment with either TPA or UVR resulted in a significant increase (~2-fold) in the number of apoptotic cells in the epidermis (Fig. 2C and D). PKC{delta} overexpression in transgenic mice enhanced TPA-induced apoptotic cells by about 4-fold (Fig. 2C). In contrast, UVR-induced sunburn cells in PKC{delta} overexpressing transgenic mice and their wild-type littermates were not significantly (P > 0.1) different at 1, 5, and 24 hours after treatment (Fig. 2D).


Figure 2
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  		Figure 2. PKC{delta} overexpression enhances TPA-induced but not UVR-induced formation of dead cells. PKC{delta} transgenic mice and their wild-type littermates were exposed to UVR (2 kJ/m2) four times (Monday, Wednesday, Friday, and Monday). In DMBA-TPA protocol, the mice were initiated with one topical treatment of 100 nmol of DMBA in 0.2 mL acetone, and after 1 week of DMBA treatment, the mice were treated with TPA (5 nmol in 0.2 mL acetone) trice (Monday, Thursday, and Monday). The mice were sacrificed to analyze the level of apoptosis at 1, 5, and 24 hours after the fourth treatment of UVR and 1 and 5 hours after the third TPA treatment in H&E-stained sections (A and B). Points, mean from three mice; bars, SE (C and D). There were six skin sections from each mouse. Two sections were scored for a total of at least eight views. Dead cells were expressed as a percentage of total epidermal cells. WT, wild-type mice; TG, PKC{delta} transgenic mice. Arrows, cell under going apoptosis.

 
PKC{delta} overexpression suppressed TPA-induced but not UVR-induced epidermal proliferative marker PCNA. We explored the possibility that PKC{delta} overexpression in transgenic mice may have differential effects on TPA-induced and UVR-induced cell proliferation. In this experiment (Fig. 3), PKC{delta} transgenic mice and wild-type littermates were exposed to UVR (2 kJ/m2) four times or treated thrice with TPA. At the indicated times after the last treatment, dorsal skin was removed and fixed in 10% formalin for the analysis of PCNA-positive cells (Fig. 3A and B). TPA or UVR treatment resulted in a significant increase (>2-fold) in the percentage of PCNA-positive cells at 1 and 5 hours as well as 1, 5, and 24 hours after the TPA and UVR treatment, respectively (Fig. 3C and D). PKC{delta} overexpression in transgenic mice significantly (P < 0.001) suppressed TPA-induced but not UVR-induced PCNA-positive cells (Fig. 3C and D).


Figure 3
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  		Figure 3. PKC{delta} overexpression inhibits TPA-induced but not UVR-induced expression of proliferative marker PCNA. Mice were treated as described in Fig. 2 legend. Mice were sacrificed at, 1, 5 and 24 hours after the fourth UVR treatment and 1 and 5 hours after the third TPA treatment for PCNA staining as described in Materials and Methods. Points, mean % PCNA-positive cells counted from 10 random areas from each mouse; bars, SE. A and B, PCNA staining. C and D, quantitation of PCNA-positive cells. Arrows, PCNA-stained cells.

 
PKC{delta} overexpression suppressed TPA-induced but not UVR-induced levels of cytokines TNF-{alpha}, GM-CSF, and G-CSF. UVR has been shown to induce epidermal keratinocytes to release proinflammatory cytokines [interleukin-1 (IL-1) and TNF-{alpha}], chemotactic cytokines (IL-6, IL-7, IL-5, GM-CSF, and TNF-{alpha}), and cytokines regulating immunity (IL-10, IL-12, and IL-18; ref. 26). We have also reported that both TPA and UVR treatments equally lead to the expression of specific epidermal cytokines (TNF-{alpha}, G-CSF, GM-CSF, IL-1{alpha}, and IL-6), which correlate with the level of expression of PKC{varepsilon} in transgenic mice (15, 16). A comparison between the sensitivity of PKC{delta} transgenic mice and wild-type mice to TPA-induced and UVR-induced expression of epidermal cytokines is shown in Fig. 4. In this experiment, epidermal cytokine levels were analyzed either at 1 and 5 hours after the third TPA application to the DMBA-initiated skin or at 1, 5, and 24 hours after the last fourth UVR exposure. Both TPA and UVR treatments significantly (P < 0.001) increased the epidermal expression of TNF-{alpha} (Fig. 4A), GM-CSF (Fig. 4B), and G-CSF (Fig. 4C) in wild-type mice. However, the level of IL-5 was not consistently changed after either UVR or TPA treatment (Fig. 4D). PKC{delta} overexpression in transgenic mice suppressed TPA-induced but not UVR-induced expression levels of TNF-{alpha}, G-CSF, and GM-CSF (Fig. 4A-C). The difference was highly significant (P < 0.001) at 5 hours after TPA treatment.


Figure 4
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  		Figure 4. PKC{delta} overexpression inhibits TPA-enhanced but not UVR-enhanced levels of epidermal cytokines in PKC{delta} transgenic mice. PKC{delta} transgenic mice and their littermates were treated as described in Fig. 3 legend. The levels of epidermal cytokines were determined as described in Materials and Methods. Points, mean of determinations from epidermal extracts from three separate mice; bars, SE.

 
Overexpression of PKC{delta} in transgenic mice suppresses TPA-induced but not UVR-induced expression of COX-2. There are two isoforms of COX, designated COX-1 and COX-2, which catalyze the first step in the synthesis of prostaglandins (PG) from arachidonic acid. COX-1 is expressed constitutively in most tissues and regulates the synthesis of PGs that control normal physiologic functions. In contrast, COX-2 is not detected in most normal tissues and is induced by a variety of mitogenic and inflammatory stimuli (27–31), which results in enhanced synthesis of PGs in neoplastic and inflamed tissues. COX-2 is overexpressed in premalignant and malignant tissues (27). COX-2 expression is linked to the induction of cancer in various experimental animal models (27). These reports (27–31) prompted us to compare the responses of PKC{delta} transgenic mice with TPA-induced and UVR-induced expression of COX-2. As shown in Fig. 5A and B, a single exposure to UVR (2 kJ/m2) or TPA (10 nmol) treatment of wild-type mice resulted in an increased expression of epidermal COX-2 protein with the peak level at 6 hours after treatment. In a separate experiment (Fig. 5C and D), the levels of COX-1 and COX-2 proteins were evaluated in repeated TPA or UVR treatment of wild-type and PKC{delta} transgenic mice. Interestingly, TPA-induced COX-2 expression in PKC{delta} transgenic mice was significantly (P < 0.001) less than wild-type mice. In contrast, both wild-type and PKC{delta} transgenic mice exposed to UVR elicited an equal increase in COX-2 protein level at 24 hours after UVR exposure. COX-1 expression was not altered after either TPA or UVR treatment of PKC{delta} transgenic mice and wild-type mice (Fig. 5C).


Figure 5
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  		Figure 5. PKC{delta} overexpression inhibits TPA-induced but not UVR-induced expression of COX-2 in PKC{delta} transgenic mice. A and B, the wild-type mice (three mice per group) were topically treated with single TPA (10 nmol in 0.2 mL acetone) or single UVR (2 kJ/m2), and the mouse epidermal extracts were prepared at the indicated times after the TPA or UVR treatment. C and D, in a parallel experiment, PKC{delta} transgenic mice and their littermates were treated thrice with TPA or four times with UVR as described in Fig. 3 legend. The mouse epidermal extract was prepared. COX-1 and COX-2 expression levels were assessed by immunoblot analysis using specific antibodies. Equal loading was confirmed by stripping the blot and reprobing it for ß-actin. The quantification of Western blots (A and C) was done by densitometry analysis using Totallab Nonlinear Dynamics Image Analysis Software (Nonlinear USA).

 
PKC{delta} overexpression in transgenic mice suppresses TPA-induced phosphorylation of Akt and p38. Akt is an important component of signal transduction pathways linked to cell survival and proliferation (32). Akt activation has been shown to be essential for COX-2 transcription induced by UVB (33) or TPA (34). Both TPA and UVR activate a variety of signal pathways, including mitogen-activated protein kinase (MAPK) family (p38 and ERK1/2; refs. 35, 36), which has been linked to the induction of COX-2 (33, 34, 37). These findings (33, 34) prompted us to explore whether TPA-induced and UVR-induced COX-2 expression in intact mouse skin in vivo involves activation of Akt and MAPKs (p38 and ERK1/2). The results are illustrated in Fig. 6, both TPA and UVR treatments increased the expression of p-Akt (Fig. 6B), p38 (Fig. 6C), ERK1/2 (Fig. 6D). PKC{delta} overexpression in transgenic mice clearly inhibited TPA-induced but not UVR-induced expression of p-Akt (Fig. 6B) and p38 (Fig. 6C).


Figure 6
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  		Figure 6. PKC{delta} overexpression in transgenic mice inhibits TPA but not UVR activation of Akt and p38. PKC{delta} transgenic mice and their wild-type littermates were exposed to single UVR (2 kJ/m2). In parallel experiment, the mice were treated with one topical application of TPA (10 nmol in 0.2 mL acetone). The mice were sacrificed at 3, 6 and 24 hours after UVR or TPA treatment. The soluble mouse epidermal extract was analyzed for the level of specific proteins [Akt and p-Akt (Ser473), p38 and p-p38, and ERK1/2 and p-ERK1/2] by immunoblot analysis. Equal loading was confirmed by stripping the blot and reprobing it for ß-actin. The quantitation (B-D) of Western blots (A) was done by densitometry analysis using Totallab Nonlinear Dynamics Image Analysis Software (Nonlinear USA).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Skin cancer is the most common human cancer (11, 12), and the multistage model of mouse skin carcinogenesis has been on the forefront to provide clues about the cellular, biochemical, and genetic events linked to the initiation, promotion, and progression steps of skin cancer formation (38). There are two commonly used protocols to induce skin cancer in mice: (a) by the initiation (DMBA) and promotion (TPA) protocol and (b) by the complete carcinogenesis regimen involving either repeated exposure to a polycylic hydrocarbon [e.g., DMBA, benzo(a)pyrene, or methylcholanthene] or UVR. However, majority of human skin cancer is linked to exposure to UVR (11, 12). We now present that PKC{delta}, a member of nPKC family (39), which suppresses induction of skin cancer promoted by TPA (6, 17), failed to affect the development of skin cancer elicited by repeated exposures to UVR. The induction of SCC by both the DMBA-TPA protocol and UVR carcinogenesis correlated with increased cell proliferation, up-regulation of cytokines (TNF-{alpha}, GM-CSF, and G-CSF), COX-2 expression, and activation of Akt and members of MAPK family (p38 and ERK1/2). We found that PKC{delta} overexpression suppressed TPA-induced but not UVR-induced levels of cell proliferative marker PCNA, apoptosis, cytokines (TNF-{alpha}, GM-CSF, and G-CSF), COX-2, and activation of Akt and p38.

To determine the role of PKC{delta} in mouse skin carcinogenesis, we generated transgenic FVB/N mouse lines expressing PKC{delta}, regulated by human keratin 14 promoter in the epidermis (6). Two independent PKC{delta} transgenic lines 16 and 37 elicited 8- and 2-fold increases in epidermal PKC{delta} protein and enzyme activity. Neither transgenic line 16 nor line 37 exhibited any phenotypic abnormalities. The presence of the transgene did not affect the litter size or the sex ratio of the litters. Consistent with the previous report (6), PKC{delta} overexpression in FVB/N transgenic mice imparted resistance to the development of SCC by DMBA initiation and TPA promotion. In contrast, PKC{delta} transgenic mice were as susceptible as wild type to the development of SCC by repeated UVR exposures (Fig. 1). These results indicate that tumor promotion by TPA and UVR may comprise unique signal transduction pathways to induction of SCC.

PKC{delta} overexpressing transgenic mice elicited different response to TPA and UVR treatment for the induction of epidermal apoptosis. UVR-induced apoptosis, compared with wild-type mice, was not enhanced in PKC{delta} transgenic mice. On the contrary, TPA-induced apoptosis in PKC{delta} transgenic mice was increased about 5-fold more than wild-type mice (Fig. 2). It is likely that endogenous PKC{delta} levels are sufficient to induce maximum UVR-induced apoptosis. PKC{delta} level was maintained in a few tumors developed in PKC{delta} transgenic mice by the DMBA-TPA protocol. However, PKC{delta} was lost in the SCC developed in PKC{delta} transgenic mice by repeated UVR exposures (Fig. 1C and D). It needs to be determined that repeated UVR exposure may lead to rapid degradation of PKC{delta}. This may explain that UVR-treated PKC{delta} overexpressing mice behave like wild-type mice.

PKC{delta} is overwhelmingly documented to induce apoptosis in a wide variety of cells cultured in vitro (18–23). The initial step in PKC{delta}-mediated apoptosis involves PKC{delta} activation. In human keratinocytes, PKC{delta} is activated by UVR via caspase-3-mediated cleavage at the third variable region (40). UVR-induced tyrosine phosphorylation of PKC{delta} has been shown to result in apoptosis in HaCaT cells (41). In addition, TPA induces translocation of PKC{delta} to mitochondria, which releases cytochrome c to initiate apoptosis (20). Activation of PKC{delta} in dendritic cells is shown by the detection of the PKC{delta}-catalytic fragment in the nuclear fragment. PKC{delta} is cleaved in cells in response to DNA-damaging agents (42). The link of PKC{delta} cleavage to the induction of apoptosis is supported by the findings that overexpression of PKC{delta} catalytic fragment results in chromatin condensation, nuclear fragmentation, and apoptosis (42). Our results indicate that PKC{delta} overexpressing transgenic mice are more sensitive than their wild-type littermates to TPA-caused but not UVR-caused increased apoptosis (Fig. 2). It is also noteworthy that PKC{delta} overexpression suppressed TPA-induced but not UVR-induced number of PCNA-positive cells. Taken together, these results (Figs. 2 and 3) indicate that PKC{delta}-caused suppression of SCC elicited by the DBMA-TPA protocol may involve both the suppression of cell proliferation and the induction of apoptosis.

Specific cytokines TNF-{alpha}, GM-CSF, and G-CSF have been documented as common molecular targets in both TPA and UVR tumor promotion (43–45). TNF-{alpha} has been reported as an endogenous tumor promoter of the mouse skin (43–45). Two independent laboratories have shown that mice deficient for TNF-{alpha} and their receptors are resistant to mouse skin tumor formation elicited by chemical carcinogenesis or by photocarcinogenesis (43, 44). We have also shown that PKC{varepsilon} overexpression in transgenic mice, which sensitizes skin to the development of SCC either by the DMBA-TPA protocol or by repeated exposure to UVR, results in increased ectodomain shedding of TNF-{alpha} in response to both TPA (46) and UVR treatment (16). GM-CSF has been shown to be linked to malignant progression. In this context, it is noteworthy that transgenic mice that overexpress GM-CSF are highly susceptible to the development of SCC (47, 48). The mechanism by which PKC{delta} suppresses TPA-induced levels of TNF-{alpha}, GM-CSF, and G-CSF is speculative at this time. PKC{delta} may either affect TPA-induced synthesis and/or the release of TNF-{alpha}. For TNF-{alpha} to exert its inflammatory responses at distant sites from its synthesis, it must be cleaved from the membrane. A specific enzyme called TNF-{alpha} Converstase (TACE) catalyzes this cleavage (49). It needs to be determined whether PKC{delta} inhibits TPA-induced levels of TNF-{alpha} by inhibiting TACE activity.

COX-2, the inducible form of COX, is the key enzyme in PG biosynthesis and is linked to the induction of skin cancer (27). For example, selective COX-2 inhibitors inhibit the induction of both basal and squamous cell skin cancers (30). In addition, COX-2 overexpression in transgenic mice sensitizes skin for carcinogenesis (31) and COX-2 deficiency in mice imparts resistance to the development of skin cancer (27). We found that PKC{delta} overexpression suppresses TPA-induced levels of COX-2 and activation of Akt and p38. Activation of Akt and p38 has been shown to mediate the induction of COX-2 by TPA and UVR (33, 34). Taken together, it seems that PKC{delta} may suppress TPA-induced expression of COX-2 by inhibiting the activation of Akt and p38 by TPA. UVR-induced activation of Akt and p38 is not sensitive to PKC{delta} overexpression (Fig. 6).

In summary, PKC{delta} overexpression did not inhibit UVR carcinogenesis while it suppressed development of SCC elicited by DMBA-TPA protocol (Fig. 1). Both TPA and UVR treatment of wild-type mice increased the expression of PCNA (Fig. 3), apoptosis (Fig. 2), stimulated the expression of cytokines (TNF-{alpha}, GM-CSF, and G-CSF; Fig. 4), and increased COX-2 expression (Fig. 5) and the expression of p-Akt, p38, ERK1, and ERK2 (Fig. 6). PKC{delta} overexpression in transgenic mice enhanced TPA-induced but not UVR-induced apoptosis and suppressed TPA-stimulated but not UVR-stimulated levels of PCNA, cytokines (TNF-{alpha}, G-CSF, and GM-CSF), COX-2, and expression of p-Akt and p38. The PKC{delta} overexpressing transgenic mice are not resistant to UVR carcinogenesis. The proapoptotic activity of PKC{delta} coupled with its ability to suppress TPA-induced expression of specific cytokines, COX-2 expression, and the phosphorylation of Akt and p38 may play roles in the suppression of TPA-promoted development of SCC. The results presented further strengthen the concept that the molecular mechanism linked to the presumed promoting component of carcinogenesis by a complete carcinogen UVR is different from those of the tumor promoter TPA (50).


   Acknowledgments
 
Grant support: NIH grants CA35368 and CA102431.

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 Nancy E. Dreckschmidt for help in breeding of PKC{delta} transgenic mice and Marybeth Wartman, Kaitlin E. Martin, and Kristin J. Ness for help in tumor induction experiments.

Received 8/ 1/05. Revised 10/26/05. Accepted 11/ 8/05.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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