K6/ODC transgenic mice as a sensitive model for carcinogen identification.

Ornithine decarboxylase (ODC), an important enzyme in the polyamine biosynthetic pathway, is aberrantly regulated in many epithelial tumors of rodents and humans. In murine skin, it has been shown that ODC overexpression provides a sufficient condition for tumor promotion. Therefore, we hypothesized that K6/ODC transgenic mice in which ODC overexpression was targeted to hair follicle keratinocytes might provide a sensitive model for identifying genotoxic carcinogens. Ten known carcinogens or noncarcinogens have been tested in the model so far and results are highly concordant with 2-year rodent bioassays (100% concordant). More importantly, each of two chemicals tested that is recognized as a human carcinogen was identified as a carcinogen in K6/ODC transgenic mice. In addition, 7, 12-dimethylbenz(a)anthracene (DMBA) dose response studies indicated that even at a very low dose, 2 nmol, a high percentage of mice (50%) had already developed tumors 8 weeks after treatment. We conclude that the K6/ODC transgenic mouse model is very sensitive to topical application of genotoxic carcinogens and could therefore be a useful mouse model for carcinogen identification and chemical risk assessment.

Analysis of ras gene mutational spectra in epidermal papillomas from K6/ODC transgenic mice.

In standard mouse strains, a high proportion (more than 90%) of epidermal tumors produced by initiation with 7,12-dimethylbenz[a]anthracene and promotion with a variety of chemical agents contain an activating mutation in codon 61 (A182-->T) of the c-Ha-ras gene. We analyzed the ras mutational spectra in 69 tumors induced by DMBA in a unique transgenic model, the K6/ODC mouse. In this model, low-dose DMBA treatment is sufficient per se for tumor induction, so tumor promotion with chemical agents is not required. In contrast to previous studies in standard mouse strains, our study showed that less than 50% of epidermal tumors from K6/ODC mice contained an activating codon 61 c-Ha-ras mutation (A182-->T). This result was obtained in mice initiated either as newborns (when the transgene is not expressed) or as adults (when the transgene is fully expressed). Analysis of other codon hot-spots and other ras genes revealed the presence of three codon 12 and 20 codon 61 (A182-->T) mutations in the c-Ki-ras gene in the 36 tumors that did not have c-Ha-ras mutations. We concluded that promotion in this model, by means of constitutive ornithine decarboxylase expression, causes the clonal expansion of a population of initiated cells not promoted by chemical agents.

Polyamines regulate expression of the neoplastic phenotype in mouse skin.

Elevated polyamine levels are characteristic of many types of neoplastic cells and tissues. We demonstrate that in transgenic mice overexpressing ornithine decarboxylase in skin, changes in tissue polyamine levels, particularly putrescine, control the development and maintenance of the neoplastic phenotype. A specific inhibitor of the transgene, alpha-difluoromethylornithine (DFMO), reversibly blocked the appearance of squamous papillomas after carcinogen treatment. Furthermore, treatment of papilloma-bearing mice with DFMO caused rapid tumor regression, also in a reversible manner. Although the rate of apoptosis in papillomas was unaffected by acute DFMO treatment, tumor cell proliferation was rapidly decreased after drug treatment. Conversely, proliferation of normal epidermal keratinocytes was unaffected by DFMO treatment. The regulatory polyamine in this model appears to be putrescine, the immediate product of ornithine decarboxylase. These results demonstrate that elevated polyamine levels are required for both the development and maintenance of the neoplastic phenotype in skin.

Ornithine decarboxylase overexpression is a sufficient condition for tumor promotion in mouse skin.

In multistage tumorigenesis models, ornithine decarboxylase (ODC) is usually dysregulated at some point during tumor promotion, an early stage of carcinogenesis. To address the question whether constitutive overexpression of ODC would be a sufficient condition for tumor promotion, mice with high levels of ODC expression targeted to epidermal keratinocytes were used in skin tumorigenesis experiments. Transgenic mice with ODC targeted to hair follicle keratinocytes were much more sensitive than littermate controls to initiation with a single low dose of carcinogen; in fact, such mice no longer required treatment with tumor promoters for tumors to develop. Targeting ODC overexpression to both interfollicular and follicular keratinocytes did not further enhance tumor yield. Our results suggest that most, if not all, target cells for chemical carcinogens in the skin reside in hair follicles, and ODC overexpression is sufficient to activate such cells to expand clonally to form epidermal tumors.

Modulation of murine hair follicle function by alterations in ornithine decarboxylase activity.

Mice that overexpress a mutated ornithine decarboxylase (ODC) transgene in outer root sheath keratinocytes of the hair follicle were used to study the role of this enzyme in regulating hair follicle structure and function. These transgenic mice have a normal first hair cycle, but lose their hair completely beginning 2-3 wk after birth. Transgene overexpression in follicular keratinocytes is first detected at day 12 after birth, coincident with the development of follicular cysts in the upper portion of the dermis. The onset of keratin 6 expression also begins around day 12; because the promoter/regulatory region of the bovine keratin 6 gene was used to target ODC transgene expression of hair follicle keratinocytes, these data demonstrate the faithful temporal and cell type-specific expression of the K6 -driven transgene. The ODC inhibitor 2-difluoromethylornithine could prevent hair loss and partially normalize skin histology if administered before the onset of ODC overexpression. 2-Difluoromethylornithine could also reactivate hair growth in animals with complete hair loss. Our results suggest that ODC is an important regulatory gene for the mouse hair follicle.

Increased frequency of spontaneous skin tumors in transgenic mice which overexpress ornithine decarboxylase.

Ornithine decarboxylase, a critical regulatory enzyme for polyamine biosynthesis, is highly inducible by growth-promoting stimuli in mouse epidermis but the enzyme level is only transiently elevated due to rapid turnover of the protein. Here we report that constitutive overexpression of the enzyme in the skin of transgenic mice causes several phenotypic abnormalities. Effects observed include development of dermal follicular cysts, excessive skin wrinkling, enhanced nail growth, alopecia, and spontaneous tumor development. These results indicate that up-regulation of polyamine biosynthesis can profoundly disturb skin homeostasis and alter susceptibility to neoplastic development.

Induction of ornithine decarboxylase in specific subpopulations of murine epidermal cells following multiple exposures to 12-O-tetradecanoylphorbol-13-acetate, mezerein and ethyl phenylpropriolate.

Single applications of 12-O-tetradecanoylphorbol-13-acetate (TPA), mezerein or ethyl phenylpropriolate (EPP) to mouse skin at appropriate doses cause similar degrees of hyperplasia and comparable levels of induction of epidermal ornithine decarboxylase (ODC) activity. Multiple (n = 5) treatments with these agents, in contrast, resulted in large differences in induced ODC activity (TPA much greater than mezerein greater than EPP) with no differences in the degree of hyperplasia or [3H]thymidine pulse-labeling among the multiple treatment groups. To attempt to explain the cellular basis for the greater ODC-inducing ability of TPA relative to mezerein and EPP in chronic exposure protocols, immunocytochemical and flow cytometric analyses were performed. Immunocytochemistry using an ODC-specific polyclonal antibody revealed substantially different pattern of ODC-positive cells in chronically exposed epidermis than observed with single exposures. TPA treatment resulted in very pronounced immunostaining of the perifollicular cells, with little evidence of specific staining in the interfollicular epidermis mezerein treatment yielded staining in both interfollicular and some perifollicular areas, while EPP treatment produced the least amount of specifically stained cells, all of which were in the interfollicular epidermis. Flow cytometric analysis of keratinocytes isolated from chronically treated skin identified three distinct subpopulations that bound varying amounts of ODC antibody. Chronic treatment of CD-1 murine epidermis with TPA appeared to cause the expansion of an intermediate sized cell subpopulation that was not apparent with EPP or mezerein. Our results suggest that chronic treatment of murine epidermis with the potent complete tumor promoter TPA leads to the selective expansion of a keratinocyte subpopulation that is hyperinducible for ODC and may be identical to the cells in the perifollicular region previously identified. These observations also suggest that the weaker tumor promoters mezerein and EPP are less capable of causing expansion of this specific subpopulation, which may be an important target cell population for neoplastic transformation in mouse epidermis.

Constitutively elevated levels of ornithine and polyamines in mouse epidermal papillomas.

Epidermal papillomas were induced in CD-1 mice by a single topical application of 7,12-dimethylbenzanthracene (DMBA) followed by twice weekly applications of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in acetone. Control groups consisted of mice treated singly or chronically with acetone or TPA. TPA induced a rapid, yet transient 500- to 1000-fold increase in ornithine decarboxylase (ODC) activity which resulted in a 2- to 8.4-fold elevation of putrescine in both singly or chronically TPA-treated mouse epidermis 4-6 h after its application. After 24 h, levels of spermidine, but not spermine, were also elevated. The ODC and arginase activities in the 11 individual papillomas studied averaged 400- and 26-fold higher respectively than basal levels in epidermis. The activity of ODC in most papillomas, unlike ODC in epidermis, could be stimulated by guanosine 5'-triphosphate (GTP). Putrescine and spermidine levels in papillomas, especially those exhibiting highly GTP-stimulated ODC, were substantially higher compared to either normal or TPA-treated epidermis. Although epidermis contains a relatively high ornithine content, its level is even further elevated in papillomas, in some cases as much as 70-fold. The consequences of the constitutively elevated polyamine levels in papillomas caused by the loss of control over the normally tightly regulated polyamine biosynthetic pathway are not known, but could be important in regulating the balance between proliferation and differentiation in this self-renewing epithelial tissue.