p53 mutations in spontaneously immortalized 3T12 but not 3T3 mouse embryo cells

We have asked whether p53 mutations are involved in the process of spontaneous immortalization of mouse embryo cells. Cells from Swiss mouse embryos were used to prepare 3T3 and 3T12 lines according to the protocol of Todaro & Green [(1963). J. Cell Biol., 17, 299-313]. After the cells emerged from crisis, p53 sequences were amplified by polymerase chain reaction (PCR) from both RNA and DNA. The sequence of the aggregated cDNA from each of six 3T3 lines showed no evidence of mutation. PCR-amplified p53 cDNA from two 3T3 lines was cloned, and individual clones in M13mp19 were partially sequenced. One cell ine showed a single, non-coding nucleotide change in 2/8 independent clones. Nine cDNA clones from the second 3T3 lines were sequenced, and no single nucleotide changes appeared more than once. The mutations which appeared only once were not detected in clones of genomic DNA. Since these apparent mutations are probably reverse transcriptase or Taq polymerase errors, we conclude that both the 3T3 lines contained only wild-type p53. In two out of three independent 3T12 lines however, missense mutations were readily observed in the aggregate cDNA sequence. Restriction fragment length polymorphism and Southern blot analyses of the genomic DNA indicated that these cells were homozygous for the mutations. The p53 protein molecules in four cell lines were analysed by immunoprecipitation: one 3T12 line showed the pattern of antibody reactivity characteristic of some p53 mutants, while the others displayed the wild-type pattern. We conclude that p53 mutations arise and are strongly selected for during immortalization according to the 3T12 but not the 3T3 protocol.