Recombinant cell-detecting RaDR-GFP in mice reveals an association between genomic instability and radiation-induced-thymic lymphoma

Abstract

In this study, we aimed to investigate how homologous recombinant (HR)-related genomic instability is involved in ionizing radiation (IR)-induced thymic lymphoma in mice. We divided five-week-old Rosa26 Direct Repeat-GFP (RaDR-GFP) transgenic mice into non-IR control and IR groups and exposed the mice in the IR group to a 7.2 Gy dose of γ-rays, delivered in 1.8 Gy fractions, once a week for four weeks. We then estimated mouse survival and recorded their body, thymus, and spleen weights. The frequency of HR events in the chromosomes of the thymus, bone marrow, and spleen cells and the phenotype of thymic lymphoma cells were analyzed using fluorescence-activated cell sorting (FACS). We found that most mice in the IR group developed thymic lymphoma, their survival rate decreasing to 20% after 180 days of IR exposure, whereas no mice died in the non-IR control group until day 400. The thymus and spleen weighed significantly more in the IR-4-month group than that in the non-IR group; however, we observed no significant differences between the body weights of the control and IR mice. FACS analysis indicated that the frequency of HR events significantly increased at two and four months after the last IR dose in the bone marrow and thymus cells, but not in the spleen cells of RaDR-GFP transgenic mice, suggesting that recombinant cells accumulated in the thymus upon IR exposure. This suggests that IR induces genome instability, revealed as increased HR, that drives the development of thymic lymphoma. Additionally, phenotypic analysis of lymphoma cells showed an increase in the CD4^-/CD8⁺ (CD8SP) cell population and a decrease in the CD4⁺/CD8^- (CD4SP) cell population in the IR-4-month group compared to that in the non-IR group, indicating that IR induces an aberrant cell phenotype characteristic of lymphoma. In conclusion, we observed a significant increase in HR events and abnormal phenotype in thymic lymphoma cells at two and four months after IR exposure in both the thymus and bone marrow tissues, suggesting that genomic instability is involved in the early stages of thymic lymphomagenesis. Our study indicates that HR-visualizing RaDR-GFP transgenic mice can help explore the links between the molecular mechanisms of genome instability and IR-induced tumorigenesis.

Keywords: Radiation; genome instability; homologous recombination (replication stress); lymphoma.

Figure 1

The overall survival rate of RaDR-GFP transgenic mice with or without TBI. Mice in the IR group were exposed to 1.8 Gy of γ-rays once a week for 4 weeks. *P<0.05 compared to the control.

Figure 2

(A) Body weight changes in RaDR-GFP transgenic mice 1, 2, and 4 months after TBI. (B) Morphological and weight changes of thymus in RaDR-GFP transgenic mice 1, 2, and 4 months after TBI. (C) Morphological and weight changes of spleen in RaDR-GFP transgenic mice 1, 2, and 4 months after TBI. (D) Histopathological changes in the thymus of non-IR and IR mice. Histopathological changes in the liver (E), pancreas (F), lung (G) and kidney (H) of non-IR and IR mice. Metastases were only seen in the liver, pancreas, lung and kidney of IR mice. Dotted circles indicate the metastasis sites in the various tissues. *P<0.05 compared to the control.

Figure 3

Representative photos of HR events (GFP-positive cells) as observed using a fluorescence microscope in the thymus of IR mice (A), non-IR control (B), and the pancreas of non-IR control (C) RaDR-GFP transgenic mice. Arrows indicate GFP-positive cells in the thymic and pancreatic tissues of RaDR-GFP transgenic mice.

Figure 4

HR frequency analysis using FACS in the thymus, bone marrow, and spleen tissues of RaDR-GFP transgenic mice with or without TBI. For each of three experiments, there were three technical repeats. Light bars represent control samples and dark bars represent IR-treated samples. *P<0.05 compared to the control.

Figure 5

Phenotypic analysis of lymphoma cells using FACS in thymic tissues of RaDR-GFP transgenic mice with or without TBI. (A) Representative flow cytometry charts illustrating CD4^-/CD8^- (DN), CD4⁺/CD8⁺ (DP), CD4⁺/CD8^- (CD4SP), and CD4^-/CD8⁺ (CD8SP) cells. Percentage changes of CD4^-/CD8⁺ (B), CD4⁺/CD8^- (C), CD4⁺/CD8⁺ (D), and CD4^-/CD8^- (E) cells in the thymus of non-IR and IR mice. For each of three experiments, there were three technical repeats. Light bars represent control samples and dark bars represent IR-treated samples. *P<0.05, #P<0.01 compared to the control.