Transgenic mice overexpressing protein kinase C epsilon in their epidermis exhibit reduced papilloma burden but enhanced carcinoma formation after tumor promotion.

To determine the role that protein kinase C epsilon (PKCepsilon) may play in skin growth, differentiation, and tumor promotion, transgenic mice were generated that overexpressed an epitope-tagged protein kinase C epsilon (T7-PKCepsilon) in their epidermis using the human keratin 14 promoter. Three independent mouse lines that overexpressed the T7-PKCepsilon 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 PKCepsilon 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-PKCepsilon 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 PKCepsilon, 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-PKCepsilon mice compared to wild-type controls. However, carcinomas developed rapidly in the T7-PKCepsilon mice treated with 7, 12-dimethylbenz[a]anthracene and TPA. These carcinomas appeared to form independently of prior papilloma development. These results demonstrate that PKCepsilon is an important regulator of skin tumor development.

Transgenic mice overexpressing protein kinase Cdelta in the epidermis are resistant to skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate.

To determine the role of protein kinase Cdelta in mouse skin carcinogenesis, we have developed transgenic FVB/N mouse lines expressing in the epidermis an epitope-tagged protein kinase Cdelta (T7-PKCdelta) regulated by the human keratin 14 promoter. The untreated T7-PKCdelta 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-PKCdelta mouse was unremarkable. Despite this mild phenotype, the effects of PKCdelta overexpression on mouse skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA) were dramatic. Two independent lines of T7-PKCdelta mice (16 and 37) expressing the T7-PKCdelta transgene were examined for responsiveness to skin tumor promotion by 7,12-dimethylbenz[a]anthracene and TPA. By immunoblot analysis, the T7-PKCdelta-16 and T7-PKCdelta-37 mice showed an 8- and 2-fold increase of PKCdelta protein. The T7-PKCdelta-16 mice averaged 300% more T7-PKCdelta activity than the T7-PKCdelta-37 mice did. The T7-PKCdelta-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-PKCdelta-16 mice relative to wild-type mice averaged 72 and 74% for males and females, respectively. The T7-PKCdelta-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-PKCdelta-16 mice. Carcinoma incidence at 25 weeks of promotion treatment was: wild-type females, 78%; T7-PKCdelta16 females, 37%; wild-type males, 45%; and T7- PKCdelta-16 males, 7%. Thus, PKCdelta when expressed at sufficient levels can suppress skin tumor promotion by TPA.

Localization of the 12-O-tetradecanoylphorbol-13-acetate response of the human ornithine decarboxylase promoter to the TATA box.

In a previous study, we narrowed the region of the human ornithine decarboxylase (ODC) promoter responsive to 12-O-tetradecanoylphorbol-13-acetate (TPA) to nt -42 to +54 around the transcription initiation site (Kim YJ, Pan H, Verma AK, Mol Carcinog 10:169-179, 1994). Here we report defining the role of the TATA box in TPA-induced transcription from the -42/+54 ODC promoter fragment. A transversion mutation at the third position of the TATA box (TATAAGT-->TAAAAGT) reduced TPA responsiveness of the reporter construct -42/+54 ODC-Luc by 49%. Electrophoretic mobility shift assays (EMSAs) using HeLa cell nuclear protein extracts revealed no differences in the binding pattern between the natural -42/+54 ODC promoter element and the -42/+54 ODC promoter element containing the T-->A mutation. However, antibodies to the general transcription factor TFIIB disrupted the DNA-protein complexes normally formed with the -42/+54 ODC promoter element in EMSAs. A consensus TATA box oligonucleotide formed two bands, with the faster mobility band displaying enhanced binding with nuclear protein extracts from TPA treated cells. Furthermore, incubation of HeLa cell nuclear extracts with an oligonucleotide containing the ODC TATA box also caused formation of two specific bands in EMSA. Both bands exhibited augmented binding to nuclear proteins from TPA-treated cells. Introduction of the T-->A transversion mutation in the ODC TATA oligonucleotide eliminated binding of the faster migrating band formed with the natural ODC TATA oligonucleotide. These results indicate that TPA modulation of the general transcription machinery may play a role in the TPA-activated transcription of the human ODC promoter.

The essential in vivo function of the herpes simplex virus UL42 protein correlates with its ability to stimulate the viral DNA polymerase in vitro.

The product of the UL42 gene of herpes simplex virus type 1 (HSV-1) is an essential protein required for viral DNA synthesis in both transient origin of replication-dependent DNA replication assays and in virus-infected cells. In vitro, UL42 has been shown to form a heterodimeric complex with the 140-kDa protein product of the viral DNA polymerase (pol) gene. Although the pol gene possesses catalytic activity in vitro in the absence of UL42, UL42 stimulates pol activity presumably by increasing its processivity. In order to investigate whether the essential in vivo function for UL42 is related to its ability to associate with and modify pol activity, we have examined the ability of a UL42 null mutant, Cgal delta 42, to induce pol activity in nonpermissive Vero cells or permissive V9 cells. No detectable high salt-resistant pol activity was observed in Vero cells, although substantial activity was induced in V9 cells. Use of temperature-sensitive and host range mutants with defects in other genes revealed that failure to induce pol activity was due to neither direct nor indirect effects caused by lack of viral DNA synthesis. Furthermore, pol protein accumulated in Cgal delta 42 virus-infected nonpermissive cells with similar kinetics and to approximately the same level as in cells infected with wild-type virus. These results suggest a direct dependence on UL42 for pol activity. We also examined whether the same domains of UL42 affected the ability of the protein to stimulate pol activity in vitro and to complement the replication of Cgal delta 42. The excellent correlation between the activities of the mutant UL42 proteins in the in vitro pol stimulation assays and in the in vivo transient complementation assay indicates that the predominant in vivo role of UL42 is to provide pol accessory function, although additional essential functions for UL42 cannot be ruled out.