Mutagenesis, Vol. 14, No. 2, 199-205,
March 1999
© 1999 UK Environmental Mutagen Society/Oxford University Press
Complex hprt deletion events are recovered after exposure of human lymphoblastoid cells to high-LET carbon and neon ion beams
1 Division of Radioisotope Technology, 2 Cellular Physiology Laboratory and 3 Cyclotron Laboratory, Institute of Physical and Chemical Research, Saitama 351-01, 4 Division of Biology, Toray Research Center Inc., Kanagawa 248, 5 Space and Particle Radiation Science Research Group, National Institute of Radiological Sciences, Chiba 263, 6 School of Medicine, Osaka University, Osaka 565 and 7 Faculty of Pharmacy, Nagasaki University, Nagasaki 852, Japan
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Abstract |
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Hypoxanthine phosphoribosyltransferase gene (hprt) mutations were induced in human TK-6 lymphoblastoid cells by irradiation at a linear energy transfer (LET) of 250 or 310 keV/µm for carbon and neon ions, respectively. At such a high level of LET, ions will lose most of their total energy and stop shortly after passing through the cell. The hprt mutations were analyzed by multiplex PCR, long-PCR and DNA sequencing of both genomic and cDNA. Over half of the C ion-induced hprt mutations (10 of 19) were point mutations, in contrast to 15% of the mutations induced by Ne ions (three of 20). The remaining 47 and 85% of the C and Ne ion-induced mutants, respectively, are deletion events. The latter events include three complex losses of multiple non-contiguous exon regions in both ion irradiation collections. We note that mutations involving the exon 6 region are frequent in the Ne ion collection: all three of the complex events retained the exon 6 region with flanking deletion of sequence and three other mutants involved deletion of this region. It may be concluded that these high-LET C and Ne ion irradiations produce different mutational spectra.
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Introduction |
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Heavy ion irradiation can be considered an important tool for studying the biological effects of radiation since the damage produced in chromosomal DNA seems to be different to that produced by conventional ionizing radiation (Brenner and Ward, 1992
; Murakami et al., 1995; Rydberg, 1996; Fukashima et al., 1997; Sasaki et al., 1997). Moreover, the kind of heavy ion as well as the beam energy also influences the nature of the DNA damage produced. Therefore, the analysis of induced mutation at the DNA sequence level may facilitate an understanding of how the characteristic energy deposition of heavy ions is reflected in the nature of mutations recovered. The basic study of mutation mechanisms may be accelerated by identification of mutations unique to the kind of radiation. This, in turn, would be useful in the estimation of radiation risk in space from heavy ion-induced mutations, since radiation protection becomes important with the recent progress in manned space flight. High-LET levels of heavy ions produced by carbon as well as other heavy ions is relevant with respect to radiation risk estimation because the body may be exposed to such high-LET ions after their passage through materials, including protective materials.
LET-dependent mutation induction in mammalian cells has been extensively studied (Kronenberg et al., 1995; Stoll et al., 1995; Suzuki et al., 1995; Zhu et al., 1996). Previously, we characterized the nature of mutations in human embryo cells induced by accelerated C ions (Kagawa et al., 1995). Point mutations were recovered as the major mutational event after exposure of primary cultured human embryo cells to C ions at a level at which they lose most of their energy and stop shortly after passing through the cell. It is of interest to determine whether this tendency is also observed with well-established human cell lines.
In this study, human TK6 lymphoblastoid cells were exposed to 250 and 310 keV/µm C and Ne ions, respectively, that had been accelerated by the Riken Ring Cyclotron. After the isolation of hprt mutant clones, the genomic DNA was extracted and analyzed by multiplex PCR and, in some cases, long-PCR. If the pattern was normal, cDNA was prepared from total RNA and the DNA was sequenced.
Striking differences in the mutational spectra obtained after C and Ne ion irradiation were observed.
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Materials and methods |
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Cell culture and exposure to heavy ions
Human TK6 lymphoblastoid cells (obtained from Dr K.Tatsumi, National Institute of Radiological Sciences, Chiba-shi, Japan) were cultured at 37°C in RPMI 1640 medium (Gibco BRL) supplemented with 1 mM
-ketoglutaric acid and 10% fetal bovine serum (Rehatsin) in a humidified atmosphere containing 5% CO2. Prior to heavy ion exposure, the cells were cultured for 2 days in RPMI medium containing CHAT (10 µM cytidine, 200 µM hypoxanthine, 0.2 µM aminopterin and 17.5 µM thymidine) and one more day in CHT medium (CHAT excluding aminopterin). After CHAT selection, the cultured cells were resuspended in fresh RPMI at 8x105 cells/ml and exposed to heavy ions as previously described (Suzuki et al., 1995). The heavy ion irradiation (Riken Ring Cyclotron, Wako-shi, Saitama, Japan) was performed at three different LET (22, 120 and 250 keV/µm for C ions and 67, 120 and 310 keV/µm for Ne ions) by decreasing the initial accelerated energy (135 MeV/µm) with the use of Lucite absorbers. Dosimetry has been described (Kanai et al., 1993, 1997). The cells were irradiated as a suspension in culture medium and this is the only departure from the previous irradiation in which the cells were attached to the inside wall of the flask.
Selection of mutant clones
After irradiation, the cells were immediately diluted 4-fold to a concentration of 2x105 cells/ml. Independent mutant clones were obtained in the following manner. Aliquots of 5 ml of the above cell suspension was dispensed to 10 6 cm diameter dishes (the expression dish) and cultured for 4–5 days in normal medium to allow phenotypic expression of the HPRT phenotype. After this expression period, the cell culture from each expression dish was separately diluted in medium with 6-thioguanine (5 µg/ml final concentration) and 0.2 ml (~4x104 cells) was added to each well of a 96-well dish (therefore 10 96-well dishes were generated, one from each expression dish). If mutants arose in more than one well from any 96-well dish they were picked for analysis; to ensure the independent nature of each mutational event only those mutants that exhibited different multiplex PCR and/or DNA sequencing profiles are included in Tables IV–VI. For spontaneous mutants, an identical selection procedure was used, except there was no expression time. Concurrent with the mutational assay, a survival assay was also performed to measure the population of viable cells among the irradiated cells. For cell survival determinations, the original cell suspensions (irradiated and unirradiated) were diluted to ~1.6 cells/well of a 96-well dish. Determination of survivor and mutant clone numbers was performed after a 2 week incubation period irrespective of the irradiation. The putative hprt mutant clones were diluted 20-fold in the selection medium and incubated for a further 5 days to confirm their hprt status.
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Extraction of RNA and genomic DNA
IsogenTM (Nippon Gene, Japan) was used for the extraction of RNA and genomic DNA from the cells of hprt mutant clones which had been amplified to ~5–10x106 cells. Isogen is a homogeneous solution composed of phenol and guanidine thiocyanate. The extraction procedure followed was as described by the supplier. The ethanol-precipitated RNA and DNA fractions obtained were stored at –80°C until use.
Multiplex PCR analysis of genomic DNA
Mutant genomic DNA (250 ng) was used as the template for the PCR reaction (GeneAmp PCR System 2400; Perkin Elmer). Other than the template DNA, the PCR reaction mixture involved ~5–10 pmol of each primer (Table Ib), 0.4 mM of each dNTP and 2.5 U ExTaq polymerase (Takara). Concentrated reaction buffer was also added to the mixture according to the protocol of Takara. The nucleotide sequence of the primers was similar to those used in our previous studies (Kagawa et al., 1995; Shimahara et al., 1995) and were based upon the sequence of the genomic hprt locus (Edwards et al., 1990). First, the reaction mixture was preheated at 94°C for 3 min. Then, the reaction proceeded for 25 cycles of the following reaction profile: denaturation at 94°C for 30 s, annealing at 59°C for 30 s and extension at 70°C for 60 s. The last extension was prolonged to 8.5 min. The following genomic DNA regions were amplified by multiplex PCR (Gibbs et al., 1990): four different regions including exons 3, 4, 5 and 9 were amplified in a single reaction; three different regions including exons 2, 6, and 7–8 were amplified in a separate reaction (see Figure 1). Exon 1 was analyzed in a separate reaction due to difficulty in its amplification. To confirm the presence of amplifiable genomic DNA in the reaction mixture a 675 nucleotide region of the autosomal aprt gene was used as a PCR control under the same reaction conditions as the multiplex PCR. All the PCR products were analyzed in a 2% agarose gel (Medium EEO; Sigma).
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Long-PCR analysis of genomic DNA
Three different regions, A (16.4 kb), B (16.4 kb) and C (19.2 kb), of the genomic DNA spanning exons 1–3, 2–4 and 4–9, respectively, are shown in Figure 1
. The LA PCR Kit v.2 (Takara) was used for the long-PCR amplification (Barnes, 1994). The long-PCR reaction mixture was essentially that of the multiplex PCR reaction except that Takara LA Taq DNA polymerase was used. The mixture was preheated at 94°C for 4 min before 35 cycles of the following reaction: denaturation for 10 s at 98°C and 20 min at 68°C for the combined annealing and extension step. The last extension reaction was prolonged to 7 min at 72°C. The amplified products were analyzed on a 0.5% agarose gel. Results are shown only for the C region (Figure 2); under these conditions and with the primers used (Table I), the A region did not provide reliable amplification.
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First strand cDNA synthesis and amplification of hprt cDNA
Total RNA was used without further purification in a reverse transcription reaction to cDNA with the First-Strand cDNA Synthesis Kit (Pharmacia). Using an appropriate amount of the synthesized cDNA and the corresponding primers (Table I
), 35 cycles of PCR reaction were performed under essentially the same condition as for the multiplex PCR. Each cycle consisted of denaturation at 94°C for 1 min, annealing at 55°C for 1.5 min and extension at 72°C for 1.5 min. The last extension was prolonged to 8.5 min.
DNA sequencing
The sequences of the PCR-amplified DNA fragments were determined by an automatic sequencer (ABI 373A DNA sequencer, Applied Biosystems Inc.) using the ABI Dye DeoxyTM Cycle Sequencing Kit (401150). Sequence analysis was performed using the SeqEd software (Applied Biosystems Inc.).
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Results |
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LET-dependent radiosensitivity
The radiosensitivity of TK6 cells was dependent on the LET (energy) of the C and the Ne ions (Table II
). The relative biological effectiveness (RBE) values for the lethal effect of the heavy ions were calculated from a comparison of the D10 dose with that for X-irradiation, which has a D10 of 1.8 Gy. The peak of RBE appeared to exist around a LET of 120 keV/µm for the C ion. Ne ion irradiation gave a relatively high RBE even at 67 keV/µm and this level was not changed at 120 keV/µm. In both ion irradiations the RBE values decreased at high LET, at which level the heavy ions stop shortly after passing through the cells due to the small energy remaining. This level of RBE at high LET levels was lower than that of the standard level for X-rays. This fact simply indicates that the beam energy was not effectively used for cell killing compared with irradiation by X-rays. The collection of hprt mutant clones was carried out at this high LET level and the values of both the surviving fraction and the mutation frequency are shown in Table III. The frequency of hprt mutation was clearly enhanced 4.5- and 7.3-fold over that of unirradiated control at this LET level of 250 keV/µm for C ions and 310 keV/µm for Ne ions, respectively. A total of 28 mutants were obtained in the C ion collection and 34 mutants in the Ne ion collection; further analysis by multiplex PCR and/or DNA sequencing revealed that within the C and Ne ion collections, 19 mutants and 20 mutants, respectively can be considered independent and it is the independent mutants that are further considered.
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Multiplex PCR analysis
Multiplex PCR analysis was used to amplify hprt exon regions from genomic DNA of the mutant clones that were isolated after exposure of TK6 cells to high-LET heavy ions. The results of the analysis of 19 independent mutants obtained after 250 keV/µm C ion treatment are compiled in Table IV
and those for 20 independent mutants after 310 keV/µm Ne ion treatment are compiled in Table V. At this level of analysis C and Ne ion irradiations clearly produced different multiplex PCR patterns (Table VI). Over half of the mutants (10 of 19) isolated after C ion irradiation exhibit a normal multiplex pattern in which each exon region was amplified. The remaining nine mutants did not amplify some region(s). In contrast, most of the mutants (17 of 20) recovered following Ne ion irradiation exhibited a pattern in which particular regions could not be amplified (Table VI). Deletions also account for ~55% of mutations in the TK6 spontaneous spectrum (Nelson et al., 1994). However, due to the level of mutation induction with C and Ne ion treatment, in conjunction with the nature of deletions (see below) and point mutations (see below) obtained after treatment, spontaneous mutations should make little contribution to the induced spectra reported here.
Analysis of mutant multiplex hprt patterns provided results of some interest. In the collection of nine exon loss mutants recovered following C ion irradiation, three mutants demonstrated distinct patterns of complex loss of non-contiguous exon regions (Table IV). Ne ion irradiation also provided a large number of exon loss mutants (17 of the total 20). Three of these mutants were shown to be complex losses of non-contiguous exon regions. Intriguingly, all these Ne ion-induced mutants retain only the exon 6 region and have suffered both 5' and 3' deletions with respect to exon 6. These striking `exon 6' events were not seen in the C ion collection. No complex deletions were seen in 78 spontaneous mutants (Nelson et al., 1994).
For the multiple exon loss mutants it was necessary to confirm the existence of amplifiable genomic DNA in the reaction tube. For this purpose, we used the aprt gene as a control PCR target as described in Materials and methods. All three gene loss mutants produced a normal PCR amplification of the aprt gene locus. In addition, we also obtained normal aprt amplification for seven more mutants in which a large part of the hprt locus was deleted (Tables IV and V).
Long-PCR analysis
Long-PCR analysis (Barnes, 1994) was applied to selected mutant clones to obtain information about the structure of the genomic hprt locus. Mutants 2Ne3-1, 2Ne3-3 and 2Ne7-2 were identified as deletions of the exon 6 region by multiplex PCR. Figure 2 shows long-PCR analysis of the 19.2 kb C region (Table I) that spans exon 6. Compared with the normal size for this region, the sizes for 2Ne3-1, 2Ne3-3 and 2Ne7-2 were estimated to be smaller by 3.4, 2.6 and 5.4 kb, respectively. Since these deletions are expected to map in the 7.9 kb region between the 3'-end of exon 5 and the 5'-end of exon 7, we attempted direct sequencing of the genomic DNA. Mutant 2Ne7-2 was found to be a deletion of 5791 bp from position 32904 to 38694. Interestingly, mutant clones 2Ne3-1 and 2Ne3-3 produced the same multiplex PCR pattern (Table V) but by long-PCR were shown to have different sizes of deletion. Therefore, although they arose in the same expression dish, these two mutants are classified as independent mutational events. The A region did not generate reliable amplification by long-PCR and this analysis is not further considered.
DNA sequencing
Mutant hprt cDNA was prepared from those mutant clones that produced a normal multiplex PCR pattern. Mutant clone 2C10-1 recovered following C ion irradiation did not produce a cDNA although it did exhibit a normal multiplex PCR pattern. Since the exon 1 region is difficult to amplify by multiplex PCR compared with other regions, we tried to obtain and succeeded in obtaining the cDNA for mutant clone 1C5-1, which exhibited a defect in the amplification of only the exon 1 region (Table IV). A similar effort was made for mutant 2Ne9-2 but a cDNA was not obtained (Table V). The results of DNA sequencing for the nine and three independent events in the C and Ne ion mutant collections, respectively, are shown in Tables VII and VIII. Amongst point mutations, base substitutions were the most frequent mutational event in both the C and Ne ion collections. Both the Ne and C ion spectra contained one splicing event, these being the loss of the entire exon 6 (2Ne3-2) and the loss of most of exon 8 (1C2-2, due to a nonsense mutation in exon 8), respectively. Interestingly, both a transition and a transversion were recovered at the same position in the C ion collection (position 580). The mutations were not found for two mutant clones in the C ion collection, 1C5-1 and 2C6-5. Mutant 2C1-2 was found to have a G
T transversion at the third position of the Val9 codon, which is the last nucleotide position of exon 1. This change does not cause an amino acid substitution, but it may not be silent in that it may reduce the amount of mature mRNA; no other change was found in the hprt reading frame. The high proportion of tranversions recovered after C ion treatment (four of six base substitutions) and Ne ion treatment (the sole base substitution mutant was actually a non-tandem GCCG event) argues in favor of their induction by the heavy ions; in the spontaneous spectrum transversions account for only 38% of base substitutions (Nelson et al., 1994). However, mutant 2C3-1 was a CT transition at a CpG site and therefore the possibility that it may be of spontaneous origin cannot be excluded.
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Discussion |
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The lethal effect in TK6 cells of C ion irradiation showed a RBE peak around 120 keV/µm (Table II
), a level similar to that observed with human embryo (HE) cells (Suzuki et al., 1995). Such a LET dependence obtained with Ne ion irradiation of TK-6 cells was essentially the same as that seen with C ion irradiation, although the RBE value of 67 keV/µm was as high as that for 120 keV/µm. This concordance is probably due to the irradiation conditions, in which the cells were suspended in a container with a 1 mm inside thickness along the direction of the heavy ion path. Since all LET values in our experiment were measured and expressed at the entrance position of the cell suspension, the real LET values of Ne ions might become higher than 67 keV/µm while moving in the suspension. The high-LET component seemed to contribute to the cell killing effect at this LET point. This interpretation is consistent with the LET dependence of the killing effect observed with Ne ion irradiation of HE cells when they form a single layer in the flask (Suzuki et al., 1997). In the latter experiment, the RBE peak was observed around 120 keV/µm and the value of RBE was relatively high even at a LET level <120 keV/µm. In the present experiment, relatively low values of RBE, ~0.5, were obtained for both ion beams at the high LET level at which we selected the hprt mutant clones (Table III). This low RBE might be accounted for by the component of ions that decreased in energy while passing through the 1 mm thickness of the cell container. This component is difficult to express as a LET in this beam-stopping region, but it is likely to be less effective in cell killing.
Multiplex PCR analysis has proven useful in the characterization of hprt– mutations. Indeed, this approach revealed characteristics of the induced hprt mutations which seem to be specific to heavy ion irradiations (Tables IV and V). The pattern of multiplex PCR amplification was found to be normal for 10 of 19 hprt mutants selected after the 250 keV/µm C ion irradiation (Table VI). This level of recovery of the normal multiplex pattern was also observed with a similar high-LET C ion irradiation of HE cells (Suzuki et al., 1995). In contrast, only three of the 20 hprt mutants exhibited the normal multiplex PCR pattern after 310 keV/µm Ne ion irradiation of TK6 cells (Table VI). Even at this level of mutant analysis it is clear that high-LET C and Ne ion irradiations produce different mutational spectra. Furthermore, among the nine exon loss mutants recovered following C ion irradiation, three mutants demonstrated distinct patterns of complex loss of multiple non-contiguous exon regions (Table IV). Such complicated patterns from multiplex PCR were also identified in three of 17 exon loss mutants after Ne ion irradiation. This high level induction of complex deletion events by high-LET irradiation has not been seen with low-LET radiation before nor has it been seen in the spontaneous spectrum (none of 78 mutants; Nelson et al., 1994). The deletion patterns for X- or 
-ray-induced hprt mutations were found to be simple as detected by Southern hybridization (Tachibana et al., 1990; Xia et al., 1994; Giver et al., 1995). Other studies using PCR and DNA sequencing analyses also found only simple patterns of hprt deletion (Morris et al., 1993, Rigaud et al., 1995; Nelson et al., 1996). In our previous analysis of -ray-induced hprt mutants, such complex patterns were also not observed amongst the deletion events (Suzuki et al., 1995). Moreover, in the Ne ion-induced complex events all three mutants retained only the exon 6 region (mutants 1Ne2-1, 1Ne4-1 and 1Ne5-1), possibly highlighting a region prone to such induced rearrangements. Indeed, three other mutants (2Ne3-1, 2Ne3-3 and 2Ne7-2) involve deletion of this region, for a total of six of the 20 mutants of the Ne ion collection involving deletion/rearrangement of this region. A more mechanistic analysis awaits the sequence junctions of these rearrangements.
The DNA base sequences were determined from the cDNA of those mutant clones that demonstrated a normal amplification pattern with multiplex PCR. Ten such independent hprt mutants were isolated after the 250 keV/µm C ion irradiation but cDNA could be obtained from only nine of these mutants. These nine mutational events were comprised of four G:CT:A (including one nonsense mutation in exon 8 that resulted in abnormal splicing and loss of 73 bp from the mRNA), two G:CA:T and one +G; two mutants cDNAs remained uncharacterized (Table VII). A possible explanation for the latter two mutants is weak hprt gene expression due to mutation of the promoter region. Point mutations were frequent events in spectra recovered from TK6 (present study) and HE cells (Kagawa et al., 1995). However, the detailed nature of these events was different: an exon skipping event was a single occurrence in the present collection but formed the majority of mutants in the previous collection. It is difficult to explain this difference, although cell line, culture conditions and selection methodology may play a role(s). However, the common character of a frequent recovery of point mutations may reflect a similar energy deposition by the C ion track at this high level of LET. A similar energy deposition may be expected to produce similar DNA damage, which may result in a similar type of mutation, which is base substitution in this instance.
Since different ion beams produce different track structures even if the LETs are the same, LET may not be the sole factor affecting the specificity of mutation. The difference between the mutational spectra of C and Ne ion beams might support this idea. The structure of the heavy ion track produced plays an important role in the determination of the biological effect, possibly including mutational specificity. High-LET radiation generally results in deposition of a large amount of energy in a small volume of DNA, leading to clusters of locally damaged sites. A combination of the spatial distribution of such DNA damage and the chromosomal structure may exert a considerable influence on the specificity of mutation.
The observed mutational spectra after C and Ne ion high-LET irradiations are clearly different. We suggest this difference may be attributed to the differences in their track structures, although the details are not yet clear.
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Acknowledgments |
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We thank the staff of the Cyclotron Laboratory in the Institute of Physical and Chemical Research for their assistance with the heavy ion irradiation. This research was supported in part by a Grant-in-Aid from the Science and Technology Agency of Japan. We thank the reviewers for their commments.
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Notes |
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8 To whom correspondence should be addressed at: Division of Radioisotope Technology, Institute of Physical and Chemical Research, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan. Tel: +81 48 467 9566; Fax: +81 48 462 4636; Email: yatagai{at}postman.riken.go.jp
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Received on June 16, 1998; accepted on October 5, 1998.
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