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 fibrocystic breast lesion. After continuous accumulation of genetic changes, the cell line was transformed from a nontumorigenic to a malignant phenotype. One of the earliest genetic aberrations is a missense mutation of codon 179 (His179Asn) in the tumor suppressor gene TP53 leading to outgrowth of a cell type expressing only the mutant form of TP53. In this report, we extend earlier investigations to reveal the genetic background for the evolution from homozygous wild type to hemizygous mutated cells. The status of the TP53 alleles was followed at different stages by fluorescence in situ hybridization (FISH) and allele-specific PCR (ASPCR) on total DNA, as well as flow-sorted chromosomes--taking advantage of a size difference between the two homologues of chromosome 17 that harbor TP53 on 17p. This further allowed us to determine on which of the two chromosomes the mutated allele was located. The results presented here show that the cells have undergone an evolution from homozygous wild type for TP53 to heterozygous (His179Asn mutation in one allele), and finally to a hemizygous mutated state (deletion of the remaining wild-type allele). The finding of a transient period in which heterozygous cells dominate the population before the eventual outgrowth of hemizygous 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.
Villadsen
R
R
Department of Tumor Endocrinology, Danish Cancer Society, Copenhagen, Denmark.
Nielsen
K V
KV
Bolund
L
L
Briand
P
P
D000483:Alleles; D001940:Breast / Q000737:chemistry / Q000166:cytology*; D002455:Cell Division; D002460:Cell Line; D002461:Cell Line, Transformed; D002886:Chromosomes, Human, Pair 17; D004247:DNA / Q000032:analysis; D004847:Epithelial Cells; D005260:Female; D005348:Fibrocystic Breast Disease / Q000235:genetics; D005434:Flow Cytometry; D016158:Genes, p53; D005838:Genotype; D006579:Heterozygote; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D020125:Mutation, Missense; D016133:Polymerase Chain Reaction; D016159:Tumor Suppressor Protein p53 / Q000502:physiology
D016159:Tumor Suppressor Protein p53; D004247:DNA