Relation of the induction of epidermal ornithine decarboxylase and hyperplasia to the different skin tumor-promotion susceptibilities of protein kinase C alpha, -delta and -epsilon transgenic mice.

To define the in vivo role of individual PKC isoforms in mouse skin carcinogenesis, we previously characterized FVB/n transgenic mice that over-expressed epitope-tagged PKC delta (T7-PKC delta) or PKC epsilon (T7-PKC epsilon) isoforms under the regulation of the human K14 promoter. In continuation of our prior PKC isoform specificity studies, we now report the generation of FVB/n transgenic mice with K14-regulated, epitope-tagged PKC alpha (T7-PKC alpha). T7-PKC alpha transgenic mice (line 115) express 8-fold more PKC alpha protein than wild-type mice. Using high-resolution immunogold cytochemistry, we determined that transgenic over-expression of T7-PKC alpha did not alter the subcellular localization of PKC alpha but that the density of PKC alpha staining increased. PKC alpha localized primarily to the cytoskeleton (tonofilaments, tight junctions) and cell membranes, with modest but definite nuclear labeling also identified. Also, PKC alpha over-expression did not alter the immunoreactive protein levels of other PKC isoforms (delta, epsilon, eta, zeta, mu) in the epidermis. Skin tumor-promotion susceptibility was compared among all 3 lines of T7-PKC transgenic mice (alpha, delta and epsilon). While T7-PKC alpha had no effect on skin tumor promotion by TPA, T7-PKC delta reduced papilloma burden by 76% compared to wild-type controls. T7-PKC epsilon further reduced papilloma burden to 93% compared to wild-type controls but still resulted in the development of squamous-cell carcinoma. To find potential mechanisms of PKC-associated differences in tumor promotion, the induction of known downstream effectors of tumor promotion, ornithine decarboxylase (ODC) activity and epidermal hyperplasia, was determined. Despite long-term papilloma inhibition in both PKC delta and PKC epsilon transgenic mice, the induction of ODC by TPA was not attenuated in PKC delta and epsilon mouse lines. Both PKC transgenic and wild-type mice exhibited sustained hyperplasia after repeated TPA treatments. However, TPA-induced epidermal hyperplasia in T7-PKC epsilon mice was significantly increased (52%) compared with T7-PKC alpha, T7-PKC delta and wild-type mice. TPA-induced ODC activity and the resultant accumulation of polyamines may play different roles (e.g., induction of apoptosis vs. proliferation) in the pathways leading to the induction of cancer in PKC alpha, PKC delta and PKC epsilon transgenic mice.

Localization of the thioredoxin system in normal rat kidney.

Components of the thioredoxin system were localized in normal rat kidney using immunoperoxidase techniques at the light microscopic level and immunogold techniques at the ultrastructural level. Results from both methods were similar. Thioredoxin, thioredoxin reductases, and peroxiredoxins showed cell-type-specific localization, with the same cell types (proximal and distal tubular epithelial, papillary collecting duct, and transitional epithelial cells) previously identified as having high amounts of antioxidant enzyme immunoreactive proteins and oxidative damage products also having high levels of proteins of the thioredoxin system. In addition, peroxiredoxins II and IV were found in high levels in the cytoplasm of red blood cells, identified in kidney blood vessels. While thioredoxin and thioredoxin reductase 1 were found in all subcellular locations in kidney cells, thioredoxin reductase 2 was found predominantly in mitochondria. Thioredoxin reductase 1 was identified in rat plasma, suggesting it is a secreted protein. Peroxiredoxins often had specific subcellular locations, with peroxiredoxins III and V found in mitochondria and peroxiredoxin IV found in lysosomes. Our results emphasize the complex nature of the thioredoxin system, demonstrating unique cell-type and organelle specificity.

Protein kinase C-epsilon transgenic mice: a unique model for metastatic squamous cell carcinoma.

Squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) are the most common forms of human skin cancer. BCC is slow growing and mostly localized, whereas SCC metastasizes to the regional lymph nodes and subsequently to distal organs. In murine skin carcinogenesis models for SCC, the incidence of metastasis is very low. We report here that FVB/N transgenic mice, which overexpress (approximately 18-fold) epitope-tagged protein kinase C-epsilon (T7-PKCepsilon) protein in the epidermis provide a unique murine model system for highly malignant/metastatic SCC. Skin tumors were developed by the initiation-promotion protocol (initiation with 100 nmol 7,12-dimethyl-benz[a]anthracene; promotion with 5 nmol 12-O-tetradecanoylphorbol-13-acetate twice weekly). T7-PKCepsilon transgenic mice showed 92% suppression of papilloma development compared with wild-type littermates after 23 weeks of tumor promotion. However, within 15-20 weeks of 12-O-tetradecanoylphorbol-13-acetate promotion, 40% of T7-PKCepsilon mice developed at least one carcinoma compared with 7% of the wild-type mice. All carcinomas from T7-PKCepsilon mice appeared without prior papilloma formation. Interestingly, 7,12-dimethyl-benz[a]anthracene alone resulted in the development of squamous cell carcinomas in 22% of T7-PKCepsilon mice, whereas wild-type littermates developed no tumors. Histopathological analysis of tumors from multiple T7-PKCepsilon mice revealed moderately differentiated SCC invading the dermal region with neoplasia appearing to originate and invade from the hair follicle. Carcinomas of T7-PKCepsilon mice rapidly metastasized to regional lymph nodes within 3 weeks of appearance. In wild-type mice, the grade of the invading tumors, originating from interfollicular epidermis, was pathologically categorized as well-differentiated SCC and remained localized to the dermis. The T7-PKCepsilon transgenic mice may provide a rapid and unique in vivo model to investigate metastatic SCC.