Complete loss of wild-type TP53 in a nontransformed human epithelial cell line is preceded by a phase during which a heterozygous TP53 mutant effectively outgrows the homozygous wild-type cells

HMT-3522 is a spontaneously immortalized cell line derived from a fibrocyst ic breast lesion. After continuous accumulation of genetic changes, the cel l line was transformed from a nontumorigenic to a malignant phenotype. One of the earliest genetic aberrations is a missense mutation of codon 179 (Hi s179Asn) in the tumor suppressor gene TP53 leading to outgrowth of a cell t ype expressing only the mutant form of TP53. In this report, we extend earl ier investigations to reveal the genetic background for the evolution from homozygous wild type to hemizygous mutated cells. The status of the TP53 al leles was followed at different stages by fluorescence in situ hybridizatio n (FISH) and allele-specific PCR (ASPCR) on fetal DNA, as well as flow-sort ed chromosomes-taking advantage of a size difference between the two homolo gues of chromosome 17 that harbor TP53 on 17p. This further allowed us to d etermine on which of the two chromosomes the mutated allele was located. Th e results presented here show that the cells have undergone an evolution fr om homozygous wild type for TP53 to heterozygous (His179Asn mutation in one allele), and finally to ct hemizygous mutated stare (deletion of the remai ning wild-type allele). The finding of a transient period in which heterozy gous cells dominate the population before the eventual outgrowth of hemizyg ous cells strongly indicates that the His179Asn mutation results in a tp53 protein with a dominant negative effect that does not totally abrogate the function of wild type TP53 in vitro. (C) 1999 Elsevier Science Inc. All rig hts reserved.