![Figure 2. Aberrant TCRδ rearrangement is required for t(12;14), but not chromosomal 14 amplifications, in murine ATM-deficient thymic lymphomas. (A) Survival curve for ATM−/− (n = 10), ATM−/−Eδ−/− (n = 11), and ATM−/−Eδ+/− (n = 8) mice with median survival at 99, 112, and 137 days, respectively. P values were calculated using the log-rank test. (B) Representative chromosome 12 (green) and chromosome 14 (red) paint analyses of a metaphase from no. 745 ATM−/−Eδ−/− lymphomas (without t(12;14)) and no. 900 ATM−/−Eδ+/− lymphomas with t(12;14) translocation. More than 20 metaphases were analyzed for each lymphoma, and t(12;14) in >50% metaphases was used as the criterion for clonal translocations. The results are summarized in panel E. (C) Southern blot mapping the rearrangements of Cδ and Jα. The diagram shows the TCRα/δ locus, EcoRI (black arrows), and probe locations (black dashed line for Southern probes and red dashes for adjacent CGH probes). A total of 10 µg EcoRI-digested genomic DNA from either tumor (T) or the corresponding kidney (K) was analyzed on each lane. Loading control probe detected a fragment of the single-copy DNA-PKcs gene. (D) CGH analyses of ATM−/−Eδ−/− thymic lymphomas 745 and 545. Each tumor DNA was analyzed with matched kidney control DNA on a 244K mouse CGH array from Agilent. The log ratio of tumor vs kidney control at each probe location is displayed in centromere-to-telomere orientation for each chromosome (from 1 to 19 then X/Y, from left to right). The red arrows and labels indicate recurrent amplification of Notch1, chromosome 14 upstream of TCRα/δ locus, and trisomy 15. Green arrows and labels indicate recurrent deletion of the telomeric portion of chromosome 12 and the Pten locus, as well as deletional rearrangement in TCR loci. (E) Summary of chromosome 12/14 paint analyses of ATM−/−Eδ−/− and ATM−/−Eδ+/− lymphomas. (F) Quantitative PCR results that measured the expression of IL7R, Jak1, Notch1, Pten, Rb1, Bcl11b, and Psmc6 genes from ATM−/−Eδ+/+ (n = 3), ATM−/−Eδ+/− (n = 4), ATM−/−Eδ−/− (n = 5) lymphomas. Psmc6 locates at the amplification area within chromosome 14 and is used as a marker for the amplification here.3 Bcl11b is within the region that is hemizygously deleted in chromosome 12.3 Briefly, total RNA was extracted using Trizol (Invitrogen, CA) from a single-cell suspension derived from ATM−/− and ATM−/−Eδ−/− thymic lymphomas as well as wild-type total thymus, and dissolved in 100 μL DEPC-H2O and verified to have A260 nm/280 nm between 1.8 and 2. Approximately 5 μg of total RNA was treated with RNase-free DNase I (Roche). The DNaseI activity was abolished by the addition of 2 µL of 25-mM EDTA and incubation at 65°C for 15 minutes. Reverse transcription was performed using SuperScript III reverse transcriptase (Invitrogen) and Oligod(T)18 (New England Biolabs) according to the manufacturer’s instructions. The resulted complementary DNA was dissolved in TE and quantified with NanoDrop (Thermo Scientific). Quantitative PCR was then performed with gene-specific primers (supplemental Table 1) for Notch1 (NM_008714.3, 138 bp across exons 25-26), Jak I (NM_146145.2, 162 bp across exons 18-20), IL7R (NM_008372.4, 149 bp across exons 5-7), Rb1 (NM_009029.2, 144 bp across exons 23-25), Bcl11b (NM_001079883.1, 126 bp across exons 3-4, within chromosome 12 telomeric deletion), Pten (NM_008960.2, 144 bp across exons 7-8), Psmc6 (NM_025959.3, 196 bp across exons 7-9, within chromosome 14 amplification), and β-actin (actb; NM_007393.3, 154 bp across exons 1-2). Reaction setups include 10 µL Sybr Select Master Mix (Applied Biosystems), 250 nM each primer, 10 ng complementary DNA, and water to a total of 20 µL. The PCR condition was 50°C (2 minutes), 95°C (10 minutes), and [95°C (15 seconds), 60°C (1 minute)] × 40, followed by the melt curve program provided by 7500 Real Time PCR System (Applied Biosystems). The results were analyzed with 7500 software package (v 2.0.6) and the ΔΔCT method (included in 7500 software) with the expression of β-actin as the reference and the expression of the specific gene in the wild-type samples as controls for normalization. Each gene in each sample was analyzed in triplicate together with H2O control. The H2O control samples always had CT beyond the range (>40) in all experiments.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/125/17/10.1182_blood-2015-01-622621/4/2665f2.jpeg?Expires=1726828475&Signature=x0jdRNtTu2DPYSsFbbav7Yb8hNgCMYCjT0igr0XlkSG9bnxjhm94ugZlTSE7m3D2UEGw1-ass0DCZ0pR0YUl16mVQNB5RFsom2zk~vmTs7gMZSglEfRvd8G~4HKBcuZN2Dtxu~UgAnyxSq1cVF2xG8oAMJwnYZaTk2kCvpUwkjDGUI5-opRyXEUOQnIkMiPJ-0vXT02lbeMM4W5KoGbb0R9ey2AFCdu02MCPh~PFWJEf~lBLIKWi0ReY~Fs49abm1qHmV3C2RLjAEn7sREdKiCxm~3dnJRQhA-7UgiTnPtI-Ocn2ERGLEp27NUFQh3WTpQMRiMHCL~yQ0Nx563cqDA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Aberrant TCRδ rearrangement is required for t(12;14), but not chromosomal 14 amplifications, in murine ATM-deficient thymic lymphomas. (A) Survival curve for ATM−/− (n = 10), ATM−/−Eδ−/− (n = 11), and ATM−/−Eδ+/− (n = 8) mice with median survival at 99, 112, and 137 days, respectively. P values were calculated using the log-rank test. (B) Representative chromosome 12 (green) and chromosome 14 (red) paint analyses of a metaphase from no. 745 ATM−/−Eδ−/− lymphomas (without t(12;14)) and no. 900 ATM−/−Eδ+/− lymphomas with t(12;14) translocation. More than 20 metaphases were analyzed for each lymphoma, and t(12;14) in >50% metaphases was used as the criterion for clonal translocations. The results are summarized in panel E. (C) Southern blot mapping the rearrangements of Cδ and Jα. The diagram shows the TCRα/δ locus, EcoRI (black arrows), and probe locations (black dashed line for Southern probes and red dashes for adjacent CGH probes). A total of 10 µg EcoRI-digested genomic DNA from either tumor (T) or the corresponding kidney (K) was analyzed on each lane. Loading control probe detected a fragment of the single-copy DNA-PKcs gene. (D) CGH analyses of ATM−/−Eδ−/− thymic lymphomas 745 and 545. Each tumor DNA was analyzed with matched kidney control DNA on a 244K mouse CGH array from Agilent. The log ratio of tumor vs kidney control at each probe location is displayed in centromere-to-telomere orientation for each chromosome (from 1 to 19 then X/Y, from left to right). The red arrows and labels indicate recurrent amplification of Notch1, chromosome 14 upstream of TCRα/δ locus, and trisomy 15. Green arrows and labels indicate recurrent deletion of the telomeric portion of chromosome 12 and the Pten locus, as well as deletional rearrangement in TCR loci. (E) Summary of chromosome 12/14 paint analyses of ATM−/−Eδ−/− and ATM−/−Eδ+/− lymphomas. (F) Quantitative PCR results that measured the expression of IL7R, Jak1, Notch1, Pten, Rb1, Bcl11b, and Psmc6 genes from ATM−/−Eδ+/+ (n = 3), ATM−/−Eδ+/− (n = 4), ATM−/−Eδ−/− (n = 5) lymphomas. Psmc6 locates at the amplification area within chromosome 14 and is used as a marker for the amplification here.3 Bcl11b is within the region that is hemizygously deleted in chromosome 12.3 Briefly, total RNA was extracted using Trizol (Invitrogen, CA) from a single-cell suspension derived from ATM−/− and ATM−/−Eδ−/− thymic lymphomas as well as wild-type total thymus, and dissolved in 100 μL DEPC-H2O and verified to have A260 nm/280 nm between 1.8 and 2. Approximately 5 μg of total RNA was treated with RNase-free DNase I (Roche). The DNaseI activity was abolished by the addition of 2 µL of 25-mM EDTA and incubation at 65°C for 15 minutes. Reverse transcription was performed using SuperScript III reverse transcriptase (Invitrogen) and Oligod(T)18 (New England Biolabs) according to the manufacturer’s instructions. The resulted complementary DNA was dissolved in TE and quantified with NanoDrop (Thermo Scientific). Quantitative PCR was then performed with gene-specific primers (supplemental Table 1) for Notch1 (NM_008714.3, 138 bp across exons 25-26), Jak I (NM_146145.2, 162 bp across exons 18-20), IL7R (NM_008372.4, 149 bp across exons 5-7), Rb1 (NM_009029.2, 144 bp across exons 23-25), Bcl11b (NM_001079883.1, 126 bp across exons 3-4, within chromosome 12 telomeric deletion), Pten (NM_008960.2, 144 bp across exons 7-8), Psmc6 (NM_025959.3, 196 bp across exons 7-9, within chromosome 14 amplification), and β-actin (actb; NM_007393.3, 154 bp across exons 1-2). Reaction setups include 10 µL Sybr Select Master Mix (Applied Biosystems), 250 nM each primer, 10 ng complementary DNA, and water to a total of 20 µL. The PCR condition was 50°C (2 minutes), 95°C (10 minutes), and [95°C (15 seconds), 60°C (1 minute)] × 40, followed by the melt curve program provided by 7500 Real Time PCR System (Applied Biosystems). The results were analyzed with 7500 software package (v 2.0.6) and the ΔΔCT method (included in 7500 software) with the expression of β-actin as the reference and the expression of the specific gene in the wild-type samples as controls for normalization. Each gene in each sample was analyzed in triplicate together with H2O control. The H2O control samples always had CT beyond the range (>40) in all experiments.
