Clonal evolution and progression of 20-methylcholanthrene-induced squamous cell carcinoma of mouse epidermis as revealed by DNA instability and other malignancy markers

Abstract

We examined the clonal evolution of skin malignant 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 immunohistochemically 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 noted in early-stage (2-6 weeks) hyperplastic epidermis, and their number increased in middle (7-11 weeks), and late-stages (12-25 weeks) of hyperplastic epidermis, indicating that they belong to the malignancy category. All papillomatous lesions and invasive carcinomas showed a positive DNA-instability test. Positive immunostaining for various biomarkers and AgNORs parameters appeared in clones with a positive DNA-instability test in earlyor middle-stage hyperplastic epidermis, and markedly increased in late-stage hyperplastic epidermis, papillomatous lesions and invasive carcinomas. The percentage of PCNA-positive vascular endothelial cells was significantly higher in VEGFpositive lesions with a positive DNA-instability test and became higher toward the late-stage of progression. Cut-woundings were made to papillomatous and invasive carcinoma lesions, and the regeneration activity of vascular endothelial cells was determined by using flash labeling with tritiated thymidine (3H-TdR). In small papillomatous lesions, vascular endothelial cells showed regenerative 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 responses 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

Differential alterations in metabolic pattern of the spliceosomal uridylic acid‐rich small nuclear RNAs (UsnRNAs) during malignant transformation of 20‐methylcholanthrene‐induced mouse CNCI‐PM‐20 embryonic fibroblasts

“…Except U1 and U6, whether p53 and any other transcription factor activates/inactivates other spliceosomal UsnRNAs is still not known. Interestingly, MCA‐induced carcinogenesis involves upregulation of P53 in chemically transformed mouse cell lines 44–48; moreover, we also observed an increased expression of P53 at preneoplastic passages which, however, subsided at late passage (P42) (unpublished data). Whether this differential expression of P53 observed at different passages of MCA‐transformed MEF, is linked with UsnRNA metabolism, is still ambiguous.…”

Ki-67 immunolabeling in pre-malignant lesions and carcinoma of the prostate. Histological correlation and prognostic evaluation

“…We consider that additional studies with anti-Ki-67 and 34ßE12 antibodies, and DNA instability tests (Hirai et al, 2001;Iwasa et al, 2001) are required to quantify the proliferation fraction in the luminal layer of this type of lesion and its correlation with the clinical evolution. In the future, the pre-therapeutic management of the expression of Ki-67 in the luminal compartment of HGPIN lesions could be important in the evaluation of the aggressiveness of these lesions and, as a result, in the selection of the proper treatment.…”

The DNA-instability test as a specific marker of malignancy and its application to detect cancer clones in borderline malignancy

“…Clonal evolution and progression of 20-methylcholanthrene-induced squamous cell carcinoma in mouse epidermis (Hirai et al, 2001) In an attempt to understand the dynamic process of carcinogenesis and cancer progression, the clonal evolutions of squamous cell carcinoma (SCC) were examined in the lesions induced by repeated topical applications of 20-methylcholanthrene (20-MC) to the mouse skin, which induces hyperplastic epidermis, papillomatous lesion and invasive carcinoma. The lesions were examined histopatholigically and immunohisto-chemically using anti-singlestranded DNA antiserum after HCl hydrolysis (DNA-instability test).…”