Molecular mechanisms of spontaneous and induced loss of heterozygosity in human cells in vitro

Abstract

The human TK6 lymphoblast cell line is heteroallelic at the thymidine kinase (TK) locus, with one functional and one nonfunctional allele. Cells that have undergone loss of heterozygosity (LOH) at TK can be selected and cloned in an in vitro assay. In order to study the extent of LOH, we have analyzed a total of 166 thymidine kinase-deficient mutants that arose either spontaneously or following induction by X-ray or ethyl methane sulfonate (EMS) using DNA probes in and around the TK gene on chromosome 17. Two distinct groups of mutants with different doubling times were identified. Among slow-growth mutants, the predominant change for both spontaneous and induced mutants was LOH that generally extended through the entire TK gene to both proximal and distal markers on 17q. While the majority of both spontaneous and X-ray-induced normal-growth mutants showed LOH, this was considerably more localized in scale for X-ray-induced mutants, which rarely involved the distal marker. LOH was rare among EMS-induced normal-growth mutants. LOH was never observed with a 17p marker, indicating that nondisjunctional events were not involved in any of the mutant clones examined. Densitometric analysis of the LOH mutants indicated mitotic recombination was a likely mechanism in more than half the spontaneous LOH mutants in both groups, whereas most induced mutants appeared to arise from simple deletions.

Interchromosomal Crossover in Human Cells Is Associated with Long Gene Conversion Tracts

“…Nevertheless, presumptive interchromosomal crossovers, in the form of chromosome-scale loss of heterozygosity (LOH), are commonly observed in mammalian cells. LOH is the major contributor to the mutational spectrum in mice and humans (31,33,47,49,78), notably at tumor suppressor loci (3,11,15,25,34,35,40,68,88,89). Crossover products have also been associated with intrachromosomal recombination between repeat sequences in mammalian cells (12,30,52,75), and similar outcomes are observed during the integration of plasmids into host cell chromosomes (9,48,90).…”

Radiation-induced Tumorigenesis

“…This LOH was frequently extended to include other genes on the chromosome, both proximal and distal to the target gene (Li et al, 1992). Although LOH was often the result of a simple deletion, it was shown, at least in some cases, to be the result of recombinational processes (Yandell et al, 1986;Li et al, 1992).…”

“…When a system was developed to study the mutational events at an autosomal locus where both copies of the gene were present, then large-scale events that involved the loss of heterozygosity (LOH) were found to be the most frequent ones (Yandell et al, 1986). This LOH was frequently extended to include other genes on the chromosome, both proximal and distal to the target gene (Li et al, 1992). Although LOH was often the result of a simple deletion, it was shown, at least in some cases, to be the result of recombinational processes (Yandell et al, 1986;Li et al, 1992).…”

Essai n° 490 : Essai In Vitro de Mutation Génique Sur Cellules de Mammifères Utilisant le Gène de la Thymidine Kinase

“…La nature moléculaire et cytogénétique des deux sortes de colonies de mutants dans l'essai MLA (petites et grandes) a été étudiée en détail (8) (10) (11) (12) (13). La nature moléculaire et cytogénétique des mutants TK6 apparaissant précocement et tardivement a aussi été largement étudiée (14) (15) (16) (18). Les mutants à la croissance lente pour les deux types de cellules ont subi des lésions génétiques impliquant le ou les gènes régulateurs présumés de la croissance situés près du locus TK, ce qui entraîne des temps de doublement plus longs et la formation de petites colonies ou de colonies apparaissant tardivement (18).…”