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[Cancer Research 60, 595-602, February 1, 2000]
© 2000 American Association for Cancer Research

Carcinogenesis

Transgenic Mice Overexpressing Protein Kinase C{epsilon} in Their Epidermis Exhibit Reduced Papilloma Burden but Enhanced Carcinoma Formation after Tumor Promotion1

Peter J. Reddig, Nancy E. Dreckschmidt, Jun Zou, Sarah E. Bourguignon, Terry D. Oberley and Ajit K. Verma2

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


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To determine the role that protein kinase C{epsilon} (PKC{epsilon}) may play in skin growth, differentiation, and tumor promotion, transgenic mice were generated that overexpressed an epitope-tagged protein kinase C{epsilon} (T7-PKC{epsilon}) in their epidermis using the human keratin 14 promoter. Three independent mouse lines that overexpressed the T7-PKC{epsilon} in their epidermis were produced. The three independent lines 206, 224, and 215 exhibited a 3-, 6-, and 18-fold elevation, respectively, in the level of PKC{epsilon} immunoreactive protein. Line 215 exhibited a 19-fold greater phosphatidylserine and 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulated kinase activity than line 224. Line 206 exhibited a low basal T7-PKC{epsilon} activity, which failed to be stimulated by phosphatidylserine and TPA. All of the line 215 transgenic mice (F0 to the F2 generation) displayed phenotypic changes in the skin. The phenotypic changes progressed gradually, starting around 4–5 months of age, with mild dryness of the tail accompanied by hair loss and inflammation at the base of the tail. Hyperproliferation and ulceration of the affected regions were observed around 7–8 months of age. The hyperproliferative epidermis from the affected regions exhibited an expansion of the suprabasal epidermal cells. Inflammation and/or ulceration were also observed in the dorsal skin, the ears, and around the eyes. The line 215 mice, which expressed the highest level of PKC{epsilon}, were evaluated for sensitivity to mouse skin tumor promotion by TPA. Tumors were elicited by the initiation (7,12-dimethylbenz[a]anthracene, 100 nmol)-promotion (TPA, 5 nmol/twice weekly) protocol. The papilloma burden was reduced by 95–96% for male and female T7-PKC{epsilon} mice compared to wild-type controls. However, carcinomas developed rapidly in the T7-PKC{epsilon} mice treated with 7, 12-dimethylbenz[a]anthracene and TPA. These carcinomas appeared to form independently of prior papilloma development. These results demonstrate that PKC{epsilon} is an important regulator of skin tumor development.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PKC3 represents a large family of PS-dependent serine/threonine kinases (1, 2, 3) . Based on structural similarities and cofactor dependence, 11 PKC isoforms have been classified into three subfamilies. The classical PKCs ({alpha}, ßI, ßII, and {gamma}) are Ca2+-dependent and are activated by diacylglycerol/TPA and PS. The novel PKCs ({delta}, {epsilon}, {eta}, and {theta}) are Ca2+- independent and require DAG/TPA and PS for activation. The atypical PKCs ({lambda} and {zeta}) require only PS for activation, although other activators, such as ceramide, have been identified (4) . PKCµ, originally classified as a novel PKC, appears to be a unique isoform (1) .

Of these 11 PKC isoforms, PKC{epsilon} appears to play an important role in cellular growth regulation. In several small cell lung carcinoma cell lines, the catalytic fragment of PKC{epsilon} was constitutively expressed, indicating that activated PKC{epsilon} may be important for the survival of these cells (5) . Overexpression of PKC{epsilon} in Rat-6 or NIH-3T3 fibroblasts led to increased growth rates, anchorage independence, and tumor formation in nude mice (6 , 7) . Additionally, PKC{epsilon} overexpression transformed nontumorigenic rat colonic epithelial cells (8) . Overexpression of PKC{epsilon} also suppressed apoptosis of interleukin-3-dependent human myeloid cells induced by removal of interleukin-3 (9) .

The mouse skin tumor promoter TPA binds and activates PKC{epsilon} (10) . However, the role PKC{epsilon} plays in mouse skin tumor promotion and epidermal cell growth and differentiation remains unclear (11 , 12) . Current evidence indicates that treatment of the mouse skin with TPA leads to a general reduction in PKC activity that persists for at least 4 days (13 , 14) . Examination of the effects of acute TPA treatment on the protein level of different PKC isoforms demonstrated decreases in PKCß and {eta} but has little or no effect on the levels of PKC{alpha}, {delta}, or {epsilon} (15 , 16) . The level of PKC activity for PKC{alpha}, ß, and {delta} was found to be reduced after acute or repeated TPA treatments, but PKC{epsilon} activity was not examined (15) . Analysis of PKC isoforms in DMBA-TPA-induced papillomas demonstrated decreases in cytosolic levels of PKC{alpha} and ßII protein, but insignificant alterations in the levels of PKC{delta}, {epsilon}, or {zeta} protein (17) . In cultured mouse skin keratinocytes, induction of differentiation by elevation of Ca2+ induces translocation of PKC{epsilon}, {delta}, and {alpha} to the membrane fraction, suggesting a role for activation of these isoforms in keratinocyte differentiation (18) .

To further define the in vivo role of PKC{epsilon} in mouse skin carcinogenesis, we have generated transgenic mice expressing an epitope-tagged PKC{epsilon} under the control of the human K14 promoter. Overexpression of PKC{epsilon} in the untreated mouse epidermis led to phenotypic abnormalities (such as inflammation, hyperkeratosis, hyperplasia, cellular hypertrophy, and ulceration), especially of the skin surrounding the tail base of older mice (~7–8 months of age). Paradoxically, two-stage tumor promotion with DMBA and TPA in the PKC{epsilon} transgenic mice exhibited significant reductions in papilloma burden compared to wild-type controls. However, carcinomas developed rapidly in the T7-PKC{epsilon} mice independently of papilloma formation.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
TPA and calpain inhibitor I were purchased from Alexis Corp. (San Diego, CA). DMBA was purchased from Aldrich Chemical Co., Inc. (Milwaukee, WI). Proteasome inhibitor Z-Leu-Leu-Leu-H (aldehyde) (MG132) was purchased from Peptide Institute, Inc. (Osaka, Japan). Horseradish peroxidase-conjugated anti-T7-Tag antibody was purchased from Novagen (Madison, WI). Rabbit polyclonal antibodies to PKC{epsilon} and actin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). DAKO immunoperoxidase LSABB+Kit was purchased from DAKO (Carpinteria, CA). Immobilized protein A/G-agarose was purchased from Pierce (Rockford, IL). Enhanced chemiluminescence and enhanced chemifluorescence Western blotting detection reagents and the PKC enzyme assay system were purchased from Amersham Life Sciences Inc. (Arlington Heights, IL). FVB/N mice, 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 mouse PKC{epsilon} cDNA in the pRSV-PKC{epsilon} vector to produce the pRSV-T7-PKC{epsilon} vector. The T7-PKC{epsilon} cDNA from pRSV-T7-PKC{epsilon} was ligated into the BamHI site of the pGEM3Z-K14 ß-globin vector by insertion of two Eco 47III fragments of the T7-PKC{epsilon} cDNA previously linked to BglII and BamHI sites to produce the pGEM3Z-K14-T7-PKC{epsilon} vector. The functional elements of pG3Z-K14-T7-PKC{epsilon} were isolated by partial digestion with HindIII and complete digestion with EheI. The purified K14-T7-PKC{epsilon} expression cassette was microinjected into the male pronuclei of one-cell fertilized embryos (FVB/N x FVB/N mice) by the University of Wisconsin’s Transgenic Mouse Facility. The transgene was detected by Southern blot analyses using genomic DNA from tail biopsies digested with EcoRV and using the radiolabeled EcoRV/BamHI fragment from pGEM3Z-K14 ß-globin vector for the probe. Founders bearing the transgene were bred to wild-type FVB/N mice to generate F1 offspring. Transgenic F1 mice were bred with other transgenic or wild-type FVB/N mice as necessary to maintain and expand the colony.

Mice.
The mice were housed in groups of three to four mice in plastic-bottomed cages in light-, humidity-, and temperature (24°C)-controlled rooms; food and water were available ad libitum. The animals were kept in a normal rhythm of 12-h light/12-h dark periods. At 8–10 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 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. The line 215 T7-PKC{epsilon} mice were used for tumor promotion. For mouse skin tumor initiation, a single 100-nmol dose of DMBA in 0.2 ml of acetone or acetone alone was applied topically to the shaved backs of the mice. Two weeks after initiation, 5 nmol of TPA in 0.2 ml of acetone or acetone alone was applied twice weekly to the skin for the duration of the experiment. The tumor incidence and burden were observed weekly starting at 4 weeks of TPA promotion. The number of mice for each experimental group was as follows: DMBA-TPA, 11 wild-type females, 15 transgenic females, 20 wild-type males, and 12 transgenic males; and DMBA-acetone, 11 wild-type females, 16 transgenic females, 19 wild-type males, 12 transgenic males; acetone-TPA, 10 wild-type females, 15 transgenic females, 19 wild-type males, and 11 transgenic males. 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.

Immunoblotting of PKC.
The mouse skin was excised and scraped to remove the s.c. tissue. The skin was ground with a mortar and pestle under liquid N2. The ground tissue was homogenized with 5 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 used as the total PKC extract. Protein concentration in the total PKC extract was determined, and 100 µg of total PKC extract protein were fractionated on a 7.5% or 10% SDS-PAGE. The proteins were transferred to 0.45 µm supported nitrocellulose membrane. The membrane was then incubated with anti-T7 Tag (1:2000 dilution) or anti-PKC{epsilon} (1:100 dilution) antibody, the bound antibody was detected using the appropriate secondary antibodies, and the detection signal was developed with Amersham’s enhanced chemiluminescence or enhanced chemifluorescence reagents. Immunoblotting of lysates from the immunocomplex kinase assays (see below) was also performed with 100 µg of total protein.

T7-PKC Immunocomplex Kinase Assay.
The dorsal skin of the mice was shaved and depilated 24 h before experimentation. The mice were euthanized, the dorsal skin was removed, and the epidermis was scrapped off on ice with a razor. The epidermis was 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, 1 mM EGTA, 100 µM benzamidine, 5 µg/ml antipain, 5 µg/ml pepstatin, 40 µM MG132, and 40 µM calpain inhibitor I], homogenized using a glass Teflon tissue homogenizer, agitated for 30 min at 4°C, and centrifuged at 14,000 rpm in a microcentrifuge for 15 min, and the supernatant of the lysate was used for IP. 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 (pH 7.4), 5 mM EDTA (pH 8.0), 10 mM EGTA (pH 7.9), 0.3% ß-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 (pH 7.4), 8 mM MgCl2, 0.136 mM 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.

Determination of the level of T7-PKC{epsilon} kinase activity of the epidermis and tumors from T7-PKC{epsilon} mice at the end of the tumor promotion experiment was performed as described above for the T7-PKC immunocomplex kinase assay, but with the following modifications. The skin was not depilated. Skin papillomas and carcinomas were excised before scraping off the uninvolved epidermis. The excised papillomas, carcinomas, and epidermis were separately homogenized and extracted in 0.5–1.0 ml of IP lysis buffer. For each treatment group, the epidermis from three mice was combined for extraction. Two to four papillomas or one to two carcinomas were excised, combined, and extracted in the IP lysis buffer. One hundred µg of the total protein extract, before IP, were used for immunoblot analysis.

Histology and 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 h in formalin and then embedded in paraffin. Four-µm sections were cut for H&E staining or immunostaining. Deparaffinized slides were used for immunostaining with the DAKO immunoperoxidase LSAB+Kit. Endogenous peroxidase activity was blocked with 30% H2O2. Nonspecific protein binding was blocked with normal swine serum. The slides were incubated overnight with the appropriate primary antibody and developed with biotinylated secondary antibody, streptavidin-conjugated horseradish peroxidase, and 3,3'-diaminobenzidine.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Construction of the K14-T7-PKC{epsilon} Expression Vector.
To target overexpression of PKC{epsilon} to the basal epidermal cells of the mouse skin, we used the human K14 promoter. This promoter has been used successfully to direct expression of several different genes to the epidermis (19) . A mouse PKC{epsilon} cDNA containing the T7 bacteriophage epitope tag at its 5' terminus was inserted into the BamHI site of the pG3Z-K14 ß-globin vector to create the pG3Z-K14-T7-PKC{epsilon} vector (Fig. 1A)Citation . The pG3Z-K14-T7-PKC{epsilon} vector expressed active T7-PKC{epsilon} assayed by immunocomplex kinase assays of CV-1 cells transiently transfected with the T7-PKC{epsilon} vector (data not shown).



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  		Fig. 1. Identification and molecular characterization of the T7-PKC{epsilon} mice. A, structure of the pG3Z-K14-T7-PKC{epsilon} vector after insertion of T7-PKC{epsilon} cDNA into the BamHI site of pG3Z-K14 ß-globin 5' of the polyadenylated tail. B, a Southern analysis to identify potential T7-PKC{epsilon} F0 mice. The genomic DNA from tail biopsies was digested with EcoRV and fractionated, and a radiolabeled EcoRV/BamHI fragment from pGEM3Z-K14 ß-globin vector was used for the probe. Genomic DNA from wild-type mice with (+) or without (-) the pG3Z-K14-T7-PKC{epsilon} plasmid was added at the level of 1 copy/genome for controls. The numbers above the lanes indicate the potential founder’s identification number. The arrowhead indicates the position of the transgene. C, immunoblot analysis. Untreated skin from line 206, 215, or 224 transgenic (+) or wild type (-) mice was homogenized in PKC extraction buffer, and 100 µg of the supernatant were used for immunoblot analysis. Monoclonal antibodies to the T7 epitope (I) or PKC{epsilon} (II) were used. The arrowheads indicate the position of PKC{epsilon}. D, T7-PKC{epsilon} activity. The epidermis from 206, 215, or 224 F1 wild-type (-) or transgenic (+), female ({square}) or male ({blacksquare}) was extracted in IP lysis buffer, and a 100 µg aliquot of protein from cleared lysates were used for IP with the T7 antibody. The immunoprecipitated T7-PKC{epsilon} protein was assayed in kinase buffer using the EGFR peptide (ERKRTLRRL) as the substrate. E, T7-PKC{epsilon} expression in mouse tissues. A transgenic line 215 F1 T7-PKC{epsilon} mouse was euthanized, and the indicated tissues were harvested. The tissues were homogenized in PKC extraction buffer, and 100 µg of the supernatant were used for immunoblot analysis with the anti-T7 antibody.

 
Generation and Characterization of T7-PKC{epsilon} Mouse Lines.
The K14-T7-PKC{epsilon} expression cassette was removed from the pG3Z-K14-T7-PKC{epsilon} vector by complete digestion with the endonuclease EheI and partial digestion with the endonuclease HindIII (Fig. 1A)Citation . The linear K14-T7-PKC{epsilon} expression cassette was purified from the contaminating vector DNA by agarose gel electrophoresis, electroelution, and ion-exchange chromatography. Once purified, the linear cassette was microinjected into the pronuclei of fertilized eggs of FVB/N mice (University of Wisconsin Biotechnology Center Transgenic Animal Facility), and the offspring were analyzed for integration of the K14-T7-PKC{epsilon} expression cassette. Genomic DNA isolated from tail biopsies of weanling mice was digested with the endonuclease EcoRV, and a Southern analysis was performed. A ~1-kb fragment of the K14 promoter ligated to the ß-globin intron isolated from the pG3Z-K14 ß-globin vector with EcoRV and BamHI was used as a probe. Nine mice exhibited transgene integration after analysis of 24 potential founder mice (Fig. 1B)Citation .

These founder mice were bred to wild-type FVB/N mice to produce F1 offspring. F1 mice from each line that was positive for the K14-T7-PKC{epsilon} transgene were examined for expression of the transgene. F1 mice were euthanized, the dorsal skin was shaved, and a Triton X-100-soluble extract was made from the dorsal skin. Immunoblots of these extracts were examined for T7-PKC{epsilon} expression by probing with either anti-T7 tag or anti-PKC{epsilon} antibodies. The blots probed with the anti-T7 antibody demonstrated that three of these lines were expressing the T7-PKC{epsilon} transgene (Fig. 1C)Citation . Examination of PKC{epsilon} levels with the anti-PKC{epsilon} antibody demonstrated that the PKC{epsilon} levels were significantly elevated in these transgenic lines (Fig. 1C)Citation . The increase in PKC{epsilon} immunoreactive protein levels was 3-, 6-, and 18-fold for line 206, 224, and 215, respectively. The T7-PKC{epsilon} expressed in these F1 mice was assayed for enzymatic activity. The epidermis was isolated from euthanized F1 mice, Triton X-100-soluble extracts were made, and the T7-PKC{epsilon} transgene was immunoprecipitated with the anti-T7 antibody. The immunoprecipitates were assayed for kinase activity by measuring the incorporation of 32P into an EGFR peptide (ERKRTLRRL) in the presence of PS and TPA. The level of overexpressed PKC{epsilon} protein was the highest in line 215, which positively correlated with the level of PKC activity (Fig. 1D)Citation . The amount of activated T7-PKC{epsilon} kinase activity detected in line 215 was approximately 19-fold greater than that observed in the line 224 mice. Line 206 displayed a low constitutive T7-PKC{epsilon} activity but exhibited no response to the presence of PS and TPA (Fig. 1D)Citation .

The pattern of expression of T7-PKC{epsilon} in different tissues was also examined. Organs from a line 215 transgenic T7-PKC{epsilon} mouse were isolated, and Triton X-100-soluble extracts were made. Immunoblotting these extracts with the anti-T7 antibody demonstrated T7-PKC{epsilon} expression in the thymus and trachea in addition to the skin (Fig. 1E)Citation .

Epidermal Expression of T7-PKC{epsilon}.
The expression pattern of the T7-PKC{epsilon} transgene was examined in the skin of T7-PKC{epsilon} mice. Formalin-fixed dorsal skin samples were taken from a wild-type mouse and a line 215 T7-PKC{epsilon} mouse. The tissues were hybridized with a polyclonal rabbit anti-PKC{epsilon} antibody. The wild-type dorsal skin sample exhibited light immunoreactivity to the anti-PKC{epsilon} antibody throughout the epidermis. Light and infrequent nuclear staining was observed (Fig. 2A)Citation . Staining with the anti-PKC{epsilon} antibody was more intense in the dorsal skin of the transgenic T7-PKC{epsilon} mouse than their wild-type littermates. PKC{epsilon} exhibited strong staining in the basal cells of the epidermis and was focally present in the suprabasal layers (Fig. 2B)Citation .



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  		Fig. 2. Immunostaining for PKC{epsilon} in the dorsal skin of the line 215 T7-PKC{epsilon} F1 mice. Dorsal skin from line 215 T7-PKC{epsilon} F1 was fixed in 10% neutral buffered formalin and embedded in paraffin, and sections (4 µm) were stained with the anti-PKC{epsilon} antibody (1:100 dilution). Sections of the dorsal skin of a wild-type F1 mouse (A) or a transgenic F1 T7-PKC{epsilon} mouse (B) were stained with the anti-PKC{epsilon} antibody. Sections of dorsal skin from a wild-type mouse (C) or a transgenic F1 T7-PKC{epsilon} mouse (D) using normal serum in place of PKC{epsilon} antibody. Bar, 100 µm.

 
PKC Isoform Expression Pattern.
The possibility was explored that PKC{epsilon} may cross-talk to other PKC isoforms by modulating their levels and their associated signals (20 , 21) . In this experiment (Fig. 3)Citation , we determined the levels of other PKC isoforms in the epidermis of T7-PKC{epsilon} mice to examine whether the elevated PKC{epsilon} levels in the epidermis altered the level of expression of other PKC isoforms. A modest increase in the levels of PKC{alpha}, ßII, and {zeta} immunoreactive proteins in the T7-PKC{epsilon} mice was observed (Fig. 3)Citation . There were no observed changes in the immunoreactive protein levels of the other PKC isoforms examined (Fig. 3)Citation .



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  		Fig. 3. PKC isoform expression in T7-PKC{epsilon} mice. Epidermis from untreated F1, line 215 T7-PKC{epsilon} (Tg) or wild-type (Wt) mice was scraped off and homogenized in IP lysis buffer. The extracts (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.

 
Phenotype and Histopathology of the T7-PKC{epsilon} Mice.
The K14-T7-PKC{epsilon} mice exhibited several phenotypic alterations. The line 215 mice exhibited the greatest penetrance, with all of the transgenic mice exhibiting phenotypic alterations. The mice were phenotypically normal at birth. The phenotypic alterations began around 4–5 months of age for the F1 and F2 mice with a mild hyperkeratosis in the tail epidermis (12 of 26 mice) around 4 months that did not persist. This was accompanied by persistent inflammation at the base of the tail in 26 of 26 (100%) mice and inflammation of the ears in 20 of 26 (77%) mice starting about 4–5 months of age. The inflammation of the tail base and ears was followed by the formation of ulcerative lesions at these sites around 7–8 months of age in 17 of 26 (65%) transgenic mice observed (Fig. 4, A and B)Citation . Mild, transient inflammation around the eyes was also observed in 7 of 26 mice (27%) in line 215 T7-PKC{epsilon} with a variable age of onset. Although not observed in the F1 mice, ulceration in the proximal dorsal skin that became evident at 5–6 months of age occurred in 7 of 20 (35%) of the F2 transgenic mice. Line 224 and 206 exhibited phenotypic abnormalities similar to that observed in line 215 mice. However, the severity of the abnormalities was greatly diminished.



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  		Fig. 4. The phenotype and histology of the line 215 T7-PKC{epsilon} mice. A and B, photographs of adult male T7-PKC{epsilon} F1 mouse from line 215 and its wild-type littermate at 8 months of age. A, wild-type littermate of the affected T7-PKC{epsilon} F1 mouse exhibited no phenotypic abnormalities. B, affected T7-PKC{epsilon} F1 mouse exhibited inflammation and ulceration at the base of the tail and inflammation of the ears. C–F, skin histology. The dorsal and tail base skin from a wild-type and a F1 line 215 T7-PKC{epsilon} mouse was fixed in 10% neutral buffered formalin and embedded in paraffin, and sections (4 µm) were stained with H&E. C, section of the dorsal skin from a wild-type mouse. D, section of the dorsal skin from a T7-PKC{epsilon} mouse. E, section of the tail base skin from a wild-type mouse. F, section of the tail base skin from a 215 T7-PKC{epsilon} mouse. Bar, 100 µm.

 
Tissue samples from the dorsal and tail base skin from wild-type and transgenic T7-PKC{epsilon} mice were examined histologically. These samples were fixed in formalin, sectioned, and stained with H&E. These mice consistently exhibited hyperkeratosis in the dorsal skin, but with no other apparent abnormalities (Fig. 4D)Citation . The grossly affected skin regions displayed several significant alterations. Hyperplasia, characterized by acanthosis, was found in the affected regions along with cellular hypertrophy. Focal regions of necrosis were also evident at sites of ulceration. Mixed inflammatory cell infiltrates were observed in the dermal layer with infiltration into the epidermis and keratinizing layers in affected regions. Prominent amongst the inflammatory cell infiltrates were neutrophils and mast cells (Fig. 4F)Citation .

Tumor Promotion.
The effect of PKC{epsilon} overexpression on mouse skin carcinogenesis in line 215 T7-PKC{epsilon} mice was determined. The mice were initiated by applying 100 nmol of DMBA to the skin in the acetone. Two weeks after initiation, the mice were promoted twice weekly with 5 nmol of TPA in acetone. Control mice were treated with acetone only. At the beginning of the experiment, the 8–10-week-old mice exhibited no phenotypic abnormalities. Treatment with DMBA and TPA elicited an average of 20 papillomas/mouse in both the wild-type females and males (Fig. 5,A and B)Citation . However, the T7-PKC{epsilon} mice averaged less than 1 papilloma/mouse for both male and female mice after treatment with DMBA and TPA. This was an average 95% reduction in papilloma burden for female and male T7-PKC{epsilon} mice. The papillomas that did form in any of the T7-PKC{epsilon} mice were small, usually less than 2 mm in diameter (Fig. 5, A and B)Citation . Both, T7-PKC{epsilon} and wild-type mice exhibited no differences in weight gain during the course of the experiment. The total survival for the wild-type mice was 92% and 88% for the T7-PKC{epsilon} mice at the end of tumor promotion.



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  		Fig. 5. Susceptibility of T7-PKC{epsilon} mice to skin tumor promotion by TPA. Mice were initiated with 100 nmol of DMBA or the acetone vehicle and then promoted with 5 nmol of TPA or the acetone vehicle. A and B, papilloma burden of female (A) and male (B) mice after tumor promotion. T7-PKC{epsilon} mice treated with DMBA and TPA ({blacktriangleup}), DMBA and acetone ({blacksquare}), or acetone and TPA (•). Wild-type mice treated with DMBA and TPA ({triangleup}), DMBA and acetone ({square}), or acetone and TPA ({circ}). The error bars indicate the SE of the papilloma burden for each data point. By the Wilcoxan rank-sum test, the papilloma burden was significantly (P < 0.001) different after 10 weeks of promotion between the DMBA-TPA-treated wild-type and T7-PKC{epsilon} mice for both males and females. C and D, representative photographs of wild-type and T7-PKC{epsilon} male mice from each treatment group after 15 weeks of promotion. C, wild-type mice treated with DMBA and TPA. D, T7-PKC{epsilon} mice treated with DMBA and TPA.

 
In spite of the low papilloma burden, the T7-PKC{epsilon} mice developed carcinomas independently of papilloma development (Fig. 5, C and DCitation ; Table 1Citation ). After 22 weeks of tumor promotion, 27% of the female and 50% of the male T7-PKC{epsilon} mice treated with DMBA and TPA developed carcinomas in the absence of prior formation of papillomas. Wild-type mice treated with DMBA and TPA developed carcinomas by this time (30% and 15% for females and males, respectively); however, all of these carcinomas developed from existing papillomas (Table 1)Citation . Additionally, 25% of the female T7-PKC{epsilon} treated with DMBA alone developed carcinomas by the twenty-first week of promotion (Table 1)Citation .


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  		Table 1 Carcinoma development in the line 215 T7-PKC{epsilon} tumor promotion experiment

 
The level of T7-PKC{epsilon} kinase activity present in papillomas and carcinomas that developed on the T7-PKC{epsilon} mice was measured to determine whether modulation of the T7-PKC{epsilon} kinase activity was necessary for the formation of these lesions. The epidermis from uninvolved skin was removed from three T7-PKC{epsilon} mice from each treatment group in the tumor promotion experiment, combined, and extracted with IP lysis buffer. Papillomas or carcinomas were also excised and extracted with IP lysis buffer. The levels of precipitable T7-PKC{epsilon} kinase activity in the epidermal extracts were very similar between each of the treatment groups at 72 and 120 h after the last treatment (Table 2)Citation . Both the basal (without PS/TPA) and the stimulated (with PS/TPA) T7-PKC{epsilon} kinase activity extracted from papillomas or carcinomas of DMBA and TPA-treated mice were greatly reduced compared to the levels found in the surrounding epidermis (Table 2)Citation . This reduction in kinase activity positively correlated with the reduced level of T7-PKC{epsilon} protein present in the total epidermal extracts used for the immunoprecipitations (Fig. 6)Citation .


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

 


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  		Fig. 6. Expression of T7-PKC{epsilon} and PKC{epsilon} protein in the epidermis (Ep.), papillomas (Pap.), and carcinomas (Car.) of mice from tumor promotion experiment. Mice were initiated with 100 nmol of DMBA (Lanes D) or acetone (Lanes A) and promoted with 5 nmol of TPA (Lanes T) or acetone (Lane A). A, after 23 weeks of tumor promotion, tissues were excised 120 h after the last treatment of male mice and homogenized in IP lysis buffer, and 100 µg of the extract were immunoblotted with the anti-T7 antibody, the anti-PKC{epsilon} antibody, and the anti-actin antibody. B, after 23 weeks of tumor promotion, tissue samples were taken 72 h after the last treatment and prepared and immunoblotted as described in A.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The introduction of elevated levels of PKC{epsilon} into the mouse epidermis led to inflammation of the tail base and ears around 4–5 months of age sometimes followed by ulceration of the tail base at around 7 months of age (Fig. 4)Citation . Histological examination of the affected mouse tail base revealed hyperplasia and inflammatory cell infiltration. Histological examination of the grossly unaffected dorsal skin from these mice revealed hyperkeratosis, but no apparent hyperplasia. This hyperkeratosis without apparent hyperplasia was also observed in K10-c-Ha-ras transgenic mice (22) . Dorsal epidermis from mice exhibiting inflammation and ulceration also displayed hyperplasia and inflammatory cell infiltrates. The majority of infiltrates appeared to be neutrophils and mast cells.

This delayed manifestation of phenotypic alterations is different from the time frame observed with the introduction of other transgenes using the human K14 promoter (23 , 24) . Introduction of TGF-{alpha} into the mouse epidermis driven by the K14 promoter led to hyperplasia of the suprabasal epidermal cells and hyperkeratosis in mice around 3–5 weeks of age. This phenotype disappeared in the TGF-{alpha} mice as they became adults. This loss of phenotype occurred despite maintenance of transgene expression (23) . Expression of an activated erB-2 receptor from the K14 promoter elicited severe phenotypic alterations in newborn mice. Most of the founder mice died immediately after birth, with the survivors exhibiting hyperplastic and dysplastic epidermal cell growth and papilloma formation (24) . Several transgenic mouse lines have demonstrated delayed development of phenotypic alterations. For example, epidermal thickening in the ear, tail, footpads, rectum, foreskin, scrotum, and vagina was observed in adult TGF-{alpha} mice (23) . Furthermore, mice expressing the complete human papilloma virus type-16 genome from the K14 promoter exhibited progressive skin abnormalities with full expression of the phenotype around 5 months of age (25) . Mice expressing an activated c-Ha-ras gene from the K10 promoter or v-fos from the human K1 promoter also exhibited a delayed appearance of phenotypic abnormalities (22 , 26) . The focal nature of the alterations in the T7-PKC{epsilon} mice has also been observed with other transgenes directed to the epidermis. For instance, the TGF-{alpha} mice exhibited focal epidermal thickening in adult mice (23) . Additionally, mice expressing the activated c-Ha-ras from the K10 promoter (K10 c-Ha-ras) developed papillomas at base of the tail, behind the ears, and on the footpads (22) . Expression of the complete human papilloma virus type-16 genome from the K14 promoter resulted in mice with ear and face hyperplasia, dysplasia, and papillomatosis with complete penetrance (25) . The delayed and focal nature of the phenotypic alterations in the T7-PKC{epsilon} mice suggests that secondary events need to occur in these regions to allow manifestation of this phenotype. The areas affected by expression of the T7-PKC{epsilon} transgene, i.e., the base of the tail and the ears, are sites that are prone to irritation. Irritation by scratching or other contact was also suggested to be the cause of the focal, late-onset skin alterations observed in K10-c-Ha-ras and K14-TGF-{alpha} transgenic mice (22 , 23) .

Transgenesis in the mouse epidermis has demonstrated that several genes are important in epidermal growth regulation. Introduction of elevated levels of TGF-{alpha}, activated erB-2, activated c-Ha-ras, v-fos, or T7-PKC{epsilon} led to papilloma formation associated with hyperplasia and/or hyperkeratosis, usually at sites of mechanical irritation (22 , 23 , 26 , 27) . Overexpression of keratinocyte growth factor also led to a thickened epidermis, but no papilloma formation (27) . Several of these genes have been shown to be important in the regulation of cell growth in culture and interact along the same signal transduction pathways. PKC{epsilon} can transform fibroblasts and epithelial cells, and this correlates with its ability to activate the c-Ras/Raf1/mitogen-activated protein kinase signaling pathways (28, 29, 30) . Additionally, in these same cells, a dominant negative Ras failed to inhibit transformation by PKC{epsilon} (28 , 30) . Thus, PKC{epsilon} may be acting downstream of the recruitment of Raf-1 to the membrane by Ras. In Rat-6 fibroblasts, elevation of PKC{epsilon} decreased c-fos mRNA levels, suggesting that PKC{epsilon} may be important in c-fos regulation (31) . Additionally, activated Ras can induce TGF-{alpha} production in keratinocytes (32) . Thus, the similarity of the phenotypes observed by overexpression of these genes in the mouse epidermis may be due to the fact that they affect similar signal transduction pathways.

Overexpression of PKC{epsilon} in the epidermis had an unexpected effect on mouse skin tumor promotion by TPA. In vitro models of cellular transformation indicated that PKC{epsilon} was a potent transforming protein when overexpressed in both fibroblasts and epithelial cells. PKC{epsilon} alone induced complete transformation of these cells, allowing tumor formation when these cells were injected s.c. in athymic mice (6, 7, 8) . This suggested that PKC{epsilon} would act to enhance the effects of tumor promotion. However, we observed a dramatic reduction in the papilloma burden when the line 215 T7-PKC{epsilon} mice were initiated with 100 nmol of DMBA and promoted with 5 nmol of TPA. Treatment of the mice with DMBA or TPA alone led to a few, small papillomas in T7-PKC{epsilon} mice, but the papilloma burdens were less than 1 papilloma/mouse. In papillomas obtained from T7-PKC{epsilon} mice 72 or 120 h after the last TPA treatment, the level of T7-PKC{epsilon} activity and protein was greatly reduced compared to the uninvolved epidermis. Thus, it appears that elevated levels of PKC{epsilon} activity may inhibit papilloma development, and this increased activity may need to be reduced for papillomas to develop. The low levels of immunoreactive PKC{epsilon} in papillomas from wild-type mice further indicate that a reduction in PKC{epsilon} levels is important for papilloma formation during tumor promotion.

The reasons for this paradoxical effect of PKC{epsilon} on mouse skin tumor promotion are unclear. Other proteins that have been termed oncogenes based on in vitro studies have also demonstrated unexpected results when tested in vivo. Homozygous deletion of c-fos did not affect the development of papillomas in skin tumor promotion experiments, but it did block the progression of papillomas to carcinomas (33) . Furthermore, E2F-1 null mice exhibited increased tumor incidences and defects in thymocyte development associated with alterations in apoptosis leading to hyperproliferation (34 , 35) . Thus, PKC{epsilon} may be able to stimulate or inhibit cell growth depending on the cellular context.

Surprisingly, in the absence of prior papilloma growth, the T7-PKC{epsilon} mice started developing carcinomas between 11 and 12 weeks of tumor promotion in DMBA and TPA-treated mice. The appearance of the T7-PKC{epsilon} mice resembled mice in a complete carcinogenesis experiment (36) . Additionally, a few of the T7-PKC{epsilon} mice initiated with DMBA and promoted with acetone developed carcinomas.

The reduction in the T7-PKC{epsilon} levels in the carcinomas indicates that elevated levels of PKC{epsilon} are not necessary for the maintenance of the carcinoma. However, the positive correlation between elevated levels of PKC{epsilon} and carcinoma formation indicates that PKC{epsilon} can induce the molecular changes necessary for carcinoma formation after treatment of the skin with a single dose of DMBA, alone or in conjunction with repeated TPA treatments.

Several proteins appear to have important roles in carcinoma development, some of which appear to be influenced by PKC. As described above, homozygous deletion of c-fos prevents the progression of papillomas to carcinomas (33) . Additionally, v-fos cooperates with v-ras to induce primary mouse keratinocytes to form squamous cell carcinomas in nude mice (37) . Connections between c-Fos and PKC{epsilon} have been previously identified. PKC{epsilon} can induce the activity of activator protein 1 response elements in reporter gene studies (38 , 39) . PKC{epsilon} also appears to be a part of the signaling cascade from c-Ha-ras that leads to activation of the c-fos promoter (20) . Thus, the interplay between c-fos and PKC{epsilon} may be important for the development of carcinomas in the T7-PKC{epsilon} mice. Regulation of p53 also appears to be critical for development of carcinomas in mouse skin. Mutations at the p53 locus have been identified in mouse skin carcinomas, but rarely in papillomas (40 , 41) . Furthermore, homozygous deletions of p53 in knockout mice did not enhance papilloma development by the DMBA/TPA tumor promotion protocol but did lead to enhanced conversion to squamous cell carcinomas (42) . Although the interaction specifically between PKC{epsilon} and p53 has not been examined, PKC does appear to play a role in the regulation of p53. PKC can phosphorylate p53 in vitro at sites that are phosphorylated in vivo, and this may regulate p53 DNA binding (43 , 44) . TPA-induced growth arrest in transformed rat fibroblasts positively correlated with enhanced p53 DNA binding and required active, wild-type p53 (43) . Therefore, p53 may be an important downstream target for PKC{epsilon}-induced carcinoma development. The proteins TGF-ß1 and TGF-ß2 appear to be important in suppressing malignant progression. Papillomas that progress with a high frequency to carcinomas do not express either TGF-ß1 or TGF-ß2. Squamous cell carcinomas do not exhibit any TGF-ß1 or TGF-ß2 expression, although these proteins are expressed in normal mouse skin (45) . Additionally, keratinocytes with the TGF-ß1 gene deleted can cooperate with v-Ha-ras to rapidly form squamous cell carcinomas in athymic mice (46) . Treatment of primary mouse keratinocytes with TPA elevates the level of TGF-ß2 expression (32) . Additionally, rat fibroblasts transformed with PKC{epsilon} exhibited elevated levels of TGF-ß2 and TGF-ß3 (47) . Thus, regulation of TGF-ß expression by PKC{epsilon} may be important for carcinoma development in T7-PKC{epsilon} mice.

In summary, PKC{epsilon} appears to be an essential enzyme for the genesis of mouse skin cancer. Elevation of PKC{epsilon} levels in the epidermis may disrupt cellular homeostasis and may push the cells to a hyperplastic, undifferentiated state. The delayed and focal appearance of abnormalities suggests that a secondary stimulus, such as mechanical irritation, may be necessary to elicit the phenotypic changes. Because PKCs are regulated at multiple levels (presence of agonist, phosphorylation, and cellular localization), the need for a secondary stimulus is predictable. The counterintuitive effects of PKC{epsilon} overexpression on tumor promotion demonstrate the importance of in vivo testing of the oncogenic capacity of genes proposed to be transforming based largely on cell culture studies. These mice will be useful for examining the mechanisms of tumor suppression and the development of carcinomas mediated by PKC{epsilon} in the mouse skin carcinogenesis model and identifying PKC{epsilon} effectors important for these responses.


   ACKNOWLEDGMENTS
 
We thank Ashok Rajput, Rachelle Stenzel, and Kathy Helmuth (University of Wisconsin, Madison, WI) for excellent technical support. We also thank Dr. Elaine Fuchs (University of Chicago, Chicago, IL) for providing the pG3Z-K14 ß-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 Supported by NIH Grant CA 35368. Back

2 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

3 The abbreviations used are: PKC, protein kinase C; PS, phosphatidylserine; TPA, 12-O-tetradecanoylphorbol-13-acetate; DMBA, 7,12-dimethylbenz[a]anthracene; FVB/N,FVB/NTacfBR; IP, immunoprecipitation; K14, Keratin 14; TGF, transforming growth factor; EGFR, epidermal growth factor receptor. Back

Received 6/30/99. Accepted 12/ 2/99.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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