Murine p53 intron sequences 5–8 and their use in polymerase chain reaction/direct sequencing analysis of p53 mutations in CD‐1 mouse liver and lung tumors

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Abstract
Inactivating point mutations and small deletions in the p53 tumor suppressor gene have been found in human liver and lung tumor—derived cell lines and tumors. However, little evidence has been reported concerning inactivation or mutation of the p53 gene in mouse primary tumors. To examine CD-1 mouse liver and lung tumors for mutations in the p53 gene, we first sequenced p53 introns 5–8 so that polymerase chain reaction amplification and sequencing primers located within the introns could be prepared. Use of these primers prevented amplification of the mouse p53 pseudogene and allowed sequencing of exons 5–8 in their entirety as well as their intron-exon junctions. DNA isolated from CD-1 mouse tumors was amplified and directly sequenced using nested primers. Nine spontaneous hepatocellular carcinomas (HCCs) and 34 chemically induced HCCs (induced by single intraperitoneal injections of N-nitrosodiethylamine [DEN] [8 HCCs], 7,12-dimethylbenz[a]anthracene [DMBA] [8 HCCs], 4-aminoazobenzene [8 HCCs], and N-OH-2-acetylaminofluorene [10 HCCs]) were examined for mutations in exons 5–8 of the p53 gene. In addition, 12 spontaneous, 10 DMBA-induced, and 13 DEN-induced lung adenocarcinomas or adenomas were analyzed for mutations. No mutations were found in any of the tumors examined. However, a mutation was demonstrated at codon 135 in the positive-control plasmid LTRp53cG(val). The results of this study suggest that inactivation of p53 is unlikely to play a major role in murine lung or liver carcinogenesis. However, inactivation of p53 may occur at a very low frequency, or it may occur as a late event and therefore be present in only a very small number of the tumor cells, rendering it undetectable by this method. Lastly, although few p53-inactivating mutations are found outside of exons 5–8 in human tumors, it is possible that these murine tumors contained mutations outside of this region and were therefore missed by our approach.