Carcinogenesis, Vol. 20, No. 9, 1689-1696,
September 1999
© 1999 Oxford University Press
Cancer Biology |
Dose-dependent mutation profile in the c-Ha-ras proto-oncogene of skin tumors in mice initiated with benzo[a]pyrene
Laboratory for Cancer Research, Department of Chemical Biology, College of Pharmacy, Rutgers, The State University of New Jersey, 164 Frelinghuysen Road, Piscataway, NJ 08854-8020 and
1 Section on Oxidation Mechanisms, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA
Female CD-1 mice were treated topically with a low (25–50 nmol) or high (800 nmol) dose of benzo[a]pyrene (BP) or acetone vehicle, followed by 5 nmol 12-O-tetradecanoylphorbol 13-acetate (TPA) twice a week for 26 weeks. Selective UV radiation fractionation followed by PCR methods were used to analyze histologically defined subsets of cells (~100–200 cells) on formalin-fixed, paraffin-embedded and H&E stained microscope sections. DNA samples from normal-appearing, hyperplastic or tumor regions from the skin of animals from each treatment group were isolated and amplified by PCR with c-Ha-ras-specific primers. Single-strand conformation polymorphism (SSCP) analyses were performed on both exon 1 and 2 products from each sample. DNA extracted from each aberrant band of SSCP analyses was amplified by PCR for further sequence analysis. The data indicate that c-Ha-ras mutations can be detected in normal-looking and hyperplastic epidermal cells as well as in tumor cells obtained from mice initiated with BP and promoted with TPA. The frequencies of c-Ha-ras mutations for normal-looking, hyperplastic and tumor samples were 3/20 (15%), 8/17 (47%) and 58/68 (85%), respectively, for the low dose group and 8/18 (44%), 10/20 (50%) and 64/86 (74%), respectively, for the high dose group. These observations indicate that there were no dose dependencies in the mutation frequencies for normal-looking, hyperplastic and tumor samples. For combined high dose and low dose samples, differences in mutation frequencies of the c-Ha-ras gene between the normal-looking, hyperplastic and tumor samples were highly significant (P < 0.0001, Fisher's exact test). All mutations detected were located at codons 12, 13 and 61 of the c-Ha-ras gene. With the numbers in parentheses indicating the nucleotide position in the coding sequence of the c-Ha-ras proto-oncogene, the distributions of mutations for G
A (35), GT (35), GC (37), GT (38), CA (181), AT (182) and AG (182) in the low dose tumors were 5, 2, 11, 74, 0, 7 and 2%, respectively, and the distribution of mutations in tumors from animals treated with a high dose of BP were 3, 7, 13, 61, 15, 1 and 0%, respectively. Differences in the global mutation spectra (site and kind of all mutations) for the c-Ha-ras gene between the high and low dose group tumors were statistically significant (P < 0.004, Fisher's exact test) and the major difference between these two groups was CA (181) base substitutions. In summary, our data indicate that: (i) 79% of the BP/TPA skin tumors in CD-1 mice had c-Ha-ras mutations for the combined data for high dose and low dose tumors; (ii) the major mutations detected in BP/TPA skin tumors were GT transversions; (iii) the global mutation profile in the c-Ha-ras proto-oncogene in skin tumors obtained after initiation with a low dose of BP was different from that obtained after initiation with a high dose of BP.
Abbreviations: BP, benzo[a]pyrene; BPDE, (+)-(7R,8S,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; B[c]PhDE, (–)-(1R,2S, 3S,4R)-3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene; DB[a,l]P, dibenzo[a,l]pyrene; DMBA, 7,12-dimethylbenz[a]anthracene; hprt, hypoxanthine (guanine) phosphoribosyltransferase gene; H&E, hematoxylin and eosin; SSCP, single-strand conformation polymorphism; SURF, selective UV radiation fractionation; TPA, 12-O-tetradecanoylphorbol 13-acetate.
2 To whom correspondence should be addressed Email: swei{at}rci.rutgers.edu
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