National Toxicology Program

Rapid Identification of Potential Carcinogens using the Tg.AC (v-Ha-ras) and the Heterozygous p53-deficient (+/-) Transgenic Mouse Models

Cancer Biology Group, Laboratory of Environmental Carcinogenesis and Mutagenesis, and National Toxicology Program, NIEHS, NIH, Research Triangle Park, NC 27709

Public Health Significance

Environmental factors and exposure to natural and synthetic chemicals, interacting through behavioral and genetic components, are known or suspected of being major determinants of cancer in humans, a multigenic and multifactorial process. Background and/or induced mutations and/or altered gene expression are believed by biomedical scientists to play critical roles in the induction of cancer, and, most likely, many other somatic diseases. The relationship between our environment and the mutagenesis and carcinogenesis of the human genome is still poorly understood. Most important, retrospective identification of relationships between exposure to hazardous agents by epidemiological research methods and environmental disease requires many years and evidence of human morbidity and death to establish. Prospective identification of chemical hazards and levels of risk is required to minimize or prevent exposure. Understanding the potential multiple pathways that our environment may induce or influence cancer and environmental disease is also important so that means of intervention and/or treatments of cancer and environmental disease may be developed.

Research Initiatives

At the present time, prospective identification of potential chemical carcinogens requires 5 to 7 years of resource intense short and long term toxicology and carcinogenesis investigation. This delays both the determination of toxic and carcinogenic potential and risk assessment. In addition, investigation of potential mechanisms for cancer induction and relevance to human cancer are difficult and, most often, lacking. We are investigating the potential use of transgenic mouse models for mutagenesis and carcinogenesis for rapid identification of environmental mutagens/carcinogens and mechanisms of tissue specific molecular toxic effects associated with the induction of experimentally induced cancers in rodents as surrogates for humans.

Principal Hypothesis: Transgenic mice, with a specific genetic alteration (inducible protooncogene and/or inactivation of a tumor suppressor gene) critical to tumorigenesis, but insufficient by itself to induce cancer, are candidate test species for rapid cancer bioassays. Exposure of such mice to transspecies carcinogens will result in the rapid induction (decreased latency) of tumors.

Background:

Tg.AC mice carry a v-Ha-ras oncogene fused to the promoter of the -globin gene (1) . The v-Ha-ras transgene has point mutations at codons 12 and 59 and the site of integration of the transgene confers on these mice the characteristic of genetically initiated skin as a target for tumorigenesis in the context of the well known, intensively studied, mouse-skin tumorigenesis model (2-7, 13). An important consideration of the Tg.AC mouse model is that the transgene is not constitutively expressed in the skin and the untreated skin appears normal when compared to the skin of the wild type FVB/N parent strain. In addition, the spontaneous incidence of skin papillomas in the dorsal skin of untreated mice is very low to zero. Dermal application of carcinogens, but not non-carcinogens, results in activation of the transgene and induction of benign skin tumors (see the list of publications below). The induction of the transgene in the skin and associated tumorigenesis acts as a reporter phenotype for the rapid identification of potential carcinogens by any potential route of application in conventional mouse models (11). The mechanistic basis for this reporter phenotype in this transgenic mouse is under investigation.

Heterozygous p53 (+/-) Deficient Mice with only a single wild type p53 allele provide a distinct target for mutagens and are analogous to humans at risk due to heritable forms of cancer, e.g., the Li-Fraumeni syndrome (8). The reduction in p53 gene dosage by this "germline first hit" increases both (a) the probability that a second mutagenic event will cause either loss of p53 tumor suppressor function or a gain of transforming activity by requiring (at minimum) only a single mutation (in the remaining functional p53 allele) and/or (b) create permissive conditions (i.e. genomic instability) for clonal expansion of cells harboring mutation(s) in other genes critical to tumorigenesis. Mice nullizygous for p53 (-/-) genes are viable (but have a high rate of early spontaneous tumors at sites apparently determined by the strain's genetic background). In the heterozygous state (one wildtype and one null allele), mice have a low background tumor incidence for up to 12 months of age (9-11), thus, allowing a sufficient period for testing free from strain specific background tumor incidence. Recent evidence suggest that the induction of cancer may indirectly (12) or directly involve the p53 tumor suppressor gene (13). This suggests that the model should have a broad range of chemically induced tumor susceptibility without being overtly sensitive.

Approach: Research efforts are presently divided along two lines of investigation:

(1) validation of the Tg.AC (v-Ha-ras) and p53-deficient (+/-) mouse models for rapid identification of potential mutagenic carcinogens by prospective testing of NTP chemical in progress for two year toxicology and carcinogenesis studies and (2) molecular biology studies designed to determine the role and function of the Tg.AC transgene in skin carcinogenesis and the role of the p53 tumor suppressor gene in mutagenesis and carcinogenesis and identification of critical genes involved in mouse carcinogenesis for comparison to human cancers of the same histogenetic type. Initial studies on the validity of using these two transgenic mouse models to identify potential carcinogens have focused on replicating two-year NCI/NTP cancer bioassays using a diverse group of mutagenic and nonmutagenic carcinogens, as well as a mutagenic noncarcinogen in rapid cancer bioassays (see Table 1 and Table 2). Concordance was very high between positive responses for both genotoxic and nongenotoxic carcinogens in the Tg.AC mouse and for mutagenic carcinogens in the heterozygous p53 deficient mouse and the two year NTP cancer bioassays.

At present two different groups of chemicals are currently under study in this mouse model, as well as the Tg.AC mouse model. The first group chosen by NIEHS/NTP scientists contain both human carcinogens as well as rodent mutagenic and nonmutagenic carcinogens to determine the range of potential responses in these two transgenic mouse lines (Table 3). The second group represents another approach to validation of the model: the prospective testing of chemicals currently being tested in the NTP cancer bioassay (Table 4). Prospective analysis provides the opportunity to remove the potential bias of selection of chemicals for validation. A high correspondence between this short-term cancer bioassay and the long-term bioassay (of both groups) would provide assurance that mutagenic carcinogens, may be identified using this mouse model. We anticipate that these studies will be completed by the beginning of the 3rd Quarter of FY97. General protocol outlines for the conduct of rapid carcinogenicity studies in these two transgenic lines are described in Table 5 and Table 6. (Information about the Study Chemicals).

Selected Publications

1. Leder, A., Kuo, A., Cardiff, R.D., Sinn, E. and Leder, P. (1990) v-Ha-ras transgene abrogates the initiation step in mouse skin tumorigenesis: Effects of phorbol esters and retinoic acid. Proc. Natl. Acad. Sci. USA, 87, 9178-9182.

2. Spalding, J.W., Momma, J., Elwell, M.R. and Tennant, R.W. (1993) Chemical induced skin carcinogenesis in a transgenic mouse line (TG*AC) carrying a v-Ha-ras gene. Carcinogenesis, 14, 1335-1341.

3. Hansen, L.A. and Tennant, R.W. (1994) Follicular origin of epidermal papillomas in v-Ha-ras transgenic TG.AC mouse skin. Proc Natl Acad Sci U S A, 91, 7822-6.

4. Hansen, L.A., C.S. Trempus, J.F. Mahler and R.W. Tennant (1996) Association of tumor development with increased cellular proliferation and transgene overexpression but not c-Ha-ras mutations, in v-Ha-ras transgenic Tg.AC mice, Carcinogenesis, 17, 1825-1833.

5. Hansen, L.A., Spalding, J.W., French, J.E. and Tennant, R.W. (1994) A transgenic mouse model (TG.AC) for skin carcinogenesis: Inducible transgene expression as a second critical event. In McClain, R.M., Slaga, T.J., LeBoeuf, R. and Pitot, H. (eds), Growth Factors and Tumor Promotion: Implications for Risk Assessment. Vol. 391. Wiley-Liss, Barton Creek, Texas, pp. 223-235.

6. Nylander-French, L. and J. French (1998) Tripropylene glycol diacrylate, but not ethyl acrylate, induces skin tumors in a twenty week short term tumorigenesis study in Tg.AC (v-Ha-ras) mice, Toxicologic Pathology, 26, 476-483.

7. Albert, R., J. French, R. Maronpot, J. Spalding and R. Tennant (1996) Mechanism of skin tumorigenesis by contact sensitizers: The effect of the corticosteroid fluocinolone acetonide on inflammation and tumor induction by 2,4-dinitro-1-fluorobenzene in the skin of the Tg.AC (v-Ha-ras) mouse, Environmental Health Perspectives, 104, 1062-1068.

8. Donehower, L.A., Harvey, M., Slagle, B.L., McArthur, M.J., Montgomery, C.A.J., Butel, J.S. and Bradley, A. (1992) Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumors. Nature, 356, 215-221.

9. Harvey, M., McArthur, M.J., Montgomery, C.A.J., Butel, J.S., Bradley, A. and Donehower, L.A. (1993) Spontaneous and carcinogen-induced tumorigenesis in p53-deficient mice. Nat. Genet., 5, 225-229.

10. Harvey, M., McArthur, M.J., Montgomery, C.A.J., Bradley, A. and Donehower, L.A. (1993) Genetic background alters the spectrum of tumors that develop in p53-deficient mice. FASEB J, 7, 938-943.

11. Tennant, R.W., French, J.E. and Spalding, J.W. (1995) Identification of chemical carcinogens and assessing potential risk in short term bioassays using transgenic mouse models. Environ Health Perspect, 103, 942-950.

12. French, J., G. Lacks, C. Trempus, J. Dunnick, J. Foley, J. Mahler, R. Tice and R. Tennant (2000) Loss of heterozygosity at the p53 locus in heterozygous p53 (+/-) mice is carcinogen dependent, Carcinogenesis, Submitted.

13. French, J., J. Spalding, L. Hansen, J. Seely, C. Trempus, R. Tice, M. Furedi-Machacek, J. Mahler and R. Tennant (2000) Benzene induces skin cancer and leukemia in Tg.AC (v-Ha-ras) transgenic mice, Carcinogenesis, In Press.