Malignant transformation of human fibroblast cell strain MSU-1.1 by N-methyl-N-nitrosourea: Evidence of elimination of p53 by homologous recombination

To determine whether N-methyl-N-nitrosourea (MNU) can induce malignant tran sformation of human fibroblasts and whether O-6-methylguanine (O-6-MeG) is involved, two populations of infinite life span cell strain MSU-1.1, differ ing only in level of O-6-alkylguanineDNA alkyltransferase, were treated wit h MNU and assayed for focus formation. MNU caused a dose-dependent increase in the frequency of foci in both groups, but the dose required was signifi cantly lower in the cells lacking O-6-alkylguanineDNA alkyltransferase, ind icating that O-6-MeG was causally involved. Of 35 independent focus-derived strains assayed for p53 transactivating ability, one was heterozygous, and 15 had lost all activity, 1 of 7 from untreated cells and 14 of 27 from MN U-treated cells. These results indicate that loss of p53 is not required fo r focus formation but may permit cells to form foci, Of 35 strains assayed for tumorigenicity, 10 formed malignant tumors with a short latency, all 10 lacked wild-type p53. The p53 heterozygous strain also formed tumors after a long latency, and the cells from those tumors lacked p53 transactivating ability. None of the 19 strains with wild-type p53 formed tumors. These re sults indicate that although loss of p53 is not sufficient for malignant tr ansformation of MSU-1.1 cells, it may be necessary. Analysis of the p53 cDN A from several focus-derived strains lacking p53 activity revealed that eac h contained the same mutation, an A to G transition at codon 215, resulting in a change from serine to glycine. Because p53 can be inactivated by muta tions at any one of a large number of sites, finding the same mutation in e ach strain assayed strongly suggests that the target population included a subpopulation of cells with this codon 215 mutation in one allele. Further analysis showed that all 15 focus-derived cells strains that lacked p53 tra nsactivating activity contained two alleles, each with the same codon 215 m utation, and that the mutant allele in the heterozygous strain also had tha t mutatation. Analysis of the p arm of chromosome 17 of the focus-derived c ell strains containing the codon 215 mutation revealed seven patterns of lo ss of heterozygosity, evidence of mitotic homologous recombination. Similar analysis of a separate series of cell strains, derived from foci induced b y cobalt-60, revealed four patterns of loss of heterozygosity, only two of which had been found with those induced by MNU. These data suggest that hom ologous mitotic recombination, induced by O-6-MeG in a subpopulation of cel ls heterozygous for p53 mutation, rendered the cells homozygous for loss of p53 activity, that this allowed the cells to form foci, and that although loss of p53 is not sufficient for malignant transformation, it predisposes cells to acquire the additional changes needed for such transformation.