Allelic loss is an important mutational mechanism in human carcinogenesis. Loss of heterozygosity (LOH) at an autosomal locus is one outcome of the repair of DNA double-strand breaks (DSBs) and can occur by deletion or by mitotic recombination. We report that mitotic recombination between homologous chromosomes occurred in human lymphoid cells exposed to densely ionizing radiation. We used cells derived from the same donor that express either normal TP53 (TK6 cells) or homozygous mutant TP53 (WTK1 cells) to assess the influence of TP53 on radiation-induced mutagenesis. Expression of mutant TP53 (Met 237 Ile)was associated with a small increase in mutation frequencies at the hemizygous HPRT (hypoxanthine phosphoribosyl transferase) locus, but the mutation spectra were unaffected at this locus. In contrast, WTK1 cells (mutant TP53) were 30-fold more susceptible than TK6 cells (wild-type TP53) to radiation-induced mutagenesis at the TK1 (thymidine kinase)locus. Gene dosage analysis combined with microsatellite marker analysis showed that the increase in TK1 mutagenesis in WTK1 cells could be attributed, in part, to mitotic recombination. The microsatellite marker analysis over a 64-cM region on chromosome 17q indicated that the recombinational events could initiate at different positions between the TK1 locus and the centromere. Virtually all of the recombinational LOH events extended beyond the TK1 locus to the most telomeric marker. In general,longer LOH tracts were observed in mutants from WTK1 cells than in mutants from TK6 cells. Taken together, the results demonstrate that the incidence of radiation-induced mutations is dependent on the genetic background of the cell at risk, on the locus examined, and on the mechanisms for mutation available at the locus of interest.

The faithful repair of DNA DSBs3is important for the maintenance of genomic integrity. Single nonrepaired DSBs can be lethal (1), and misrepaired DSBs can lead to chromosome aberrations (2, 3), mutations, or cell death (4, 5). DSBs can be introduced into DNA as a consequence of oxidative damage, mechanical stress, endonuclease activity, or exposure to IR. Eukaryotes have at least two distinct mechanisms for the repair of DSBs: illegitimate (nonhomologous)end-joining, and homologous recombination (reviewed in Ref.6).

IR influences the transcription of many genes in mammalian cells(7). Several early-response genes are transcribed at high levels shortly after exposure to IR (8). Some of these early-response genes encode transcription factors that direct the cellular responses to IR, including cell cycle arrest, induction of DNA repair, and apoptosis. The tumor suppressor gene TP53is among the early-response genes. After DNA damage, TP53 functions in the induction of cell cycle arrest (9), the regulation of gene amplification (10), and the induction of apoptosis(11, 12). Although many biochemical functions of the TP53 protein have been identified (13), the precise mechanisms by which TP53 regulates DNA repair, genomic stability, and mutagenesis remain to be elucidated.

We tested the effect of a specific “gain-of-function” TP53 mutation(14) on the incidence of mutations arising after exposure to densely IR, and asked whether the mutational spectra were affected by this particular form of mutant TP53 (Met 237 Ile). We used two male human B-lymphoblastoid cell lines, TK6 and WTK1, that were derived from the same donor (15). Both DNA fingerprint analysis and karyotypic analysis confirmed the syngeneic origin of the two cell lines (16). Five markers on the long arm of chromosome 17 showed the same pattern of heterozygous alleles in the two cell lines(17). In contrast, two short arm markers that were heterozygous in TK6 cells were homozygous in the WTK1 cells. Thus, WTK1 cells are homozygous for at least a part of chromosome 17p. We cannot exclude the possibility that other, unidentified genetic differences may exist between these cell lines.

WTK1 and TK6 cells are each heterozygous for the autosomal TK1 locus located on chromosome 17q21–23(18, 19, 20). The same TK1 allele in each cell line was inactivated by exposure to the frameshift mutagen ICR-191(21, 22). Aneuploidy of chromosome 17 is rare in both TK6 and WTK1 cells, with 2% or less of cells showing trisomy 17(23). The same is true for the WI-L2-NS cell line, the progenitor of WTK1 cells that shares the same homozygous mutation in the TP53gene.4The male-derived cell lines are hemizygous for the HPRTlocus located at chromosome Xq26 (24). Whereas TK6 cells express only wild-type TP53, WTK1 cells have two identical TP53 mutations (a transition of ATG to ATA in codon 237,producing a Met to Ile amino acid substitution; Refs.25, 26, 27).

We report that WTK1 cells were less sensitive to the cytotoxic effects and more prone to the mutagenic effects of Fe ions. Gene dosage analysis and microsatellite mapping were combined to assess the mechanisms of mutagenesis at the TK1 locus. Mitotic recombination between homologous chromosomes (allelic recombination)was significantly elevated in Fe ion-exposed WTK1 cells as compared with similarly exposed TK6 cells. Furthermore, multilocus LOH events were significantly longer in mutants derived from WTK1 cells. In contrast, the Fe ion-induced mutation spectra at the HPRTlocus in TK6 and WTK1 cells could not be distinguished from each other. Thus, mutation induction is dependent on the locus examined, the possible mechanism of mutation at each locus, and the genetic background of the cell at risk.

Cell Culture, Irradiation, and Determination of Cellular Survival and Mutation Frequencies.

TK6 and WTK1 cells were grown at 37°C in suspension cultures in a humidified 5% CO2 atmosphere in RPMI 1640 supplemented with 10% heat-inactivated horse serum, 100 units/ml penicillin, and 100 mg/ml streptomycin (Life Technologies, Inc., Grand Island, NY). Cells were kept at densities of 1–10 × 105 cells/ml to maintain them in exponential growth. Four to 5 days prior to irradiation, cells were pretreated with CHAT (10 μm deoxycytidine, 200 μmhypoxanthine, 0.2 μm aminopterin, and 17.5μ m thymidine) to remove preexisting TK1- and HPRT-deficient mutants from the population (28). Two days after CHAT treatment, cells were resuspended in standard growth medium plus THC (CHAT without aminopterin). Cells were exposed to graded doses of 1 GeV/amu Fe ions at the Alternating Gradient Synchrotron (AGS) at Brookhaven National Laboratory. Replicate cultures were exposed in T-25 tissue culture flasks (up to 1.5 × 108 cells/group) to a given dose to ensure that there were sufficient numbers of surviving mutants for good statistics. Control cultures were treated in an identical manner except for the exposure to the Fe ion beam. An aliquot of each culture was immediately seeded into 96-well plates at 1–20 cells/well to determine the surviving fraction. Colonies were scored after 11 days of growth, and relative surviving fractions were calculated according to standard methods (28, 29, 30, 31). After irradiation, cultures were grown for 3 days (TK1) or 6 days (HPRT) in nonselective medium to allow for phenotypic expression.

For selection of TK1-deficient mutants, cells were seeded into 96-well microtiter plates in the presence of TFT (2.0 mg/ml; Sigma). TK6 cells were seeded into medium containing TFT at a density of 4 × 104 cells/well whereas WTK1 cells were seeded into medium containing TFT at densities of 5–10 × 102 cells/well. Cells from each culture were also seeded into 96-well dishes at 1 cell/well in the absence of TFT to determine the plating efficiency. The plates were incubated for 11 days prior to scoring early-arising TK1-deficient mutants. The mutant fractions were calculated according to standard methods(29). To collect a series of independent TK1-deficient mutants, we irradiated 50 separate cultures of TK6 cells and 50 separate cultures of WTK1 cells, each at a dose of 94.5 cGy. Fifty early-arising colonies (one mutant per culture) of TK1-deficient mutants that arose in each cell line were picked from the mutation dishes after 11 days of incubation, and these cultures were expanded for DNA extraction. The same microwell dishes were refed with fresh TFT and incubated for an additional 7 days to obtain late-arising TK1-deficient mutants. Fifty late-arising TK1 mutants were picked for each cell line and expanded prior to DNA extraction.

For selection of HPRT-deficient mutants, cells were seeded in the presence of 0.5 mg/ml 6-TG. Cells were seeded into microwell dishes in the presence of the selective agent at a density of 4 × 104 cells/well. Cells from each culture were also plated at 1 cell/well in the absence 6-TG for evaluation of the plating efficiency. All of the plates were incubated 11 days prior to scoring HPRT mutants according to standard methods(28). Fifty mutant colonies were picked for each cell line and expanded for DNA extraction.

DNA Extraction and Analysis of TK1 deficient Mutants.

DNA extractions were performed using the G-NOME-kit (BIO 101)according to the manufacturer’s description. DNA from the TK6 and WTK1 parent cell lines and from each TK1 mutant was subjected to Southern analysis at the TK1 locus according to standard methods with minor modifications (30). Briefly, 10 μg of genomic DNA were digested to completion with the restriction enzyme SacI (New England Biolabs), fractionated by electrophoresis in a 0.8% agarose gel, transferred to a nylon membrane(Hybond-N+, Amersham), and fixed by UV-cross-linking (Stratalinker, Stratagene). The membrane was hybridized overnight with gel-purified cDNA probes labeled with[α-32P]dATP by random priming (Amersham). The cDNA probe for the TK1 gene (pTK11) was kindly provided by Dr. P. Deininger (Louisiana State University, Baton Rouge, LA)(32). After hybridization, the membranes were washed and exposed to Hyperfilm MP (Amersham) for 2–7 days at −80°C. SacI digestion revealed two unique bands in the parental cell lines, a 14.8 kb band corresponding to the active TK1allele and an 8.4 kb band corresponding to the silent TK1allele. Three additional restriction fragments were common to both alleles. To determine whether one or two copies of the silent TK1 allele remained in a given TK1-deficient mutant, we standardized the loading of each lane by stripping the blots and hybridizing them to the cDNA of a gene (BCL-2) located on another chromosome. The BCL-2 gene is located on chromosome 18 (33). The cDNA probe for BCL-2 was kindly provided by Dr. S. Korsmeyer (Dana Farber Cancer Institute, Boston,MA) (34). SacI digestion revealed three nonpolymorphic restriction bands, one of which was approximately 8.4 kb in size. This band was used to normalize for the DNA loading of each lane of a given filter. Densitometric analysis established an intensity ratio for the restriction fragment linked to the silent TK1allele versus the 8.4 kb BCL-2 restriction fragment. The TK1/BCL-2 intensity ratio for the lane containing the parental cell line on each filter defined the presence of a single TK1 allele. We used the following criteria to discriminate between LOH by recombination and LOH by deletion: if the TK1/BCL-2 intensity ratio for a given mutant was ≥1.8 times that of the control cell line on the same filter, the mutation occurred by recombination, generating two silent alleles. If the TK1/BCL-2 intensity ratio was <1.8 times that of the control, the mutation occurred via deletion. The quantitation of signals was performed by densitometric analysis of exposed Hyperfilm MP (Amersham) using the Molecular Dynamics Personal Densitometer SI and ImageQuant software (version 1.11).

Intragenic Analyses of HPRT-deficient Mutants.

The intragenic analysis of HPRT-deficient mutants was performed by multiplex PCR. The nine exons of the X-linked HPRT gene were amplified using eight primer pairs (exons 7 and 8 were coamplified)according to standard methods (35). To ensure that the DNA from each mutant was of suitable quality for PCR amplification, we included a set of primers for the APRT locus (located on chromosome 16) as an internal control in each multiplex PCR reaction(36).

Analyses of Multilocus LOH Events.

Primer pairs for microsatellite markers were obtained from Research Genetics. The chromosomal locations of analyzed loci relative to the TK1 locus are summarized in Table 1(37, 38, 39, 40). Microsatellite markers were amplified individually by PCR according to the manufacturer’s description. Genotype analysis of the corresponding marker loci was performed as described elsewhere with minor modifications (37). Products obtained in the PCR reactions were detected after hybridization to one of the terminally[α-32P]dATP-labeled primers, used in the PCR,to the nylon membrane. Filters were exposed to Hyperfilm MP (Amersham)overnight at −80°C.

Cytotoxic Response to Densely Ionizing Fe Ions.

TK6 and WTK1 cells were exposed to graded doses of densely IR (1 GeV/amu Fe ions). TK6 cells were more sensitive to the cytotoxic effects of Fe ions than were WTK1 cells (Fig. 1). We determined D0 values for each cell line. The D0 is defined as the dose required to reduce the number of surviving cells to 37% of their former value using data from an exponential survival curve. The D0 values were 51 cGy and 75 cGy for TK6 cells and WTK1 cells, respectively.

TK1 Mutation Induction in TK6 and WTK1 Cells.

TK1-deficient mutants were induced in both cell lines as a linear function of dose (Fig. 2). Densely ionizing Fe ions were almost 30-fold more mutagenic at the TK1 locus in WTK1 cells than in TK6 cells. The induced mutant fractions were 5.02 ± 0.35 × 10-7/cGy (mean ± 1 SE; r2, 0.937) for TK6 cells and 144.4 ± 7.58 × 10-7/cGy (mean ± 1 SE; r2, 0.955) for WTK1 cells. The average background mutation frequencies were 12.81 ± 0.15 × 10-6 for TK6 cells and 159.33 ± 34.0 × 10-6 for WTK1 cells. In the TK1mutation assay, two phenotypic classes of mutants were observed (see“Materials and Methods”): early-arising mutants with growth rates that corresponded to the growth-rate of the parental cells (doubling times, 13–17 h), and late-arising mutants that grew at a slower rate (doubling times, >21 h). The growth rates of individual mutant clones were evaluated at least 30 generations post-selection to verify their doubling times (data not shown). All of the late-arising clones exhibited protracted doubling times. The proportion of late-arising mutants observed after exposure to densely ionizing Fe ions was higher in TK6 cells (65%) than in WTK1 cells (39%).

Molecular Characterization of the TK1 Mutation Spectra for Fe Ion-induced TK1-deficient Mutants.

Genomic DNA isolated from TK1-deficient mutants of WTK1 and TK6 cells was subjected to Southern blot analysis using a human TK1cDNA probe (pTK11). A SacI polymorphic restriction site in the 3′-flanking region of the human TK1 gene distinguishes the active allele from the silent allele in both TK6 and WTK1 cells. Hybridization reveals two SacI restriction fragments of 14.8 kb and 8.4 kb in size corresponding to the functional and the nonfunctional TK1 allele, respectively (Fig. 3). Three additional bands are common to both alleles (Fig. 3). Mutants were classified as follows: no detectable alterations (both alleles retained) wherein the loss of TK1 activity is likely attributable to point mutations or small insertions or deletions(changes <200 bp) that inactivate the functional TK1allele, partial deletions or rearrangements in the functional allele that lead to the appearance of novel restriction fragments, and LOH mutations leading to loss of the 14.8 kb band, with no new restriction fragments appearing. LOH was the most prevalent type of mutation at the TK1 locus after exposure to 94.5 cGy Fe ions, irrespective of the growth rate of the mutant or the cell of origin (Table 2). A very small fraction of early-arising mutants of each cell line had small intragenic mutations, partial deletions, or rearrangements. All but one of the late-arising TK1 mutants of TK6 cells and WTK1 cells were the result of LOH events (Table 2).

Mechanism of Radiation-induced LOH Formation at the TK1 Locus: More Recombinational LOH in TP53 Mutant Cells.

Gene dosage analysis was performed to establish whether individual LOH events retained one silent TK1 allele or two copies of the silent TK1 allele. We have classified the mutants with one silent allele as deletion mutants based on a combination of their gene dosage and their microsatellite marker analysis (see next page). Most mutants that retained two silent copies were classified as having arisen by recombination based on the microsatellite marker analysis(see next page). Rare mutants with two silent copies of TK1and LOH tracts ≥64 cM may have arisen by either recombination or nondisjunction. We observed both deletional and recombinational LOH in TK1-deficient mutants of TK6 and WTK1 cells (Fig. 3 and Table 2). Deletion mutants of TK6 cells had TK1/BCL-2intensity ratios of 1.00 ± 0.02 (mean ± 1 SE), whereas deletion mutants of WTK1 cells had mean intensity ratios of 1.10 ± 0.02. On the basis of our confidence in the measurements of those mutants that had one copy of the silent TK1 allele, ratios ≥1.8 were classified as having two silent copies of the TK1 gene. The TK1:BCL-2 intensity ratios for TK6-derived mutants with two silent TK1 alleles were 2.31 ± 0.18, whereas for the WTK1-derived mutants, the mean intensity ratios were 2.45 ± 0.07 (mean ± 1 SE). Recombinational LOH was rare in early-arising mutants derived from TK6 cells but was more prevalent among the early-arising mutants derived from WTK1 cells (χ2, 1 df,11.27; P < 0.001; see Table 2). The same was true for the late-arising mutants: more recombinational LOH was seen in mutants derived from WTK1 cells(χ2, 2 df, 10.9; P < 0.005; see Table 2). Thus, the predominating mutational mechanism underlying LOH formation differed in the two cell lines. Mitotic recombination was much more common in mutants that arose in the WTK1 cells that express only the mutant form of TP53. Deletions were more common in the mutants that arose in the TK6 cells that express wild-type TP53.

Extent of Multilocus LOH Events Associated with TK1-deficiency and Assessment of LOH Tract Length as a Function of Mechanism.

A series of 14 polymorphic microsatellite markers that span 64 cM inclusive of the TK1 locus was used to determine the extent of LOH in individual TK1 mutants (Table 1). TK6 and WTK1 cells have the same genotype for these 14 markers on the long arm of chromosome 17q. For a given TK1 mutant, one of two types of events might be observed at each microsatellite marker: retention of heterozygosity (banding pattern same as the control) or LOH (loss of the allele linked to the previously active TK1 allele). An example of the genotype analyses of a series of TK1mutants and for one of the polymorphic markers (D17S785) is shown in Fig. 4.

LOH tracts were grouped into size classes on the basis of their occurrence among the mutants of each cell line (Table 3). Early-arising mutants of TK6 cells had shorter LOH tracts than similar mutants derived from WTK1 cells: most were shorter than 5 cM(χ2, 3 df, 29.5; P < 0.001). In contrast, the LOH tract lengths were very variable among the early-arising mutants of WTK1 cells.

Late-arising mutants of both cell lines exhibited longer LOH tracts than did early-arising mutants (Table 3). For late-arising TK6 mutants,12 of 48 exhibited LOH tracts <20 cM whereas 36 of 48 mutants showed multilocus LOH events spanning 20 cM or more. In contrast, short LOH tracts were rare in late-arising TK1 mutants derived from WTK1 cells: only 1 of 49 mutants had an LOH tract length <20 cM,whereas 48 of 49 mutants exhibited multilocus LOH events encompassing>20 cM. Late-arising TK1 mutants of WTK1 cells exhibited significantly longer LOH tracts than those seen in similar mutants that arose in TK6 cells (χ2, 2 df, 15.3; P < 0.001).

The genotypic analysis of LOH tract lengths was done by a PCR-based method that is not sensitive enough to determine gene copy number at a given locus. However, the mechanism of LOH for each of the microsatellite markers could be inferred from the gene dosage analysis at the TK1 locus for a given mutant. The combined results are shown in Table 4. Several features should be noted: (a) the centromeric breakpoints were randomly distributed in LOH mutants that arose by deletion; (b) for those LOH mutants that arose by mitotic recombination, the position of the most centromeric marker involved in the exchange was also randomly distributed; (c)the telomeric breakpoints in the deletion mutants were variable;(d) nearly all of the mutants (46 of 48) that arose by mitotic recombination had long LOH tracts (≥21 cM) that extended to the most telomeric marker; and (e) only 2 of 48 mutants that arose by mitotic recombination retained heterozygosity at the most telomeric marker (D17S928). Most of the recombination events appeared to be single cross-overs.

Detailed analysis of LOH tract lengths and the mechanism of mutation helped highlight other distinctions in mutant spectra as a function of the cell at risk. Deletion tract lengths were shorter among early-arising TK1 mutants of TK6 cells than for similar mutants of WTK1 cells (χ2, 2 df,11.75; P < 0.001; see Table 4, upper part). A second difference was seen in recombination tract lengths in the early-arising TK1 mutants: the two recombination-mediated early-arising mutants in TK6 cells had short recombination tracts that did not extend to the telomere (one, ≤1 cM,and one, ≤10 cM), whereas 12 of 15 early-arising recombination-mediated mutants in WTK1 cells had LOH tracts extending to the telomere (≥21 cM). Three early-arising WTK1 mutants had two copies of TK1 and LOH tracts ≥64 cM. These rare mutants may have arisen either by recombination or nondisjunction. We also assessed correlations between LOH tract length and the mechanisms of mutagenesis for the late-arising TK1 mutants (Table 4, lower part). Deletion tract lengths were again shorter for the TK6-derived mutants (χ2, 2 df, 6.77; P < 0.05). Among the late-arising deletion mutants of TK6 cells, 22% had LOH tracts ≤10 cM. In contrast, no mutants of this type were found in WTK1 cells: all of the deletion mutants were >10 cM. We did not see a difference in the LOH tract lengths in late-arising mutants of TK6 and WTK1 cells that retained two silent TK1 alleles (χ2, 2 df, 0.613; P > 0.5). All of the late-arising TK6- and WTK1-derived recombination mutants showed LOH tracts that were >21 cM and extended to the most telomeric marker. Three late-arising TK6 mutants and six late-arising WTK1 mutants had two copies of TK1 and LOH tracts ≥64 cM. These mutants may have arisen either by recombination or nondisjunction.

HPRT Mutation Induction and Mutation Spectra in TK6 and WTK1 Cells.

HPRT-deficient mutants arose as a linear function of dosage after the exposure of TK6 or WTK1 cells to densely ionizing Fe ions. The dose-response relationships are shown in Fig. 5. In contrast to what was observed for the TK1 locus, HPRT mutation induction was only slightly enhanced(<2-fold) in WTK1 cells expressing mutant TP53. The induced mutant fractions were 1.16 ± 0.08 × 10-7/cGy (mean ± 1 SE; r2, 0.953) for TK6 cells and 2.28 ± 0.20 × 10-7/cGy (mean ± 1 SE; r2, 0.901) for WTK1 cells. The average background mutation frequencies at the HPRT locus were 2.96 ± 0.27 × 10-6 for TK6 cells and 7.65 ± 2.25 × 10-6 for WTK1 cells. The PCR analysis of individual HPRT-deficient mutants is illustrated in Fig. 6, and the results for 84 mutants derived from TK6 and WTK1 cells exposed to 94.5 cGy of Fe ions are summarized in Table 5. HPRT mutants were classified into three categories based on the PCR analysis: total deletion (loss of all of the HPRT-specific bands with retention of the APRT-specific band as a PCR control), partial deletion/rearrangement (absence of some HPRT-specific bands or a change in the size of a particular HPRT-specific band),or no detectable alteration (changes too small to be visible, e.g., <20 bp, in any of the HPRT-specific bands). The intragenic HPRT mutation spectra for the two cell lines could not be distinguished (χ2, 2 df, 0.584; P > 0.5).

In summary, these results suggest that the expression of mutant TP53(Met 237 Ile) is associated with a small increase in mutation fraction at the X-linked hemizygous HPRT locus and with a large increase in mutation fraction at the autosomal TK1 locus in cells exposed to densely ionizing Fe ions. The marked elevation in TK1 mutagenesis in WTK1 cells is associated with an increase in the incidence of mitotic recombination.

TK6 cells and WTK1 cells are human lymphoblastoid cell lines derived from the progenitor cell line WI-L2 (15). The two cell lines cannot be distinguished from each other by either cytogenetic or DNA fingerprinting analysis (16, 17). These cell lines, however, differ in TP53 status. WTK1 cells have a homozygous mutation (Met 237 Ile) in the TP53 gene that is associated with a high level of expression of mutant TP53 protein(25, 26, 27). This missense mutation is located in the sequence-specific DNA binding domain of TP53, and missense mutations within this region are frequently observed in human tumors(41). The mutant form of TP53 found in WTK1 cells adopts a wild-type conformation (42) and has also been detected in human lymphoid tumors (43, 44).

WTK1 cells and TK6 cells are known to differ in their sensitivity to cell killing and the induction of TK1 mutations after exposure to sparsely ionizing X-rays or low energy α particles(16, 17, 45). There was no difference found between the two cell lines in the progression of irradiated cells out of G1 phase and into S phase and G2 phase of the cell cycle (26). Furthermore, TK6 cells and WTK1 cells each have an intact G2-M checkpoint after irradiation(46). Therefore, it is unlikely that differences in the cell cycle regulation account for the large increase in radiation-induced TK1 mutagenesis that has been reported for WTK1 cells. TK6 and WTK1 cells differ in their susceptibility to X-ray-induced apoptosis (25, 26, 47). In earlier studies,we have shown that the suppression of programmed cell death in TK6 cells by overexpression of the antiapoptotic proteins BCL-2 or BCL-XL can explain part, but not all, of the 30-to 50-fold increase in X-ray-induced TK1 mutagenesis seen in WTK1 cells (48). Overexpression of either BCL-2 or BCL-XL was associated with a more modest increase in X-ray-induced TK1 mutagenesis in TK6-derived cell lines than is seen in nonoverexpressing WTK1 cells. Of particular importance,TP53-null TK6 cells do not show the high levels of spontaneous or X-ray-induced mutations seen in WTK1 cells (14). Instead,TP53-null TK6 cells had very similar levels of X-ray-induced TK1 mutations to TK6 cells that express their normal amount of TP53. Taken together, these observations show that cell cycle regulation, failed apoptosis, or loss of other TP53 activities cannot entirely account for the high levels of radiation-induced mutagenesis seen in WTK1 cells. These observations support the suggestion that some forms of mutant TP53, including the one found in WTK1 cells, may act to promote radiation-induced TK1 mutagenesis and that they might represent gain-of-function mutants (14).

In the present study, we compared the response of TK6 and WTK1 cells to high energy Fe ions and showed that WTK1 cells were less sensitive to cell killing and more prone to mutagenesis after exposure to this type of densely IR. We assessed mutation induction at two endogenous loci that produced very different results: WTK1 cells were only slightly more susceptible (<2-fold) to mutation at the X-linked, hemizygous HPRT locus than were TK6 cells, whereas a very large increase (30-fold) in mutation susceptibility was observed for WTK1 cells at the autosomal TK1 locus compared with TK6 cells. The differences in mutation susceptibility at the two loci are likely attributable to: (a) the loss of viability of cells with deletions >1.3Mbp telomeric to the HPRT locus(49, 50, 51) as compared with the tolerance for very large deletions inclusive of the TK1 locus (40); and(b) the possibility for allelic recombination that exists at the TK1 locus but not at the hemizygous HPRTlocus.

Most of the TK1-deficient mutants isolated after exposure to Fe ions have lost the active TK1 allele. Allelic loss can be a critical event in human cancer, and LOH involving a tumor suppressor locus is common in a wide variety of human tumors. LOH can occur by different mechanisms: chromosome loss, chromosome loss and reduplication of the homologous chromosome, deletion of one allele(hemizygous LOH), or mitotic recombination between homologous alleles(homozygous LOH). Whole chromosome loss is generally thought to be unlikely because of functional hemizygosity in the human genome. Similarly, chromosome loss and reduplication (nondisjunction) is a rare event in carcinogenesis (52, 53).

In our study, exposure to densely ionizing Fe ions led to both deletional LOH and LOH by mitotic recombination. Chromosome loss can be excluded on the basis of fluorescence in situ hybridization analysis of TK1 deletion mutants with an additional marker(HsRAD51C) proximal to D17S806.5Recombination between homologous chromosomes was particularly elevated in WTK1 cells expressing the mutant form of TP53 (Met 237 Ile),irrespective of the growth-rate of the individual mutants. A small subset (6%) of mutants arose either by recombination or nondisjunction. To discriminate between these types of events would require the analysis of additional markers proximal to D17S806. We speculate that wild-type TP53 plays a role in limiting recombination and that the (Met 237 Ile) mutant form of TP53 found in WTK1 cells facilitates promiscuous recombination.

Using the combined approaches of gene dosage analysis and microsatellite mapping, we found that mitotic recombination inclusive of the TK1 locus was nearly always associated with a single exchange between the homologous chromosomes that extended to the most telomeric marker on chromosome 17q. Our studies were performed in asynchronous cells. If the exchange events took place in G1 phase, they may have occurred via chromosome break-induced replication, as has been shown to occur in diploid,G1 phase Saccharomyces cerevisiae(54). This mechanism led to LOH of all of the chromosomal markers telomeric to a single site-specific DSB and resembles what we have observed in the majority of TK1-deficient mutants that arose by recombination. Alternatively, if the exchanges leading to LOH at TK1 and the linked marker loci took place in late S phase or G2, they may reflect a single exchange followed by segregation of the sister chromatids at mitosis (55). Those rare mutants that had two copies of the silent TK1allele and short LOH tracts must represent double-exchange events.

Our results on the extent of LOH in TK6 and WTK1 cells exposed to densely ionizing Fe ions can be compared with results on spontaneous and X-ray-induced TK1 mutations derived from TK6 cells(40). Spontaneous TK1-deficient mutants tended to have large LOH tracts, whereas small LOH tracts (1–10 cM) were more prevalent among mutants arising after X-rays. We also observed some small LOH tracts among early-arising TK1-deficient mutants isolated after the exposure of TK6 cells to Fe ions; however, most of the mutants that arose in TK6 cells that were exposed to Fe ions were late-arising mutants that showed more extensive LOH tracts. Similarly,LOH tracts in Fe ion-exposed WTK1 cells were generally >10 cM.

Mitotic recombination has been thought of as a minor mechanism of mutagenesis in mammalian cells, in part because of the lack of good model systems to detect what is generally a conservative form of DNA repair (55). Gene conversion events were detected at low frequency at the TK1 locus of human lymphoblasts after X-ray exposure (20). A high frequency of mitotic recombination was observed at the APRT locus in cells from people who are obligate heterozygotes with characterized germ-line mutations(56). Linkage analysis using microsatellite markers was used to define the mechanisms of mutagenesis, and it was shown that 76% of the mutants had LOH tracts that sometimes extended to the end of chromosome 16q. These events were classified as having occurred by mitotic recombination. Long LOH tracts were also observed after X-irradiation at the MmAPRT locus in the mouse embryonal carcinoma cell line P19H22, which carries one chromosome 8 derived from Mus musculus domesticus, and one from a feral mouse(57). It was suggested that the LOH events that extended to the most telomeric marker arose by mitotic recombination, but no gene dosage studies were performed to confirm the presence of two MmAPRT alleles. Mitotic recombination also appeared to play a role in spontaneous mutagenesis at the MmAPRT locus in heterozygous mice (58). Our results using both linkage analysis and gene dosage analysis confirm that mitotic recombination occurs in human cells. These events frequently extend from a random exchange point centromeric to the target locus to the most telomeric marker available.

Our results are in general agreement with studies that indicated that spontaneous and X-ray-induced mutation frequencies were elevated at the TK1 locus in cells expressing only the (Met 237 Ile) mutant form of TP53 (16, 17). Mutant TP53 (Met 237 Ile) was associated with an increased frequency of LOH-type mutations and enhanced allelic recombination (17, 59). Recombination between plasmid substrates was elevated in WTK1 cells compared with TK6 cells (59). Transfection studies showed that overexpression of either mutant TP53 (Met 237 Ile) or mutant TP53 (Val 143 Ala) in TK6 cells led to enhanced recombination between plasmid substrates and to elevated spontaneous and X-ray-induced TK1mutation frequencies (60). In Jurkat cells, the overexpression of three different forms of mutant TP53 was associated with increased frequencies of X-ray-induced T-cell receptor rearrangements (61). The COOH-terminal portion of TP53 was shown to be essential for the suppression of recombination(62). In addition, a recent study demonstrated that the inactivation of wild-type TP53 in TK6 cells by a gene-targeting approach did not lead to an increase in spontaneous or radiation-induced mutagenesis (14). In summary, several studies suggested that certain mutant forms of TP53, including that expressed in WTK1 cells, might act as gain-of-function mutants,promoting both mitotic recombination and strictly homologous recombination.

Other studies have suggested a second role for TP53 in limiting the incidence of nonhomologous end-joining. Cells lacking wild-type TP53,including WI-L2-NS and TK6-E6, showed elevated frequencies of end-joining using plasmid substrates (63). The elevated frequencies of unbalanced translocations seen in X-irradiated WTK1 cells were thought to reflect an increased incidence of illegitimate recombination (16), although the sequence fidelity at the site of the translocation was not addressed directly. Another possibility is that the translocations occurred between repetitive DNA sequences via promiscuous recombination. Recombination between homologous sequences on heterologous mouse chromosomes has been demonstrated to occur after a single DSB, occasionally leading to a translocation (64).

The machinery of homologous recombination in human cells is a subject of intense investigation (65). TP53 may help regulate the process of homologous recombination. TP53 has been shown to interact physically and functionally both with HsRAD51, the major strand transfer protein in human cells (66), and with the prokaryotic homologue RecA (67). Two domains of the TP53 molecule were shown to be required for the interaction with HsRAD51:residues 94–160, and residues 264–315 (68). It has been postulated that wild-type TP53 disrupts the homo-oligomerization of HsRAD51, blocking the formation of filaments that promote recombination(68). Other lines of evidence suggest that wild-type TP53 can act to suppress homologous recombination. TP53 bound to synthetic Holliday junctions and was preferentially located at the Holliday junction as opposed to the free DNA arms or DNA ends (69). Overexpression of wild-type TP53 suppressed an intrinsically high level of homologous recombination in cells that naturally do not express wild-type TP53 (70). Several mutant forms of TP53 that fail to induce a G1 phase arrest were competent for the suppression of homologous recombination, which suggests a separation of functions (71). Evidence supporting a role for TP53 in the suppression of recombination between intrachromosomal repeat sequences has recently been reported (72).

In this study, we observed an association between the expression of wild-type TP53 in TK6 cells and a relatively low incidence of radiation-induced mutations, including less recombination-mediated mutations. Biochemical studies are in progress to characterize the physical and functional interactions in the TP53-HsRAD51 protein complexes in TK6 and WTK1 cells.

In summary, we showed that expression of mutant TP53 in WTK1 cells was associated with enhanced mutagenesis at an autosomal locus and an elevated frequency of mitotic recombination. The recombination-mediated LOH events were generally ≥21 cM in length and extended to the most telomeric marker available. We speculate that this mutant form of TP53(Met 237 Ile) promotes recombination, as previously suggested by others(14). The observed differences in the TK1mutation spectra in TK6 and WTK1 cells suggest an additional role for TP53 in the maintenance of genomic stability and in preventing the genetic changes that lead to human cancer.

Fig. 1.

Cell killing as a function of the dose of densely ionizing Fe ions for TK6 and WTK1 cells. Data points represent three independent experiments. The results were analyzed by zero-intercept linear regression using the StatView 4.5 statistical package (Abacus Concepts,Berkeley, CA).

Fig. 1.

Cell killing as a function of the dose of densely ionizing Fe ions for TK6 and WTK1 cells. Data points represent three independent experiments. The results were analyzed by zero-intercept linear regression using the StatView 4.5 statistical package (Abacus Concepts,Berkeley, CA).

Close modal
Fig. 2.

Fe ion-induced mutation induction at TK1. Induced mutation frequencies at the TK1 locus in TK6(slope = 5.02 ± 0.35 × 10-7/cGy; r2, 0.937) and WTK1(slope = 144.4 ± 7.58 × 10-7/cGy; r2, 0.955) cells. The slopes represent the mean of three independent experiments ± 1 SE. The results were analyzed by zero-intercept linear regression using the StatView 4.5 statistical package (Abacus Concepts,Berkeley, CA).

Fig. 2.

Fe ion-induced mutation induction at TK1. Induced mutation frequencies at the TK1 locus in TK6(slope = 5.02 ± 0.35 × 10-7/cGy; r2, 0.937) and WTK1(slope = 144.4 ± 7.58 × 10-7/cGy; r2, 0.955) cells. The slopes represent the mean of three independent experiments ± 1 SE. The results were analyzed by zero-intercept linear regression using the StatView 4.5 statistical package (Abacus Concepts,Berkeley, CA).

Close modal
Fig. 3.

Southern Blot analysis of SacI-digested genomic DNA from WTK1 cells (LanesW) and 16 TK1-deficient mutants of WTK1 cells (Lanes 1–16)obtained after exposure to Fe ions. Left panel, after hybridization to the TK1 cDNA probe (pTK11) only the 8.4 kb restriction fragment carrying the silent allele remained in this particular group of mutants. Right panel, after stripping, the identical blot was hybridized to a BCL-2cDNA probe. Densitometric analysis establishes an intensity ratio of the 8.4 kb TK1- and BCL-2 signals for control cells and for each mutant.

Fig. 3.

Southern Blot analysis of SacI-digested genomic DNA from WTK1 cells (LanesW) and 16 TK1-deficient mutants of WTK1 cells (Lanes 1–16)obtained after exposure to Fe ions. Left panel, after hybridization to the TK1 cDNA probe (pTK11) only the 8.4 kb restriction fragment carrying the silent allele remained in this particular group of mutants. Right panel, after stripping, the identical blot was hybridized to a BCL-2cDNA probe. Densitometric analysis establishes an intensity ratio of the 8.4 kb TK1- and BCL-2 signals for control cells and for each mutant.

Close modal
Fig. 4.

Analysis of the representative microsatellite marker D17S785 for TK6 cells and for a series of late-arising TK1 mutants of TK6 cells that arose after Fe ion exposure. TK6 control cells and mutants 6, 8, 9, 10, 13, 22, 24, and 25 are heterozygous for D17S785 (alleles a and b are retained). All of the other mutants have lost allele a, which is linked to the active TK1 gene.

Fig. 4.

Analysis of the representative microsatellite marker D17S785 for TK6 cells and for a series of late-arising TK1 mutants of TK6 cells that arose after Fe ion exposure. TK6 control cells and mutants 6, 8, 9, 10, 13, 22, 24, and 25 are heterozygous for D17S785 (alleles a and b are retained). All of the other mutants have lost allele a, which is linked to the active TK1 gene.

Close modal
Fig. 5.

Induced mutation frequencies at the HPRTlocus in TK6 cells (slope = 1.16 ± 0.08 × 10-7/cGy; r2, 0.953) and WTK1 cells (slope = 2.28 ± 0.20 × 10-7/cGy; r2, 0.901). The slopes represent the mean of 3 independent experiments ± 1 SE. The results were analyzed using zero-intercept linear regression using the StatView 4.5 statistical package (Abacus Concepts,Berkeley, CA).

Fig. 5.

Induced mutation frequencies at the HPRTlocus in TK6 cells (slope = 1.16 ± 0.08 × 10-7/cGy; r2, 0.953) and WTK1 cells (slope = 2.28 ± 0.20 × 10-7/cGy; r2, 0.901). The slopes represent the mean of 3 independent experiments ± 1 SE. The results were analyzed using zero-intercept linear regression using the StatView 4.5 statistical package (Abacus Concepts,Berkeley, CA).

Close modal
Fig. 6.

PCR analysis of intragenic alterations in HPRT-deficient mutants that arose after exposure to 94.5 cGy of Fe ions. Lane W, WTK1 cells; lanes 1–4, mutants derived from WTK1 cells; left and right margins, the eight exon-specific amplification products. Mutant 1, no detectable alterations; mutant 2, total deletion; mutant 3, exons 1 through 3 are deleted; mutant 4, exons 2 through 6 are deleted. A fragment of the APRT gene was coamplified as a positive control for each PCR reaction.

Fig. 6.

PCR analysis of intragenic alterations in HPRT-deficient mutants that arose after exposure to 94.5 cGy of Fe ions. Lane W, WTK1 cells; lanes 1–4, mutants derived from WTK1 cells; left and right margins, the eight exon-specific amplification products. Mutant 1, no detectable alterations; mutant 2, total deletion; mutant 3, exons 1 through 3 are deleted; mutant 4, exons 2 through 6 are deleted. A fragment of the APRT gene was coamplified as a positive control for each PCR reaction.

Close modal

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by NASA Grant T-964W (to A. K.). Lawrence Berkeley National Laboratory is operated under Department of Energy contract DE-AC03-76SF00098 (to the University of California).

3

The abbreviations used are: DSB,double-strand break; IR, ionizing radiation; HPRT, hypoxanthine phosphoribosyl transferase; TK1, thymidine kinase; APRT, adenosine phosphoribosyl transferase; TFT, trifluorothymidine; 6-TG,6-thioguanine; amu, atomic mass unit(s); cGy, centigray.

4

A. J. Grosovsky, personal communication.

5

A. Kronenberg, S. Bailey, and E. Goodwin,unpublished results.

Table 1

Microsatellite marker loci included in the LOH tract length analyses and their relative position to TK1

LocusaPosition relative to TK1Distance from TK1b (cM)
D17S806 Centromeric 43 
D17S787 Centromeric 35 
D17S808 Centromeric 27 
D17S948 Centromeric 22 
D17S807 Centromeric 20 
D17S840 Centromeric 14 
D17S929 Centromeric 
D17S801 Centromeric 
D17S785 Centromeric 
D17S937 Centromeric 0.1 
TK1c   
D17S802 Telomeric <1 
D17S836 Telomeric 
D17S784 Telomeric 10 
D17S928d Telomeric 21 
LocusaPosition relative to TK1Distance from TK1b (cM)
D17S806 Centromeric 43 
D17S787 Centromeric 35 
D17S808 Centromeric 27 
D17S948 Centromeric 22 
D17S807 Centromeric 20 
D17S840 Centromeric 14 
D17S929 Centromeric 
D17S801 Centromeric 
D17S785 Centromeric 
D17S937 Centromeric 0.1 
TK1c   
D17S802 Telomeric <1 
D17S836 Telomeric 
D17S784 Telomeric 10 
D17S928d Telomeric 21 
a

Primer pairs for analyzed loci were obtained from Research Genetics (Huntsville, AL). Only informative DNA markers have been included in the analysis; analyzed loci are listed in order from the most centromeric to the most telomeric.

b

The genetic distances between these loci were taken from Gyapay et al.(37).

c

The TK1 gene was positioned in a 1 cM interval between microsatellite markers D17S937 and D17S802 (38, 39).

d

D17S928 was the most distal marker available for this study and was estimated to be 10–29 cM from the telomere (37).

Table 2

Fe ion-induced mutation spectra for early- and late-arising TK1 mutants

The mutant classification was performed by Southern blot analysis using a TK1 cDNA probe and densitometric analysis of TK1 gene dosage.

Mutant classificationTK6 early-arisingWTK1 early-arisingTK6 late-arisingWTK1 late-arising
LOH 42 45 49 49 
Partial deletion/rearrangement 
No detectable alterations 
Total 50 49 50 50 
LOH via deletion (1 silent allele) 40 30 41 26 
LOH via recombination (2 silent alleles) 12 17 
LOH via recombination or nondisjunction (2 silent alleles) 
Total 42 45 49 49 
 χ2 for LOH type,a 1 df, 11.27; P < 0.001  χ2 for LOH type, 2 df, 10.9; P < 0.005  
Mutant classificationTK6 early-arisingWTK1 early-arisingTK6 late-arisingWTK1 late-arising
LOH 42 45 49 49 
Partial deletion/rearrangement 
No detectable alterations 
Total 50 49 50 50 
LOH via deletion (1 silent allele) 40 30 41 26 
LOH via recombination (2 silent alleles) 12 17 
LOH via recombination or nondisjunction (2 silent alleles) 
Total 42 45 49 49 
 χ2 for LOH type,a 1 df, 11.27; P < 0.001  χ2 for LOH type, 2 df, 10.9; P < 0.005  
a

χ2 analysis compares deletion mutants versus nondeletion mutants.

Table 3

LOH tract length analysis in TK1−/− mutants arising after exposure to densely ionizing Fe ions

Genotypes of 14 TK1-linked microsatellite markers spanning a region of 64 cM were determined by PCR to evaluate the extent of LOH surrounding TK1.

Length of LOH tracts (cM)TK6 early-arisingWTK1 early-arising
<5 28 13 
5–20 13 
20–40 13 
40–64 12 
Totala 42 45 
Length of LOH tracts (cM)TK6 early-arisingWTK1 early-arising
<5 28 13 
5–20 13 
20–40 13 
40–64 12 
Totala 42 45 
TK6 late-arisingWTK1 late-arising
<20 12 
20–40 27 25 
40–64 23 
Totalb 48 49 
TK6 late-arisingWTK1 late-arising
<20 12 
20–40 27 25 
40–64 23 
Totalb 48 49 
a

χ2, 3 df, 29.5; P < 0.001.

b

χ2, 2 df,15.3; P < 0.001.

Table 4

Correlation of the mechanism of LOH formation and LOH tract length in TK1-deficient mutants of TK6 and WTK1 cells

The mechanism of LOH formation at the TK1 locus was determined by gene dosage blotting using SacI-digested DNA of TK1-deficient mutants of TK6 and WTK1 cells. Mutants that arose via deletion retain one silent copy of the TK1 gene, and mutants that arose via recombination retain two silent copies of the TK1 gene. The LOH tract lengths were determined using TK1-linked microsatellite markers spanning a region of 64 cM around TK1.

LOH formation by (cM)TK6 early-arisingWTK1 early-arising
Deletion   
≤1 24 
2–10 13 
>10 12 
Total 40 30 
 χ2, 2 df, 11.75; P < 0.001  
Recombinationa   
≤20 
21–63 12 
≥64b 
Total 15 
LOH formation by (cM)TK6 early-arisingWTK1 early-arising
Deletion   
≤1 24 
2–10 13 
>10 12 
Total 40 30 
 χ2, 2 df, 11.75; P < 0.001  
Recombinationa   
≤20 
21–63 12 
≥64b 
Total 15 
TK6 late-arisingWTK1 late-arising
Deletion   
≤10 
11–28 19 14 
29–64 13 12 
Total 41 26 
 χ2, 2 df, 6.77; P < 0.05  
Recombination   
21–28 
29–63 
≥64c 
Total 23 
 χ2, 2 df, 0.613; P < 0.001  
TK6 late-arisingWTK1 late-arising
Deletion   
≤10 
11–28 19 14 
29–64 13 12 
Total 41 26 
 χ2, 2 df, 6.77; P < 0.05  
Recombination   
21–28 
29–63 
≥64c 
Total 23 
 χ2, 2 df, 0.613; P < 0.001  
a

Statistical analysis could not be performed because of the restricted sample size.

b

Mutants with LOH tracts ≥64 cM may have arisen either by recombination or nondisjunction.

c

Mutants with LOH tracts ≥64 cM may have arisen by recombination or nondisjunction.

Table 5

Characterization of HPRT-deficient mutants of TK6 and WTK1 cells arising after exposure to 94.5 cGy of 1 GeV/amu Fe ions

The intragenic analysis of HPRT-deficient mutants of TK6 and WTK1 cells was performed by PCR. HPRT mutants were classified as follows:(a) total deletion: loss of all HPRT exons but retention of the PCR control fragment for APRT;(b) partial deletion or rearrangement: loss or change in size of one but not all HPRT exons; and (c) no detectable alterations: all HPRT exons present with no visible change in size (<10–20 bp).

Mutant classificationTK6WTK1
Total deletion 14 20 
Partial deletion/rearrangement 13 
No detectable alteration 14 15 
Total 36 48 
 χ2, 2 df, 0.584; P > 0.5  
Mutant classificationTK6WTK1
Total deletion 14 20 
Partial deletion/rearrangement 13 
No detectable alteration 14 15 
Total 36 48 
 χ2, 2 df, 0.584; P > 0.5  

A substantial effort in support of this project was provided by members of the staff of the Brookhaven National Laboratory (BNL), where the Fe ion irradiations were performed. We would like to thank the Alternating Gradient Synchrotron (AGS) operations staff at BNL and the LBNL/AGS dosimetry group for assistance with the beam delivery and beam characterization at the AGS facility. The support of the BNL medical department staff was also greatly appreciated. We also thank Drs. Ed Goodwin and Susan Bailey (Los Alamos National Laboratory) for assistance with cytogenetics.

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