Abstract
Interleukin-7 (IL-7) is an essential T-cell survival cytokine. IL-7 receptor (IL-7Rα) deficiency severely impairs T-cell development due to substantial apoptosis. We hypothesized that IL-7Rαnull-induced apoptosis is partially contributed by an elevated p53 activity. To investigate the genetic association of IL-7/IL-7Rα signaling with the p53 pathway, we generated IL-7Rαnullp53null (DKO) mice. DKO mice exhibited a marked reduction of apoptosis in developing T cells and an augmented thymic lymphomagenesis with telomere erosions and exacerbated chromosomal anomalies, including chromosome duplications, breaks, and translocations. In particular, Robertsonian translocations, in which telocentric chromosomes fuse at the centromeric region, and a complete loss of telomeres at the fusion site occurred frequently in DKO thymic lymphomas. Cellular and molecular investigations revealed that IL-7/IL-7Rα signaling withdrawal diminished the protein synthesis of protection of telomere 1 (POT1), a subunit of telomere protective complex shelterin, leading to telomere erosion and the activation of the p53 pathway. Blockade of IL-7/IL-7Rα signaling in IL-7-dependent p53null cells reduced POT1 expression and caused telomere and chromosome abnormalities similar to those observed in DKO lymphomas. This study underscores a novel function of IL-7/IL-7Rα during T-cell development in regulating telomere integrity via POT1 expression and provides new insights into cytokine-mediated survival signals and T-cell lymphomagenesis.
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Main
Interleukin-7 (IL-7) is an essential and nonredundant cytokine for T-cell development.1 Humans and mice deficient in IL-7 receptor (IL-7Rα) incur severe T-cell development defects, manifesting SCID.1, 2, 3, 4 Early studies have shown that unbalanced survival signals of the Bcl-2 family members contribute to the impaired thymopoiesis in IL-7Rα deficiency.1 Nevertheless, ectopic expression of Bcl-2 or inactivation of Bcl-2-associated X protein in IL-7Rαnull mice only partially rescues the thymopoietic defect,5, 6, 7 suggesting the possible involvement of the other pathways.
The tumor-suppressor p53 is a crucial transcription factor in controlling the cell cycle and apoptosis of cells under genotoxic stresses.8 Early studies show that p53 participates in critical thymopoiesis checkpoints related to T-cell receptor rearrangement and DNA damage repair.9, 10, 11 Emerging evidence suggests that p53 is a crucial regulatory factor of normal physiological processes, such as maintenance of stem cell state, development, tissue homeostasis, and autoimmunity.12, 13, 14, 15 We hypothesized that p53 activation also contributes to the apoptosis and impaired thymopoiesis in IL-7Rα deficiency. However, the interplay between the IL-7/IL-7Rα signaling and the p53 pathway has not been demonstrated.
To decipher the potential genetic association of the IL-7Rα signaling with the p53 pathway, we crossed IL-7Rαnull mice with p53null mice. Intriguingly, genetic deletion of p53 in IL-7Rαnull background (IL-7Rαnullp53null, DKO mice) not only markedly reduced the apoptosis of developing T cells, but also significantly increased the incidence and accelerated the onset of thymic lymphoma compared with p53null mice. Furthermore, p53 inactivation permitted the survival of IL-7Rαnull thymocytes that incurred telomere dysfunction and encouraged chromosome instability, leading to exacerbated lymphomagenesis in DKO mice. Additionally, we demonstrated that IL-7/IL-7Rα signaling has a crucial role in maintaining telomere integrity and genomic stability during thymopoiesis by regulating the expression of protection of telomere 1 (POT1), a crucial component of telomere protective complex shelterin.
Results
Thymopoiesis defect in IL-7Rαnull mice is associated with a marked increased in p53 activity
IL-7Rα deficiency results in a 99% to 99.9% reduction in thymic cellularity.4 Besides the documented imbalance of pro-survival and pro-apoptotic signals of the Bcl-2 family members,4, 5, 6, 7 we postulated that elevated p53 activity also contributes to the thymopoietic defect in IL-7Rαnull mice. Our immunofluorescence staining for p53 confirmed a preferential increase in the percentage of p53-positive thymocytes in IL-7Rαnull mice as compared with those in wild-type (WT) and p53null mice (Figure 1a). Because p53 phosphorylation is associated with its activation,16 we determined its levels. Indeed, phosphorylation of p53 at serines 23 (p53Ser23) and 18 (p53Ser18) in the thymocytes of IL-7Rαnull mice was markedly increased as compared with those of WT and p53null mice (Figure 1b). This enhanced p53 activity in IL-7Rαnull thymocytes was confirmed as marked upregulation of p53 downstream targets, such as p53 upregulated modulator of apoptosis (puma) and p21, by western blotting and quantitative RT-PCR (Figures 1b and c). Similarly, an increase in Bcl-2-associated X protein mRNA with a concurrent reduction in Bcl2 mRNA in IL-7Rαnull thymocytes was also observed (Figure 1c). To determine the thymocyte subsets that incurred elevated p53 activity, we employed intracellular staining of p53Ser18 and found that CD4−8− subpopulation of both WT and IL-7Rαnull mice sustained the highest level of p53Ser18 staining (Supplementary Figure S1A). Some CD4+ and CD8+ thymocytes of IL-7Rαnull mice also incurred elevated p53Ser18 activity (Supplementary Figure S1A). Consistent with a previous report,9 p53 was not activated in the thymi of Rag1null mice whose impaired thymopoiesis was caused by a failure to initiate T-cell receptor rearrangement (Supplementary Figure S1B). Together, these results suggest that the activated p53 pathway is a specific contributing factor to the impaired thymopoiesis in IL-7Rαnull mice.
Genetic inactivation of p53 reduces the apoptosis of developing thymocytes in IL-7Rαnull mice with a dire consequence of augmented thymic lymphomagenesis
To explore whether genetic deletion of p53 in IL-7Rαnull mice prevents apoptosis and restores thymopoiesis, we generated DKO mice by crossing IL-7Rαnull and p53null mice in the C57BL/6 background. As expected, the thymocyte composition of CD4+8−, CD4+8+ (DP), CD4−8−, and CD4−8+ subpopulations in DKO mice was largely restored to a comparable level to that of WT thymocytes with a more than 30-fold increase in thymic cellularity (Figure 2a). The cellularity of all four pro-T subsets, DN1 to DN4, was markedly increased in DKO mice, although those of CD44+25− (DN1) and CD44−25+ (DN3) were more profound (Figure 2b). Because all p53null mice succumbed to tumorigenesis and died within 40 weeks with a median survival of 23 weeks (Figure 2c),17 we explored whether IL-7Rαnull imposed any effects on T-cell lymphomagenesis of the p53null background by monitoring cohorts of WT, IL-7Rαnull, p53null, and DKO mice for 40 weeks. Strikingly, DKO mice developed tumors significantly earlier than p53null mice with 100% mortality by 25 weeks and a median survival of 18 weeks (Figure 2c). Neither WT nor IL-7Rαnull mice developed tumors during this period (Figure 2c). Pathological and flow cytometry (FACS) examinations revealed that more than 80% of the DKO mice, as compared with 42% of the p53null mice, developed thymic lymphomas (Supplementary Table 1). These thymic lymphomas from both DKO and p53null mice appeared to similarly arise from immature CD4−CD8+CD24+ ISP and CD4+CD8+CD24+ DP thymocytes (Figure 2d). Interestingly, close to 60% of thymic lymphomas in DKO mice disseminated systemically to both lymphoid, such as the spleen and lymph nodes, and non-lymphoid tissues, such as the liver and kidneys, whereas only 13% of the tumors from p53null mice showed limited dissemination to the lymphoid but not non-lymphoid tissues (Figure 2e and Supplementary Table 2). FACS analysis further confirmed thymic origin of the disseminated tumors in DKO mice (Figure 2e). These results clearly demonstrate that inactivation of p53 partially, but markedly, restored the thymopoiesis defects of IL-7Rαnull with the dire consequence of augmented thymic lymphomagenesis. Our study provides direct evidence of the functional interplay between IL-7Rα signaling and the p53 pathway during thymopoiesis.
Thymic lymphomas in DKO mice incur exacerbated genomic instability
Genomic instability, such as aneuploidy and translocation, is a hallmark and potential cause of tumorigenesis.18, 19 To investigate whether the augmented lymphomagenesis and enhanced dissemination in DKO mice were associated with increased genomic instability, we performed DNA content analysis via FACS. Fresh thymic lymphomas from p53null mice contained aneuploid and polyploid (>4n, Figure 3a) cells, similar to previous observations,20, 21 whereas the frequency of irregular DNA contents was greatly increased in DKO thymic lymphomas (Figure 3a). Moreover, within each tumor sample, a significantly higher percentage of polyploidy was observed in DKO tumors (Figure 3b, DKO versus p53null, P=0.017, two-tailed Mann–Whitney rank sum test). Cytogenetic analyses with thymic lymphomas from eight DKO and four p53null mice further confirmed exacerbated genomic instability in DKO tumors: 30% of 50 metaphases examined from the DKO lymphomas maintained diploid chromosome number of 40 (=2n), whereas more than 65% of the 50 metaphases from p53null tumors showed diploid chromosome number (Figure 4a). More strikingly, over 80% metaphases from DKO tumors contained at least one translocation involving centromeric fusions between the telocentric chromosomes (Robertsonian translocation), among which approximately 40% contained 2–4 translocations/cell (Figure 4b). In sharp contrast, translocations in tumors from p53null mice were very rare and none of the metaphases contained two or more translocations per cell (Figure 4b), similar to previous reports.20, 21 To further explore other chromosome aberrations in the thymic lymphomas, we employed spectral karyotyping (SKY) analyses. Indeed, DKO tumors frequently showed unbalanced near-triploid chromosome amplification, besides chromosome breaks and non-clonal unbalanced translocations (Figure 4c and Supplementary Table 3), whereas chromosome structural aberrations were rarely observed in p53null tumors (Supplementary Figure S2).20, 21 To determine whether chromosome instability is observed prior to tumor development, we karyotyped thymocytes from 4–6-week-old DKO mice before overdevelopment of tumors, defined as aberrant ISP and DP composition via FACS. Indeed, chromosome duplications and fusions were observed in these pre-malignant DKO thymocytes (Supplementary Figure S3A), indicating that chromosome instability preceded overdevelopment of tumor. To further confirm their lack of early emerging lymphomas, thymocytes of 4–6-week-old DKO mice were also transferred to syngeneic Ragnull mice that lacked mature T cells. CD4+ and CD8+ mature T cells were observed in the peripheral blood lymphocytes of Ragnull mice that received DKO thymocytes over the 3-month period of observation (Supplementary Figure S3B). These transferred thymocytes did not develop into lymphomas in Ragnull mice, like those of ISP or DP phenotypes in the spleen and thymus of DKO mice (Supplementary Figure S3C and Figures 2d and e). Together, these results confirm that genomic abnormality occurred in pre-malignant DKO thymocytes and strongly support the notion that IL-7Rα deficiency in combination with p53 deficiency permits the survival of developing thymocytes and encourages genomic instability, thereby exacerbating lymphomagenesis.
IL-7Rαnull deficiency is associated with telomere dysfunction, which activates DNA damage signals that coincide with telomere signals
Chromosome translocations and fusions can be triggered by various mechanisms, such as T-cell receptor rearrangement, DNA damage-induced double-strand DNA breaks, telomere dysfunction, and DNA repair defects.10, 18, 20, 22, 23 Robertsonian translocations frequently occur in cells incurring telomere dysfunction.22, 24 We, therefore, hypothesized that IL-7Rαnull results in telomere dysfunction leading to the exacerbated lymphomagenesis in DKO mice. Telomeric fluorescence in situ hybridization (Telo-FISH) revealed complete loss of telomeres at the centromeric fusions of all Robertsonian translocations and some of non-fused chromosomes in DKO lymphomas (Figure 5a), whereas no obvious telomere loss was observed in p53null tumors (Figure 5a). Telo-FISH analyses with fresh unmanipulated thymocytes from 4–6-week-old DKO and p53null mice confirmed that telomere dysfunction occurred in pre-malignant DKO thymocytes as multi-telomere loss (up to 3/metaphase) in about 27% thymocytes examined as compared with the occasional (7%) undetectable telomeres (not >1/metaphase) in p53null thymocytes (Figure 5b). Likewise, Telo-FISH analysis on fresh IL-7Rαnull thymocytes also confirmed the frequent loss of telomeres and an overall reduction in the intensity of telomere hybridization signals (Supplementary Figure S4). Because of the difficulty in obtaining sufficient number of metaphases from IL-7Rαnull thymocytes, we employed a flow cytometry-based analysis of Telo-FISH signal (Flow-FISH) to determine the mean fluorescence intensity of telomere signals of cells that possessed identical DNA content determined by propidium iodide staining. As shown in Figure 5c, Flow-FISH analyses confirmed that telomere signals of IL-7Rαnull and DKO thymocytes were significantly reduced compared with those of WT and p53null thymocytes with identical DNA content of 2n, which were G0/G1 phase cells. These results demonstrate that developing thymocytes fail to maintain telomere integrity in the absence of IL-7Rα.
Telomere dysfunction, either via telomere uncapping or shortening, is sensed as double-strand DNA breaks, which trigger DNA damage responses and activate the checkpoint pathways.19, 25, 26, 27 Consequently, these lead to the localization of DNA damage response factors, such as γH2AX and 53BP1, at the site of dysfunctional telomeres called telomere-induced DNA damage foci (TIF).25, 27, 28 To validate the effects of IL-7Rαnull in inducing telomere dysfunction, we examined TIFs in IL-7Rαnull and DKO thymocytes by immunofluorescence staining of γH2AX followed by Telo-FISH (immunofluoresent fluorescence in situ hybridization (IF-FISH)). Confocal microscopic image analyses of >50 thymocytes from each genotype revealed that over 70% of IL-7Rαnull and DKO thymocytes incurred >4 TIF/cell, whereas only 15–30% of WT and p53null thymocytes contained >4 TIF/cell (Figure 5d). In contrast, irradiated WT thymocytes did not show distinct TIFs despite a massive upregulation of γH2AX induced by the irradiation (Supplementary Figure S5). Among the thymocytes examined, not all DNA damage signals colocalized with telomeres, suggesting the existence of other DNA damages during thymopoiesis (Figure 5d). To verify that IL-7/IL-7Rα signaling withdrawal caused the increases in TIF, we performed IF-FISH analysis using an established IL-7-dependent p53null thymic cell line D1Bcl2 that was resistant to apoptosis because of constitutive expression of Bcl2.29, 30 IL-7 withdrawal from D1Bcl2 cells resulted in cell cycle arrest within 24 h without apparent induction of apoptosis up to 72 h.30 However, it greatly enhanced the DNA damage signals revealed by co-staining with anti-γH2AX and 53BP1 antibodies, the majority of which also colocalized with telomeres as TIFs (Figure 5e). Together, these results strongly suggest that the lack of IL-7/IL-7Rα signaling in developing thymocytes leads to telomere dysfunction, which triggers DNA damage responses, activates the p53 pathway, and induces apoptosis. Genetic deletion of p53 allows the survival of those thymocytes-accruing telomere dysfunction, DNA damages, and chromosome abnormalities, which initiate and mark the early transition towards tumorigenesis.
IL-7 signaling blockade in p53null D1 cell line recapitulates telomere dysfunction and chromosomal anomalies observed in DKO lymphomas
To recapitulate our in vivo results of the telomere dysfunction and genomic instability observed in DKO thymocytes, we analyzed telomere signals at various time points after IL-7 withdrawal from D1 cells via Flow-FISH. As expected, IL-7 withdrawal resulted in a significant reduction (∼25%) in telomere signal at as early as 16 h (Figure 6a). At 20–24 h after IL-7 withdrawal, the telomere signal was reduced by about 50%, leading to a complete loss of telomere signal and apoptosis by 30–40 h (Figure 6a). To verify that the observed drastic reduction in telomere signals via Flow-FISH reflected telomere shortening, we assessed telomere length by Southern blot analysis of terminal restriction fragments (TRFs) (Figure 6b). Similar to Flow-FISH results, IL-7 withdrawal from D1 cells resulted in a reduction of telomere length to 18.3 Kb within 16 h from 32 Kb and a further decrease to 12.7 Kb by 20 h post-IL-7 withdrawal (Figure 6b). Together, these results confirm that IL-7 withdrawal results in telomere erosion and genomic instability in IL-7-dependent thymocytes.
As IL-7 withdrawal from D1 cells induced rapid apoptosis in 30–40 h, which prevented us from examining chromosome abnormalities induced by gradual or partial IL-7 withdrawal (Supplementary Figure S6A), we explored whether an appropriate dose of a JAK3 inhibitor could block D1 proliferation with minimal induction of apoptosis. JAK3 inhibitor VI blocked the IL-7-mediated proliferation at 2 μM without inducing massive apoptosis during the first 40 h (Supplementary Figure S6). DNA content analyses revealed that the JAK3 inhibitor treatment induced a G2/M phase cell cycle arrest and a marked increase in polyploidy (>4n) to 10–20% as compared with a <0.5% polyploidy in untreated D1 cells (Figure 6c). Further examination on chromosome integrity via karyotyping and Telo-FISH confirmed that 10–20% of the cells became polyploid, possessing diplo- and quadruple-chromosomes by 40 h of the JAK3 inhibitor treatment (Figures 6d and e). Strikingly, this observed chromosome numerical abnormality was also associated with telomere attrition, chromosome breaks, Robertsonian translocations (Figure 6e, inserts), and reminiscence of the abnormalities observed in our DKO thymic lymphomas. Altogether, these results strongly support the notion that IL-7Rα deficiency results in telomere dysfunction, which leads to chromosome abnormalities in p53null cells.
IL-7/IL-7Rα signaling protects telomeres by maintaining protein synthesis of POT1
Telomeres are protected by a specialized protein complex shelterin, consisting of TRF1 (telomeric repeat binding factor 1 or TERF1), TRF2, TIN2 (TERF1-interacting nuclear factor 2), TPP1 (or adrenocortical dysplasia protein homolog), repressor activator protein 1, and POT1.26 Deficiency in either TRF2 or POT1, which binds double-strand or single-strand telomeric DNA, respectively, induces telomere dysfunction that activates DNA damage signals and the p53 pathway.26, 31, 32 Therefore, we explored the effects of IL-7 withdrawal from D1 cells on shelterin subunits. Intriguingly, at 20 h post-IL-7 withdrawal, POT1 protein expression was greatly diminished, associated with an elevated γH2AX (Figure 7a). The level of TPP1 was modestly reduced. However, the expression of other shelterin subunits TFR1, TRF2, and TIN2 was unaffected by the IL-7 withdrawal (Figure 7a). Similar reduction of POT1 expression also occurred in vivo in IL-7Rαnull thymocytes as compared with that of WT thymocytes (Figure 7b). Only modest reduction in the expression of TPP1, TRF2, and TIN2 was observed, whereas that of TRF1 and repressor activator protein 1 was unaffected in IL-7Rαnull thymocytes (Figure 7b). Further, kinetic analyses upon IL-7 withdrawal demonstrated an inverse correlation between the levels of POT1 and γH2AX in D1 cells (Figure 7c). Similarly, the Jak3 inhibitor greatly suppressed IL-7-mediated STAT5 phosphorylation and POT1 expression, but not that of TIN2, with a concomitant increase in γH2AX (Figure 7d). These results strongly suggest that IL-7/IL-7Rα signaling is essential for proper POT1 expression and the maintenance of telomere integrity in IL-7-dependent cells.
To better understand the regulatory mechanism of POT1 by IL-7, we examined the mRNA levels of both POT1a and POT1b, two murine orthologs, in D1 cells upon IL-7 withdrawal and found that neither RNA transcript was affected (Supplementary Figure S7). As POT1a and POT1b proteins have an identical molecular size and the antibody we used does not distinguish these two proteins, our analyses on POT1 protein were not meant to discriminate them. We examined whether POT1 protein stability was affected by the IL-7/IL-7Rα signaling in D1 cells in the presence of a protein synthesis inhibitor cycloheximide. However, the presence or absence of IL-7 did not alter the kinetics of POT1 degradation in D1 cells (Supplementary Figure S8), suggesting that IL-7 signaling did not regulate POT1 protein stability. To further determine whether POT1 protein synthesis was altered in the absence of IL-7, we performed metabolic labeling upon IL-7 withdrawal and found that the level of newly synthesized POT1, measured by the 35S-Methoinine labeling, was greatly diminished within 12 h of IL-7 withdrawal (Figure 7E), indicating that IL-7 regulates POT1 at the protein synthesis level. Finally, to test whether enforced expression of either POT1a or POT1b would rescue the telomere attrition by IL-7 withdrawal in D1 cells, we subjected D1Bcl2 cells overexpressing POT1a or POT1b to IL-7 withdrawal. Interestingly, either POT1a or POT1b alone did not prevent IL-7 withdrawal-induced telomere attrition, but co-expression of POT1a and POT1b prevented the telomere loss induced by IL-7 withdrawal (Figure 7F). Taken together, these results strongly suggest that IL-7/IL-7Rα signaling protects telomere integrity by maintaining POT1expression at the protein synthesis level in both IL-7-dependent D1 cells and primary thymocytes.
Discussion
Our study reveals a previously unappreciated interaction between IL-7/IL-7Rα signaling and the p53 pathway, as well as the crucial role of IL-7/IL-7Rα in maintaining genomic stability during thymopoiesis. We demonstrate that in the absence of IL-7/IL7Rα signaling, IL-7-dependent cells fail to maintain POT1 protein synthesis and telomere integrity, leading to rapid telomere attrition, activation of DNA damage signal, and activation of the p53 pathway. Genetic deletion of p53 in IL-7Rαnull background permits the survival of thymocytes-accruing telomere dysfunction, but encourages chromosome instability and exacerbates lymphomagenesis. Our results clearly demonstrate that activation of the p53 pathway is another contributing pro-apoptotic signal in IL-7Rαnull thymocytes, besides the well-documented unbalanced signals of the Bcl-2 family members.1, 4 Therefore, multiple pro-survival pathways are involved in the essential and nonredundant function of IL-7/IL-7Rα signaling during T-cell development.
Recent evidence demonstrates that POT1 is essential for protecting telomeres from rapid degradation.31, 32, 33 Our study is the first that demonstrates the essential role of POT1 in cytokine-mediated T-cell development by suppressing telomeric DNA damage signals. Although the consequence of POT1null to T-cell development is not previously documented because of embryonic lethality of POT1anull mice, the observed chromosome abnormalities in our DKO thymic lymphomas are similar to those reported in mouse embryonic fibroblasts lacking POT1a/b, especially those with simultaneous inactivation of p53.32, 33 Our results with ectopic expression of POT1a/b in D1 cells and those of the above-referenced reports of POT1a/bnull mouse embryonic fibroblast strongly suggest that both POT1a and POT1b are required to prevent telomeric DNA damage signals and the loss of both promotes polyploidy, chromosome fusions, and breaks.32, 33 Moreover, our study also suggests that IL-7 regulates POT1 expression at the protein synthesis level via the JAK3-STAT5 pathway because JAK3 inhibitor treatment of D1 cells suppresses POT1 expression and mimics chromosome abnormalities incurred in DKO lymphomas. This notion also agrees with a previous report on JAK3 inhibitor-induced endoreduplication in leukemia cells.34 Nevertheless, IL-7 deficiency-induced telomere dysfunction as rapid telomere erosion in D1 cells, and IL-7Rαnull and DKO thymocytes was not reported in POT1a/bnull mouse embryonic fibroblast.32, 33 The differences between our study and previous observations are likely related to the cellular context, that is, intrinsic properties of different cell type and the potential contribution of other IL-7-dependent effects, which will be investigated in future studies.
Recent observations reveal that about 10% T-cell acute lymphoblastic leukemia carry gain-of-function mutations of IL-7Rα gene, which promote oncogenic transformation via constitutive activation of JAK1, but not JAK3.35, 36 Nevertheless, this T-cell acute lymphoblastic leukemia is not linked to poor prognosis,35, 36 suggesting a different tumorigenic process from that of our DKO mice. As IL-7Rα deficiency causes lymphopenia, which was modestly improved in DKO mice by p53 inactivation but to a lesser extent than that by Bcl2 overexpression,5, 6, 7 it is also plausible to propose that the lymphopenia environment promotes vigorous compensatory proliferation of stem cells and progenitors, and encourages accrued mutations and genomic instability, thereby enhancing their risk of oncogenesis like those observed in recent studies.37, 38 Therefore, IL-7 signaling should be exquisitely controlled to ensure proper thymopoiesis while preventing lymphomagenesis.
In summary, this is the first study illustrating the functional interplay of the IL-7Rα signaling with the p53 pathway in maintaining genomic stability during thymopoiesis. Our study underscores a novel function of IL-7Rα signaling during thymopoiesis in maintaining POT1 expression and telomere integrity, which may shed light on future studies on immune deficiency and lymphomagenesis. Moreover, this DKO mouse may represent a unique model for studying cytokine withdrawal-induced chromosome instability in tumorigenesis.
Materials and Methods
Mice
DKO mice were generated by intercrossing IL-7Rαnull (B6 129S7-Il7rtm1Imx/J) p53null mice for more than five generations. All mice, including Rag1null (B6.129S7-Rag1tm1Mom/J), were purchased from the Jackson Laboratories (Bar Harbor, ME, USA). All mice were bred and kept under specific pathogen-free conditions in the animal care facility of LSUHSC following protocols approved by the Institutional Animal Care and Use Committee. Genotypes were confirmed by PCR.
Cell lines and retroviral transduction
The IL-7-dependent thymocyte cell line D1 established from p53 KO mice were maintained in RPMI 1640 supplemented with 10% fetal bovine serum (Sigma, St. Louis, MO, USA), 2 mM L-glutamine, 50 μM β-mercaptoethanol (Invitrogen, Carlsbad, CA, USA), and 50 μg/ml murine recombinant IL-7 (PeproTech, Rocky Hill, NJ, USA). The D1 cells were treated with 2 μ M JAK3 inhibitor VI (EMD Biosciences, Darmstadt, Germany) for various durations in the presence of 50 μg/ml IL-7. D1 or D1Bcl2 cells were transduced with retroviruses pLPCN-MycPOT1a, pLPCN-MycPOT1b (kindly provided by Dr. Titia de Lange), or both and selected with puromycin (5 μg/ml) in the presence of IL-7 for 7 days.
Flow cytometry analysis
All antibodies for FACS analyses were purchased from BD Biosciences (San Jose, CA, USA), unless otherwise specified. Thymocytes and thymic lymphomas were harvested and processed to make single-cell suspensions for cell-surface marker analysis. Flow cytometric acquisition was performed using a FACSCalibur (BD Biosciences) and analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA).
Histology and immunofluorescence analyses
Fresh tissues were fixed in methanol-free formaldehyde for H&E and immunofluorescent staining. p53+ subpopulation was calculated as the percentage of area covered by p53+ cells over that of total thymocytes (DAPI+).
Intracellular staining of p53ser18
Fresh thymocytes in single-cell suspension were first stained for cell-surface markers and fixed in cold 70% EtOH at −20°C for 4 h. They were subsequently stained with rabbit anti-p53ser18 antibody (100 × dilution; Cell Signaling Technology, Boston, MA, USA) and PE-conjugated goat anti-rabbit antibody in FACS buffer (PBS containing 2 mM EDTA, 2% FBS, and 0.01% sodium azide) in the presence of phosphatase inhibitor (EMD Chemicals, San Diego, CA, USA) at 4°C. The level of p53ser18 signal was determined via FACS.
Tumorigenesis and pathological analyses
Cohorts of 26–36 mice/genotype were observed for tumor development. A portion of the tumors was preserved in buffered zinc formalin fixative (Anatech Ltd, Battle Creek, MI, USA), and sectioned for H&E staining. The other portion of the tumors was dissociated to single-cell suspension for surface marker staining and FACS analysis. H&E sections were viewed by a licensed pathologist to determine the tumor type in reference to their cell-surface marker expression and anatomic location of the tumors.
Western blotting
Fresh thymocytes or cultured D1 cells were lysed in RIPA buffer containing phosphatase inhibitors and protease inhibitors (EMD Chemicals). Cell lysate containing 20–100 μg protein was loaded onto the NuPAGE 4–12% Bis-Tris or 7% Tris-Acetate gel (Invitrogen), separated by electrophoresis, transferred to the PVDF membrane (Invitrogen), and blotted with antibodies against specific proteins. The primary antibodies are p53 (CM5; Novocastra, Newcastle upon Tyne, UK); p53Ser15, p53Ser20, puma, p21, γH2AX, Stat5-Tyr694, Stat5, and repressor activator protein 1 (Cell Signaling); Vinculin and TRF2 (Santa Cruz, Santa Cruz, CA, USA); β-actin (Sigma); and POT1, TIN2, and TRF1 (Abcam, Cambridge, MA, USA).
Metaphase preparation and karyotyping
Primary thymic lymphomas from p53null and DKO mice were expanded in the presence of recombinant IL-2 (20 U/ml) and treated with 100 ng/ml Colcemid (Invitrogen) for 1 h before harvesting for metaphase preparation. For metaphase preparation of fresh thymocytes, cells from 6–8-week-old mice were harvested 2 h after they were treated i.p. with Colcemid at 30 μg/kg body weight. Metaphase preparations were performed following standard procedures.39 Karyotypes of metaphase images were captured using an Applied Imaging Model ER-3339 cooled CCD camera (Applied Spectral Imaging Inc., Vista, CA, USA) mounted on top of a Nikon Eclipse E400 with CytoVision version 3.1 image-capture software (Applied Spectral Imaging). Telomere in situ hybridization and SKY analyses were performed using Cy3-labeled telomere PNA (CCCTAA)3 probe (Panagene Inc., Daejeon, Korea) and 20-color mouse SKY paint kit (ASI), respectively, following standard procedures and manufacturer's instructions.39 SKY images were acquired using a SD301 SpectraCubeTM system (ASI) mounted on top of an epifluorescence microscope Axioplan 2 (Zeiss, Oberkochen, Germany). Images were analyzed using Spectral Imaging 4.0 acquisition software (ASI). G-banding was simulated by electronic inversion of DAPI counterstaining.
IF-FISH
IF-FISH was performed with primary thymocytes or established cell lines. Cells in suspension were fixed in 2% paraformaldehyde at RT for 10 min, cytospun onto coated microscope slides (Shandon, Pittsburgh, PA, USA) followed by acetone treatment at −20°C for 5 min, and 30 min incubation with blocking solution (1% BSA and 5% goat serum in PBS). Primary antibodies, γH2AX (mouse monoclonal, JBW301; Millipore, Billerica, MA, USA) and 53BP1 (rabbit polyclonal, 100-304, Novus Biologicals, Littleon, CO, USA) were incubated in blocking solution at RT for 2 h, followed by 30 min incubation with Alexa-568-labeled goat anti-rabbit and Alexa-647-labeled goat anti-mouse antibodies (Invitrogen). The slides were completely dehydrated by pass through 75%, 95%, and 100% ethanol and air-dried for 5 min. Telomere in situ hybridization was performed with 140 nM FAM-labeled telomere PNA (CCCTAA)3 probe (Panagene Inc.) in hybridization buffer (70% formamide, 20 mM Tris-HCl, 10 mM Na2HPO4, and 10 mM NaCl) at 80°C for 5 min, followed by 1 h hybridization in dark at RT. The slides were washed with PBS containing 0.1% Tween-20 at 57°C for 20 min, followed by 2 × SSC containing 0.1% Tween 20 at RT for 5 min, DAPI counterstained and mounted in ProLong Gold (Invitrogen).
Flow-FISH
Flow cytometry-based analysis of telomere signal (Flow-FISH) was performed following a standard protocol40 with slight modification. Briefly, cells were incubated with 28 nM FAM-labeled telomere PNA (CCCTAA)3 probe in hybridization buffer at RT for 10 min, followed by a 10 min incubation at 87°C, and a 90-min incubation at RT in dark. After two washes with PBS containing 0.1% Tween-20 and two washes with 2 × SSC containing 0.1% Tween 20, they were incubated in 350 μl of RNase/propidium iodide-staining buffer (BD Biosciences) at RT for 15 min and used for FACS analysis. FAM-labeled telomere signal was detected at FL1 and cells with the same DNA content were compared.
TRF assay
A Southern blot-based TRF analysis was performed using a TeloTAGGG telomere Length Assay kit (Roche, Basel, Switzerland) as per manufacturer's instruction. Briefly, 2 μg of genomic DNA harvested from D1 cells cultured in the presence or absence was digested with Hinf I and Rsa I at 37°C for 4 h and electorphoresed in 0.7% agarose gel at 25 V for 20 h. The gel was then treated with 0.25 N HCl for 30 min, denatured with 0.5 N NaOH for 20 min, and transferred onto Hybond-N+ membrane (GE Healthcare, Waukesha, WI, USA) with 0.4 N NaOH for 4 h. The membrane was subsequently hybridized with DIG-labeled telomere probe at 42°C for 3 h, followed by two washes with 2 × SSC containing 0.1% SDS at RT and 0.2 × SSC containing 0.1% SDS at 50°C for 15 min each. The telomere signal was revealed by incubating the membrane with AP-conjugated anti-DIG antibody, followed by AP substrate addition, and acquired via a BioRad VersaDoc imaging system. The telomere length of each sample was determined by first measuring the signal density (OD) for each of the 35–40 equally sized squares over the entire gel length and calculated as TRF=Σ(ODi)/Σ(ODi/Li), where ODi is the signal density of each individual square and Li is the length of the TRF at the position i.
Analyses of protein synthesis
To examine de novo protein synthesis of POT1 upon IL-7 withdrawal, D1 cells cultured in the absence or presence of IL-7 for 12 h were transferred to Methionine-free RPMI (Invitrogen) medium for 30 min followed by pulsing with 35S Methionine at 20 mCi/ml (1175 Ci/mmol, PerkinElmer, Waltham, MA, USA) for 2 h. Cell lysate containing 100 μg of the total protein was used for IP with 3 μg of rabbit anti-POT1 antibody (Abcam) and Protein G Agarose beads at 4°C o/n. Eluted proteins were separated via eletrophoresis with an 8% Tris-Glycine NuPAGE gel (Invitrogen), transferred onto PVDF membrane, and 35S Methionine-labeled proteins were detected by exposing to a Biomax MR film.
Statistical analysis
The differences in cell survival and gene expression between different samples and/or treatments were analyzed via two-tailed Student's t-tests using SigmaPlot (Systat Software Inc., Chicago, IL, USA). Statistical significance was set at P<0.05, unless otherwise stated in the text.
Abbreviations
- IL-7Rα:
-
IL-7 receptor α chain
- POT1:
-
protection of telomere 1
- DN:
-
CD4−8− double negative
- DP:
-
CD4+8+ double positive
- ISP:
-
CD4−CD8+ CD24+ immature single positive
- SKY:
-
spectral karyotyping
- Telo-FISH:
-
telomeric fluorescence in situ hybridization
- Flow-FISH:
-
flow cytometry-based analysis of Telo-FISH
- TIF:
-
telomere-induced DNA damage foci
- IF-FISH:
-
immunofluorescent fluorescence in situ hybridization
- TRF:
-
terminal restriction fragment
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Acknowledgements
We thank Drs. Alistair Ramsay, Wanguo Liu, Jay K Kolls, and Drew Pardoll for constructive suggestions; Ms. Heidi Davis for editorial assistance; Drs. Beatriz Finkel-Jimenez, Sandra Burkett, Marilyn Li, and Kong Chen for technical assistance; and Ms. Yunping Huang for animal breeding. This work is supported in part by funds from the LGTRC, LCRC, NIH grants to YC (CA112065 and P20RR021970) and TI (P20RR020152).
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Kibe, R., Zhang, S., Guo, D. et al. IL-7Rα deficiency in p53null mice exacerbates thymocyte telomere erosion and lymphomagenesis. Cell Death Differ 19, 1139–1151 (2012). https://doi.org/10.1038/cdd.2011.203
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DOI: https://doi.org/10.1038/cdd.2011.203
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