Dose-dependent mutation profile in the c-Ha-ras proto-oncogene of skin tumors in mice initiated with benzo[a]pyrene.
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 (approximately 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), G-->T (35), G-->C (37), G-->T (38), C-->A (181), A-->T (182) and A-->G (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 C-->A (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 G-->T 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.
Wei
S J
SJ
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, USA.
Chang
R L
RL
Merkler
K A
KA
Gwynne
M
M
Cui
X X
XX
Murthy
B
B
Huang
M T
MT
Xie
J G
JG
Lu
Y P
YP
Lou
Y R
YR
Jerina
D M
DM
Conney
A H
AH
D000279:Administration, Cutaneous; D019943:Amino Acid Substitution; D000818:Animals; D001564:Benzo(a)pyrene / Q000008:administration & dosage / Q000633:toxicity*; D002273:Carcinogens / Q000008:administration & dosage / Q000633:toxicity*; D002278:Carcinoma in Situ / Q000139:chemically induced / Q000235:genetics / Q000473:pathology; D003043:Cocarcinogenesis; D003062:Codon / Q000235:genetics; D004247:DNA / Q000235:genetics; D004252:DNA Mutational Analysis; D004273:DNA, Neoplasm / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D004817:Epidermis / Q000187:drug effects / Q000473:pathology; D005091:Exons / Q000235:genetics; D005260:Female; D011905:Genes, ras; D006965:Hyperplasia; D007636:Keratoacanthoma / Q000139:chemically induced / Q000235:genetics / Q000473:pathology; D051379:Mice; D010212:Papilloma / Q000139:chemically induced / Q000235:genetics* / Q000473:pathology; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics*; D012878:Skin Neoplasms / Q000139:chemically induced / Q000235:genetics* / Q000473:pathology; D013755:Tetradecanoylphorbol Acetate / Q000633:toxicity
D002273:Carcinogens; D003062:Codon; D004273:DNA, Neoplasm; D001564:Benzo(a)pyrene; D004247:DNA; D016283:Proto-Oncogene Proteins p21(ras); D013755:Tetradecanoylphorbol Acetate