RAG-Mediated V(D)J Recombination Is Not Essential for Tumorigenesis in Atm-Deficient Mice

Atm-deficient mice die of malignant thymic lymphomas characterizedby translocations within the Tcr ${alpha}$ / ${delta}$ locus, suggesting that tumorigenesisis secondary to aberrant responses to double-stranded DNA (dsDNA)breaks that occur during RAG-dependent V(D)J recombination.We recently demonstrated that development of thymic lymphomain Atm^-/- mice was not prevented by loss of RAG-2. Thymic lymphomasthat developed in Rag2^-/- Atm^-/- mice contained multiple chromosomalabnormalities, but none of these involved the Tcr ${alpha}$ / ${delta}$ locus. Thesefindings indicated that tumorigenesis in Atm^-/- mice is mediatedby chromosomal translocations secondary to aberrant responsesto dsDNA breaks and that V(D)J recombination is an important,but not essential, event in susceptibility. In contrast to thesefindings, it was recently reported that Rag1^-/- Atm^-/- micedo not develop thymic lymphomas, a finding that was interpretedas demonstrating a requirement for RAG-dependent recombinationin the susceptibility to tumors in Atm-deficient mice. To testthe possibility that RAG-1 and RAG-2 differ in their roles intumorigenesis, we studied Rag1^-/- Atm^-/- mice in parallel toour previous Rag2^-/- Atm^-/- study. We found that thymic lymphomasoccur at high frequency in Rag1^-/- Atm^-/- mice and resemblethose that occur in Rag2^-/- Atm^-/- mice. These results indicatethat both RAG-1 and RAG-2 are necessary for tumorigenesis involvingtranslocation in the Tcr ${alpha}$ / ${delta}$ locus but that Atm deficiency leadsto tumors through a broader RAG-independent predisposition totranslocation, related to a generalized defect in dsDNA breakrepair.

INTRODUCTION

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Introduction

Ataxia telangiectasia (AT) is an autosomal recessive diseasecaused by mutation of the ataxia telangiectasia mutated (ATM)gene (16). Absence of the ATM protein, which plays an importantrole in cellular responses to the presence of double-strandedDNA (dsDNA) breaks and induction of cell cycle checkpoints (6,18), results in a pleiotrophic phenotype characterized by progressivecerebellar ataxia, ocular telangiectasias, immunodeficiencies,increased sensitivity to ionizing radiation, and increased incidenceof lymphoid cancers (2, 17, 20). While the exact pathophysiologicalmechanism relating the genetic defect to the clinical manifestationsof the disease has not been elucidated, it is plausible thatseveral components of the AT phenotype, including the predispositionto development of lymphoid malignancies, result from aberrantrecognition and repair of dsDNA breaks such as those that normallyoccur during V(D)J recombination in lymphoid cells (15).

Support for this hypothesis comes from studies of the murinemodel of AT. Karyotypic analysis of the thymic lymphomas thatcharacteristically develop in Atm-deficient mice (1, 3, 22)has identified consistent translocations within the Tcr ${alpha}$ / ${delta}$ locus,suggesting the involvement of V(D)J recombination in the processof lymphomagenesis (11). Because V(D)J recombination is dependentupon the recombinase-activating genes (Rag1 and Rag2) (13, 19),Liao and Van Dyke generated Rag1^-/- Atm^-/- mice to assess therequirement for V(D)J recombination in tumorigenesis (8). Consistentwith the hypothesis that V(D)J recombination is involved intumorigenesis in Atm-deficient mice, Liao and Van Dyke reportedthat no thymic lymphomas developed in Rag1^-/- Atm^-/- mice, whereasall Rag1^+/- Atm^-/- mice died of thymic lymphoma. In contrast,an analogous study conducted by our group observed that Rag2^-/-Atm^-/- mice did develop thymic lymphomas and did so througha mechanism that did not involve Tcr ${alpha}$ / ${delta}$ translocation (14). Giventhat absence of either RAG-1 or RAG-2 eliminates V(D)J recombination(13, 19), the disparity in the results of these two tumor incidencestudies was surprising. A difference in lymphoma susceptibilitybetween Rag1^-/- Atm^-/- mice and Rag2^-/- Atm^-/- mice could signifypreviously unidentified differences in RAG-1 and RAG-2 function.Alternatively the difference in results of previously reportedstudies of Rag1^-/- Atm^-/- and Rag2^-/- Atm^-/- mice could be attributableto experimental variables such as differences in genetic backgroundand/or environmental differences in mouse colonies used in thesestudies. To more directly compare the roles of RAG-1 and RAG-2in the generation of thymic lymphomas, we have studied Rag1^-/-Atm^-/- mice under the conditions previously used in characterizingsusceptibility to lymphomas in Rag2^-/- Atm^-/- mice.

MATERIALS AND METHODS

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Materials and Methods

Mice. The creation of Atm-deficient mice (allele designation Atm^ins5790neo)has been described previously (1). Progeny of heterozygote matingswere genotyped by PCR (9). Rag1-deficient mice (C57BL/6J-sv/129;Jackson Labs) were identified phenotypically by flow cytometricanalysis of peripheral blood lymphocytes as described previously(14). To generate mice deficient for both Rag1 and Atm, doubleheterozygotes were crossed and progeny were typed as describedabove.

Tumor incidence and histopathological analyses. As described previously (14), mice were examined daily for evidenceof morbidity and mortality. Mice were sacrificed when deathwas judged to be imminent and prepared for necropsy. Organsfrom mice that were found dead were similarly preserved. Tissueswere fixed, sectioned, stained, and examined by light microscopy(L. G. Luna, Histopathological methods and color atlas of specialstains and tissue, American Histolabs, Inc., Publications Division,Gaithersburg, Md.).

Statistical analysis. Kaplan-Meier survival curves were generated as previously described(7, 14). Statistical significance was determined by the Mantel-Haenszelmethod (12). Individual P values are provided after adjustmentfor multiple comparisons according to the method of Hochberg(4). All P values are two-tailed.

Thymic lymphoma cell culture. Thymic lymphomas were harvested, and single-cell suspensionswere cultured in complete medium supplemented with 100 IU ofhuman recombinant interleukin 2 per ml and 6 ng of recombinantmurine interleukin 7 per ml as described previously (14). TailDNA was isolated from all Rag1^-/- Atm^-/- mice developing thymiclymphomas and genotyped by PCR for both Rag1 and Rag2 to verifygenotypes. Rag2 PCR typing was carried out as previously described(5), and Rag1 typing was carried out under identical conditions,with substitution of Rag1-specific primers as follows: RAG-1forward, CAA CAT CTG CCT TCA CTT C; RAG-1 reverse, TAC CCT GAGCTT CAG TTC TG; neo, TAT CAG GAC ATA GCG TTG GCG.

SKY and fluorescent in situ hybridization (FISH). Metaphase chromosomes were prepared from tumor cell lines atearly-passage numbers (0 to 2) as described previously (14).Spectral karyotyping (SKY) of the tumor cell lines was performedfollowing published procedures (10, 14, 21). Six to ten metaphaseswere analyzed for each tumor. BAC clones (Genome Systems, St.Louis, Mo.) for the TcrCa and TcrVd were labeled with digoxigenin-dUTPand biotin-dUTP (Roche Biomolecular, Indianapolis, Ind.) anddetected with fluorescein- and rhodamine-conjugated avidin andantibody as described previously (11). Images were acquiredwith a Leica DMRXA microscope (Leica, Wetzlar, Germany) andQ-FISH software (Leica Imaging Systems, Cambridge, United Kingdom).

RESULTS AND DISCUSSION

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Results and Discussion

Incidence of malignant thymic lymphomas in mice deficient in both Atm and Rag1. As in our previous study, Rag1^-/- Atm^-/- mice, like Rag2^-/-Atm^-/- mice, were found to develop malignant thymic lymphomas(Fig. 1). Of 24 Rag1^-/- Atm^-/- mice monitored, 11 (46%) werediagnosed with malignant thymic lymphoma at necropsy comparedwith 0 of 33 Rag1⁺ Atm^+/+ mice (P < 0.001 by Mantel-Haenszeltest comparing overall probability as a function of time). Medianage at death for Rag1^-/- Atm^-/- mice was 8.3 months (range oftimes to death, 3.75 to 14.25 months). As expected, Rag1⁺ Atm^-/-littermate controls also developed malignant thymic lymphomas,with 33 of 48 Rag1⁺ Atm^-/- mice (69%) having this diagnosisat necropsy (Fig 1) and a median age at death of 7.0 months(range, 2.75 to 17.5 months).

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FIG. 1. Kaplan-Meier analysis of tumor incidence. Thymic lymphoma-free survival is plotted versus time in months for Rag1^-/- Atm^-/- mice and littermate controls. A drop in the curve represents the sacrifice or death of an animal diagnosed at necropsy with malignant thymic lymphoma. Tick marks represent the sacrifice or death of an animal that was not subsequently diagnosed with malignant thymic lymphoma. As no Rag1^-/- Atm^+/+ or Rag1⁺ Atm^+/+ animals were diagnosed with malignant thymic lymphomas, the curves for these two groups are at 100% event-free survival.

In addition to malignant thymic lymphomas, Atm-deficient micealso showed an increased incidence of nonthymic tumors comparedwith control mice. Four of 24 Rag1^-/- Atm^-/- mice (17%) and6 of 48 Rag1⁺ Atm^-/- mice (13%) developed various nonthymictumors (carcinomas and sarcomas) compared to 0 of 33 Rag1⁺ Atm^+/+mice (P = 0.0002 and P < 0.0001, respectively, based on actuarialprobability of developing nonthymic tumors and after adjustmentfor both comparisons). The occurrence of tumors other than thymiclymphomas was similar to our previously reported observationwith Rag2^-/- Atm^-/- mice.

To investigate potential mechanisms of tumorigenesis in Rag1-deficientmice, molecular cytogenetic analysis using SKY was employedto identify chromosomal aberrations in early-passage cells culturedfrom three Rag1^-/- Atm^-/- thymic lymphomas. Thymic lymphomacells from Rag1^-/- Atm^-/- mice had multiple translocations ineach tumor (Table 1). A representative metaphase from tumorno. 20154 is shown in Fig. 2. This tumor had a translocation[T(12;4)] in addition to other chromosomal aberrations. Remarkably,each of three Rag1^-/- Atm^-/- tumors analyzed had a chromosomaltranslocation involving chromosome 12, bands D and E. This samebreakpoint region has been identified in both Atm^-/- and Rag2^-/-Atm^-/- thymic lymphomas (11, 14). Hybridization using a FISHprobe containing the Igh-6 gene (at 12F1) confirmed that theregion distal to the breakpoint was lost (data not shown). Onetumor (no. 20343) revealed a complex translocation involvingchromosome 14. In order to exclude the involvement of the Tcrlocus in this rearrangement, we used FISH with BAC clones forthe constant and variable regions of the TCR ${alpha}$ / ${delta}$ locus, respectively.All metaphase spreads and interphase nuclei analyzed (n = 19)showed colocalization of the differentially labeled probes.We can therefore exclude the possibility that intrachromosomalrearrangements, such as inversions, disrupt the integrity ofthe Tcr locus.

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TABLE 1. Chromosomal aberrations in Rag1^-/- Atm^-/- thymic lymphomas

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FIG. 2. Spectral karyotyping analysis of a metaphase cell from Rag1^-/- Atm^-/- thymic lymphoma no. 20154 displaying chromosome aberrations. The entire metaphase is shown in display colors in panel A, with arrows indicating translocated chromosomes. The spectrum-based classification karyotype is shown in panel B, with each pseudocolored chromosome positioned next to its inverted 4',6'-diamidino-2-phenylindole-banded image. The T(12;4) is enlarged in panel C next to the G-band ideogram for chromosome 12, to highlight the band region involved in the translocation. The full karyotype of this metaphase is 42, X, T(4D;9A), Del(9B-F), T(12D-E;4D), Dp(15B-D), +4, +11, +12, +14, -18, -Y. Note that this representative metaphase for tumor no. 20154 does not contain every clonal aberration that is described in Table 1.

The results of this study further support our previous findingthat V(D)J recombination is not required for tumorigenesis inAtm-deficient mice (14). Rag1^-/- Atm^-/- mice, like Rag2^-/- Atm^-/-mice, develop malignant thymic lymphomas. The basis for thedifference between the findings reported here and those of Liaoand Van Dyke (8), who failed to observe thymic lymphomas inRag1^-/- Atm^-/- mice, is not clear but may reflect variablesincluding genetic differences in the strains employed and/orenvironmental conditions under which animals were housed. Tumorsfrom Rag1^-/- Atm^-/- thymic lymphomas resemble those from Rag2^-/-Atm^-/- thymic lymphomas in several aspects, including cell surfacephenotype (variable levels of CD4 and CD8 expression and absenceof TCR ${alpha}$ /ß expression; data not shown), the presenceof multiple translocations in individual tumor cells, and theabsence of Tcr ${alpha}$ / ${delta}$ locus translocation—a translocation whichis consistently seen in Atm^-/- thymic lymphomas (11, 14). Itthus appears that the mechanism of thymic lymphoma developmentin Rag1^-/- Atm^-/- mice, as in Rag2^-/- Atm^-/- mice, is relatedto abnormal repair of dsDNA breaks that occur secondary to processesother than V(D)J recombination. The observance of multiple chromosomalaberrations as well as the lack of translocations within theTcr ${alpha}$ / ${delta}$ locus in Rag1^-/- Atm^-/- thymic lymphomas is consistentwith this hypothesis. Of note is the frequent translocationsite at chromosome 12D-E seen in thymic lymphomas from Atm^-/-mice, Rag2^-/- Atm^-/- mice, and Rag1^-/- Atm^-/- mice. Furtherstudies to elucidate the genes involved in this region may helpto define the mechanism of tumorigenesis in Atm-deficient mice.

ACKNOWLEDGMENTS

L.K.P., Z.W., and M.V. contributed equally to this work.

We thank Genevieve Sanchez, Amy Werling, Wendy Davis, and staffmembers at Bioqual for providing excellent animal care and CorinneEpperly for expert technical assistance. Sincere appreciationis extended to Andre Nussenzweig for critical reading of themanuscript.

FOOTNOTES

* Corresponding author. Mailing address: National Institutes of Health, Building 31, Room 5C35, Bethesda, MD 20892. Phone: (301) 496-3129. Fax: (301) 496-0887. E-mail: richard_hodes{at}nih.gov.

Present address: Avalon Pharmaceuticals, Gaithersburg, Md.

Present address: Laboratory of Genetics, The Salk Institutefor Biological Studies, La Jolla, CA 92037.

REFERENCES

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