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

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

L K Petiniot et al. Proc Natl Acad Sci U S A. 2000.

Abstract

The majority of Atm-deficient mice die of malignant thymic lymphoma by 4-5 mo of age. Cytogenetic abnormalities in these tumors are consistently identified within the Tcr alpha/delta locus, suggesting that tumorigenesis is secondary to aberrant responses to double-stranded DNA breaks that occur during V(D)J recombination. Since V(D)J recombination is a recombinase-activating gene (RAG)-dependent process, we generated Rag2(-/-)Atm(-/-) mice to assess the requirement for RAG-dependent recombination in thymic lymphomagenesis. In contrast to expectation, the data presented here indicate that development of malignant thymic lymphoma in Atm(-/-) mice is not prevented by loss of RAG-2 and thus is not dependent on V(D)J recombination. Malignant thymic lymphomas in Rag2(-/-)Atm(-/-) mice occurred at a lower frequency and with a longer latency as compared with Atm(-/-) mice. Importantly, cytogenetic analysis of these tumors indicated that multiple chromosomal abnormalities occurred in each tumor, but that none of these involved the Tcr alpha/delta locus. Nonmalignant peripheral T cells from TCR-transgenic Rag2(-/-)Atm(-/-) mice also revealed a substantial increase in translocation frequency, suggesting that these translocations are early events in the process of tumorigenesis. These data are consistent with the hypothesis that the major mechanism of tumorigenesis in Atm(-/-) mice is via chromosomal translocations and other abnormalities that are secondary to aberrant responses to double-stranded DNA breaks. Furthermore, these data suggest that V(D)J recombination is a critical, but not essential, event during which Atm-deficient thymocytes are susceptible to developing chromosome aberrations that predispose to malignant transformation.

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Figures

Figure 1

Figure 1

Kaplan–Meier analysis of tumor incidence studies. Thymic lymphoma-free survival is plotted versus time in months for _Rag2_−/−_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. For purposes of statistical analysis Rag2_−/−_Atm+/+ and Rag2_−/−_Atm+/− mice were considered as one group denoted Rag2_−/−_Atm+ (n = 17); likewise, Rag2+Atm+/+ and Rag2+Atm+/− were considered as one group denoted Rag2+Atm+ (n = 13). As no animals from either of these groups were diagnosed with malignant thymic lymphomas, the curves for these two groups are at 100% event-free survival. To distinguish between Rag2_−/−_Atm+ and Rag2+Atm+ mice, dots above the tick marks were used to indicate the death of Rag2_−/−_Atm+ mice.

Figure 2

Figure 2

SKY analysis of a metaphase cell from _Rag2_−/−_Atm_−/− 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.

Figure 3

Figure 3

FISH analysis of the Tcr α/δ locus in HyTcrTg+_Rag2_−/−_Atm_−/− thymic lymphoma 3013. (a) FISH was performed on tumor 3013 with BAC probes to variable region α (Vα) and constant region α (Cα) in the Tcr α/δ locus. The Vα signals are shown in green and the Cα signals are shown in red. Chromosomes are counterstained with 4′,6-diamidino-2-phenylindole (blue). The signals are colocalized, indicating that the Tcr α/δ locus is not disrupted. The extra green signal on one chromosome 14 is due to integration of the (prespliced) HyTcr transgene (Tg) at that site. (b) The same metaphase as in a labeled with a chromosome 14 painting probe (yellow), confirming the chromosomal location of the Tcr α/δ BAC probes. The chromosome 14 breakpoint in the T(14;1) (indicated by the arrow) is not within the Tcr α/δ locus.

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