Different capacities for recombination in closely related human lymphoblastoid cell lines with different mutational responses to X-irradiation.

Abstract

WIL2-NS and TK6 are two distinct human lymphoblast cell lines derived from a single male donor. WIL2-NS cells are significantly more resistant to the cytotoxic effects of X-irradiation but considerably more sensitive to induced mutation. In an effort to determine the mechanistic basis for these differences, we analyzed the physical structures of thymidine kinase (tk)-deficient mutants isolated after X-ray treatment of tk heterozygotes derived from TK6 and the more mutable WIL2-NS. Southern analysis showed that while 84% of TK6-derived mutants had arisen by loss of heterozygosity (LOH), all 106 mutants from WIL2-NS derivatives arose with LOH at tk and all but one showed LOH at other linked loci on chromosome 17. We adapted a fluorescence in situ hybridization technique to distinguish between LOH due to deletion, which results in retention of only one tk allele, and LOH due to a mechanism involving the homologous chromosome (e.g., recombination), which results in the retention of two alleles. Among the LOH mutants derived that were analyzed in this way, 9 of 26 from WIL2-NS and 11 of 17 from TK6 cell lines arose by deletion. The remaining mutants retained two copies of the tk gene and thus arose by a mechanism involving the homologous allele. Since many of these mutants arising by a homologous mechanism retained partial heterozygosity of chromosome 17, they must have arisen by recombination or gene conversion, and not chromosome loss and reduplication. Finally, the recombinational capacities of WIL2-NS and TK6 were compared in transfection assays with plasmid recombination substrates. Intermolecular recombination frequencies were greater in WIL2-NS than in TK6. These data are consistent with a model suggesting that a recombinational repair system is functioning at a higher level in WIL2-NS than in TK6; the greater mutability of the tk locus in WIL2-NS results from more frequent inter-and intramolecular recombination events.Mutations that activate oncogenes or inactivate tumor suppressors are intimately involved in carcinogenic pathways. Therefore, it is important to study factors that affect mutation rates in human cells, because this will improve our understanding of cancer risk from environmental exposures and spontaneous processes. A number of studies suggest that the frequencies at which mutations are observed at various gene targets vary tremendously, depending on their location in the genome (reviewed in reference 56). Single gene mutations that inactivate a genetic locus are theoretically equally likely to occur at hemizygous loci (+/0: one active allele, no homologous allele) or heterozygous loci (+/-: one active and one inactive allele). Very large deletions may have a higher probability of being lethal if they span hemizygous regions of the genome and include an essential locus, while loss of one copy of such loci from chromosomal regions that exist in duplicate may not be lethal. Homologous recombination or gene conversion, processes that can convert a heterozygous locus to a homozygous state (-/-, with...

Abrogation of p53 function by HPV16 E6 gene delays apoptosis and enhances mutagenesis but does not alter radiosensitivity in TK6 human lymphoblast cells

“…In the present study, a moderate increase in both the spontaneous and radiation-induced mutation frequencies at the tk locus was observed in the TK6-E6-5E cells. For the tk locus, an increased mutation frequency may result from an increase in inter-chromosomal recombinational events, as has been implicated in WTK1 cells (Xia et al, 1994). The increased mutation frequency at the tk locus in the TK6-E6-5E cells thus may not be unexpected since loss of p53 function by various means, including gene loss, dominant negative p53 mutation, or transfection with SV40 large T antigen or the HPV16 E6 gene, has been shown to increase the recombination rate in a plasmid based assay by more than 100-fold in some human tumor cell lines (Mekeel et al, manuscript submitted).…”

“…WTK1 cells are less sensitive to radiation-induced cell killing but more susceptible to radiation-induced gene mutations at the hypothanthine-guanine phosphoribosyl transferase (hprt) and the thymidine kinase (tk) loci than TK6 cells (Amundson et al, 1993). The radioresistance in the WTK1 cells has been attributed to a delayed apoptotic response while the greater mutability of the autosomal tk locus is thought to result from more frequent interand intramolecular recombination events (Xia et al, 1994(Xia et al, , 1995. It has been proposed that these di erences are related to the di ering p53 status of the two cell lines (Xia et al, 1995).…”

Dissociation of p53-mediated suppression of homologous recombination from G1/S cell cycle checkpoint control

“…Previous studies on p53 and HR were based on various means of wild-type p53 inactivation, i.e., by SV40 large T antigen, by HPV-E6, by a dominant-negative mutant or by allelic deletion, which led not only to loss of trans-activation function, but also to disruption of the presumptive downstream pathway or regulatory protein domain responsible for suppression of HR (Meyn et al, 1994;Xia et al, 1994;WiesmuÈ ller et al, 1996;Bertrand et al, 1997;Mekeel et al, 1997). Mechanistic insight has come from WiesmuÈ ller and colleagues who studied changes in HR frequencies as a result of altered recombination substrates (DuddenhoÈ er et al, 1998).…”

“…p53 has also been reported to be involved in control of the G2/M checkpoint and a variety of other aspects of cell proliferation and DNA metabolism (Ko and Prives, 1996;Levine, 1997;Janus et al, 1999a). Suppression of spontaneous homologous recombination (HR) has recently been established as a new endpoint of wild-type p53 function (Meyn et al, 1994;Xia et al, 1994;WiesmuÈ ller et al, 1996;Bertrand et al, 1997;Mekeel et al, 1997;DuddenhoÈ er et al, 1998). In a DNA repair pathway, recombinational processes may act to maintain genetic stability, but if deregulated or increased, genomic instability and malignant transformation can result.…”

p53 and recombination intermediates: role of tetramerization at DNA junctions in complex formation and exonucleolytic degradation

“…When expressed at high levels, it transcriptionally transactivates approximately 50 genes, such as the G1 arrest gene p21 WAF1/CIP1 , and thereby inhibits growth and induces apoptosis under certain cellular conditions (Lane, 1992;Tokino et al, 1994;Levine, 1997). At both basal and elevated protein levels, wtp53 down-regulates homologous recombination processes, to avoid detrimental rearrangements such as the unrestrained loss of heterozygosity (Xia et al, 1994;Meyn et al, 1994;WiesmuÈ ller et al, 1996;Honma et al, 1997;Bertrand et al, 1997;Mekeel et al, 1997). Separation of function mutations were identi®ed, which served to demonstrate that wtp53 regulates spontaneous and radiation-induced homologous recombination independently of its activities in transcription and growth control (Saintigny et al, 1999;DudenhoÈ er et al, 1999;Willers et al, 2000).…”