Rapid induction of skin tumors in human but not mouse c-Ha-ras proto-oncogene transgenic mice by chemical carcinogenesis

Abstract

The rasH2 transgenic mice carry human c-Ha-ras proto-oncogene, and are highly susceptible to chemical carcinogenesis. Previous studies showed that the mutation of c-Ha-ras induced by DMBA in the tumors of rasH2 were detected only in transgenes. To examine if the difference between the codons of the c-Ha-ras gene in human and mouse contributed to the tissue-specific sensitivity to DMBA, we generated a line of transgenic mice, mras, carrying mouse c-Ha-ras genome with its own promoter. Western blot analysis showed that the protein expression of H-RAS in the skin was increased in both rasH2 and mras compared with wild-type. Chemical skin carcinogenesis was induced by DMBA and TPA. In rasH2 mice, the latency of tumor formation was shorter than wild-type littermates. Both the number and the volume of skin tumors were increased in rasH2 than those of wild-type. However, in mras mice, enhancement of tumor formation was not observed as compared with wild-type. The mean number of tumors and the latency of tumor development was almost the same between mras and wild-type littermates. Mutational analysis showed only A to T transversion in human c-Ha-ras transgenes at codon 61 but not in murine endogenous c-Ha-ras gene in the tumors of rasH2. In the tumors of wild-type littermates and mras, A to T transversion in murine c-Ha-ras at codon 61 were detected. These results indicate that the differences in the codon of the c-Ha-ras gene between mouse and human might contribute to the tissue-specific sensitivity of DMBA.

Figure 1

The genomic structure of transgenes for rasH2 mice (a) and mras mice (b). B, BamH I; S, Sac I; Xh, Xho I; Xb, Xba I.

Figure 2

Elevated expression of H‐RAS protein in mras and rasH2 mouse dorsal skin.

Figure 3

The gross appearance of tumor formation on the back skin of mice 25 weeks after the treatment with DMBA. (a) DMBA and TPA‐induced skin tiny papilloma in a control wild‐type mouse; (b) DMBA and TPA‐induced skin papilloma and squamous cell carcinoma in a rasH2 mouse; (c) DMBA and TPA‐induced skin papilloma in an mras mouse.

Figure 4

Average number of papillomas/mouse. Time dependence of skin tumor development on the back skin in male mice treated with DMBA and TPA. (•) rasH2 (transgenic mouse [Tg]); (○) rasH2 (wild‐type mouse [Wt]); (▪) mras (Tg); (□) mras (Wt).

Figure 5

Histopathology of representative lesions of back skin tissue (hematoxylin–eosin stain). (a) Normal skin tissue in a control wild‐type mouse at 25 weeks; (b) DMBA and TPA‐induced skin hyperplasia in a wild‐type mouse at 25 weeks; (c) DMBA and TPA‐induced skin papilloma in a wild‐type mouse at 25 weeks; (d) normal skin tissue in a rasH2 mouse at 25 weeks; (e) DMBA and TPA‐induced skin hyperplasia in a rasH2 mouse at 25 weeks; (f) DMBA and TPA‐induced skin papilloma in a rasH2 mouse at 25 weeks; (g) DMBA and TPA‐induced skin squamous carcinoma in a rasH2 mouse at 25 weeks. Note the invasion into the deep subcutaneous and smooth muscle layers; (h) normal skin tissue in an mras mouse at 25 weeks; (i) DMBA and TPA‐induced skin papilloma in an mras mouse at 25 weeks.

Figure 6

(a) Mutation analyses of human c‐Ha‐ras transgene at codon 61. A to T transversion at the second base of transgenic human c‐Ha‐ras codon 61 in papillomas detected by oligonucleotide hybridization. H, human genomic DNA; NS, skin of non‐treatment; T, tumor; TS, skin of DMBA and TPA treatment; (b) direct sequencing showed no mutation in papillomas of murine c‐Ha‐ras transgenes at codon 61.

Figure 7

PCR‐direct sequencing of c‐Ha‐ras at codon 61 in mras mice. In the tumors of wild‐type littermates and in mras mice after DMBA and TPA treatment, A to T transversion in murine c‐Ha‐ras transgenes at codon 61 were detected (arrows). (a) Non‐neoplastic regions of mras; (b) papilloma in mras; (c) non‐neoplastic regions of wild‐type; (d) papilloma in wild‐type.