Irradiation promotes V(D)J joining and RAG-dependent neoplastic transformation in SCID T-cell precursors

Abstract

Defects in the nonhomologous end-joining (NHEJ) pathway of double-stranded DNA break repair severely impair V(D)J joining and selectively predispose mice to the development of lymphoid neoplasia. This connection was first noted in mice with the severe combined immune deficient (SCID) mutation in the DNA-dependent protein kinase (DNA-PK). SCID mice spontaneously develop thymic lymphoma with low incidence and long latency. However, we and others showed that low-dose irradiation of SCID mice dramatically increases the frequency and decreases the latency of thymic lymphomagenesis, but irradiation does not promote the development of other tumors. We have used this model to explore the mechanistic basis by which defects in NHEJ confer selective and profound susceptibility to lymphoid oncogenesis. Here, we show that radiation quantitatively and qualitatively improves V(D)J joining in SCID cells, in the absence of T-cell receptor-mediated cellular selection. Furthermore, we show that the lymphocyte-specific endonuclease encoded by the recombinase-activating genes (RAG-1 and RAG-2) is required for radiation-induced thymic lymphomagenesis in SCID mice. Collectively, these data suggest that irradiation induces a DNA-PK-independent NHEJ pathway that facilitates V(D)J joining, but also promotes oncogenic misjoining of RAG-1/2-induced breaks in SCID T-cell precursors.

FIG. 1

Effect of irradiation on coding end processing in wild-type thymic lymphoma cells. VL3-3M2 cells were transfected with pDR42, at various times before (B) or after (C) treatment with 0 (A) or 100 cGy of γ-irradiation, as indicated. After 48 h of culture, plasmid DNA was recovered and used to transform E. coli. pDR42 recombinant plasmids were purified from chloramphenicol-resistant colonies, and the coding junctions were sequenced. Shown are the 5′ P (single underline), N, and 3′ P (double underline) additions (P/N/P) at the CJ for each recombinant pDR42 clone. The number of nucleotides deleted from the 5′ (Δ5′) and 3′ (Δ3′) coding flanks are also indicated. P nucleotides were identified based on palindromy with the 3′ end of the 5′ coding flank (5′-ACAGGAAACAGGATC-3′) or the 5′ end of the 3′ coding flank (5′-GATGATATCGTCGAC-3′) sequences. For junctions where nucleotides could be assigned either to the coding flank or as P additions, the assignment was made to minimize the degree of P additions. The data represent independent clones derived from two independent transfections. The frequencies of independent recombinants sequenced were 93% (A), 100% (B), and 100% (C).

FIG. 2

Effect of irradiation on coding end processing in SCID thymic lymphoma cells. LK6.2 cells were transfected with pDR42 3 h (B) or 0.5 h (C) before treatment with 0 (A) or 100 cGy of γ-irradiation, as indicated. Recombinant pDR42 clones were isolated and sequenced as described for Fig. 1. The frequency of independent recombinants sequenced was 64% (A), 83% (B), and 65% (C). B, before treatment.

FIG. 3

Effect of RAG-2 function on thymic lymphomagenesis in irradiated SCID mice. SCID (RAG-2^+/+ or RAG-2^+/−) and RAG-2^−/− SCID mice were irradiated (100 cGy) within 48 h of birth. RAG-2^−/− mice were irradiated as newborns (400 cGy) or adults (750 cGy) as previously described (50). Depicted is the percentage of tumor-free mice of each genotype up to 20 weeks postirradiation. Mice showing no signs of morbidity were sacrificed at 16 weeks (newborn RAG-2^−/−), 18 weeks (SCID and RAG-2^−/− SCID), or 20 weeks (adult RAG-2^−/−) and showed no evidence of lymphoma by gross pathology. N, number of mice analyzed in each group.

FIG. 4

Endogenous TCRβ expression in TCRβ-SCID mice. Thymus RNA from individual 2-week-old untreated or irradiated TCRβ-SCID mice was reverse transcribed into cDNA. RT-PCR was performed using eight different Vβ-specific sense primers coupled with a TCR-Cβ anti-sense primer. The products were Southern blotted and hybridized with a TCR-Cβ probe. Note that, as expected, Vβ8 transcripts were detectable in all thymus samples from SCID mice expressing the Vβ8.2 transgene. Thymocyte RNA obtained from 4- to 6-week-old nontransgenic SCID and BALB/c mice were included as negative and positive controls, respectively, for the presence of TCRβ transcripts.

FIG. 5

Southern blot detection of TCRδ coding end breaks and rearrangements in TCRβ-SCID thymocytes. (A) Genomic DNA was extracted from thymocyte cell suspensions prepared from 4 individual 6-week-old TCRβ-SCID mice and a nontransgenic littermate, as well as from the thymus and kidney of a 4-week-old wild-type C57BL/6 animal. The DNA was digested with EcoRI, electrophoresed, and Southern blotted. The membrane was hybridized with probe no. 4, which detects a single germline fragment between Jδ1 and Jδ2 and allows detection of rearrangement to Jδ1 (82). The positions of germline fragments, Dδ2 coding end breaks (CE), DJδ or D-DJδ partial rearrangements, and complete V-D-Jδ rearrangements are indicated. Molecular size standards are included in the far right lane, with sizes in kilobases. (B) Partial TCRδ locus map. The position of probe no. 4 within the TCRδ locus is displayed (adapted from reference 82). EcoRI sites are indicated, as are the CE breaks and germline fragment detected in this assay.

FIG. 6

Impact of TCRβ transgene on T-cell development in SCID mice. Thymocytes from two 4-day-old TCRβ-SCID mice and a nontransgenic littermate were evaluated for CD4 and CD8 expression by flow cytometry. One TCRβ-SCID mouse treated with 100 cGy of γ-irradiation 24 h prior to this analysis is also shown for comparison. The cellularity of each thymus is indicated. The absolute number of CD25⁺ DN cells in each sample did not vary by more than twofold.