SUMMARY

We examined the clonal evolution of skin malig-nant lesions by repeated topical applications of 20-methylcholanthrene (20-MC) to the skin, which induces hyperplastic epidermis, papillomatous lesion and invasive carcinoma in mice. The lesions were examined histologically and immunohisto-chemically with anti-single-stranded DNA after acid hydrolysis (DNA-instability test), p53, VEGF, DFF45, PCNA and AgNORs parameters analyses. Multiple clones with increased DNA instability comparable to that of invasive carcinoma were not-ed in early-stage (2-6 weeks) hyperplastic epider-mis, and their number increased in middle (7-11 weeks), and late-stages (12-25 weeks) of hyperplas-tic epidermis, indicating that they belong to the malignancy category. All papillomatous lesions and invasive carcinomas showed a positive DNA-insta-bility test. Positive immunostaining for various bio-markers and AgNORs parameters appeared in clones with a positive DNA-instability test in early-or middle-stage hyperplastic epidermis, and markedly increased in late-stage hyperplastic epi-dermis, papillomatous lesions and invasive carcino-mas. The percentage of PCNA-positive vascular 319 endothelial cells was significantly higher in VEGF-positive lesions with a positive DNA-instability test and became higher toward the late-stage of pro-gression. Cut-woundings were made to papilloma-tous and invasive carcinoma lesions, and the regen-eration activity of vascular endothelial cells was determined by using flash labeling with tritiated thymidine (3H-TdR). In small papillomatous lesions, vascular endothelial cells showed regener-ative response, but the response was weak in large lesions. No such response was noted in invasive carcinomas; rather, cut-wounding induced collapse of blood vessels, which in turn induced massive coagulative necrosis of cancer cells. These respons-es can be interpreted to reflect exhausted vascular growth activity due to excessive stimulation by VEGF-overexpression, which was persistently seen from hyperplastic epidermis to invasive carcinoma