J Recombination Is Not Essential for Tumorigenesis in Atm-Deficient Mice

Recombinase-activating gene (RAG) 2-mediated V(D)J recombination is not essential for tumorigenesis in Atm-deficient mice

Lisa K. Petiniot*†‡, Zoe¨ Weaver‡§, Carrolee Barlow‡¶ሻ, Rhuna Shen*†‡, Michael Eckhaus**, Seth M. Steinberg††, Thomas Ried§, Anthony Wynshaw-Boris¶‡‡, and Richard J. Hodes*§§¶¶

*Experimental Immunology Branch, §Genetics Department, and ††Biostatistics and Data Management Section, Ofﬁce of the Director of the Division of Clinical Sciences, National Cancer Institute; ¶Genetic Disease Research Branch, National Human Genome Research Institute; **Veterinary Resources Program, Ofﬁce of Research Services; §§National Institute on Aging, National Institutes of Health, Bethesda, MD 20892; and †Howard Hughes Medical Institute, National Institutes of Health Research Scholars Program, One Cloister Court, Bethesda, MD 20892

Edited by Michael Potter, National Institutes of Health, Bethesda, MD, and approved March 20, 2000 (received for review December 28, 1999) The majority of Atm-deficient mice die of malignant thymic lym- that these lymphomas displayed translocations detected by spec- phoma by 4–5 mo of age. Cytogenetic abnormalities in these tral karyotyping (SKY) (5), and recent analysis demonstrated tumors are consistently identified within the Tcr ␣/␦ locus, sug- that 100% of thymic lymphomas from AtmϪ/Ϫ mice had trans- gesting that tumorigenesis is secondary to aberrant responses to locations in chromosome 14. Notably, these translocations were double-stranded DNA breaks that occur during V(D)J recombina- shown by fluorescent in situ hybridization (FISH) to occur tion. Since V(D)J recombination is a recombinase-activating gene consistently within both alleles of the T cell receptor (Tcr) ␣/␦ ,RAG)-dependent process, we generated Rag2؊/؊Atm؊/؊ mice to locus (M. Liayanage, C.B., Z.W., A. Coleman, D. G. Pankratz) assess the requirement for RAG-dependent recombination in thy- S. Anderson, A.W.-B., and T.R., unpublished work). The con- mic lymphomagenesis. In contrast to expectation, the data pre- sistent involvement of the Tcr ␣/␦ locus suggests a mechanism of sented here indicate that development of malignant thymic lym- tumorigenesis in which dsDNA breaks occurring within this phoma in Atm؊/؊ mice is not prevented by loss of RAG-2 and thus locus during V(D)J recombination are not efficiently handled in is not dependent on V(D)J recombination. Malignant thymic lym- Atm-deficient cells during T cell development. The dsDNA phomas in Rag2؊/؊Atm؊/؊ mice occurred at a lower frequency and breaks and increased chromosomal instability typically seen in with a longer latency as compared with Atm؊/؊ mice. Importantly, AT (reviewed in ref. 1) predispose to development of malignant cytogenetic analysis of these tumors indicated that multiple chro- thymic lymphoma. mosomal abnormalities occurred in each tumor, but that none of In a recent study designed to test this hypothesis, Liao and Van these involved the Tcr ␣/␦ locus. Nonmalignant peripheral T cells Dyke (8) used the fact that V(D)J recombination is a recombi- from TCR-transgenic Rag2؊/؊Atm؊/؊ mice also revealed a substan- nase-activating gene (RAG)-dependent process and generated tial increase in translocation frequency, suggesting that these Rag1Ϫ/ϪAtmϪ/Ϫ mice to assess tumor incidence. If tumorigenesis translocations are early events in the process of tumorigenesis. is secondary to faulty V(D)J recombination, no tumors would be These data are consistent with the hypothesis that the major expected to develop in these double knockout mice. Consistent -mechanism of tumorigenesis in Atm؊/؊ mice is via chromosomal with this hypothesis, development of malignant thymic lym Ϫ/Ϫ Ϫ/Ϫ translocations and other abnormalities that are secondary to ab- phoma did not occur in nine Rag1 Atm mice, whereas five ϩ/Ϫ Ϫ/Ϫ errant responses to double-stranded DNA breaks. Furthermore, of five Rag1 Atm control mice died of malignant thymic these data suggest that V(D)J recombination is a critical, but not lymphomas by 7 mo of age. essential, event during which Atm-deficient thymocytes are sus- We had independently initiated a similar series of experiments Ϫ/Ϫ Ϫ/Ϫ ceptible to developing chromosome aberrations that predispose to in which we generated Rag2 Atm mice for assessment of malignant transformation. tumor susceptibility. Surprisingly, in contrast to expectation and the results reported by Liao and Van Dyke (8), we observed development of malignant thymic lymphomas in Rag2Ϫ/ϪAtmϪ/Ϫ taxia telangiectasia (AT) is an autosomal recessive disease mice, although at a lower frequency and with a longer latency Acharacterized by ataxia, ocular telangiectasias, humoral and than in AtmϪ/Ϫ mice. Strikingly, cytogenetic evaluation of these cellular immunodeficiencies, increased incidence of cancers, and Rag2Ϫ/ϪAtmϪ/Ϫ thymic lymphomas revealed an absence of the increased sensitivity to ionizing radiation (reviewed in ref. 1). Tcr ␣/␦ locus translocations that were seen in lymphomas from The genetic defect causing this disease is mutation of the gene AtmϪ/Ϫ mice with intact RAG function. These findings indicate encoding the AT mutated (ATM) protein (2). ATM appears to that rearrangement of the Tcr ␣/␦ locus is a critical, but not play an important role in cellular responses to the presence of double-stranded DNA (dsDNA) breaks, probably due to its function in cell cycle checkpoint control (3). Several components This paper was submitted directly (Track II) to the PNAS office. of the AT phenotype, such as sensitivity to ionizing radiation, Abbreviations: AT, ataxia telangiectasia; Atm, ataxia telangiectasia mutated; RAG, recom- infertility, immunodeficiency, and lymphoreticular malignancies binase-activating gene; Tcr, T cell receptor; SKY, spectral karyotyping; FISH, fluorescent in with characteristic translocations, plausibly result from aberrant situ hybridization; ds, double stranded; BAC, bacterial artificial chromosome. responses to dsDNA breaks. More specifically, it has been ‡L.K.P. and Z.W. contributed equally to this work. suggested that immunodeficiency and the increased incidence of ࿣Present address: The Salk Institute for Biological Studies, Laboratory of Genetics, 10010 lymphoid cancers seen in this disease are secondary to aberrant North Torrey Pines Road, La Jolla, CA 92037. recognition and repair of dsDNA breaks that normally occur ‡‡Present address: University of California, San Diego School of Medicine, 9500 Gilman during V(D)J recombination and isotype switching in lymphoid Drive, La Jolla, CA 92093-0627. cells (3, 4). ¶¶To whom reprint requests should be addressed at: Building 10, Room 4B10, National Institutes of Health, 10 Center Drive, MSC 1360, Bethesda, MD 20892-1360. E-mail: Support for this hypothesis comes from studies of the murine [email protected]. Ϫ/Ϫ model of AT. It has been observed that the majority of Atm The publication costs of this article were defrayed in part by page charge payment. This mice die from malignant thymic lymphoma by 4–5 mo of age article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. (5–7). The initial characterization of AtmϪ/Ϫ mice demonstrated §1734 solely to indicate this fact.

6664–6669 ͉ PNAS ͉ June 6, 2000 ͉ vol. 97 ͉ no. 12 Downloaded by guest on September 30, 2021 essential, event during which Atm-deficient thymocytes are Thymic Lymphoma Cell Culture. Thymic lymphomas were harvested susceptible to developing translocations that predispose to ma- and single-cell suspensions were cultured in RPMI 1640 (Bio- lignancy. Furthermore, the data indicate that the role of ATM Whittaker) supplemented with 15% FCS (Biofluids, Rockville, is not limited to recognition and repair of dsDNA breaks MD), sodium pyruvate (1%), nonessential amino acids (1%), initiated by V(D)J recombination, but rather that ATM func- L-glutamine (0.5%), 2-mercaptoethanol (5 ϫ 10Ϫ5), penicillin tions more broadly in predisposing to translocation events and (0.5%), streptomycin (0.5%), and either 20 IU/ml human tumorigenesis. recombinant IL-2 (AtmϪ/Ϫ lymphomas) or 100 IU/ml human recombinant IL-2 and 6 ng/ml rmIL-7 (Rag2Ϫ/ϪAtmϪ/Ϫ Materials and Methods lymphomas). Flow Cytometric Analysis. The following conjugated antibodies were used for three-color flow cytometric analysis: anti-CD8 SKY and FISH. Metaphase chromosomes were prepared from FITC, anti-Thy 1.2 FITC, anti-CD4 phycoerythrin (Becton tumor cell lines at early passage numbers (passages 0–2). Cells Dickinson Immunocytometry Systems), anti-CD3 FITC, anti- were incubated in 0.1 ␮g/ml Colcemid (GIBCO/BRL) for 30–60 B220 phycoerythrin, anti-H57 biotin, and anti-V␤8 biotin min and then lysed in 0.075 M KCl. Chromosomes were fixed in (PharMingen). Anti-Leu-4 FITC, anti-Leu-4 phycoerythrin, and 3:1 methanol:acetic acid and dropped onto glass slides. Thymo- anti-Leu-4 biotin (Becton Dickinson Immunocytometry Sys- cyte chromosomes were prepared by culturing single-cell thy- tems) were used as negative staining controls. Biotin-conjugated mocyte suspensions for2hin0.1␮g/ml Colcemid and then lysing antibodies were revealed with streptavidin-CyChrome (Phar- and fixing as described above. Splenocyte chromosomes were Mingen). Samples were analyzed on a FACScan (Becton Dick- prepared from splenocytes cultured for 48 h in RPMI 1640 ϩ inson Immunocytometry Systems) using CellQuest software. 20% FCS with 6 ␮g/ml concanvalin A to preferentially stimulate T cells. After an additional 30-min incubation in 0.1 ␮g/ml Breeding and Typing Mice. The creation of Atm-deficient mice Colcemid, cells were harvested and metaphase chromosomes (allele designation Atmins5790neo) has been described previously were prepared as described. SKY of the tumor cell lines was (5). Progeny of heterozygote matings were genotyped by PCR performed as described previously (16, 17). Raw spectral images (9). Rag2-deficient mice (10) were identified phenotypically by were visualized by assigning red, green, or blue colors to specific flow cytometric analysis of peripheral blood lymphocytes as spectral ranges. Chromosomes were then unambiguously iden- described above. Rag2Ϫ/Ϫ cells were CD4ϪCD8ϪB220Ϫ; Rag2ϩ tified using a spectral classification algorithm that results in the cells were CD4ϩ, CD8ϩ, or B220ϩ. To generate mice deficient assignment of a separate classification color to all pixels with for both Rag2 and Atm, double heterozygotes were crossed and identical spectra by use of Sky View software (Applied Spectral progeny were typed as described above. To create Imaging). Aberrations were defined using the nomenclature HyTcrTgϩRag2Ϫ/ϪAtmϪ/Ϫ mice, HyTcrTgϩ mice (11) were first rules from the Committee on Standardized Genetic Nomencla- crossed with Rag2Ϫ/ϪAtmϩ/Ϫ mice. These mice were typed for ture for Mice (see the Mouse Genome Informatics web site at the presence of transgene by flow cytometric analysis as de- http://www.informatics.jax.org). Six to 10 metaphases were an- scribed above. Peripheral blood lymphocytes from alyzed for each tumor, and 10–12 metaphases were analyzed for HyTcrTgϩRag2Ϫ/Ϫ mice were B220Ϫ, but had a population of Thy each splenocyte or thymocyte preparation. Bacterial artificial 1.2ϩV␤8ϩ cells, whereas peripheral blood lymphocytes from HyT- chromosome (BAC) clones for FISH analysis were labeled by IMMUNOLOGY crTg-Rag2Ϫ/Ϫ mice were Thy 1.2ϪB220Ϫ. HyTcrTgϩRag2Ϫ/ϪAtmϩ/Ϫ nick-translation with biotin-dUTP (C␣) and Spectrum Orange- mice were then crossed with HyTcrTg-Rag2Ϫ/ϪAtmϩ/Ϫ mice. dUTP (V␣ and C␦), whereas the whole chromosome 14 paint was labeled with digoxin-dUTP and detected with mouse anti- Tumor Incidence Studies and Histopathological Analysis. Mice were digoxin followed by sheep anti-mouse IgCy5.5. FISH results were examined daily for evidence of morbidity or mortality. Mice were imaged and analyzed using QFISH software (Leica, Cambridge, killed when death was judged to be imminent; thoracic and U.K.). abdominal organs were removed en bloc and preserved in 10% neutral buffered Formalin (Sigma) for necropsy. Organs from Results mice that were found dead were similarly preserved. Fixed Incidence of Malignant Thymic Lymphomas in Mice Deficient in Both tissues were embedded in paraffin blocks, sectioned, and stained ATM and RAG-2. To determine whether tumorigenesis in Atm- with hematoxylin and eosin by American Histology Labs (12), deficient mice is dependent on V(D)J recombination, we inter- and sections were examined by light microscopy. crossed Rag2ϩ/ϪAtmϩ/Ϫ mice to generate Rag2Ϫ/ϪAtmϪ/Ϫ mice and littermate controls as described in Materials and Methods. Statistical Analysis. Survival time of mice was calculated from date Kaplan–Meier analysis of tumor-free survival was determined. If of birth until date of death or date last known alive. All animals tumorigenesis is secondary to aberrant recognition of dsDNA in which death was felt to be imminent were killed, and the breaks that occur during V(D)J recombination of the Tcr ␣/␦ underlying morbidity was recorded as the cause of mortality. locus, it would be expected that preventing V(D)J recombination Kaplan-Meier survival curves were generated separately for by deleting the Rag2 gene would prevent the development of death due to malignant thymic lymphoma and death due to malignant thymic lymphoma in Rag2Ϫ/ϪAtmϪ/Ϫ mice. malignancy other than malignant thymic lymphoma (13). Ani- In contrast to this prediction, however, Rag2Ϫ/ϪAtmϪ/Ϫ mice mals were considered to have failed in the analysis of a given end did in fact develop malignant thymic lymphomas (Fig. 1). Of 24 point if a diagnosis of that end point was made; animals that died Rag2Ϫ/ϪAtmϪ/Ϫ mice followed, 6 (25%) were diagnosed with due to a cause other than that end point had their follow-up time malignant thymic lymphoma at necropsy as compared with 0 of censored in that curve at the date of death. Three mice were 13 Rag2ϩAtmϩ/ϩ mice (P ϭ 0.0018 by Mantel–Haenszel test excluded from analysis because their survival durations were comparing overall probabilities as a function of time). Age at unknown. The statistical significance of differences between death ranged from 7–17.5 mo, with the mean age at death being pairs of Kaplan–Meier curves, each representing a different 12 mo. Rag2ϩAtmϪ/Ϫ littermate controls had a higher frequency group of animals, was determined by the Mantel–Haenszel of tumorigenesis and a shorter latency to tumor development method (14). Individual, unadjusted P values are reported and all (Fig. 1). Seven of 12 Rag2ϩAtmϪ/Ϫ mice (58%) developed were determined to indicate statistical significance at the 0.05 malignant thymic lymphoma as compared with 0 of 13 level in view of the multiple comparisons being made according Rag2ϩAtmϩ/ϩ mice (P ϭ 0.0012 by Mantel–Haenszel test) or as to the method of Hochberg (15). All P values are two-tailed. compared with 6 of 24 Rag2Ϫ/ϪAtmϪ/Ϫ mice (P ϭ 0.036 by

Petiniot et al. PNAS ͉ June 6, 2000 ͉ vol. 97 ͉ no. 12 ͉ 6665 Downloaded by guest on September 30, 2021 compared with control mice. Seven of 24 Rag2Ϫ/ϪAtmϪ/Ϫ mice (29%) and 2 of 12 Rag2ϩAtmϪ/Ϫ mice (17%) developed various forms of nonthymic malignancy as compared with 0 of 13 Rag2ϩAtmϩ/ϩ mice (P ϭ 0.0134 and P ϭ 0.0072, respectively, by Mantel–Haenszel test). These malignancies included two cases of disseminated lymphoma (Rag2Ϫ/ϪAtmϪ/Ϫ mice), a variety of sarcomas (Rag2Ϫ/ϪAtmϪ/Ϫ and AtmϪ/Ϫ mice), and ovarian gran- ulosa cell tumors (Rag2Ϫ/ϪAtmϪ/Ϫ and AtmϪ/Ϫ mice). No mice in control groups (Rag2Ϫ/ϪAtmϩ and Rag2ϩAtmϩ mice) developed any malignant tumors. As might be expected, the early morbidity and mortality of Rag2Ϫ/ϪAtmϩ mice were often attributable to infectious causes such as pneumonia, myocarditis, candidal esophagitis, subcutaneous abscess, or colitis secondary to Helicobacter infection. The majority of Rag2ϩAtmϩ (8/13) mice were alive when this study was terminated.

-Flow Cytometric Analysis of Rag2؊/؊Atm؊/؊ Tumors. Flow cytomet ric analysis of Rag2Ϫ/Ϫ AtmϪ/Ϫ and HyTcrTgϩRag2Ϫ/ϪAtmϪ/Ϫ Fig. 1. Kaplan–Meier analysis of tumor incidence studies. Thymic lymphoma- malignant thymic lymphomas was performed to evaluate surface Ϫ/Ϫ Ϫ/Ϫ free survival is plotted versus time in months for Rag2 Atm mice and phenotypes. Although nearly all AtmϪ/Ϫ thymic lymphomas have littermate controls. A drop in the curve represents the sacriﬁce or death of an been reported to have a CD4ϩCD8ϩ phenotype (5–6, 8), analysis animal diagnosed at necropsy with malignant thymic lymphoma. Tick marks Ϫ/Ϫ Ϫ/Ϫ represent the sacriﬁce or death of an animal that was not subsequently of Rag2 Atm tumors revealed variable profiles. All tumors diagnosed with malignant thymic lymphoma. For purposes of statistical anal- were negative for surface TCR (C␤ and/or CD3) as expected in Ϫ Ϫ ysis Rag2Ϫ/ϪAtmϩ/ϩ and Rag2Ϫ/ϪAtmϩ/Ϫ mice were considered as one group Rag2 / mice; however, surface expression of CD4 and CD8 denoted Rag2Ϫ/ϪAtmϩ (n ϭ 17); likewise, Rag2ϩAtmϩ/ϩ and Rag2ϩAtmϩ/Ϫ differed among tumors (data not shown). One tumor had a were considered as one group denoted Rag2ϩAtmϩ (n ϭ 13). As no animals CD4ϪCD8Ϫ phenotype (1107); one was primarily CD4ϪCD8Ϫ, from either of these groups were diagnosed with malignant thymic lympho- with a subpopulation of CD8ϩ cells (2865); one was predomi- mas, the curves for these two groups are at 100% event-free survival. To ϩ ϩ ϩ Ϫ/Ϫ ϩ ϩ ϩ nantly CD4 with a subpopulation that was CD4 CD8 (8039); distinguish between Rag2 Atm and Rag2 Atm mice, dots above the tick ϩ ϩ marks were used to indicate the death of Rag2Ϫ/ϪAtmϩ mice. and two tumors were CD4 CD8 (2998, 8097). The HyTcrTgϩRag2Ϫ/ϪAtmϪ/Ϫ thymic lymphomas analyzed also ex- pressed varying levels of CD4 and/or CD8 and were TCR V␤8ϩ, Mantel–Haenszel test). Age at death ranged from 4–7.75 mo, consistent with expression of the HyTcr transgene (data not Ϫ Ϫ ϩ Ϫ Ϫ Ϫ Ϫ with a mean age at death of 6.4 mo. shown). All Rag2 / and HyTcrTg Rag2 / Atm / thymic lym- Since T cell development is arrested at the CD4ϪCD8Ϫ stage phomas were Thy 1.2ϩ, verifying that they were of T cell origin. in Rag2Ϫ/Ϫ mice (10), it was possible that the occurrence of The expression of CD4 and/or CD8 on the cell surface of thymic lymphomas in Rag2Ϫ/ϪAtmϪ/Ϫ mice is related to the multiple Rag2Ϫ/ϪAtmϪ/Ϫ thymic lymphomas was unanticipated developmental arrest in Rag2-deficient thymocytes. To test this as RAG-2 is necessary for V(D)J recombination and is therefore possibility, we generated HyTcrTgϩRag2Ϫ/ϪAtmϪ/Ϫ mice, in normally required for T cell development past the CD4ϪCD8Ϫ which the arrest in T cell development due to Rag2 deficiency is stage (10). These results are similar to the previous finding that rescued by Tcr transgene expression (18), and monitored these CD3ϪCD4ϩCD8ϩ thymic lymphomas develop in Rag1Ϫ/Ϫp53Ϫ/Ϫ mice for development of thymic lymphoma. Preliminary study of and Rag2Ϫ/Ϫp53Ϫ/Ϫ mice (19). To further clarify the circum- HyTcrTgϩRag2Ϫ/ϪAtmϪ/Ϫ mice revealed that malignant thymic stances under which CD4 and/or CD8 expression might be lymphomas do develop in these mice and thus demonstrates that occurring, we analyzed thymocytes from non-tumor-bearing lymphomagenesis in Atm-deficient mice is independent of T cell Rag2Ϫ/ϪAtmϪ/Ϫ mice by flow cytometry. Thymocytes of three developmental arrest. Rag2Ϫ/ϪAtmϪ/Ϫ mice had a CD3ϪCD4ϪCD8Ϫ phenotype as In addition to malignant thymic lymphomas, Atm-deficient expected in mice with a Rag2-deficient background, whereas mice also showed an increased incidence of nonthymic tumors as thymocytes of three other Rag2Ϫ/ϪAtmϪ/Ϫ mice had a

Fig. 2. SKY analysis of a metaphase cell from Rag2Ϫ/ϪAtmϪ/Ϫ thymic lymphoma 2998 displaying chromosome aberrations. The entire metaphase is shown in display colors with arrows indicating aberrant chromosomes next to the pseudocolors generated by spectra-based classiﬁcation (see Materials and Methods). The full karyotype of this metaphase is 40, XX, T(12;15), T(Dp16;12). Note that this representative metaphase for tumor 2998 does not contain every clonal aberration that is described in Table 1.

6666 ͉ www.pnas.org Petiniot et al. Downloaded by guest on September 30, 2021 Table 1. Summary of structural aberrations in Rag2؊/؊ Atm؊/؊ and HyTcrTg؉Rag2؊/؊ Atm؊/؊ thymic lymphomas identiﬁed by SKY and FISH analysis SKY-detected clonal Tumor Genotype aberrations* TCR Vϰ/Cϰ FISH†

2593 AtmϪ/Ϫ T(9;14)‡ V/C rearranged T(12;9) ϩ15 2865 Rag2Ϫ/Ϫ T(9;17) V/C colocalization AtmϪ/Ϫ T(12;9) T(18;12) ϩ15 1107 Rag2Ϫ/Ϫ T(2;14) V/C colocalization AtmϪ/Ϫ T(7;4) T(14;2;1) 8097 Rag2Ϫ/Ϫ T(17;1) V/C colocalization AtmϪ/Ϫ T(8;14), T(14;8) ϩ15 2998 Rag2Ϫ/Ϫ T(12D-E;15A) V/C colocalization AtmϪ/Ϫ Dic(12E;15E) Dp(14) T(Dp16;12) T(17;1) 8039 Rag2Ϫ/Ϫ T(4;9) V/C colocalization AtmϪ/Ϫ Is(9;4) T(12;15) Fig. 3. FISH analysis of the Tcr ␣/␦ locus in HyTcrTgϩRag2Ϫ/ϪAtmϪ/Ϫ thymic Dp(14E–F) lymphoma 3013. (a) FISH was performed on tumor 3013 with BAC probes to Dp(16) variable region ␣ (V␣) and constant region ␣ (C␣)intheTcr ␣/␦ locus. The V␣ Dp(17) signals are shown in green and the C␣ signals are shown in red. Chromosomes ϩ4 are counterstained with 4Ј,6-diamidino-2-phenylindole (blue). The signals are ϩDel(5) colocalized, indicating that the Tcr ␣/␦ locus is not disrupted. The extra green Ϫ9 signal on one chromosome 14 is due to integration of the (prespliced) HyTcr IMMUNOLOGY transgene (Tg) at that site. (b) The same metaphase as in a labeled with a 3013 HyTCRTgϩ T(14;1) V/C colocalization chromosome 14 painting probe (yellow), confirming the chromosomal loca- Ϫ/Ϫ Rag2 tion of the Tcr ␣/␦ BAC probes. The chromosome 14 breakpoint in the T(14;1) AtmϪ/Ϫ (indicated by the arrow) is not within the Tcr ␣/␦ locus. 3021 HyTCRTgϩ T(14;ln14;16) V/C colocalization Ϫ/Ϫ Rag2 ϩ Ϫ/Ϫ Ϫ/Ϫ AtmϪ/Ϫ HyTcrTg Rag2 Atm thymic lymphomas. SKY analysis was used to identify chromosome structural aberrations, whereas *Structural aberrations were considered clonal if present in two or more FISH analysis using gene-specific probes for the variable and metaphases. Gains and losses were considered clonal if present in at least two constant regions of the Tcr ␣/␦ locus was carried out to deter- or three metaphases, respectively. Aberrations involving chromosomes 12 or mine the presence or absence of translocations within this locus. 14 are shown in bold. At least 10 metaphase cells were analyzed per tumor. Ϫ/Ϫ Ϫ/Ϫ ϩ Ϫ/Ϫ Ϫ/Ϫ †Rearrangement within the Tcr ␣ locus was detected by FISH with two BAC SKY analysis of Rag2 Atm and HyTcrTg Rag2 Atm clones, one each from the variable and constant regions of the gene, as tumors revealed multiple translocations (Fig. 2), with chromo- described in Materials and Methods. some 14 being involved in several of these tumors (Table 1). ‡T, translocation (the chromosome donating the centromere is listed first); Dic, However, unlike AtmϪ/Ϫ tumors, Rag2Ϫ/ϪAtmϪ/Ϫ and dicentric chromosome; Dp, duplication of a chromosome segment; Is, inser- HyTcrTgϩRag2Ϫ/ϪAtmϪ/Ϫ tumors exhibited colocalization of the tion (the inserted segment is listed first); Del, interstitial deletion of a chro- variable and constant region Tcr ␣/␦ locus FISH probes, indi- mosome segment; In, an inverted chromosome segment. cating an absence of translocations within this locus (Fig. 3 and Table 1). Interestingly, although no consistent translocation can be identified in Rag2Ϫ/ϪAtmϪ/Ϫ and/or HyTcrTgϩRag2Ϫ/Ϫ Ϫ Ϫ CD3 CD4 CD8dull phenotype (data not shown). This unex- AtmϪ/Ϫ tumors studied, every tumor did exhibit a translocation pected phenotype of dull CD8 expression could signify a bypass involving either chromosome 14 or chromosome 12, both of in Rag2Ϫ/ϪAtmϪ/Ϫ mice of the blockade in thymocyte develop- which are homologous to human chromosome 14. Moreover, the ment imposed by the absence of RAG-2. Interestingly, nonma- breakpoints approximate the same band regions (14D and lignant thymocytes from Rag1Ϫ/Ϫp53Ϫ/Ϫ and Rag2Ϫ/Ϫp53Ϫ/Ϫ 12D–F) as has been observed in AtmϪ/Ϫ tumors (Fig. 2; Table 1). mice have also been shown to have an unexpected CD4ϩCD8ϩ The predilections for translocations within these chromosomes phenotype (19). may be related to the conformation of chromatin structure. If the chromatin is in a more open conformation in these regions, as is Cytogenetic Analysis of Rag2؊/؊Atm؊/؊ Tumors. To investigate chro- necessary for efficient V(D)J recombination (20), these loci mosomal alterations that might contribute to underlying mech- might be more susceptible to chromosomal translocations. anisms of tumorigenesis, we performed cytogenetic analysis on If the chromosomal abnormalities observed in these early passage cells cultured from multiple Rag2Ϫ/ϪAtmϪ/Ϫ and Rag2Ϫ/ϪAtmϪ/Ϫ and HyTcr TgϩRag2Ϫ/ϪAtmϪ/Ϫ thymic lympho-

Petiniot et al. PNAS ͉ June 6, 2000 ͉ vol. 97 ͉ no. 12 ͉ 6667 Downloaded by guest on September 30, 2021 Table 2. Chromosome aberrations in HyTcrTg؉Rag2؊/؊ Atm؊/؊ of differences in genetic background that were created with the premalignant thymus and spleen* introduction of the Rag2Ϫ/Ϫ gene (C57BL/6 background) into No. of aberrant Atm-deficient mice (129/SvEv background) and/or environ- metaphases/ mental differences in mouse colonies. It should be noted that, Mouse Genotype Thymus Spleen total analyzed although not as common as lymphomas and leukemias, nonlymphomatous malignancies have also been seen in human 8046 HyTcrTgϩ T(11;9) T(3;10),T(3;1) Thymus: patients with AT (21). Rag2Ϫ/Ϫ T(2;11),T(11;2) T(12;14) 2/10 ϭ 20% The findings reported here help to further define the AtmϪ/Ϫ T(12;15)† Spleen: mechanism by which tumorigenesis occurs in Atm-deficient Rb[T(11;9).4]‡ 6/12 ϭ 50% mice. V(D)J recombination is a universal event in thymocyte Rb[X.Del(X)] development that involves the production of dsDNA breaks 8644 HyTcrTgϩ T(12;4)§ Not analyzed Thymus: and which predisposes Atm-deficient cells to undergo malig- Rag2Ϫ/Ϫ T(12;15;4)¶ 8/10 ϭ 80% nant transformation due to the insertion of oncogenic genes AtmϪ/Ϫ into active TCR loci in T cells (22). However, although V(D)J recombination is a critical event in thymic lymphoma devel- ϩ 8645 HyTcrTg None Not analyzed Thymus: opment in Atm-deficient mice, our data indicate that it is not Ϫ/Ϫ Rag2 0/10 ϭ 0% essential. In its absence, dsDNA breaks from other causes can ϩ/ϩ Atm predispose Atm-deficient cells to undergo malignant transfor- *Structural aberrations were determined by SKY analysis. Most aberrations mation, as is evidenced by the occurrence of thymic lympho- Ϫ/Ϫ Ϫ/Ϫ were not clonal (see below for exceptions). mas bearing multiple translocations in Rag2 Atm mice. †This translocation was present in two metaphase cells. The lack of a single type of translocation in Rag2Ϫ/ϪAtmϪ/Ϫ ‡Rb, Robertsonian translocation (centromere–centromere dicentric). tumors suggests that heterogeneous or stochastic processes §This translocation was present in four metaphase cells. are responsible for these dsDNA breaks. As would be expected ¶This translocation was present in four metaphase cells. in such a model, a lower frequency and longer latency of thymic lymphoma development was observed in Rag2Ϫ/ Ϫ Ϫ ϩ Ϫ Ϫ ϪAtm / mice when compared with Rag2 Atm / mice. Ad- mas are early events that occur in precursors of thymic ditionally, the occurrence of nonlymphomatous malignancies lymphomas and predispose to malignant transformation, one in Rag2Ϫ/ϪAtmϪ/Ϫ and older AtmϪ/Ϫ mice is consistent with the would expect to see similar chromosomal abnormalities in model. mature T cells from nonmalignant mice of these genotypes. To Nonmalignant peripheral T cells from Atm-deficient mice have investigate this possibility, SKY analysis of nonmalignant ϩ Ϫ/Ϫ Ϫ/Ϫ been shown to harbor a greater than 20-fold increase in chromo- spleen and thymus T cells from HyTcrTg Rag2 Atm mice some 14 structural abnormalities as compared with peripheral T was performed. A high frequency of chromosomal aberrations cells from wild-type mice (M. Liyanage, C.B., Z.W., A. Coleman, D. was observed in two of three mutant mice studied (Table 2). G. Pankratz, S. Anderson, A.W.-B., and T.R., unpublished work), Although most chromosomal aberrations were not clonal, suggesting that the translocations within the Tcr ␣/␦ locus seen in thymocytes from one mouse (8644) did harbor recurrent Atm-deficient thymic lymphomas occur early in the process of chromosomal aberrations. No aberrations were found in ϩ Ϫ/Ϫ ϩ/ϩ tumorigenesis. To address the same question in the development of spleen or thymus T cells from a HyTcrTg Rag2 Atm thymic lymphomas in Rag2Ϫ/ϪAtmϪ/Ϫ mice, we analyzed thymo- mouse (Table 2), and SKY analysis of T cells from wild-type cytes from HyTcrTgϩRag2Ϫ/ϪAtmϪ/Ϫ mice. If the chromosomal mice did not reveal any chromosome aberrations in over 30 structural abnormalities observed in thymic lymphomas from metaphases from three experiments (data not shown). Rag2Ϫ/ϪAtmϪ/Ϫ mice reflect predisposing events in malignant Discussion transformation of lymphocytes, we would expect an increased frequency of thymic or peripheral T cells with similar chromosomal The results of the study reported here clearly indicate that V(D)J aberrations in HyTcrTgϩRag2Ϫ/ϪAtmϪ/Ϫ mice. Analysis of nonma- recombination is not essential for tumorigenesis; both lignant T cells from HyTcrTgϩRag2Ϫ/ϪAtmϪ/Ϫ mice indeed revealed Ϫ/Ϫ Ϫ/Ϫ ϩ Ϫ/Ϫ Ϫ/Ϫ Rag2 Atm and HyTcrTg Rag2 Atm mice develop ma- multiple chromosome aberrations in thymocytes and splenocytes. lignant thymic lymphomas. Interestingly, however, the thymic These data suggest that the chromosomal structural abnormalities Ϫ/Ϫ Ϫ/Ϫ lymphomas that develop in Rag2 Atm and seen in Rag2Ϫ/ϪAtmϪ/Ϫ thymic lymphomas represent early events in ϩ Ϫ/Ϫ Ϫ/Ϫ HyTcrTg Rag2 Atm mice have distinct characteristics the pathway of tumorigenesis and can predispose to selective Ϫ Ϫ when compared with thymic lymphomas that develop in Atm / expansion of lymphocyte clones, as has been observed in the human Ϫ Ϫ Ϫ Ϫ mice. Most importantly, cytogenetic analysis of Rag2 / Atm / disease (4, 23). ϩ Ϫ Ϫ Ϫ Ϫ and HyTcrTg Rag2 / Atm / thymic lymphomas revealed an Because both RAG-1 and RAG-2 are required for V(D)J absence of translocations involving the Tcr ␣/␦ locus which are recombination, with the elimination of either resulting in the consistently present in AtmϪ/Ϫ thymic lymphomas from Rag2- absence of V(D)J recombination, one might expect tumor competent mice. This finding is consistent with the fact that these susceptibility in Rag1Ϫ/ϪAtmϪ/Ϫ mice to be similar to that tumors originate in mice that lack the RAG-2 protein and observed in Rag2Ϫ/ϪAtmϪ/Ϫ mice. However, recent results therefore cannot generate the dsDNA breaks that initiate V(D)J reported by Liao and Van Dyke (8) indicated that malignant recombination. thymic lymphomas were not observed in Rag1Ϫ/ϪAtmϪ/Ϫ mice, Additionally, Atm-deficient mice were found in our study to in marked contrast to the results reported here of tumor have an increased incidence of nonlymphoid malignancies as occurrence in Rag2Ϫ/ϪAtmϪ/Ϫ mice. This unexpected discrep- compared with Atm-competent mice. The occurrence of ma- ancy may be accounted for by the fact that the majority of lignancies other than thymic lymphomas in Atm-deficient mice Rag1Ϫ/ϪAtmϪ/Ϫ mice in the study by Liao and Van Dyke (8) has not been previously reported, possibly because AtmϪ/Ϫ died or were killed by 9 mo of age, whereas nearly all of the mice die of malignant thymic lymphoma before other types of tumors that developed in Rag2Ϫ/ϪAtmϪ/Ϫ mice in the study malignancies have time to develop. In the study reported here, described here appeared at a later age. Alternatively, a dif- a lower frequency of Rag2ϩAtmϪ/Ϫ mice (58%) developed ference in lymphoma susceptibility between Rag1Ϫ/ϪAtmϪ/Ϫ malignant thymic lymphoma than was previously reported by and Rag2Ϫ/ϪAtmϪ/Ϫ mice could signify previously undescribed Barlow et al. (100%; ref. 5). This difference could be the result differences in RAG-1 and RAG-2 function or could be

6668 ͉ www.pnas.org Petiniot et al. Downloaded by guest on September 30, 2021 attributable to differences in genetic background and/or en- We extend sincere thanks for the excellent animal care provided by vironmental differences in mouse colonies used in these Genevieve Sanchez, Amy Werling, and the staff at Bioqual, Inc. We are studies. A comparison of Rag1Ϫ/ϪAtmϪ/Ϫ and Rag2Ϫ/ϪAtmϪ/Ϫ also grateful to Andre Nussenzweig and Al Singer for careful reading of mice has been initiated to address these questions. this manuscript and constructive comments.

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