In the proposed studies, the opossum Monodelphis domestica will be used to investigate activation of Ki-ras and Ha- ras oncogenes during the development of ultraviolet radiation (UVR)-induced corneal tumors. Activation of the N-ras gene will also be investigated, if the gene proves to be expressed in corneal tumors, and material suitable for future studies of UVR-induced alterations in the p53 tumor suppressor gene will be collected. The major goal is to identify the role of a specific UVR- induced DNA lesion, the cyclobutane pyrimidine dimer, in activation of ras oncogenes. M. domestica is particularly suitable for studies of the role of pyrimidine dimers, because the animal is capable of photoreactivation, a light- dependent enzymatic reaction that specifically repairs cyclobutane pyrimidine dimers in DNA. Since post-UVR exposure to photoreactivating light (PRL) removes pyrimidine dimers but not other DNA photoproducts, UVR effects due to pyrimidine dimers can be identified by comparing UVR effects in animals exposed only to UVR with UVR effects in animals exposed to UVR followed by PRL. Virtually all M. domestica chronically exposed to UVR develop corneal stromal tumors with the characteristics of fibrosarcomas. Tumors develop gradually; non-neoplastic proliferation of stromal fibroblasts precedes frank neoplasia. Post-UVR exposure to PRL delays the development and reduces the incidence of UVR- induced corneal tumors in M. domestica, but does not abolish tumor formation. A mutationally activated Ki-ras oncogene has been identified in a UVR-induced opossum corneal tumor. The coding sequence of the opossum Ki-ras oncogene has been determined. The nucleotide sequence of the opossum Ha-ras and, possibly, N-ras genes will be similarly determined. The present studies propose to examine in detail the incidence and mechanism of ras oncogene activation in UVR-induced corneal tumors of M. domestica. Eyes at different stages of tumor development will be collected from UVR-exposed opossums, both from animals exposed to UVR alone and from those exposed to UVR followed by PRL Ras oncogene amplification in corneal lesions will be assessed. Exons 1 and 2 of the Ki-ras, Ha-ras, and, possibly, N-ras oncogenes will be amplified from corneal DNA by the polymerase chain reaction (PCR), and point mutations in codons 12, 13, and 61 in these genes will be identified by direct nucleotide sequence determination. For ras oncogenes with suitable nucleotide sequences, activating mutations in subpopulations of cells will be identified by newly developed techniques of PCR amplification and differential endonuclease cleavage. These studies will determine the nature and incidence of ras oncogene activation in UVR-induced corneal tumors in M. Domestica and will indicate when during the course of tumor development activation occurs. Most importantly, these studies will correlate mechanisms of ras activation with UVR-induced DNA lesions and will thus identify the precise role of the pyrimidine dimer in UVR activation of ras oncogenes. Collection and storage of material for future studies of p53 tumor suppressor gene alterations in these tumors will expand and verify the conclusions. These studies will provide valuable information about the mechanism of tumor induction by UVR and the relative importance of the pyrimidine dimer in tumorigenesis. Elucidation of mechanisms of UVR carcinogenesis, in turn, will help in the prevention and treatment of UVR-induced skin tumors, the most common form of human cancer and the cancer type that will be most dramatically affected by the anticipated diminution of the earth's protective ozone layer.
| Kusewitt, D F; Preble, N E; Bonnett, C D (2000) Photoreactivation does not alter ras and p53 mutation spectra in ultraviolet radiation-induced corneal sarcomas of Monodelphis domestica. Mol Carcinog 27:117-24 |
| Kusewitt, D F; Sherburn, T E; Miska, K B et al. (1999) The p53 tumor suppressor gene of the marsupial Monodelphis domestica: cloning of exons 4-11 and mutations in exons 5-8 in ultraviolet radiation-induced corneal sarcomas. Carcinogenesis 20:963-8 |
| Kusewitt, D F; Dyble, J; Sherburn, T E et al. (1998) Altered UV resistance and UV mutational spectrum in repair-proficient murine fibroblasts expressing endonuclease V. Mutat Res 407:157-68 |
| Kusewitt, D F; Gale, J M; Sherburn, T E et al. (1997) H-ras oncogene activation in invasive UVR-induced corneal sarcomas of the opossum Monodelphis domestica. DNA Cell Biol 16:1217-22 |
