Radiation-induced Thymic Lymphoma Research Articles

Mechanisms Underlying the Development of Murine T-Cell Lymphoblastic Lymphoma/Leukemia Induced by Total-Body Irradiation.

Exposure to ionizing radiation is associated with an increased risk of hematologic malignancies in myeloid and lymphoid lineages in humans and experimental mice. Given that substantial evidence links radiation exposure with the risk of hematologic malignancies, it is imperative to deeply understand the mechanisms underlying cellular and molecular changes during the latency period between radiation exposure and the emergence of fully transformed malignant cells. One experimental model widely used in the field of radiation and cancer biology to study hematologic malignancies induced by radiation exposure is mouse models of radiation-induced thymic lymphoma. Murine radiation-induced thymic lymphoma is primarily driven by aberrant activation of Notch signaling, which occurs frequently in human precursor T-cell lymphoblastic lymphoma (T-LBL) and T-cell lymphoblastic leukemia (T-ALL). Here, we summarize the literature elucidating cell-autonomous and non-cell-autonomous mechanisms underlying cancer initiation, progression, and malignant transformation in the thymus following total-body irradiation (TBI) in mice.

Characterizing the role of Phlda3 in the development of acute toxicity and malignant transformation of hematopoietic cells induced by total-body irradiation in mice

The tumor suppressor p53 is a transcriptional factor that plays a crucial role in controlling acute toxicity and long-term malignant transformation of hematopoietic cells induced by genotoxic stress such as ionizing radiation. Among all transcriptional targets of p53, one gene that is robustly induced by radiation is the pleckstrin homology domain-only protein Phlda3. However, the role that Phlda3 plays in regulating the response of hematopoietic cells to radiation is unknown. Here, using isogenic cell lines and genetically engineered mouse models, we showed that radiation induces Phlda3 in human leukemia cells and mouse normal hematopoietic cells in a p53-dependent manner. However, deletion of the Phlda3 gene did not ameliorate radiation-induced acute hematologic toxicity. In addition, distinct from mice that lose p53, loss of Phlda3 did not alter the latency and incidence of radiation-induced thymic lymphoma in mice. Remarkably, whole-exome sequencing data showed that lymphomas in irradiated Phlda3+/+ mice harbor a significantly higher number of single nucleotide variants (SNVs) and indels compared to lymphomas in irradiated Phlda3+/− and Phlda3−/− littermates. Together, our results indicate that although deletion of Phlda3 does not accelerate the development of radiation-induced thymic lymphoma, fewer SNVs and indels are necessary to initiate lymphomagenesis after radiation exposure when Phlda3 is silenced.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Whole-Exome Sequencing of Radiation-Induced Thymic Lymphoma in Mouse Models Identifies Notch1 Activation as a Driver of p53 Wild-Type Lymphoma.

Mouse models of radiation-induced thymic lymphoma are widely used to study the development of radiation-induced blood cancers and to gain insights into the biology of human T-cell lymphoblastic leukemia/lymphoma. Here we aimed to identify key oncogenic drivers for the development of radiation-induced thymic lymphoma by performing whole-exome sequencing using tumors and paired normal tissues from mice with and without irradiation. Thymic lymphomas from irradiated wild-type (WT), p53+/-, and KrasLA1 mice were not observed to harbor significantly higher numbers of nonsynonymous somatic mutations compared with thymic lymphomas from unirradiated p53-/- mice. However, distinct patterns of recurrent mutations arose in genes that control the Notch1 signaling pathway based on the mutational status of p53. Preferential activation of Notch1 signaling in p53 WT lymphomas was also observed at the RNA and protein level. Reporter mice for activation of Notch1 signaling revealed that total-body irradiation (TBI) enriched Notch1hi CD44+ thymocytes that could propagate in vivo after thymocyte transplantation. Mechanistically, genetic inhibition of Notch1 signaling in immature thymocytes prevented formation of radiation-induced thymic lymphoma in p53 WT mice. Taken together, these results demonstrate a critical role of activated Notch1 signaling in driving multistep carcinogenesis of thymic lymphoma following TBI in p53 WT mice. SIGNIFICANCE: These findings reveal the mutational landscape and key drivers in murine radiation-induced thymic lymphoma, a classic animal model that has been used to study radiation carcinogenesis for over 70 years.

The Japanese Wild-Derived Inbred Mouse Strain, MSM/Ms in Cancer Research.

Simple SummarySince human studies on tumor susceptibility alleles require vast numbers of DNA samples from both cancer patients and well-matched controls, their investigation and identification in humans have been complemented using mouse models. However, a number of confounding factors are associated with this type of research, including heterogeneity, weak genetic interactions, and lifestyle habits. Inbred strains relatively recently established from wild mice are often more resistant to carcinogenic stimulation and various pathogens than standard inbred mouse strains. A Japanese wild-derived inbred mouse strain, MSM/Ms has been used to map tumor resistance loci as well as other Quantitative Trait Loci (QTL) in Japan. Furthermore, genetic tools have been developed with MSM/Ms. MSM/Ms genomic sequences are currently available, which have greatly promoted the identification of tumor resistance loci as well as genes controlling quantitative variations and provide a more detailed understanding of gene function. MSM/Ms is a unique inbred mouse strain derived from the Japanese wild mouse, Mus musculus molossinus, which has been approximately 1 million years genetically distant from standard inbred mouse strains mainly derived from M. m. domesticus. Due to its genetic divergence, MSM/Ms has been broadly used in linkage studies. A bacterial artificial chromosome (BAC) library was constructed for the MSM/Ms genome, and sequence analysis of the MSM/Ms genome showed approximately 1% of nucleotides differed from those in the commonly used inbred mouse strain, C57BL/6J. Therefore, MSM/Ms mice are thought to be useful for functional genome studies. MSM/Ms mice show unique characteristics of phenotypes, including its smaller body size, resistance to high-fat-diet-induced diabetes, high locomotive activity, and resistance to age-onset hearing loss, inflammation, and tumorigenesis, which are distinct from those of common inbred mouse strains. Furthermore, ES (Embryonic Stem) cell lines established from MSM/Ms allow the MSM/Ms genome to be genetically manipulated. Therefore, genomic and phenotypic analyses of MSM/Ms reveal novel insights into gene functions that were previously not obtained from research on common laboratory strains. Tumorigenesis-related MSM/Ms-specific genetic traits have been intensively investigated in Japan. Furthermore, radiation-induced thymic lymphomas and chemically-induced skin tumors have been extensively examined using MSM/Ms.

Analysis of miRNA-mRNA Crosstalk in Radiation-Induced Mouse Thymic Lymphomas to Identify miR-486 as a Critical Regulator by Targeting IGF2BP3 mRNA.

Ionizing radiation is one of the common environmental carcinogens. miRNAs play critical roles in the processes of tumor occurrence, development, metastasis. However, the relationship between radiation-induced carcinogenesis and miRNA rarely reported. This study is aimed to investigate the effect of miRNAs on radiation-induced carcinogenesis. In this study we established the radiation-induced thymic lymphoma mice model. By using miRNA array of RTL tissue and predicting for miRNAs target genes, a miRNA-mRNA crosstalk network was established. Based on this network, we identified a critical miRNA, miR-486, which was the most down-regulated in the radiation-induced carcinogenesis. Then the function of miR-486 was confirmed by using knockout mice and cellular experiments. As a result, miR-486 could inhibit proliferation of mouse lymphoma cells by targeting IGF2BP3 mRNA. The adenovirus over-expression miR-486 vector reduced tumorigenesis in vivo. MiR-486 knockout mice have a strong tendency of radiation-induced carcinogenesis. In conclusion, miR-486 inhibits the proliferation of lymphoma cells and tumorigenesis induced by radiation through targeting IGF2BP3.

Transplantation of Unirradiated Bone Marrow Cells after Total-Body Irradiation Prevents the Development of Thymic Lymphoma in Mice through Niche Competition.

Mouse models of radiation-induced thymic lymphoma are commonly used to study the biological effects of total-body irradiation (TBI) on the formation of hematologic malignancies. It is well documented that radiation-induced thymic lymphoma can be inhibited by protecting the bone marrow (BM) from irradiation; however, the mechanisms underlying this phenomenon are poorly understood. Here, we aimed to address this question by performing transplantation of BM cells from genetically engineered mice that have defects in tumor immunosurveillance or occupying different thymic niches. We found that BM cells from mice that have impaired tumor immunosurveillance, by deleting tumor necrosis factor alpha (TNFα), interferon gamma (IFNγ) or perforin-1 (PRF1), remained sufficient to suppress the formation of radiation-induced thymic lymphoma. On the other hand, BM cells from Rag2-/-; γc-/- mice and Rag2-/- mice, which have defects in occupying thymic niches beyond double negative (DN2) and DN3, respectively, failed to inhibit radiation-induced lymphomagenesis in the thymus. Taken together, based on our findings, we propose a model where unirradiated BM cells suppress radiation-induced lymphomagenesis in the thymus by competing with tumor-initiating cells for thymic niches beyond the DN3 stage.

Loss of Setd4 delays radiation-induced thymic lymphoma in mice

Radiation-induced lymphomagenesis results from a clonogenic lymphoid cell proliferation due to genetic alterations and immunological dysregulation. Mouse models had been successfully used to identify risk and protective factors for radiation-induced DNA damage and carcinogenesis. The mammalian SETD4 is a poorly understood putative methyl-transferase. Here, we report that conditional Setd4 deletion in adult mice significantly extended the survival of radiation-induced T-lymphoma. However, in Tp53 deficient mice, Setd4 deletion did not delay the radiation-induced lymphomagenesis although it accelerated the spontaneous T-lymphomagenesis in non-irradiated mice. The T-lymphomas were largely clonogenic in both Setd4flox/flox and Setd4Δ/Δ mice based on sequencing analysis of the T-cell antigen β receptors. However, the Setd4Δ/Δ T-lymphomas were CD4+/CD8+ double positive, while the littermate Setd4flox/floxtumor were largely CD8+ single positive. A genomic sequencing analysis on chromosome deletion, inversion, duplication, and translocation, revealed a larger contribution of inversion but a less contribution of deletion to the overall chromosome rearrangements in the in Setd4Δ/Δ tumors than the Setd4flox/flox tumors. In addition, the Setd4flox/flox mice died more often from the large sizes of primary thymus lymphoma at earlier time, but there was a slight increase of lymphoma dissemination among peripheral organs in Setd4Δ/Δ at later times. These results suggest that Setd4 has a critical role in modulating lymphomagenesis and may be targeted to suppress radiation-induced carcinogenesis.

Down regulation of miR-143 promotes radiation – Induced thymic lymphoma by targeting B7H1

MicroRNA-143 has been implicated in tumor metastasis by directly targeting Bcl-2, and microRNA-143 expression is decreased in several human tumors. However, the expression and targets of miR-143 in radiation carcinogenesis remain unclear. We found that the expression of miR-143 is down-regulated and the expression of B7H1 (Pdcd1) is up-regulated in radiation-induced thymic lymphoma model in BALB/c mice. Additionally, overexpression of miR-143 strongly inhibited cell proliferation and increased cell apoptosis and its down-regulation promoted cell proliferation and reduced cell apoptosis. We also determined that there is an inverse correlation between miR-143 expression and B7H1 protein expression in radiation-induced thymic lymphoma samples, and miR-143 targets B7H1 in a 3′UTR-dependent manner. In addition, we found that adenovirus over-expression of pre-miR-143 reduced tumorigenesis in vivo. Finally, we conclude that down-regulated expression of miR-143 and up-regulation of its direct target B7H1 may indicate a novel therapeutic method for radiation-induced thymic lymphoma by increased expression of miR-143 or inhibition of B7H1.

Modification of radiation-induced murine thymic lymphoma incidence by curcumin

Introduction: Curcumin is a known antioxidant, preventing radiation damage including carcinogenesis. However, concentration and feeding schedule of curcumin in modification of radiation induced thymic lymphoma incidence in vivo model has not been studied. Materials and Methods: We report here modification of incidence of γ-radiation-induced thymic lymphoma in mice fed with different doses of curcumin (0.05 to 1*) in diet. Results: Female Swiss mice (6-8 weeks) fed with normal diet and exposed to 3 Gy whole body60Co γ-irradiation (WBI) showed 85 * incidence of thymic lymphoma (TL) at 120 days post-irradiation. A concentration of 1 * curcumin was found the most effective in TL incidence prevention than other fed concentrations. The TL incidence was remarkably reduced when curcumin was fed to the mice before than after the radiation exposure. The incidence of TL was reduced to 63* in mice fed with 1* curcumin in diet for 3 weeks after exposure to WBI. On the other hand, when animals were fed with same concentration of curcumin for 2 weeks and 3 weeks before WBI, the TL incidence was reduced to 55* and 35*, respectively. Curcumin feeding resulted in significant prevention in micronucleus formation in the bone marrow, which was corroborated with inhibition in DNA damage quantified by comet assay. Moreover, significant prevention in DNA damage was also observed in the peripheral blood cells in curcumin fed and irradiated mice, which however, was not prominent in thymus. Curcumin was able to prevent apoptotic death in thymus and bone marrow 4 h after irradiation, which however, got attenuated at longer post-irradiation period (24 h). Conclusion: These results suggest modification of TL incidence by curcumin in irradiated mice involving DNA damage and apoptotic death mechanisms.

Nucks1 synergizes with Trp53 to promote radiation lymphomagenesis in mice.

NUCKS1 is a 27 kD vertebrate-specific protein, with a role in the DNA damage response. Here, we show that after 4 Gy total-body X-irradiation, Trp53+/− Nucks1+/− mice more rapidly developed tumors, particularly thymic lymphoma (TL), than Trp53+/− mice. TLs in both cohorts showed loss of heterozygosity (LOH) of the Trp53+ allele in essentially all cases. In contrast, LOH of the Nucks1+ allele was rare. Nucks1 expression correlated well with Nucks1 gene dosage in normal thymi, but was increased in the majority of TLs from Trp53+/− Nucks1+/− mice, suggesting that elevated Nucks1 message may be associated with progression towards malignancy in vivo. Trp53+/− Nucks1+/− mice frequently succumbed to CD4- CD8- TLs harboring translocations involving Igh but not Tcra/d, indicating TLs in Trp53+/− Nucks1+/− mice mostly originated prior to the double positive stage and at earlier lineage than TLs in Trp53+/- mice. Monoclonal rearrangements at Tcrb were more prevalent in TLs from Trp53+/− Nucks1+/− mice, as was infiltration of primary TL cells to distant organs (liver, kidney and spleen). We propose that, in the context of Trp53 deficiency, wild type levels of Nucks1 are required to suppress radiation-induced TL, likely through the role of the NUCKS1 protein in the DNA damage response.

Mouse models for radiation-induced cancers.

Potential ionising radiation exposure scenarios are varied, but all bring risks beyond the simple issues of short-term survival. Whether accidentally exposed to a single, whole-body dose in an act of terrorism or purposefully exposed to fractionated doses as part of a therapeutic regimen, radiation exposure carries the consequence of elevated cancer risk. The long-term impact of both intentional and unintentional exposure could potentially be mitigated by treatments specifically developed to limit the mutations and precancerous replication that ensue in the wake of irradiation The development of such agents would undoubtedly require a substantial degree of in vitro testing, but in order to accurately recapitulate the complex process of radiation-induced carcinogenesis, well-understood animal models are necessary. Inbred strains of the laboratory mouse, Mus musculus, present the most logical choice due to the high number of molecular and physiological similarities they share with humans. Their small size, high rate of breeding and fully sequenced genome further increase its value for use in cancer research. This chapter will review relevant m. musculus inbred and F1 hybrid animals of radiation-induced myeloid leukemia, thymic lymphoma, breast and lung cancers. Method of cancer induction and associated molecular pathologies will also be described for each model.

Expression profiling reveals transcriptional regulation by Fbxw7/mTOR pathway in radiation-induced mouse thymic lymphomas

The tumor suppressor gene FBXW7 is deleted and mutated in many different types of human cancers. FBXW7 primarily exerts its tumor suppressor activity by ubiquitinating different oncoproteins including mTOR. Here we used gene transcript profiling to gain a deeper understanding of the role of FBXW7 in tumor development and to determine the influence of mTOR inhibition by rapamycin on tumor transcriptome and biological functions. In comparison to tumors from p53 single heterozygous (p53+/-) mice, we find that radiation-induced thymic lymphomas from Fbxw7/p53 double heterozygous (Fbxw7+/-p53+/-) mice show significant deregulation of cholesterol metabolic processes independent of rapamycin treatment, while cell cycle related genes were upregulated in tumors from placebo treated Fbxw7+/-p53+/- mice, but not in tumors from rapamycin treated Fbxw7+/-p53+/- mice. On the other hand, tumors from rapamycin treated Fbxw7+/-p53+/- mice were enriched for genes involved in the integrated stress response, an adaptive mechanism to survive in stressful environments. Finally, we demonstrated that the Fbxw7 gene signatures identified in mouse tumors significantly overlap with FBXW7 co-expressed genes in human cancers. Importantly these common FBXW7 gene signatures between mouse and human are predictive for disease-free survival in human colon, breast and lung adenocarcinoma cancer patients. These results provide novel insights into the role of FBXW7 in tumor development and have identified a number of potential targets for therapeutic intervention.

Radiation-driven lipid accumulation and dendritic cell dysfunction in cancer.

Dendritic cells (DCs) play important roles in the initiation and maintenance of the immune response. The dysfunction of DCs contributes to tumor evasion and growth. Here we report our findings on the dysfunction of DCs in radiation-induced thymic lymphomas, and the up-regulation of the expression of the lipoprotein lipase (LPL) and the fatty acid binding protein (FABP4), and the level of triacylglycerol (TAG) in serum after total body irradiation, which contribute to DCs lipid accumulation. DCs with high lipid content showed low expression of co-stimulatory molecules and DCs-related cytokines, and were not able to effectively stimulate allogeneic T cells. Normalization of lipid abundance in DCs with an inhibitor of acetyl-CoA carboxylase restored the function of DCs. A high-fat diet promoted radiation-induced thymic lymphoma growth. In all, our study shows that dysfunction of DCs in radiation-induced thymic lymphomas was due to lipid accumulation and may represent a new mechanism in radiation-induced carcinogenesis.

MiR-467a is upregulated in radiation-induced mouse thymic lymphomas and regulates apoptosis by targeting Fas and Bax.

It has been reported dysregulation of certain microRNAs (miRNAs / miRs) is involved in tumorigenesis. However, the miRNAs associated with radiocarcinogenesis remain undefined. In this study, we validated the upregulation of miR-467a in radiation-induced mouse thymic lymphoma tissues. Then, we investigated whether miR-467a functions as an oncogenic miRNA in thymic lymphoma cells. For this purpose, we assessed the biological effect of miR-467a on thymic lymphoma cells. Using miRNA microarray, we found four miRNAs (miR-467a, miR-762, miR-455 and miR-714) were among the most upregulated (>4-fold) miRNAs in tumor tissues. Bioinformatics prediction suggests miR-467a may potentially regulate apoptosis pathway via targeting Fas and Bax. Consistently, in miR-467a-transfected cells, both proliferation and colony formation ability were significantly increased with decrease of apoptosis rate, while, in miR-467a-knockdown cells, proliferation was suppressed with increase of apoptosis rate, indicating that miR-467a may be involved in the regulation of apoptosis. Furthermore, miR-467a-knockdown resulted in smaller tumors and better prognosis in an in vivo tumor-transplanted model. To explain the mechanism of apoptosis suppression by miR-467a, we explore the expression of candidate target genes (Fas and Bax) in miR-467a-transfected relative to negative control transfected cells using flow cytometry and immunoblotting. Fas and Bax were commonly downregulated in miR-467a-transfected EL4 and NIH3T3 cells, and all of the genes harbored miR-467a target sequences in the 3'UTR of their mRNA. Fas and Bax were actually downregulated in radiation-induced thymic lymphoma tissues, and therefore both were identified as possible targets of miR-467a in thymic lymphoma. To ascertain whether downregulation of Fas and / or Bax is involved in apoptosis suppression by miR-467a, we transfected vectors expressing Fas and Bax into miR-467a-upregulated EL4 cells. Then we found that both Fas- and Bax-overexpression decreased cell viability with increase of apoptosis rate, indicating that downregulation of Fas and Bax may be at least partly responsible for apoptosis suppression by miR-467a. These data suggest that miR-467a may have oncogenic functions in radiation-induced thymic lymphoma cells and that its increased expression may confer a growth advantage on tumor cells via aberrant expression of Fas and Bax.

Quantifying initial cellular events of mouse radiation lymphomagenesis and its tumor prevention in vivo by positron emission tomography and magnetic resonance imaging

Radiation-induced thymic lymphoma (RITL) in mice is induced by fractionated whole-body X-irradiation (FX) and has served as a useful model for studying radiation carcinogenesis. In this model, the initial postirradiation cellular events in the thymus and bone marrow (BM) are critically important for tumorigenesis, and BM transplantation (BMT) prevents RITL. However, direct assessment of these events is so far restricted by the lack of noninvasive monitoring techniques. Here, we have developed positron emission tomography (PET) and magnetic resonance imaging (MRI) methods to quantify the events critical for RITL development and the effects of BMT in living animals. Apparent diffusion coefficients (ADCs) were calculated from diffusion-weighted MRI to evaluate the changes in the BM of mice receiving FX. ADC values dramatically changed in the irradiated BM, corresponding to pathological findings of the irradiated BM, returning to normal levels following BMT sooner than with spontaneous recovery. PET with 4ʹ-[methyl-11C]thiothymidine, a novel tracer for cell proliferation, revealed that the irradiated thymus showed significantly higher tracer uptake than the unirradiated thymus 1 week after FX. Interestingly, its increased uptake was completely abolished by BMT, even with very few donor-derived cells in the thymus. Thereafter, the thymus receiving BMT had significantly increased tracer uptake. These findings suggest that BMT first suppresses FX-induced aberrant thymocyte proliferation and then accelerates thymic regeneration. This study demonstrates the feasibility of using PET and MRI for noninvasive monitoring of tumorigenic cellular processes in an animal model of radiation-induced cancer.

Increased p16 DNA methylation in mouse thymic lymphoma induced by irradiation.

DNA methylation is an important part of epigenetics. In this study, we examined the methylation state of two CpG islands in the promoter of the p16 gene in radiation-induced thymic lymphoma samples. The mRNA and protein levels of P16 were significantly reduced in radiation-induced thymic lymphoma tissue samples. Twenty-three CpG sites of the CpG islands in the p16 promoter region were detected, and the methylation percentages of −71, −63, −239, −29, −38, −40, −23, 46 CpG sites were significantly higher in radiation-induced thymic lymphoma tissue samples than those in matched non-irradiated thymus tissue samples. This study provides new evidence for the methylation state of p16 in the radiation-induced thymic lymphoma samples, which suggests that the methylation of these CpG sites in the p16 promoter may reduce its expression in the thymic lymphoma after irradiation.

Down Regulation of miR200c Promotes Radiation‐Induced Thymic Lymphoma by Targeting BMI1

The miR-200c has recently been implicated in the epithelial to mesenchymal transition (EMT) process by directly target the EMT related transcriptional factors ZEB1 and ZEB2. The expression of this miRNA is inversely correlated with tumorgenecity and invasiveness in several human cancers. However, little is known about the expression and targets of the miR-200c in radiation carcinogenesis. Here in this study, using a split radiation induced thymic lymphoma (RITL) model in BALB/c mice, we found that miR-200c is down-regulated in RITL samples. Cell death and apoptosis in lymphoma cells was induced by miR-200c mimic while decreased by miR-200c inhibitor. Computational analysis found a putative target site of miR-200c in the 3'UTR of one of the polycomb group (PcG) protein BMI1 mRNA, which was verified by a luciferase reporter assay. Forced over-expression of miR-200c decreased the level of BMI1 protein and moreover, over-expression of BMI1 rescued the biological effects of miR-200c, indicating BMI1 is a direct mediator of miR-200c functions. Furthermore, the BMI1 expression level was up-regulated and inversely correlated with miR-200c in RITL samples. Finally, our data also indicates that Adenovirus over-expression of pre-miR-200c reduced tumorgenesis in vivo. Taken together, we conclude that down-regulated expression of miR-200c and up-regulation of its direct target BMI1 in radiation-induced thymic lymphoma, which may indicate a novel therapeutic method for RITL through induction of miR-200c or inhibition of BMI1.

Illegitimate V(D)J recombination-mediated deletions in Notch1 and Bcl11b are not sufficient for extensive clonal expansion and show minimal age or sex bias in frequency or junctional processing

Illegitimate V(D)J recombination at oncogenes and tumor suppressor genes is implicated in formation of several T cell malignancies. Notch1 and Bcl11b, genes involved in developing T cell specification, selection, proliferation, and survival, were previously shown to contain hotspots for deletional illegitimate V(D)J recombination associated with radiation-induced thymic lymphoma. Interestingly, these deletions were also observed in wild-type animals. In this study, we conducted frequency, clonality, and junctional processing analyses of Notch1 and Bcl11b deletions during mouse development and compared results to published analyses of authentic V(D)J rearrangements at the T cell receptor beta (TCRβ) locus and illegitimate V(D)J deletions observed at the human, nonimmune HPRT1 locus not involved in T cell malignancies. We detect deletions in Notch1 and Bcl11b in thymic and splenic T cell populations, consistent with cells bearing deletions in the circulating lymphocyte pool. Deletions in thymus can occur in utero, increase in frequency between fetal and postnatal stages, are detected at all ages examined between fetal and 7 months, exhibit only limited clonality (contrasting with previous results in radiation-sensitive mouse strains), and consistent with previous reports are more frequent in Bcl11b, partially explained by relatively high Recombination Signal Information Content (RIC) scores. Deletion junctions in Bcl11b exhibit greater germline nucleotide loss, while in Notch1 palindromic (P) nucleotides are more abundant, although average P nucleotide length is similar for both genes and consistent with results at the TCRβ locus. Non-templated (N) nucleotide insertions appear to increase between fetal and postnatal stages for Notch1, consistent with normal terminal deoxynucleotidyl transferase (TdT) activity; however, neonatal Bcl11b junctions contain elevated levels of N insertions. Finally, contrasting with results at the HPRT1 locus, we find no obvious age or gender bias in junctional processing, and inverted repeats at recessed coding ends (Pr nucleotides) correspond mostly to single-base additions consistent with normal TdT activity.

Up-Regulated Expression of miR-23a/b Targeted the Pro-Apoptotic Fas in Radiation-Induced thymic Lymphoma

Background: MicroRNAs (miRNAs) are small, single-stranded, noncoding RNAs, which usually bind to the 3'-untranslated region of target mRNAs and are capable of inducing posttranscriptional gene regulation by blocking translation or by degrading the target mRNA. However, the expression level of miR-23 in radiation induced carcinogenesis is largely unknown. Methods: Radiation induced thymic lymphoma model in BALB/c mice was set up. miR-23a & miR-23b miRNA levels in different tissues and cells were detected by real-time qPCR. miR-23a/b inhibitor and miR-23a/b mimics were transfected to lymphoma cells and the target of miR-23a/b was identified by microRNA target prediction and Luciferase assays. Results: We found that miR-23a & miR-23b were up-regulated in radiation induced thymic lymphoma tissue samples. Cell death and apoptosis were increased by miR-23a/b inhibitor and decreased by miR-23a/b mimics in lymphoma cells. Computational analysis found a putative target site of miR-23a/b in the 3′UTR of Fas mRNA, which was verified by luciferase reporter assay. Forced over-expression of miR-23a/b decreased the level of Fas protein. Moreover, over-expression of Fas rescued the pro-proliferation effect of miR-23, indicating Fas is a direct mediator of miR-23 functions. Furthermore, contrast to miR-23a/b which was up regulated, the Fas expression level was down-regulated and inversely correlated with miR-23 in split radiation induced lymphoma tissue samples. Finally, our data also indicates that miR-23a could repress Fas much more potent than miR-23b and the additional region besides conserved seed pairing enables miR-23a's higher regulation. Conclusions: In this study, using a radiation induced thymic lymphoma model in BALB/c mice, We conclude that the expression of miR-23a/b is up-regulated in radiation-induced thymic lymphoma and it maybe a novel therapeutic target of that cancer.