High frequency and heterogeneous distribution of p53 mutations in aflatoxin B-1-induced mouse lung tumors

Inactivation of the p53 tumor suppressor gene Is one of the most frequent g enetic alterations observed in human lung cancers. However, p53 mutations a re more rarely detected in chemically induced mouse lung tumors. In this st udy, 62 female AC3F1 (A/J x C3H/HeJ) mice were treated with anatoxin B-1 (A FB(1); 150 mg/kg i.p. divided into 24 doses over 8 weeks). At 6-14 months a fter dosing, mice were killed, and tumors were collected. A total of 71 AFB (1)-induced lung tumors were examined for overexpression of p53 protein by immunohistochemical staining. Positive nuclear p53 staining was observed in 79% of the AFB(1)-induced tumors, but the pattern was highly heterogeneous . In approximately 73% of the positively stained tumors, fewer than 5% of c ells demonstrated positive staining; in the other 27%, between 10% and 60% of the cells stained positively, with staining localized to the periphery o f the tumors in many cases. Single-strand conformational polymorphism analy sis of the evolutionarily conserved regions of the p53 gene (exons 5-8) fro m APB(1)-induced whole lung tumor DNA revealed banding patterns consistent with point mutations in 20 of 76 (26%) tumors, with 85% of the mutations in exon 7 and 15% of the mutations in exon 6. Identification of point mutatio ns could not be confirmed by direct sequence analysis because bands represe nting putative mutations appeared only weakly on autoradiograms. This was p resumably due to the heterogeneous nature of the DNA analyzed. Single-stran d conformational polymorphism analysis of DNA from laser capture microdisse cted cells of paraffin-embedded AFB(1)-induced tumor tissue sections staine d for p53 produced banding patterns consistent with point mutations in 18 o f 30 (60%) DNA samples. Direct sequencing of the microdissected samples rev ealed mutations at numerous different codons in exons 5, 6, and 7. Of 26 mu tations found in microdissected regions from adenomas and carcinomas, 9 wer e G:C-->A:T transitions, II were A:T-->G:C transitions, and 5 were transver sions (2 G:C-->T:A, 2 T:A-->A:T, and 1 A:T-->C:G), whereas 1 deletion mutat ion was identified. The concordance between immunostaining and molecular de tection of p53 alterations was 72% when laser capture microdissection was u sed versus 17% based on whole tumor analysis. The high mutation frequency a nd heterogeneous staining pattern suggest that p53 mutations occur relative ly late in APB(1)-induced mouse lung tumorigenesis and emphasize the value of analyzing different staining regions from paraffin-embedded mouse lung t umors.