In order to study the oncogenesis of melanocytes, transgenic mouse lines were established that express a mutated human Ha-ras (TPras) gene in pigment producing cells. The ras transgenic mice exhibit an altered phenotype, including melanocytic hyperplasia and a muted agouti coat, indicative of hyperproliferative melanocytes. These mice and their wild-type littermates have been subjected to a variety of carcinogenesis protocols, including 7, 12-dimethylbenz-[a]anthracene (DMBA), 12-O-tetradecanoylphorbol-13-acetate (TPA) and UV radiation exposure. Topical DMBA treatment of TPras mice resulted in a high incidence of melanomas. Metastatic lesions were observed in skin, lungs and lymph nodes. TPA treatment of TPras mice induced a small number of papillomas but no nevi or melanomas. UV light exposures induced papillomas in negative littermate and melanomas in some albino TPras mice. These results show that melanocytes expressing an activated Ha-ras in the TPras transgenic mice are susceptible to induction of melanoma by DMBA.

The TP-ras transgenic mouse line expresses an activated human T24 Ha-ras gene with a mutation in codon 12, regulated by a mouse tyrosinase promoter. The transgene is expressed in melanocytes of the skin, eyes, and brain. The mice develop cutaneous melanoma when treated with 7,12-dimethylbenz[a]anthracene. Cell lines have been generated from the cutaneous tumors and metastatic lesions. By using fluorescence in situ hybridization with mouse whole chromosome paints, the cell lines were characterized for chromosomal abnormalities. Key findings in the tumor cells included translocations of chromosome 4 and alterations in chromosome 6. One tumor cell line contained a double translocation involving chromosomes 3 and 6. To extend the results of the chromosome 4 painting, Southern analysis of the p15INK4B, p16INK4A, and p19INK4D genes was performed. Our data indicated that there were homozygous and partial allelic deletions and polymorphisms in the region of chromosome 4 containing these genes, resulting in the absence or reduced expression of the p16 product. These findings are similar to those reported for human melanoma, and the TP-ras transgenic mouse may therefore be a valuable model for studying novel strategies for melanoma prevention and treatment.

Chemopreventive and antitumor properties of perillyl alcohol (POH) studied preclinically indicate that topical POH inhibits both ultraviolet B (UVB)-induced murine skin carcinogenesis (squamous cell tumor models) and DMBA-induced murine melanoma (transgenic models involving tyrosinase-driven Ras). A previous Phase 1 clinical trial in participants with normal-appearing skin demonstrated that topical POH cream was well tolerated at a dose of 0.76% (w/w). Here we performed a three month, double-blind, randomized, placebo-controlled Phase 2a trial of two different doses of topical POH in individuals with sun-damaged skin. Participants applied POH cream twice daily to each dorsal forearm. Baseline and end-of-study biopsies were taken from each participant to evaluate whether the topical application of POH was effective in reversing actinic damage as evidenced by normalization of quantitative skin histopathologic scores and change in nuclear chromatin pattern measured by karyometric analysis. There was a borderline reduction in the histopathologic score of the lower dose of POH compared to placebo (p = 0.1), but this was not observed in the high dose group. However, in the high dose group, a statistically significant reduction in the proportion of nuclei deviating from normal was observed using karyometric analysis (p< 0.01). There was no statistical significance demonstrated in the lower dose group. No changes were observed in p53 expression, cellular proliferation (by PCNA expression), or apoptosis in either treatment group compared to placebo. These results suggest that while our karyometric analyses can detect a modest effect of POH in sun-damaged skin, improved delivery into the epidermis may be necessary.