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Recent work from our laboratory has shown that elevated src kinase activity enhances tumor promotion, malignant progression, and metastasis during multistage skin carcinogenesis. In this study, we have generated "gene-switch" src(530) transgenic mice to further analyze the role of this nonreceptor tyrosine kinase in multistage carcinogenesis. Target transgenic mice that have an activated form of the human c-src (src(530)) gene fused with GAL4 binding sites upstream of the thymidine kinase (TK) promoter were generated. Two lines of epidermis-specific transactivator mice were used that targeted the expression of GLVPc or GLp65 transactivators, fusion molecules containing a truncated progesterone receptor with a GAl4-DNA binding domain, with either a mouse loricrin (ML) or human keratin 14 (HK14) promoter, respectively. The transactivator mice (ML.GLVPc or HK14.GLp65) and the target mice (TK.src(530)) were mated to generate bitransgenic mice, and src(530) transgene expression was induced by topical application of RU486 (mifepristone, a progesterone receptor antagonist). In both ML.GLVPc/TK.src(530) and HK14.GLp65/TK.src(530) bitransgenic mice, histological analysis revealed that only the bitransgenic mice had marked epidermal hyperplasia and hyperkeratosis after treatment with RU486. Neither the nontransgenic mice nor the mice hemizygous for either the transactivator transgene or the target transgene alone showed any response to treatment with RU486. In addition, no differences were observed in the skin of the bitransgenic mice versus nontransgenic littermates without treatment of RU486. Interestingly, in HK14.GLp65/TK.src(530) bitransgenic mice, squamous cell carcinomas (SCCs) arose along the periphery of the area of the punch biopsies in 25% of the bitransgenic mice several weeks after taking the biopsy and subsequent to RU486 treatment. Collectively, the data support a role of c-src activation in epidermal hyperproliferation. Furthermore, the data support the conclusion that src activation can substitute for an initiating event in the presence of a tumor promoting stimulus (i.e., wounding). Finally, inducible src(530) transgenic mice provide a new tool for dissecting the role of src activation in multistage carcinogenesis by allowing temporal control of the expression of this oncogene.

The erbB family of receptor tyrosine kinases, which consists of the epidermal growth factor receptor (EGFr/erbB1), erbB2 (neu), erbB3 and erbB4, has been shown to be important for both normal development as well as neoplasia. The expression of rat erbB2 was targeted to the basal layer of mouse epidermis with the bovine keratin 5 promoter. Overexpression of wild type rat erbB2 in the basal layer of epidermis led to alopecia, follicular hyperplasia and sebaceous gland enlargement as well as hyperplasia of the interfollicular epidermis. Spontaneous papillomas, some of which converted to squamous cell carcinomas, arose in homozygous erbB2 transgenic mice as early as 6 weeks of age with >90% incidence by 6 months. Analysis of several proliferation/differentiation markers indicated that erbB2 overexpression led to epidermal hyperproliferation and a possible delay in epidermal differentiation. Transgenic mice were also hypersensitive to the proliferative effects of the skin tumor promoter, 12-0-tetradecanoylphorbol-13-acetate (TPA) and were more sensitive to two-stage carcinogenesis. Elevations in EGFr and erbB2 protein as well as erbB2:EGFr and erbB2:erbB3 heterodimers were observed in skin of the erbB2 transgenic mice. Phosphotyrosine levels of the EGFr, erbB2 and erbB3 proteins were also elevated. These results indicate an important role for erbB2 signaling in epidermal growth, development and neoplasia. Oncogene (2000) 19, 4243 - 4254

Transgenic mice overexpressing insulin-like growth factor-1 (IGF-1) in the basal layer of skin epidermis were generated using the bovine keratin 5 promoter (BK5). Neonatal transgenic mice were slightly smaller at birth and exhibited early ear unfolding, wrinkled and thickened skin, and slightly enlarged ears compared with nontransgenic littermates. Morphological evaluation of the skin revealed that persistent overexpression of IGF-1 in the basal layer of the epidermis resulted in epidermal hyperplasia, hyperkeratosis, and an increased labeling index that persisted in adult mice. Phenotypic changes observed in skin were associated with transgene expression in the basal layer of the epidermis and activation of the IGF-1 receptor. Squamous papillomas (some of which converted to carcinomas) developed in a significant proportion (approximately 50%) of older BK5.IGF-1 mice. Treatment of BK5.IGF-1 transgenic mice with multiple topical applications of the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, in the absence of tumor initiation led to the development of additional skin papillomas. Furthermore, treatment of BK5.IGF-1 transgenic mice with an initiating dose of 7,12-dimethylbenz[a]anthracene only led to the formation of additional papillomas in the absence of promotion. In two-stage carcinogenesis experiments, BK5.IGF-1 transgenic mice developed 7-fold more papillomas than nontransgenic littermates. Phosphatidylinositol-3-kinase and protein kinase B (Akt) activities were elevated (3-4-fold), and mitogen-activated protein kinase activity was elevated approximately 1.7-fold in the epidermis of transgenic mice compared with nontransgenic mice. In addition, UV light-induced epidermal apoptosis was significantly suppressed in BK5.IGF-1 transgenic mice. These data suggest that persistent activation of IGF-1 receptor signaling pathways in basal epithelial cells leads to spontaneous tumor promotion and that up-regulation of both mitogenic and cell survival signaling pathways may play an important role in the action of IGF-1 in this model system.

The present study has examined changes in activities and levels of four protein kinase C (PKC) isozymes (PKC alpha, PKC beta, PKC gamma and PKC delta) detectable in mouse epidermal preparations following both single and multiple treatments with 12-O-tetradecanoylphorbol-13-acetate (TPA). In addition, PKC epsilon and PKC eta protein levels were monitored by immunoblotting following TPA application. Finally, PKC isozyme activity profiles were also examined in epidermal preparations from mice treated with single applications of two non-phorbol ester tumor promoters: chrysarobin (CHRY) and okadaic acid (OA). Fifteen minutes following topical treatment with a tumor promoting dose of TPA (3.4 nmol), the activities of PKC beta and PKC gamma decreased in the epidermal cytosol to 30% and 50% of control values, respectively, while these activities were increased in the epidermal particulate fraction by approximately 50%. PKC delta activity, found predominantly in the particulate fraction of control epidermis, was greatly diminished in both subcellular fractions at 15 min while PKC alpha activity was translocated approximately 20% from cytosol to particulate fraction. Significant reductions in all four detectable PKC isozyme activities in both particulate and cytosol fractions were observed 48 h after a single treatment with TPA, although particulate PKC alpha activity appeared to be less affected at this point in time compared to the other PKC isozymes. Immunoblotting analyses of PKC isozyme protein levels after TPA treatment followed the changes in activity for cytosolic PKC alpha, PKC beta and PKC gamma. However, particulate PKC delta and PKC epsilon protein levels remained relatively unchanged while particulate PKC eta protein levels were significantly down-regulated after a single TPA treatment. Multiple topical treatments (twice-weekly for 2 weeks) with TPA produced a pattern of loss followed by only partial recovery of total PKC activity. Furthermore, all four PKC isozyme activities examined by hydroxylapatite (HA) chromatography were significantly reduced, including PKC alpha, after four applications of TPA. Cytosolic PKC alpha, PKC beta and PKC gamma protein levels as determined by immunoblotting again followed the activity profiles; particulate PKC eta protein levels were significantly reduced, whereas particulate PKC delta and PKC epsilon levels again appeared relatively unchanged. Fifteen minutes after topical application of 220 nmol CHRY, an approximately 25% decrease in particulate associated with PKC alpha activity was observed while particulate activities associated with PKC beta, PKC gamma and PKC delta were unaffected by CHRY at this time point.(ABSTRACT TRUNCATED AT 400 WORDS)

In the current study, the protein kinase C (PKC) isozymes present in mouse epidermis have been identified using immunological and chromatographic methods. Six PKC isozymes, PKC alpha, PKC beta, PKC gamma, PKC delta, PKC epsilon, and PKC zeta, were identified in unfractionated epidermal preparations by protein immunoblotting. The subcellular distribution and presence of these isozymes was further verified by hydroxyapatite (HA) chromatography with the exception of PKE epsilon, which could not be detected following HA chromatography. The five PKC isozymes recovered following HA chromatography were detected in both epidermal cytosol and particulate fractions, although PKC delta was found in a much higher proportion relative to the other PKC isozymes in the particulate fraction using histone H1 as the substrate. The biochemical properties of the epidermal PKC isozymes partially purified by HA chromatography agreed with those reported for other tissues and further supported their immunological identification in epidermal preparations. The activities of HA chromatography peaks corresponding to PKC alpha, PKC beta, and PKC gamma were found to be dependent on both Ca2+ and phosphatidylserine (PtdSer), whereas, the activities of HA peaks corresponding to PKC delta and PKC zeta were Ca(2+)-independent but PtdSer-dependent. The HA peak corresponding to PKC gamma also displayed a characteristic biphasic modulation by arachidonic acid (activation at low, inactivation at high concentrations) and inactivation by preincubation with PtdSer. PKC zeta activity was also characteristic, in that it was dependent on PtdSer and was not increased by the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate. Some differences in substrate specificity were also observed between the epidermal PKC isozymes. The presence of multiple isozymes of PKC in mouse epidermis suggests that the different isozymes may play distinct roles in signal transduction and tumor promotion in this tissue.