Involvement of V(D)J recombinase in the generation of intragenic deletions in the Rit1/Bcl11b tumor suppressor gene in
-ray-induced thymic lymphomas and in normal thymus of the mouse
Jun Sakata1,2,
Jun Inoue1,
Hiroyuki Ohi1,
Hitomi Kosugi-Okano1,
Yukio Mishima1,3,
Katsuyoshi Hatakeyama2,
Ohtsura Niwa4 and
Ryo Kominami1,3,5
1 Department of Molecular Genetics and 2 Department of Regenerative and Transplant Medicine, Graduate School of Medical and Dental Sciences and 3 Center for Transdisciplinary Research, Niigata University, Asahimachi 1-757, Niigata 951-8122, Japan and 4 Radiation Biology Center, Kyoto University, Yoshida-Konoecho, Sakyou-Ku, Kyoto, 606-8315, Japan
5 To whom correspondence should be addressed at: Department of Molecular Genetics, Graduate School of Medical and Dental Sciences, Niigata University, Asahimachi 1-757, Niigata 951-8122, Japan Email: rykomina{at}med.niigata-u.ac.jp
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Abstract
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Mouse thymic lymphomas induced by
-irradiation exhibited homozygous deletions of the Rit1/Bcl11b tumor suppressor gene on chromosome 12 at high frequencies. Internal deletions of one allele were frequently accompanied by loss of the other allele. In order to elucidate the mechanism of these internal deletions, the sites of breakage and rejoining were examined by PCR mapping and sequencing. The 5' site of the deletions clustered within an
5 kb region of intron 1 and the 3' site was confined to a site in intron 3. These sites contained P and/or N nucleotides and cryptic sequences recognizable by the RAG1/2 recombinase in the vicinity. This suggests that the Rit1 intragenic deletions were generated by endogenous illegitimate V(D)J recombinase activity and such aberrant recombination was also detected by nested PCR of DNA from the thymus of unirradiated mice but not of RAG2-deficient mice. A rough estimate indicated that there reside as many as 103104 thymocytes having Rit1 deletions, assuming the presence of 108 thymocytes in the thymus of unirradiated mice. Moreover, the recombination frequency was not affected by
-irradiation. These results show no effect of radiation on Rit1 mutations and suggest an indirect mechanism for its role in lymphomagenesis.
Abbreviations: DSBs, double-strand breaks; MNU, N-methyl-N-nitrosourea
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Introduction
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Radiation is one of the agents that induces genomic instability by producing DNA double-strand breaks (DSBs). DSBs are repaired mainly by a non-homologous end joining process that is error prone and, hence, multiple DSBs in a cell may facilitate the generation of large-scale DNA changes such as chromosomal rearrangements and deletions (14). These genetic alterations can activate oncogenes or inactivate tumor suppressor genes to disregulate cell growth and facilitate survival of target cells during initiation or progression of carcinogenesis. Lymphocyte precursors have an intrinsic system to induce genomic instability, i.e. the developmentally regulated process of V(D)J recombination that generates functional immunoglobulin and T cell receptor genes (57). V(D)J recombination involves site-specific DNA cleavages mediated by the RAG1/2 recombinase and subsequent processing and rejoining events by a complex of enzymes, V(D)J recombinase (68). These reactions, although usually confined within a given immunoglobulin or T cell receptor locus, can also operate on any positions comprising the RAG1/2 recognition sequence or cryptic sequences (915).
We previously performed genome-wide allelic loss analysis of
-ray induced mouse thymic lymphomas and subsequently isolated a novel tumor suppressor gene, Rit1/Bcl11b, on mouse chromosome 12 (1618). Rit1 comprises four exons and encodes a zinc finger protein that is a key regulator of
ß T cell development (19). The locus showed allelic loss in as high as 70% of the lymphomas and Rit1 exhibited frequent biallelic DNA changes. Interestingly, most of the biallelic changes consisted of internal deletions spanning exons 2 and 3 of one allele and allelic loss of the remaining allele. These deletions could be a result of genomic instability generated either by
-irradiation or by illegitimate V(D)J recombinase activity.
To elucidate the mechanism of these internal deletions in thymic lymphomas, we examined the sites of breakage and rejoining by PCR mapping and sequencing. Analyses were also made of thymic lymphomas induced by N-methyl-N-nitrosourea (MNU), an alkylating agent that can modify and change guanine bases in DNA to adenine bases after DNA replication (20). In this paper we demonstrate that the sites of deletions are highly clustered, with characteristic sequence motifs, suggesting that these deletions involve an illegitimate V(D)J recombinase activity in both radiation- and MNU-induced mouse thymic lymphomas. Furthermore, such aberrant recombination was detected in normal thymus. Interestingly, frequency of the deletion was not increased by
-irradiation.
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Materials and methods
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Mice, irradiation and lymphomas
-Ray-induced thymic lymphomas used in this study were those described previously (1618). In brief, male MSM mice carrying a p53-deficient allele were mated with BALB/c female mice and the F1 hybrid mice generated were subjected to fractionated
-irradiation, starting at age 4 weeks. MNU-induced thymic lymphomas were obtained as follows. F1 hybrid mice were given a single i.p. dose (75 mg/kg body wt) of MNU (Nakarai Co., Kyoto) when aged 6 weeks (21). Development of thymic lymphoma was diagnosed by the presence of labored breathing and existence of tumors was confirmed upon autopsy of the mice.
BALB/c mice aged 4 weeks were irradiated with 2.5 Gy
-rays and killed 0, 4 and 24 h and 4 and 14 days after irradiation. Genomic DNA was isolated from the thymus and also from the thymus of unirradiated mice aged 4 and 6 weeks for comparison. Isolation of genomic DNA including tail and lymphoma DNA was carried out by standard protocols.
PCR and primer sequences
PCR and separation of PCR products by gel electrophoresis were performed as described previously (1618). Primers used for analysis of Rit1 internal deletions are listed in Figure 1.

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Fig. 1. (A) Genomic structure of the Rit1 gene and positions of primers. F1, F1-2, R1-2 and R1 were used for nested PCR assays. (B) Primer sequences.
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Nested PCR assay and estimation of deletion frequency
We developed a nested PCR assay for the Rit1 internal deletion of the major breakpoint region and the sensitivity and reproducibility were analyzed. The lymphoma DNA with the intragenic deletion was serially diluted with 600 ng DNA prepared from mouse tail. These 600 ng DNA preparations were amplified in 30 µl of buffer with outer primers F1 and R1 for 30 cycles, and 1 µl of these first PCR products was transferred to a 10 µl nested PCR for another 30 cycles with inner primers F1-2 and R1-2 (see Figure 1B). The F1 and F1-2 primers are homologous to the 5' sequence of the major breakpoint region in intron 1, and primers R1 and R1-2 are homologous to the 3' sequence of the major breakpoint region in intron 3. Reaction mixtures were then analyzed by electrophoresis on agarose gels and the presence of an ethidium bromide stained band of the appropriate size was taken as evidence that a given DNA preparation was amplifiable. Sequence analysis confirmed that DNA in the band consisted of Rit1 recombinant molecules. DNA prepared from tail was never amplifiable in >10 trials and the level of detection sensitivity paralleled that by others (12,13).
Theoretically, PCR detection of a positive signal becomes stochastic in a tube containing 6 pg lymphoma DNA, at a concentration of about a single deletion molecule, in a background of 200 000 normal haploid genomes. Positive rates of detection in 30 µl samples containing less than one recombinant molecule follow the Poisson distribution and reflect the concentration of recombinant molecules in the sample solution tested (12,13). Five or ten replicates of 600 ng DNA (105 cell equivalents) and of 60 ng DNA (104 cell equivalents) from each DNA preparation were assayed by nested PCR. As a control, DNA preparations were used that contained 6 pg lymphoma DNA and 600 ng tail DNA in 30 µl. The frequency of cells bearing the Rit1 deletion in thymus was estimated by comparison of positive rates between the control preparations and samples containing 60 ng thymus DNA.
DNA sequencing
DNA products were gel purified and subjected to direct sequencing with Dichloro-Rhodamine Dye Terminators (ABI) and an ABI 310 sequencer.
Western blotting
Rabbit anti-Rit1 antibodies, anti-Rit1-Z, were generated against purified recombinant Rit1 proteins (amino acids 496617) as described previously (19). Thymus and lymphoma cells were suspended in a solution of 40 mM TrisHCl (pH 7.5), 0.25 M sucrose, 5 mM MgCl2, 25 mM KCl and mixed with an equal volume of lysis buffer, 0.125 M TrisHCl (pH 6.8), 10% sucrose, 10% SDS, 10% 2-mercaptoethanol and 0.004% bromophenol blue. The extract (200 µg) was electrophoresed in 8% SDSPAGE gels and blotted onto Hybond membranes (Amersham Pharmacia Biotech, Piscataway, NJ). Protein bands were visualized using chemiluminescent detection (ECL plus; Amersham Pharmacia Biotech).
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Results
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Intragenic deletions of Rit1 in lymphomas
Our previous study demonstrated that deletions in the Rit1 gene in thymic lymphomas are restricted to exons 2 and 3, but not exon 1 or 4 (18). In order to analyze the breakage and rejoining sites of internal deletions in the Rit1 gene, we designed 14 forward primers in intron 1 and six reverse primers in intron 3. Figure 1 shows a diagram of the Rit1 gene and the positions of the seven PCR primers used for the present analysis. The positions of forward and reverse primers are
2030 kb apart in the gene and hence pairs of the primers do not yield any amplification products with normal tissue samples. Fifty-nine lymphomas, induced by
-irradiation in p53(+/+) F1 mice, with allelic loss at the Rit1 locus were examined. PCR analysis using different combinations of forward and reverse primers identified several forward primers and one reverse primer that yielded PCR products at high frequencies (data not shown). As summarized in Table IA, 11 of the 59 lymphomas yielded PCR products. This is consistent with our previous result that homozygous deletions were found in 12 of the same 59 lymphomas by PCR and Southern blotting (18). One lymphoma that failed to yield PCR products may be ascribed to a relatively large deletion extending outside from intron 1 or intron 3 regions. A single pair of primers (F1 and R1, see Materials and methods) succeeded in amplifying seven products out of the 11 positive samples. These results indicated that breakpoints were clustered in the regions 3' to the F1 primer sequence and 5' to the R1 primer sequence.
Table IA includes the results of thymic lymphomas developed in p53(+/) F1 mice. Our previous p53 genotyping of these lymphomas showed that most of them were p53-null due to loss of the wild-type p53 allele (17,18). Deletion in the Rit1 gene was detected in only one of the 76 lymphomas, and the frequency was significantly lower than that in p53(+/+) mice. p53 genotyping showed that this lymphoma was p53-null. We also examined lymphomas that were induced by an alkylating agent, MNU, in p53(+/+) and p53(+/) F1 mice. Internal deletions were found among four of the 12 lymphomas in p53(+/+) mice and none of 19 lymphomas in p53(+/) mice (Table IA).
Deletions by V(D)J recombination activity
Sequencing of the PCR product was carried out and the features of the junction regions between introns 1 and 3 were revealed. Table IIA summarizes the results of the PCR products with the F1 and R1 primer set. DNA sequences flanking the rejoining sites contained the RAG1/2 recognition heptamer sequence, CACAGTG, in intron 3 and a cryptic RAG1/2 recognition sequence, CACA, in intron 1, although there were no obvious nonamer-like sequences (915). The additional nucleotides found in the junction sites were N and/or P nucleotides, which may have been generated through extension of the sequence by terminal deoxynucleotidyltransferase or through trimming by RAG1/2 enzyme (8). These sequence features were reminiscent of V(D)J recombination of the T cell receptor genes during T cell development (68). In all 10 internal deletions found, the cleavage site in intron 3 was located at the same position and this cleavage preference might be ascribed to the presence of the complete heptamer recognition sequence. Similar features were revealed for the recombinant DNA detected by other sets of primers (data not shown). These findings strongly suggest that V(D)J recombinase activity is involved in generation of the internal deletions of Rit1 and thereby contributes to radiation-induced thymic lymphomagenesis.
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Table II. DNA sequence of the breakpoint junctions of intragenic Rit1 deletions and the corresponding wild-type regions of introns 1 and 3
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Preference for deletions in p53 wild-type lymphomas
Allelic loss at the Rit1 locus was found in
70% of mouse thymic lymphomas (17,18). We next examined internal deletions in the lymphomas that retained heterozygosity at this locus. Deletions were detected in 10 of the 35 radiogenic lymphomas and in four of the 13 MNU-induced lymphomas that developed in p53(+/+) mice (Table IB). Sequence analysis of the products identified similar recombination structures in lymphomas with allelic loss. No difference was found in the deletion frequency between lymphomas with and without allelic loss. Of the lymphomas in p53(+/) F1 mice, only one lymphoma exhibited an internal deletion and the lymphoma was p53-null by p53 genotyping. This low frequency was also similar to that of lymphomas with allelic loss.
Internal deletions of Rit1 were detected in lymphomas without allelic loss, which indicates that expression of the wild-type allele may be suppressed by mechanisms other than loss of the allele. Therefore, protein expression of Rit1 was examined in these lymphomas by western blotting using anti-Rit1 antibodies. Approximately 40% (70/171) of the lymphomas examined the expression and another 50% of the lymphomas exhibited expression lower than that in the thymus (data not shown). Figure 2 shows some examples of western blots. As expected, all eight lymphomas with both an internal deletion and allelic loss did not show any expression, whereas seven of the 10 lymphomas having an internal deletion without allelic loss showed no expression and two exhibited lower expression. This might suggest a relationship between the deletions and loss of expression of Rit1, although the mechanism of loss of expression of the wild-type allele in lymphomas is unknown. Sequence analysis of the wild-type allele revealed a deletion of one cytosine in exon 4 in one lymphoma, resulting in truncation of Rit1 protein at position 812 between the fourth and fifth zinc finger domains (data not shown).

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Fig. 2. Western blot analysis of thymus and thymic lymphomas using anti-Bcl11b-Z antibodies. ID(+) and ID(), lymphomas with and without Rit1 internal deletions, respectively. Arrows indicate Rit1 proteins and bars display the position of size markers: 150, 100 and 75 kDa from top to bottom.
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Intragenic deletions in normal thymocytes
The implication of illegitimate V(D)J recombinase activity in generating internal deletions in Rit1 in lymphomas raises the possibility that there exist thymocytes with the deletions even in unirradiated mice. We tested this possibility by designing a set of nested PCR primers for the region of the F1-R1 internal deletion and examined the PCR conditions that could detect one recombinant DNA molecule in a test tube. Three independent samples were prepared that contained 600 ng normal tail DNA alone (105 cell equivalents), normal tail DNA with 60 pg lymphoma DNA with the deletion (10 cell equivalents) and normal tail DNA with 6 pg lymphoma DNA (1 cell equivalent). Five replicates containing 30 µl of the three samples from each pool were then subjected to PCR amplification. Replicates of the 60 pg lymphoma DNA yielded all positives in the three samples. On the other hand, detection in three preparations with the 6 pg lymphoma DNA was stochastic, and two positives, two positives and one positive were obtained in five trials, i.e. the positive rate was one-third in total. No positive signals were detected in tail DNA alone. Figure 3A shows a representative result. This indicated that the conditions of nested PCR allowed us to detect one recombinant DNA molecule in a test tube. We assumed that if a given sample provides a one-third positive rate in this assay it contains one recombinant DNA molecule in the 30 µl solution.

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Fig. 3. Nested PCR of Rit1 intragenic deletions in the thymus from unirradiated and irradiated mice. (A) Reconstitution test. A known number of the F1-R1 deletions of Rit1 were serially diluted into 600 ng of tail DNA and amplified by the nested PCR method in replicates. Aliquots of 60 pg and 6 pg of DNA contain 10 molecules and one molecule on average, respectively. (B) Detection of Rit1 deletions in thymus DNA.
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We then examined 600 and 60 ng of thymocyte DNA (105 and 104 cell equivalents, respectively) from unirradiated mice (Figure 3B). Amplification of 600 ng thymocyte DNA of BALB/c, MSM and F1 mice provided positive signals in almost all trials and that of 60 ng thymocyte DNA gave positive signals in about one-third of the reactions. Since positive rates follow a Poisson distribution and reflect the concentration of recombinant molecules in the sample solution, they can provide rough estimates of the concentration. Table IIIA summarizes the positive rate per DNA sample and the prevalence of cells having recombinant DNA in the thymus. Since the estimated prevalences ranged from about three times more to three times less, considering the 95% confidence interval, this suggests the existence of at least 103104 thymocytes with internal deletions in Rit1 in the thymus, which is assumed to contain 108 thymocytes. PCR analysis of DNA from thymocytes of RAG2-knockout mice failed to give any positives and that of SCID mice lacking the catalytic subunit of DNA-dependent protein kinase gave positives at low frequencies (Table IIIA). Each of the PCR products was subjected to direct sequencing. Some of the products gave complex ladder patterns due to the presence of more than two different recombinant molecules in 60 ng DNA, but most of them were of monoclonal origin and able to be sequenced. The results showed features of deletions due to V(D)J recombination, similar to those found in thymic lymphomas (Table IIB).
Effect of irradiation on intragenic deletions
Irradiation may increase the frequency of recombination leading to internal deletions. The frequency of recombinational deletions was therefore determined in thymocytes various times (from 4 h to 2 weeks) after irradiation. DNA was extracted from the thymus of approximately 4- to 6-week-old mice that had received 2.5 Gy ionizing radiation. Results of nested PCR are presented in Table IIIB. The rates of detecting deletions were >0.8 for 600 ng thymus DNA and about one-tenth to one-third for 60 ng DNA. Essentially no difference in the deletion frequency was observed among thymocytes before and after irradiation, although thymuses 1 and 4 days after irradiation tended to give lower rates. The results suggest that irradiation does not increase the generation of Rit1 intragenic deletions.
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Discussion
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Mouse thymic lymphomas induced by
-irradiation exhibited homozygous deletions of Rit1 at high frequencies, loss of one allele being due to internal chromosomal deletions (18). In this paper we have performed sequence analyses of internal deletion sites of the Rit1 gene. The analyses revealed clustering of recombination sites, almost all starting within an
5 kb region of intron 1 and ending at a site in intron 3. Subsequent sequencing showed the presence of cryptic sequences recognizable by the RAG1/2 recombinase near the breakpoints and the insertion of P and/or N nucleotides in the rejoining sites. These sequence features, reminiscent of V(D)J recombination sites, indicate that cleavage was by the RAG1/2 recombinase that could recognize cryptic sequences present 3' to the intron 1 joining sites and 5' to the intron 3 joining sites (24,25). These results implicate an illegitimate V(D)J recombinase activity in the development of thymic lymphomas, consistent with previous results on mutations found in human T cells and T cell leukemia (915). Analysis of MNU-induced thymic lymphomas provided similar results, although the two agents have different modes of action in carcinogenesis, i.e. mainly large mutations with ionizing radiation and base substitutions with MNU.
The intragenic deletions of Rit1 were found in p53 wild-type lymphomas at a much higher frequency than in p53-deficient lymphomas. Only two p53-deficient lymphomas carried the deletions among the 132 samples examined. This is consistent with our previous results suggesting an association between Rit1 and p53, although it is not clear what this association reflects (18). Thus, loss of the Rit gene may contribute to oncogenesis only in p53-proficient lymphocytes or, if it contributes in p53-deficient lymphocytes, the influence may be masked by p53 deficiency in lymphomagenesis. This interpretation may be consistent with the finding that inactivation of V(D)J recombination has no effect on the development of thymic lymphomas in p53-deficient mice (26,27).
Interestingly, the intragenic deletions of Rit1 were not limited to thymic lymphomas with allelic loss but also found in lymphomas that retained both Rit1 alleles. Deletions in these lymphomas also had a strong bias for p53 wild-type lymphomas and only one was in a p53-deficient lymphoma. This indicates a role of the deletion in lymphoma development even in those lymphomas (Table IB). Western blot analysis using anti-Rit1 antibodies revealed loss of expression in most of these lymphomas. Therefore, some epigenetic mechanism such as DNA methylation might be involved in inactivation of the wild-type allele of Rit1 (28).
Intragenic deletion of Rit1 was detected in the thymus of unirradiated mice. Thymocytes having a Rit1 deletion in one allele seem to be prone to malignancy and one such cell could develop into thymic lymphoma. This is consistent with our preliminary result showing that Rit1(KO/+) mice developed radiation-induced thymic lymphomas at a higher frequency than Rit1 wild-type mice (unpublished results). A rough estimate indicated that there are as many as 103104 thymocytes with Rit1 deletions, assuming the presence of 108 thymocytes in the mouse thymus. This deletion is induced or triggered by illegitimate V(D)J recombinase activity, because deletions were not found in the thymus of RAG2-deficient mice and sequences in the vicinity of recombination sites possessed the same feature found in lymphomas. Of importance is that irradiation did not increase the frequency of intragenic deletions. Transplantation studies of thymocytes at various times after exposure to fractionated radiation revealed the presence of prelymphoma cells in the irradiated thymus (29,30). The thymic prelymphoma cells were assumed to be preneoplastic cells that required a period of residence in the thymus microenvironment to evolve into frank thymic lymphomas. Thymic lymphomas developed in 4 and 26% of recipient mice that were transplanted with thymocytes 4 days and 2 weeks, respectively, after irradiation (29,30). This indicated that thymic prelymphoma cells first develop within 4 days of irradiation. Our results show, however, no increase in intragenic deletions in the thymus 4 days and 2 weeks after irradiation. This suggests that thymic prelymphoma cells differ in nature from thymocytes with the intragenic deletion or represent a small subpopulation of those thymocytes.
It is known that V(D)J recombinase activity gives rise to trans-rearrangements between two different T cell receptor loci in mice (31,32) and the frequency of trans-rearrangements increases on irradiation in SCID mice but not in wild-type mice (32). This is consistent with the failure to induce Rit1 mutations by irradiation. It is possible that irradiation leads to Rit1 inactivation by directly inducing loss of the remaining wild-type allele of Rit1 in T cell precursors, but we think this possibility less likely. Our previous experiment on Rit1/Bcl11b-deficient mice showed severe apoptosis of thymocytes after birth, suggesting that thymocytes having the Rit1 intragenic deletion may undergo apoptosis at this stage if they lose the wild-type allele due to irradiation (19). Our current hypothesis is that Rit1 inactivation contributes to lymphoma development when it occurs in premalignant cells that can escape apoptosis.
The effects of irradiation may include mutation of some other oncogenes by direct or by non-targeted mechanisms (1,2). However, we prefer another possibility. Classic studies of radiation induction of mouse thymic lymphomas demonstrated that an unirradiated thymus can contribute to the development of malignancy when transplanted into the kidney capsule of irradiated mice, demonstrating that irradiation of the target cells in the thymus is not a prerequisite for the development of lymphomas (33). It was also shown that lymphomagenesis can be suppressed by shielding a very small portion of bone marrow from radiation or by bone marrow transplantation shortly after irradiation (3436). Potworowski et al. (37) reported that dendritic cells, but not thymocyte precursors, supplied to the thymus by bone marrow play a key role in the prevention of lymphoma development after irradiation. Fractionated whole-body irradiation causes thymic atrophy and depletion of bone marrow cells, resulting in regeneration and differentiation arrest of the surviving thymocytes. Treatment of the bone marrow is known to ensure a rapid recovery of thymus volume in irradiated mice, probably leading to restoration of the microenvironment (35, 38). In the absence of recruitment of some cell components from bone marrow, primitive T cells or T cell precursors are assumed to undergo preneoplastic changes (3840). Based on the results of our present study, V(D)J recombinase activity is responsible for the induction of mutations in the target tumor suppressor gene and radiation is likely responsible for modifying the tissue microenvironment in such a way as to give the mutated cells a growth advantage. Radiation may contribute to carcinogenesis in part by its capacity to induce mutation. However, another role of radiation in affecting the body's microenvironments is suggested in this study. Indeed, the importance of tissue microenvironment in carcinogenesis is now well recognized (41).
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Acknowledgments
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We thank K.Nakamura for advice of statistical analysis. This work was supported by grants-in-aid for scientific research of the Ministry of Education, Science, Art and Sports and for Cancer Research from the Ministry of Health, Labor and Welfare of Japan.
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Received November 5, 2003;
revised December 28, 2003;
accepted January 14, 2004.