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[Cancer Research 59, 5710-5718, November 1, 1999]
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[Cancer Research 59, 5710-5718, November 15, 1999]
© 1999 American Association for Cancer Research

Carcinogenesis

Transgenic Mice Overexpressing Protein Kinase C{delta} in the Epidermis Are Resistant to Skin Tumor Promotion by 12-O-Tetradecanoylphorbol-13-acetate1

Peter J. Reddig, Nancy E. Dreckschimdt, Helga Ahrens, Robita Simsiman, Ching-Ping Tseng2, Jun Zou, Terry D. Oberley and Ajit K. Verma3

Department of Human Oncology, Medical School, University of Wisconsin, Madison, Wisconsin 53792 [P. J. R., N. E. D., H. A., R. S., J. Z., A. K. V.], and Department of Pathology and Laboratory Medicine, Veterans Administration Hospital and Medical School, University of Wisconsin, Madison, Wisconsin 53792 [T. D. O.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To determine the role of protein kinase C{delta} in mouse skin carcinogenesis, we have developed transgenic FVB/N mouse lines expressing in the epidermis an epitope-tagged protein kinase C{delta} (T7-PKC{delta}) regulated by the human keratin 14 promoter. The untreated T7-PKC{delta} mice displayed excessive dryness in the skin of the tail with a variable penetrance over time. Histologically, the tail skin showed hyperplasia with evidence of hyperkeratosis. The epidermis of the rest of the T7-PKC{delta} mouse was unremarkable. Despite this mild phenotype, the effects of PKC{delta} overexpression on mouse skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA) were dramatic. Two independent lines of T7-PKC{delta} mice (16 and 37) expressing the T7-PKC{delta} transgene were examined for responsiveness to skin tumor promotion by 7,12-dimethylbenz[a]anthracene and TPA. By immunoblot analysis, the T7-PKC{delta}-16 and T7-PKC{delta}-37 mice showed an 8- and 2-fold increase of PKC{delta} protein. The T7-PKC{delta}-16 mice averaged 300% more T7-PKC{delta} activity than the T7-PKC{delta}-37 mice did. The T7-PKC{delta}-37 mice did not manifest any difference in tumor burden or incidence. However, the reduction in papilloma burden at 25 weeks of promotion for the T7-PKC{delta}-16 mice relative to wild-type mice averaged 72 and 74% for males and females, respectively. The T7-PKC{delta}-16 mice reached 50% papilloma incidence between 12 and 13 weeks of promotion compared with 8 weeks for wild-type mice. Furthermore, the carcinoma incidence was also reduced in T7-PKC{delta}-16 mice. Carcinoma incidence at 25 weeks of promotion treatment was: wild-type females, 78%; T7-PKC{delta}-16 females, 37%; wild-type males, 45%; and T7-PKC{delta}-16 males, 7%. Thus, PKC{delta} when expressed at sufficient levels can suppress skin tumor promotion by TPA.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The multistage model of mouse skin carcinogenesis is a useful system in which biochemical events unique to initiation, promotion, or progression can be studied and related to cancer formation (1 , 2) . TPA,4 a component of croton oil, is a potent mouse skin tumor promoter (3) . TPA has been used extensively to study skin tumor promotion (1 , 2) . However, the exact molecular mechanisms of tumor promotion by TPA remain to be defined.

PKC, which is ubiquitous in eukaryotes, is a major intracellular receptor for TPA (4) . PKC forms part of the signal transduction system involving the turnover of inositol phospholipids and is activated by DAG, which is produced as a consequence of this turnover (5) . On the basis of the structural similarities and cofactor requirements, the PKC isoforms have been grouped into three subfamilies of enzymes: the conventional PKCs ({alpha}, {beta}I, {beta}II, and {gamma}), which are dependent on Ca2+, PS, and DAG/TPA; the nPKCs ({epsilon}, {delta}, {eta}, and {theta}), which require only PS and DAG/TPA; and the atypical PKCs ({iota}/{lambda} and {zeta}), which retain PS dependence but have no requirement for Ca2+ or DAG/TPA for activation [PKCµ, which is usually classified as a nPKC, is not easily grouped with any of the other isoforms (6 , 7) ].

PKC is an important component of the signal transduction pathways controlling cell proliferation and tumorigenesis (1 , 5 , 8) . The positive correlation between the affinity of different types of phorbol esters for PKC and their tumor-promoting efficacy suggests that the activation of PKC may be a critical step in the promotion of mouse skin tumor formation (9) . Also, DAG, an endogenous activator of PKCs, promotes mouse skin tumor formation (10 , 11) . Taken together, these results implicate PKC activation as an essential step in mouse skin tumor promotion. However, several groups have demonstrated that repeated applications of TPA depress PKC activity and protein levels (12, 13, 14) . These results indicate that both loss of PKC activity and degradation of PKC may be important for mouse skin tumor promotion by TPA. Studies on the effect of TPA treatment on epidermal PKC{delta} levels have produced variable results. Leibersperger et al. (15) detected a reduction in PKC{delta} protein levels after a single TPA treatment, as well as in TPA-induced papillomas. However, others observed little change in the total PKC{delta} protein levels in the epidermis or papillomas after single or repeated TPA treatments (12 , 16 , 17) . Despite the lack of effect of a single treatment or repeated TPA treatments on epidermal PKC{delta} protein levels, the PKC{delta} kinase activity was significantly reduced by these treatments (12) . Thus, a reduction in the level of epidermal PKC{delta} protein or activity may be important for tumor formation.

The distinct role that each individual PKC isoform plays in the signal transduction pathways to mouse skin tumor promotion by TPA is under extensive investigation but remains ambiguous. To evaluate the in vivo role of PKC{delta} in mouse skin tumor promotion by TPA, we generated transgenic mice overexpressing an epitope-tagged PKC{delta} (T7-PKC{delta}) under the control of the human keratin 14 promoter. The T7-PKC{delta} mice were healthy and fertile and exhibited only mild phenotypic alterations in the absence of TPA treatment. However, the T7-PKC{delta} mice were found to be extremely resistant to mouse skin tumor promotion by TPA.


   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 Company, Inc. (Milwaukee, WI). The anti-T7-Tag HRP-conjugated antibody was purchased from Novagen (Madison, WI). The restriction endonucleases were purchased from New England Biolabs (Beverly, MA). Genescreen was purchased from NEN Life Science Products (Boston, MA). The random-primed DNA labeling was purchased from Boehringer Mannheim (Indianapolis, IN). The acrylamide, bisacrylamide, SDS, 0.45 µm supported nitrocellulose membrane, Bio-Rad Protein Assay, Bio-Rad DC Protein Assay, and SDS-PAGE standards were purchased from Bio-Rad Laboratories (Hercules, CA). Monoclonal antibodies to PKC{delta}, {epsilon}, {lambda}, µ, {theta}, and {zeta} were purchased from Transduction Laboratories (Lexington, KY). Polyclonal antibodies to PKC{beta}I, {beta}II, {gamma}, {epsilon}, {eta}, and actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). An affinity-purified anti-PKC{alpha} was purchased from Life Technologies, Inc. (Grand Island, NY). The immobilized protein A/G agarose was purchased from Pierce (Rockford, IL). The ECL and ECF Western blotting detection reagents and the protein kinase C enzyme assay system were purchased from Amersham Life Sciences Inc. (Arlington Heights, IL). FVB/NTacfBR mice (FVB/N), 7–9 weeks of age, were purchased from Taconic (Germantown, NY).

Transgene Construction and Generation of Transgenic Lines.
The BglII/SalI fragment from the pET-21c(+) vector, containing the T7-Tag open reading frame, was ligated to the NH2 terminus of the mouse PKC{delta} cDNA in the pRSV-PKC{delta} vector to produce the pRSV-T7-PKC{delta} vector (18) . The T7-PKC{delta} cDNA was ligated into the pGEM3Z-K14 {beta}-globin vector by cloning fragments of T7-PKC{delta} from pRSV-T7-PKC{delta} into pGEM-I and pGL2-Basic multicloning sites to place BamHI compatible sites at the 5' and 3' ends of the of T7-PKC{delta} cDNA. These fragments were subcloned into the BamHI site of the pGEM3Z-K14 {beta}-globin vectors to produce the pG3Z-K14-T7-PKC{delta} vector. The functional elements of pGEM3Z-K14-T7-PKC{delta} were isolated by partial digestion with EheI and HindIII and purified by electroelution and ion exchange chromatography after agarose gel electrophoresis. The purified K14-T7-PKC{delta} expression cassette was microinjected into the male pronuclei of one-cell fertilized embryos (FVB/N x FVB/N) by the University of Wisconsin’s Transgenic Mouse Facility. Genomic DNA from tail biopsies was digested with EcoRV, fractionated on a 0.7% agarose gel, and immobilized on a Genescreen. The radiolabeled EcoRV/BamHI fragment from pGEM3Z-K14 {beta}-globin vector, encompassing ~1 kb of the K14 promoter and the entire {beta}-globin intron, was implemented as the probe for detection of integrated K14-T7-PKC{delta} DNA. The transgene was detected by Southern blot analyses using genomic DNA digested with EcoRV. An EcoRV/BamHI fragment from the pGEM3Z-K14 {beta}-globin vector labeled with 32P using the random-primed DNA labeling kit from Boehringer Mannheim was used as a probe.

Mice.
Transgenic mice were maintained by mating transgenic siblings or mating transgenic mice with wild-type FVB/N mice. The generation of mice for the tumor promotion experiments was performed by mating F1 and F2 hemizygous T7-PKC {delta} mice with wild-type FVB/N mice. At 7–9 weeks of age, the dorsal skins of the mice were shaved 3–4 days before treatment, and those mice in the resting phase of their hair cycle were used for experimentation. The solutions of TPA and DMBA were prepared in acetone and were applied to the shaved backs of individual mice in a volume of 0.2 ml.

Tumor Induction Experiments.
Mouse skin tumors were induced by the initiation-promotion regimen. For mouse skin tumor initiation, a single 100-nmol dose of DMBA in 0.2 ml of acetone was applied topically to the shaved backs of both male and female, line 16 and line 37, wild-type and transgenic mice. Two weeks after initiation, 5 nmol of TPA in 0.2 ml acetone or acetone alone was applied twice weekly to skin for the duration of the experiment (25 weeks). The tumor incidence and burden was observed weekly starting at 7 weeks of TPA promotion. The number of mice at 25 weeks of promotion for each experimental group was as follows: line 16 DMBA-TPA (wild type females, n = 15; transgenic females, n = 18; wild-type males, n = 15; transgenic males, n = 14); DMBA-TPA line 37 (wild type females, n = 19; transgenic females, n = 7; wild-type males, n = 11; transgenic males, n = 11); DMBA-acetone line 16 (wild-type females, n = 18; transgenic females, n = 20; wild-type males, n = 18; transgenic males, n = 15); and DMBA-acetone line 37 (wild-type females, n = 16; transgenic females, n = 9; wild-type males, n = 19; transgenic males, n = 14). Mice were removed from the experiment if they were wounded from fighting. Carcinomas were recorded grossly as downward-invading lesions, a subset of which was examined histologically, and malignancy was confirmed as invading the panniculus carnosus. Carcinoma-bearing mice were killed shortly after diagnosis. The Wilcoxan rank sum test was used to determine the value of P for the papilloma burden.

Immunoblotting of PKC Isoforms.
Mice were shaved and depilated before experimentation. The mouse skin was excised and scraped to remove the s.c. fat. The skin was either pulverized with a mortar and pestle under liquid N2, or the epidermis was removed and homogenized. The ground skin or epidermis was homogenized with five volumes of PKC extraction buffer [20 mM Tris-HCl (pH 7.4), 0.3% Triton X-100, 2 mM EDTA, 10 mM EGTA, 0.25 M sucrose, 1 mM DTT, 10 µg/ml leupeptin, and 10 µg/ml aprotinin]. The homogenate was centrifuged at 100,000 x g for 60 min at 4°C, and the supernatant was the total PKC extract. Protein concentration in the total PKC extract was determined, and 50–100 µg of total PKC extract protein were fractionated on a 7.5 or 10% SDS-polyacrylamide gel. The proteins were transferred to 0.45 µm supported nitrocellulose membrane. The membrane was then incubated with the appropriate primary and secondary antibodies, and the detection signal was developed with ECL or ECF reagents (Amersham). Monoclonal antibodies to PKC {alpha}, {beta}II, {gamma}, {delta}, {epsilon}, {theta}, µ, {lambda}, and {zeta} and T7-Tag or a polyclonal antibody to PKC {eta} were used to detect the respective PKC isoforms at dilutions of 1:200, 1:1000, 1:500, 1:500, 1:500, 1:500, 1:2000, 1:500, 1:1000, 1:2000, and 1:1000, respectively.

PKC Immunocomplex Kinase Assay.
The dorsal skin of mice was shaved and depilated 24 h before experimentation. The mice were euthanized, the dorsal skin was removed, and the epidermis was scraped off on ice with a razor blade. The epidermis from two to three mice were pooled and placed in 0.5 ml of IP lysis buffer [50 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 200 µM Na3VO4, 200 µM NaF, and 1 mM EGTA], homogenized in a glass Teflon tissue homogenizer, and agitated for 30 min at 4°C. After centrifugation for 15 min at 14,000 rpm, the supernatant was used for immunoprecipitation. The lysate was preabsorbed with 5 µl of protein A/G agarose for 10 min at 4°C. Five µg of anti-T7-Tag antibody and 10 µl of protein A/G agarose were added to the lysate, and the volume of the lysate was adjusted to 1 ml with lysis buffer. The mixture was incubated for 2–4 h at 4°C with agitation. The immunoprecipitate was pelleted at 14,000 rpm in a microcentrifuge, washed, and resuspended in 300 µl of assay buffer [50 mM Tris-HCl (pH 7.4), 5 mM EDTA (pH 8.0), 10 mM EGTA (pH 7.9), 0.3% {beta}-mercaptoethanol, 5 µg/ml aprotinin, 5 µg/ml leupeptin, and 50 µg/ml phenylmethylsulfonyl fluoride]. Twenty-five µl of the immunoprecipitate were assayed in kinase buffer containing 50 mM Tris-HCl (pH 7.4), 8 mM MgCl2, 0.136 mM ATP, 0.2 µCi [{gamma}-32P]ATP, 100 µM EGFR peptide (ERKRTLRRL), 3 mM DTT, 34 µg/ml of L{alpha}-phosphatidyl-L-serine, 3 µg/ml TPA, and 1 mM EGTA. The reaction was incubated at 37°C for 15 min, stopped with 10 µl of 300 mM H3PO4, spotted onto filter discs, washed with 75 mM H3PO4, and counted.

PKC Immunocomplex Kinase Assay of TPA-treated Mice.
Mice were euthanized 72 h after the last TPA treatment. The uninvolved epidermis or papillomas were removed from four mice and pooled and placed in IP lysis buffer. The cleared supernatant was used for immunoprecipitation. The T7-PKC{delta} protein was precipitated with 5 µg of anti-T7-Tag antibody for 2–4 h at 4°C. The pelleted immunoprecipitate was resuspended in assay buffer and assayed as described above.

Histology.
The tissue to be examined was excised promptly after euthanasia and immediately placed in 10% neutral buffered formalin. The tissue was fixed for 1 h in the formalin and then embedded in paraffin. Sections of 4 µm were cut for H&E staining or immunostaining. Deparaffinized slides were used for immunostaining.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Susceptibility of FVB/N Mice to TPA Tumor Promotion and Skin PKC Isoform Composition.
We used the FVB/N inbred mouse strain for transgenesis. The FVB/N mice are an excellent strain for the production of transgenic mice (19) . A detailed TPA dose-response study was performed with the FVB/N mice to determine the appropriate dose of TPA for mouse skin tumor promotion. The mice were initiated by topically applying a single 100-nmol dose of DMBA to the dorsal skin and then were treated twice weekly with different doses of TPA. The first papillomas appeared after 8 weeks of promotion with 5 and 10 nmol of TPA at incidences of 4 and 33%, respectively. After 11 weeks of promotion, mice treated with 2 nmol of TPA developed papillomas with an incidence of 4%. At the end of 19 weeks of promotion with 10, 5, or 2 nmol of TPA, the incidence was 100, 94, and 50%, respectively (Fig. 1A)Citation . The papilloma burden was 15.0 ± 0.8, 6.6 ± 0.8, and 0.8 ± 0.2 at 19 weeks of promotion with 10, 5, or 2 nmol of TPA, respectively (Fig. 1B)Citation . No papillomas were observed after treatment of the initiated skin with the vehicle acetone or 0.1 or 1.0 nmol of TPA (Fig. 1B)Citation . The FVB/N mice also exhibited a high incidence of carcinomas. The first carcinomas appeared at 15 weeks with 10 nmol of TPA. At the end of 19 weeks of tumor promotion, the carcinoma incidence was 92, 52, and 4% with 10, 5, or 2 nmol of TPA, respectively (data not shown).



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  		Fig. 1. Effects of TPA dose on the promotion of mouse skin tumor formation in female FVB/N mice and PKC isoforms in the FVB/N skin. Mice initiated with 100 nmol of DMBA were treated twice weekly for 19 weeks with TPA (x, 0; {square}, 0.1 nmol; {triangleup}, 1.0 nmol; •, 2.0 nmol; {diamondsuit}, 5.0 nmol; or {blacksquare}, 10.0 nmol) in 0.2 ml of acetone. The tumor incidence and multiplicity were counted biweekly for the 24 mice/treatment. A, papilloma incidence. B, papilloma burden. Each value is the mean number of papillomas ± SE for 24 mice. C, PKC isoform composition of FVB/N mouse skin. Immunoblot analysis was performed using total mouse skin extract (50 µg). Monoclonal antibodies to PKC {alpha}, {beta}II, {gamma}, {delta}, {epsilon}, {theta}, µ, {lambda}, and {zeta}, or polyclonal antibodies to PKC {gamma} and {eta} were used to detect the respective PKC isoforms. Arrowheads, position of each PKC isoform. PKC extracts from mouse brain (M. Brain) or mouse lung (Lung) and or whole-cell lysates of A431 epidermoid carcinoma cells (A431) or Jurkat cells were used as positive controls as indicated. The positive controls for PKC{alpha} and PKC{beta}II are bacterially expressed, mouse PKC{alpha} and PKC{beta}II.

 
As a prelude to the analysis of the role PKC{delta} in skin tumor promotion, we determined the PKC isoform composition in the FVB/N mouse skin. PKC{alpha} and PKC{beta}II were the only conventional Ca2+-dependent isoforms identified in the FVB/N skin extracts. The presence of PKC{beta}I in the skin could not be confirmed because of the cross-reactivity of the anti-PKC{beta}I antibodies with PKC{beta}II (data not shown). The nPKC isoforms {delta}, {epsilon}, {eta}, {theta}, and µ were all detected in the mouse skin. The atypical PKC isoforms {lambda} and {zeta} were also detected in skin extracts from FVB/N mice (Fig. 1C)Citation .

Construction of the K14-T7-PKC{delta} Expression Vector.
The human keratin 14 promoter was used to direct expression of PKC{delta} to the basal cells of the epidermis (20) . The human keratin 14 promoter has been successfully used to overexpress several proteins in the mouse epidermis such as TGF-{alpha}, tumor necrosis factor-{alpha}, and keratinocyte growth factor (21, 22, 23) . The mouse cDNA for PKC{delta}, tagged at its NH2 terminus with 21 amino acids from the T7 bacteriophage major coat protein, was inserted at the BamHI site of the pGEM3Z-K14 {beta}-globin vector to create the pGEM3Z-K14-T7-PKC{delta} vector (Fig. 2A)Citation . This construct expressed active T7-PKC{delta} protein in in vitro PKC immunocomplex kinase assays after transient transfection into cultured cells (data not shown).



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  		Fig. 2. Molecular characterization of the T7-PKC{delta} Mice. A, structure of the pG3Z-K14-T7-PKC{delta} after insertion of T7-PKC{delta} cDNA into the BamHI site of pGEM3Z-K14 {beta}-globin. B, Southern blot using genomic DNA from tail biopsies digested with EcoRV. A restriction fragment containing 1 kb from the 3' end of the K14 promoter and the {beta}-globin intron was used for identification of F0 mice with an integrated K14-T7-PKC{delta} expression cassette. The numbers above the lanes are the potential founder’s identification numbers. Arrowhead, position of the transgene. C, expression of PKC{delta} protein. The skin from a line 16 (I and II) or a line 37 (III and IV) mouse was homogenized in PKC extraction buffer, and the cleared supernatant (100 µg) was used for immunoblot analysis. Monoclonal antibodies to the T7 epitope (I and III) or PKC{delta} (II and IV) were used. Arrowheads, position of PKC{delta}. D, PKC{delta} activity. To detect T7-PKC{delta} activity, the epidermis was harvested from two to three F1 female (I) or male (II) mice and extracted with IP lysis buffer. The T7-PKC{delta} protein was immunoprecipitated with the anti-T7-Tag antibody and assayed in kinase buffer using the EGFR peptide as a substrate in the presence (+) or absence (-) of PS and TPA. E, expression of T7-PKC{delta} in mouse tissues. The indicated tissues were harvested from a transgenic line 16 F1 T7-PKC{delta} mouse and homogenized in PKC extraction buffer, and 100 µg of extract were used for immunoblot analysis using the anti-T7-HRP antibody and the polyclonal anti-actin antibody.

 
Generation and Isolation of Founder T7-PKC{delta} Mice.
The linear K14-T7-PKC{delta} expression cassette was removed from the pGEM3Z-K14-T7-PKC{delta} by digestion with EheI and HindIII (Fig. 2A)Citation . Transgenic mice were generated from one-cell embryos that were microinjected with the K14-T7-PKC{delta} expression cassette (University of Wisconsin Biotechnology Center Transgenic Animal Facility). A Southern analysis of this DNA was performed by treatment of the genomic DNA with the endonuclease EcoRV and by hybridizing the fractionated, membrane-bound DNA with a probe containing ~1 kb of the 3' end of the K14 promoter and the {beta}-globin intron from the pG3Z-K14-{beta} globin vector. The Southern analysis identified nine potential founder mice (Fig. 2B)Citation .

The founders were bred to wild-type FVB/N mice to produce F1 mice that were examined for expression of the transgene by immunoblot analysis (Fig. 2C)Citation . Individual adult F1 carrier mice from each line of potential T7-PKC{delta} mice were euthanized, the skin was removed, and total Triton X-100 soluble PKC extracts were made. Expression of the transgene was detected by immunoblotting with a monoclonal antibody to the T7 epitope of T7-PKC{delta} or a monoclonal antibody to PKC{delta}. Two mouse lines with high (line 16) and low (line 37) T7-PKC{delta} expression levels were selected for further expansion (Fig. 2C)Citation . Offspring of founders 3 and 32 also exhibited expression of the transgene and a similar phenotype but were not expanded for further study (data not shown).

Characterization of the T7-PKC{delta} Mice.
The relative levels of immunoprecipitable T7-PKC{delta} kinase activity in the epidermis of transgenic mice (lines 16 and 37) are shown in Fig. 2DCitation . In this experiment, the epidermis was scraped from the skin of two mice and homogenized in a 1% Triton X-100 lysis buffer, and the soluble fraction was used for immunoprecipitation with the anti-T7-Tag antibody. The immunoprecipitates were assayed for PKC activity in the presence or absence of the PKC activators PS and TPA. The average level of PS- and TPA-stimulated T7-PKC{delta} kinase activity in line 16 mice was 3-fold greater than line 37 mice. Male and female mice had similar T7-PKC{delta} kinase activity within each transgenic line (Fig. 2D)Citation . Immunoblots of the total extracts probed with the anti-PKC{delta} antibody displayed an 8-fold increase in PKC{delta} protein levels in line 16 mice and a 2-fold increase in line 37 mice when compared with the endogenous level of PKC{delta} protein (data not shown).

The expression of transgenes regulated by the human K14 promoter was not limited to the epidermis. Tissue samples were isolated from a transgenic line 16 F1 mouse and were extracted with a PKC extraction buffer containing 0.3% Triton X-100. Probing the membrane-bound PKC extracts with the anti-T7 antibody identified expression of T7-PKC{delta} in the thymus and stomach in addition to the skin (Fig. 2E)Citation . Expression of the K14 promoter has been observed previously in these tissues (22) .

Neither transgenic line 16 nor line 37 exhibited any significant phenotypic abnormalities (Fig. 3A)Citation . The presence of the transgene did not affect litter size or the sex ratio of the litters. Neither pups nor adult mice exhibited any gross phenotypic abnormalities in the dorsal epidermis. H&E-stained dorsal skin sections demonstrated no difference in the morphology of the wild-type and the transgenic mouse epidermis (Fig. 3, C and D)Citation . The only phenotype that was observed in all four of the lines was hyperkeratosis of the tail. H&E-stained sections from the untreated tail skin of wild type and T7-PKC{delta} mice were examined. T7-PKC{delta} mice exhibited hyperplasia of the tail epidermis (Fig. 3, D and E)Citation . The onset of the hyperkeratosis in the tail epidermis occurred around 2–4 weeks of age. This phenotype exhibited an incomplete penetrance, which varied over time. Adult mice monitored weekly over a 2-month period exhibited an average penetrance of 38 and 40% in line 16 females and males, respectively. In line 37, the penetrance of the hyperkeratosis was 25 and 50% for females and males, respectively. The hyperkeratosis was temporary, persisting from <1 week to several weeks, depending on the mouse.



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  		Fig. 3. Phenotype of the T7-PKC{delta} mice. A, photographs of female F1 line 16 T7-PKC{delta} (Tg) and wild-type (Wt) mice. Littermates are shown from the F1 generation of the T7-PKC{delta} line 16 mice. Formalin-fixed sections stained with H&E of the dorsal skin from wild-type (B) and T7-PKC{delta} (C) mice and the tail skin from wild-type (D) and T7-PKC{delta} (E) mice are shown. B–E: bars, 100 µm.

 
Overexpression of T7-PKC{delta} in the epidermis may induce compensatory alterations in the levels of other PKC isoforms that could modulate the effects of elevated PKC{delta} levels. The relative levels of various PKC isoforms in the epidermis of wild-type and line 16 T7-PKC{delta} mice were determined by immunoblot analysis. The protein levels of epidermal PKC{alpha}, {epsilon}, {lambda}, µ, and {zeta} were not affected in T7-PKC{delta} mice (Fig. 4)Citation . However, the levels of PKC{beta}II and PKC{theta} protein were reduced in T7-PKC{delta} mice. Additionally, the level of PKC{eta} protein was elevated in the epidermis from T7-PKC{delta} mice (Fig. 4)Citation .



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  		Fig. 4. PKC isoform expression in T7-PKC{delta} mice. Untreated epidermis was removed from line 16 F4 T7-PKC{delta} mice (Tg) or wild-type (Wt) littermates and homogenized in PKC extraction buffer. The extracts (50 and 100 µg) were immunoblotted, and the individual PKC isoforms were detected with the appropriate antibody. The level of actin was also determined as a control for gel loading variations.

 
Mouse Skin Tumor Promotion.
The T7-PKC{delta} transgenic mice were evaluated for their sensitivity to TPA skin tumor promotion. In these experiments, transgenic T7-PKC{delta} mice and wild-type littermates were initiated by application of a single 100-nmol dose of DMBA and were then treated twice weekly with 5 nmol of TPA in acetone. No papillomas were observed in any of the mice initiated with DMBA and promoted with acetone alone (data not shown). The reduction in both papilloma and carcinoma burden for both male and female T7-PKC{delta} line 16 mice was dramatic (Fig. 5, B and DCitation ; Table 1Citation ). The papilloma burden was reduced relative to the wild-type mice by 72% in the male T7-PKC{delta} mice and by 74% in the female mice after 25 weeks of tumor promotion (Fig. 5DCitation ; Table 1Citation ). A second experiment with the line 16 mice elicited similar reductions in papilloma burden in the transgenic mice (data not shown). The line 16 T7-PKC{delta} mice also exhibited a delay in tumor incidence relative to the wild-type control mice (Fig. 5CCitation ; Table 1)Citation . Both male and female FVB/N mice surpassed 50% papilloma incidence by the eighth week of promotion, whereas the T7-PKC{delta} mice did not exceed 50% incidence until weeks 12 and 13 of promotion for females and males, respectively. Tumor incidence of 100% was achieved by the tenth week of promotion for both male and female wild-type mice. In contrast, the female T7-PKC{delta} mice did not reach 100% incidence over the 25 weeks of tumor promotion, whereas the male T7-PKC{delta} mice reached 100% incidence only after 23 weeks of promotion (Fig. 5CCitation ; Table 1Citation ). The T7-PKC{delta} line 37 mice, which expressed 4-fold less T7-PKC{delta} than line 16, were tested for their responsiveness to mouse skin tumor promotion by TPA. In the experiment with the line 37 mice, both male and female transgenic mice exhibited no significant reduction in papilloma burden or incidence compared with the wild-type mice (Fig. 5, E and F)Citation .



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  		Fig. 5. Mouse skin tumor promotion in T7-PKC{delta} mice. Wild-type and T7-PKC{delta} mice were initiated with a single 100-nmol dose of DMBA and promoted with 5 nmol of TPA. Photographs of representative wild-type (A) and line 16 T7-PKC{delta} mice (B) mice from the experiments illustrated in C and D after 25 weeks of TPA promotion. In each experiment, wild-type males ({blacksquare}), wild-type females (•), T7-PKC{delta} males ({diamondsuit}), and T7-PKC{delta} females ({triangleup}) were treated in parallel. The tumor incidences during 25 weeks of tumor promotion are shown for line 16 (C) and line 37 (E). The tumor burdens during 25 weeks of tumor promotion are shown for line 16 (D; P < 0.001 for both males and females after 10 weeks of treatment) and line 37 (F). Bars, SE of the papilloma burden for each papilloma count.

 

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  		Table 1 Summary of tumor at 25 weeks of promotion treatment for line 16 T7-PKC{delta} mice

 
At the end of 25 weeks of tumor promotion in the T7-PKC{delta} line 16 mice, the carcinoma incidence in the female mice was 78 and 37% for the wild-type and transgenic mice, respectively. Similarly, the carcinoma incidence for the males in this experiment was 45% for the wild-type mice and 7% for the transgenic mice. With respect to the wild-type controls, the average latency period for carcinoma development was delayed by 3 weeks for both male and female T7-PKC{delta} line 16 transgenic mice. Carcinoma development in the tumor promotion experiment with the T7-PKC{delta} line 37 mice, as compared with wild-type littermates, was not consistently altered by the presence of the transgene compared with wild type controls (data not shown).

T7-PKC{delta} Activity and Protein Levels after Tumor Promotion.
To determine whether papilloma formation in the T7-PKC{delta} mice required the loss of T7-PKC{delta}, the levels of T7-PKC{delta} activity and total PKC{delta} protein were examined at the end of the 25 weeks of tumor promotion. Seventy-two h after the last treatment, four mice from each treatment group were euthanized, and the dorsal, uninvolved epidermis and papillomas were removed. The samples were solubilized in IP lysis buffer, immunoprecipitated with the anti-T7-Tag antibody, and assayed for PKC activity with an EGFR peptide substrate. In two separate experiments, T7-PKC{delta} kinase activity could be detected in extracts from all of the treatment groups (Table 2)Citation . A comparison of the T7-PKC{delta} kinase activity in the dorsal epidermis from acetone or TPA-treated mice revealed that TPA treatment modestly reduced the level of immunoprecipitable T7-PKC{delta} kinase activity after chronic TPA treatment (Table 2)Citation . Additionally, the level of T7-PKC{delta} kinase activity in papillomas as compared with the levels in the uninvolved TPA-treated epidermis was not consistently altered.


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  		Table 2 PKC immunocomplex kinase assay of T7-PKC{delta} after tumor promotion

 
These extracts were also examined for the levels of total PKC{delta} protein present in each treatment group (Fig. 6)Citation . The level of PKC{delta} remained constant in the epidermis of wild-type mice even after 25 weeks of biweekly TPA treatment. In the epidermal extracts from the TPA-treated T7-PKC{delta} mice, minor reductions in the level of total PKC{delta} were observed with respect to the levels of PKC{delta} in the acetone-treated controls. Expression of endogenous PKC{delta} in papillomas from wild-type mice were consistently reduced to extremely low levels as compared with the levels in the uninvolved epidermis of TPA-treated mice. Unlike papillomas examined from wild-type mice, total PKC{delta} protein was maintained in papillomas from T7-PKC{delta} mice at variable levels as compared with the surrounding, uninvolved epidermis (Fig. 6)Citation .



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  		Fig. 6. The expression pattern of PKC{delta} after 25 weeks of tumor promotion. Line 16 T7-PKC{delta} (Tg) and wild-type (Wt) mice from the tumor promotion experiment were euthanized 72 h after the last acetone or TPA treatment. The extracts (100 µg) from acetone- or TPA-treated epidermis and papillomas from the kinase assay were immunoblotted. PKC{delta} was detected with the monoclonal anti-PKC{delta}. As a control for loading variation, actin levels were determined with a polyclonal goat anti-actin antibody.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
PKC{delta} has been shown to be an important component of the signaling pathways involved in the regulation of cell growth, differentiation, and apoptosis (24) . Overexpression of PKC{delta} in NIH 3T3 cells and CHO cells reduced their growth rates (25 , 26) . Furthermore, expression of a constitutively active PKC{delta} inhibited colony formation of both wild-type and c-Ha-ras-transformed NIH 3T3 cells (27) . Additionally, the transformation of c-Src overexpressing rat fibroblasts could be dramatically enhanced by the inhibition of PKC{delta} using pharmacological inhibitors or a dominant-negative PKC{delta} mutant (28) . Specific cleavage of PKC{delta} by caspase-3 occurs during UV or ionizing radiation-induced apoptosis. This indicates that PKC{delta} may play a central role in the execution of apoptosis (29 , 30) . However, conclusive evidence defining the in vivo role of individual PKC isoforms in tumor promotion has been elusive (1 , 8) . Using directed overexpression of PKC{delta} in the mouse epidermis, we found that the PKC{delta} isoform inhibited tumor promotion by TPA. Thus, PKC{delta} may be a key regulator of mouse skin tumor promotion by TPA.

The expression of elevated levels of PKC{delta} in the epidermis of FVB/N mice did not alter the normal regulation of skin development or differentiation. The lack of phenotypic alterations may have occurred for several reasons. The endogenous PKC{delta} is readily detectable in the mouse epidermis (15 , 16) . This suggests that a high level of PKC{delta} protein may be required for normal epidermal differentiation and that the PKC{delta} elevation attained in our transgenic lines was not sufficient to disrupt epidermal differentiation. The T7-PKC{delta} transgene exhibited little constitutive activity; thus, activation of T7-PKC{delta} would have been dependent on endogenous activators, like DAG. The level of DAG, or other activators, is most likely regulated upstream of T7-PKC{delta} (5) . Hence, the rate-limiting step in PKC{delta} signaling in normal epidermal homeostasis may not be at the level of PKC{delta} protein but at the level of activators present.

The only abnormality observed in the T7-PKC{delta} mice was the sporadic hyperproliferation and hyperkeratosis of the tail epidermis. This type of localized phenotypic alterations has been observed by several investigators who have used this heterologous human K14 promoter for transgene production (21 , 31) . Mice overexpressing TGF-{alpha} from the human K14 promoter exhibited thickening of the epidermis in the ear, tail, footpads, rectal epithelium, foreskin, scrotum, and vaginal epithelium (21) . Targeted expression of the human papillomavirus type-16 genome with the K14 promoter led to ear and face hyperplasia with dysplasia and papillomatosis with complete penetrance (31) . A simple explanation for these regional alterations in these transgenic mouse lines may be that the affected sites are areas prone to mechanical irritation (21 , 32) . The sporadic nature of the dry tail phenotype of the T7-PKC{delta} mice suggests that irritation may elicit this phenotype. Other investigators have suggested mechanical irritation as the probable cause for focal, regional phenotypes in mice harboring epidermal transgenes (21 , 31 , 32) Another potential cause for this restricted phenotype could be regional skin sensitivities to the different transgenes. This has been observed with the EGF treatment of adult mice, resulting only in the hyperproliferation in the epidermis of the tail and the footpads of treated mice (33) .

Recent investigations have demonstrated cross-regulation between different PKC isoforms. Overexpression of PKC{alpha} in lymphoid cell lines specifically induces a reduction in PKC{delta} mRNA and protein (34) . Analysis of the PKC signaling cascade, resulting in the activation of the c-fos promoter in mouse mammary epithelial cells, revealed that PKC{lambda} was upstream of PKC{epsilon} and PKC{zeta} was downstream of PKC{epsilon} (35) . Thus, overexpression of an individual PKC isoform may affect the regulation of other isoforms. Using immunoblot analysis, we found the levels of epidermal PKC{alpha}, {epsilon}, {lambda}, µ, and {zeta} were unaltered by overexpression of PKC{delta}, whereas the levels of PKC{beta}II and PKC{theta} were reduced and PKC{eta} was elevated. Thus, overexpression of PKC{delta} does not induce major alterations in the PKC isoform composition of the epidermis. The specific modulation PKC{beta}II, {eta}, and {theta}, but not the other isoforms, suggests that the effects of elevated epidermal PKC{delta} are specific and not an artifact of the model system.

The results of mouse skin tumor promotion with the T7-PKC{delta} mice were dramatic. The T7-PKC{delta} line 16 mice averaged a 73% reduction in papilloma burden for both male and female mice. The appearance of tumors on the T7-PKC{delta} line 16 mice was also delayed by 4 weeks on average. The carcinoma incidence was also reduced in the line 16 mice. The low number of papillomas and the delay in their formation in the T7-PKC{delta} line 16 mice suggest that a second event may need to occur to allow the formation of papillomas in the line 16 mice. The reduction in carcinoma incidence may be a direct effect of PKC{delta} on papilloma progression or simply the result of the reduced papilloma burden. Additional studies need to be performed to determine whether this is a direct or indirect effect of PKC{delta} overexpression. The T7-PKC{delta} line 37 mice exhibited lower levels of T7-PKC{delta} protein and activity compared with line 16 and did not display any alterations in sensitivity to mouse skin tumor promotion. The inhibition of tumor promotion in the line 16 mice and the lack of alterations in the line 37 mice imply that the inhibition of papilloma formation by treatment with DMBA/TPA requires a threshold level of PKC{delta} activity.

Reduced levels of PKC{delta} protein and activity have been observed after single or multiple TPA treatments of the mouse skin (12 , 15) . The level of PKC{delta} protein was also found to be significantly reduced in papillomas (15) . However, these reductions in PKC{delta} protein levels were not consistently observed (12 , 16 , 17) . The level of T7-PKC{delta} activity and total PKC{delta} protein levels were analyzed at the end of the 25-week carcinogenesis experiment to determine whether loss of PKC{delta} was important for papilloma formation in the wild-type and transgenic mice. In the T7-PKC{delta} line 16 mice, the level of T7-PKC{delta} kinase activity in the TPA-treated epidermis was only modestly reduced compared with acetone-treated transgenic mice. T7-PKC{delta} kinase activity was detected in papillomas from transgenic mice, and the level of activity did not change consistently compared with the activity in the surrounding, uninvolved epidermis. However, examination of the PKC{delta} protein levels in wild-type mice demonstrated a consistent loss of endogenous PKC{delta} protein in the papillomas from wild-type mice. In contrast, PKC{delta} was detected in papillomas from T7-PKC{delta} mice at variable levels. The observed loss of detectable PKC{delta} from wild-type papillomas implies that decreased PKC{delta} protein normally facilitates papilloma formation. The retention of T7-PKC{delta} activity in papillomas formed in transgenic mice suggests that the loss of T7-PKC{delta} activity is not necessary for the formation of papillomas on the transgenic mice. The observed delay in papilloma incidence and reduction in papilloma burden suggests that a secondary event was needed for papilloma formation in the transgenic mice. Activation of other signal transduction pathways may allow the cells to overcome the inhibition of papilloma formation by elevated PKC{delta} levels.

The ability of PKC{delta} to suppress papilloma formation implies that its activation may block epidermal keratinocyte proliferation and/or transformation. These results are consistent with the role of PKC{delta} in in vitro cell culture studies that have shown PKC{delta} to be an inhibitor of cell growth and transformation. Overexpression of PKC{delta} inhibited the growth of fibroblasts, vascular smooth muscle cells, and endothelial cells by delaying passage through different phases of the cell cycle, depending on the cell type (25 , 26 , 36 , 37) . Inhibition of vascular smooth muscle cell proliferation by elevated PKC{delta} levels correlated with decreased expression of cyclins D1 and E (36) . In the mouse epidermis, TPA-induced proliferation correlated with up-regulation of both cyclin D1 and cyclin E (38) . Additionally, the homozygous deletion of cyclin D1 reduced the papilloma burden in response to DMBA-TPA treatment of the mouse skin (39) . These proteins may be important mediators of the TPA response mediated by PKC{delta} in the mouse epidermis (Fig. 7)Citation . To begin to examine the role of PKC{delta} in the regulation of epidermal proliferation, the effect of a single treatment with 5 nmol of TPA on epidermal hyperplasia was examined over a 48-h period. No differences in hyperplasia were detected between wild-type and transgenic epidermis after TPA treatment (data not shown). Thus, the effects of PKC{delta} overexpression in the skin may be more complex than simply blocking cell proliferation; however, further study will be required to prove this.



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  		Fig. 7. Proposed molecular model for the effects of overexpression of PKC{delta} on mouse skin tumor promotion. TPA or DAG, generated from the hydrolysis of 4,5-inosine bisphosphate (PIP2) by phospholipase C-{gamma} (PLC-{gamma}), in conjunction with PS, activates PKC{delta} at the plasma membrane. Active PKC{delta} may act to suppress tumor growth by induction of downstream effectors like AP-1, cyclin D1, or cyclin E. Initiated keratinocytes bearing activated c-Ha-ras gene exhibit elevated levels of TGF-{alpha} production. TGF-{alpha} activation of the EGFR negatively regulates PKC{delta} activity through the activation of the Src family tyrosine kinases c-Src or c-Fyn. Activation of these kinases in keratinocytes leads to tyrosine phosphorylation of PKC{delta} and a reduction in its kinase activity. Reduced levels of active PKC{delta} may be permissive for keratinocyte growth. Elevated levels of PKC{delta} may overcome this inhibitory signal and allow suppression of tumor promotion in the T7-PKC{delta} mice.

 
Transient cotransfections of PKC{delta} with AP-1-regulated reporter genes in NIH3T3 cells have demonstrated that PKC{delta} can activate the AP-1 response elements (27) . Furthermore, the activation of AP-1 by PKC{delta} positively correlated with its inhibition of activated c-Ha-ras-mediated transformation of NIH3T3 cells. In mouse keratinocytes, induction of terminal differentiation by TPA correlates with the activation of AP-1 (40 , 41) . Induction of terminal differentiation in initiated cells would prevent their expansion into papillomas. Thus, activation of AP-1 may also be important for tumor suppression in T7-PKC{delta} mice (Fig. 7)Citation .

Introduction of an activated c-Ha-ras gene into primary mouse keratinocytes leads to a block in their ability to differentiate in response to elevated Ca2+ and increased susceptibility to papilloma formation in skin grafts (42 , 43) . The transformation of keratinocytes by c-Ha-ras overexpression correlates with increased levels of TGF-{alpha} and reduced PKC{delta} activity (44, 45, 46) . The reduction in PKC{delta} activity coincides with tyrosine phosphorylation of PKC{delta} by the Src tyrosine kinases c-Src or c-Fyn. This appears to be mediated by TGF-{alpha} activation of the EGFR signaling pathway (46 , 47) . Because activation of the c-Ha-ras locus is the initiating event in ~90% of the papillomas formed on DMBA-treated skin, down-regulation of PKC{delta} by this mutation may be important for papilloma formation (48) . Elevation of the intracellular level of PKC{delta} may prevent complete suppression of PKC{delta} activity. This retention of PKC{delta} activity may then be able to prevent the development of papillomas from initiated cells (Fig. 7)Citation .

Activation of PKC by TPA must impart a positive growth signal to promote tumor formation in wild-type mice. Because PKC{delta} appears to inhibit papilloma formation, other isoforms may be transducing this positive signal. This supports the hypothesis that individual PKC isoforms have unique roles in cell growth regulation. Furthermore, the ability of PKC{delta} to inhibit papilloma formations suggests that PKC{delta} may truly be an in vivo suppressor of tumorigenic transformation.


   ACKNOWLEDGMENTS
 
We thank Ashok Rajput, Sarah Bourguinon, Rachelle Stenzel, and Kathy Helmuth for excellent technical support. We also thank Dr. Elaine Fuchs at the University of Chicago for providing the pGEM3Z-K14 {beta}-globin vector.


   FOOTNOTES
 
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.

1 This work was supported by NIH Grant CA35368. Back

2 Present address: School of Medical Technology, Chang Gung University, Taiwan, Republic of China. Back

3 To whom requests for reprints should be addressed, at Department of Human Oncology, K4/532 Clinical Sciences Center, 600 Highland Avenue, Madison, WI 53792. Phone: (608) 263-9136; Fax: (608) 262-6654. Back

4 The abbreviations used are: TPA, 12-O-tetradecanoylphorbol-13-acetate; DAG, diacylglycerol; PS, phosphatidylserine; DMBA, 7,12-dimethylbenz[a]anthracene; PKC, protein kinase C; nPKC, novel PKC; EGFR, epidermal growth factor receptor; HRP, horseradish peroxidase; TGF, transforming growth factor. Back

Received 6/18/99. Accepted 9/21/99.


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Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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