Manganese superoxide dismutase gene dosage affects chromosomal instability and tumor onset in a mouse model of T cell lymphoma

Abstract

Increased reactive oxygen species (ROS) such as superoxide have been implicated as causal elements of oncogenesis. A variety of cancers have displayed changes in steady-state levels of key antioxidant enzymes, with the mitochondrial form of superoxide dismutase (MnSOD) being commonly implicated. Increasing MnSOD expression suppresses the malignant phenotype in various cancer cell lines and suppresses tumor formation in xenograft and transgenic mouse models. We examined the impact of MnSOD expression in the development of T cell lymphoma in mice expressing proapoptotic Bax. Lck-Bax38/1 transgenic mice were crossed to mice overexpressing MnSOD (Lck-MnSOD) as well as MnSOD+/- mice. The effects of MnSOD on apoptosis, cell cycle, chromosomal instability (CIN), and lymphoma development were determined. The apoptotic and cell cycle phenotypes observed in thymocytes from control and Bax transgenic mice were unaffected by variations in MnSOD levels. Remarkably, increased gene dosage of MnSOD significantly decreased aneuploidy in premalignant thymocytes as well as the onset of tumor formation in Lck-Bax38/1 mice. The observed effects of MnSOD support a role for ROS in CIN and tumor formation in this mouse model of T cell lymphoma.

Figure 1. Increased DHE oxidation in Lck-Bax38/1 transgenic mice

Thymocytes from control and Lck-Bax38/1 mice were isolated and stained with DHE as described in the Materials and Methods. Contour plots of viable (based on FSC-SSC staining) Thy1.2-positive lymphocytes is shown. The plots illustrate the two populations that are observed in these experiments and the gating strategy use to analyze the DHE high population. (B) Representative histogram overlay of the DHE positive population from the Lck-Bax38/1 sample (heavy dark line) and the control sample (light gray line). (C) The FL2-H mean fluorescence intensity (DHE-MFI) values (Mean +/− SEM) from four independent experiments is shown. In all four experiments, thymocytes from Lck-Bax38/1 mice had increased DHE staining relative to control mice.*P <0.01 using a two-way analysis of variance (ANOVA).

Figure 2. Altered antioxidant levels in Lck-Bax38/1 thymic tumors

Protein levels of key antioxidants were assessed in thymi isolated from control and MnSOD +/+ Lck-Bax38/1 premalignant mice and thymic tumors from these mice. Immunoblot analysis for MnSOD, CuZnSOD, and Catalase from pre-malignant thymi (A) and thymic tumors (C) are shown and compared to samples from three control non-transgenic thymi. Quantification of antioxidant protein expression in pre-malignant thymi (B) or thymic tumors (D) from Lck-Bax38/1 mice is normalized to Actin. Each diamond corresponds to the relative amount of protein expression from an individual thymus or thymic tumor. Statistical significance for differences in antioxidant expression in both Lck-Bax38/1 pre-malignant thymi and thymic tumors were assessed by a two-tailed Student’s t test. The mean value for all three enzymes were not statistically different between pre-malignant Lck-Bax38/1 mice and control thymi while the mean values in the tumors were significantly different from the controls (*p<0.05).

Figure 3. Increased expression and activity of MnSOD in transgenic mice

Thymocyte lysates were isolated from three control and three Lck-MnSOD transgenic mice as described in the Materials and Methods. (A) Immunoblot analysis for MnSOD from transgene negative (MnSOD +/+) and transgene positive mice is shown. (B) Quantification (Mean ± SD) of MnSOD thymocyte protein expression normalized to Actin is shown. (C) MnSOD activity (Mean ± SD) in thymocyte lysates from three transgene negative (MnSOD +/+) and three Lck-MnSOD mice is shown.

Figure 4. MnSOD gene dosage does not affect thymic cellularity and apoptosis

(A) The total number of thymocytes from mice of the indicated genotypes is shown. Each diamond corresponds to the total number of viable thymocytes observed from an individual mouse. (B) The graph shows in vitro thymocyte viability (trypan blue exclusion) from mice of the indicated genotypes either 12 hours after treatment with 1 μM dexamethasone (Black bars) or 48 hours in culture in the absence of any treatment (Gray bars). The data shown are the mean ± SD from two independent experiments.

Figure 5. MnSOD gene dosage does not alter thymocyte cell cycle in control or Lck-Bax38/1 transgenic mice

(A) Shown are representative DNA content profiles or propidium iodide stained cells from mice of the indicated genotypes. The percentage of proliferating (%S/G2/M) cells for each panel is indicated. (B) The % S/G2/M (mean ± SD) of thymocytes from 6–12 week old mice of the indicated genotype is shown. A minimum of four mice was examined in each group. While Lck-Bax38/1 transgenic mice had a higher fraction of dividing cells, MnSOD gene dosage had no significant effect on thymocyte proliferation in either control or Lck-Bax38/1 mice.

Figure 6. MnSOD gene dosage affects thymic aneuploidy in Lck-Bax38/1 transgenic mice

Shown are the percentage of aneuploid thymocytes from mice between 6 and 12 weeks of age. Each diamond represents the percentage of aneuploid cells observed in a minimum of 50 thymic metaphase spreads from each individual mouse. Statistical significance for ploidy analysis was assessed by Fisher’s exact test as described in the Materials and Methods. *p<0.01 vs transgene negative control mice. Lck-MnSOD expression attenuated aneuploidy in Lck-Bax38/1 mice. †p<0.05 vs MnSOD +/+ Lck-Bax38/1 mice. ‡p<0.01 vs MnSOD +/− Lck-Bax38/1 mice.

Figure 7. MnSOD gene dosage affects the onset of tumorigenesis in Lck-Bax38/1 transgenic mice

Kaplan-Meier lymphoma free survival analysis of Lck-Bax38/1 mice on backgrounds expressing various levels of MnSOD is shown. MnSOD +/+ (●), MnSOD +/− (□), and Lck-MnSOD (♦) mice were followed for lymphoma free survival as described in the Materials and Methods. Lymphoma formation in Lck-Bax38/1 mice was significantly (P=0.015) accelerated in MnSOD +/− mice compared to Lck-MnSOD transgene positive mice. Tumor formation of the MnSOD +/+ Lck-Bax38/1 was not significantly different from either the MnSOD +/− (P=0.062) or Lck-MnSOD (P=0.400) but survival of these mice was intermediate between the groups supporting a dose response relationship between tumor formation and MnSOD expression levels.

Figure 8. MnSOD protein expression is retained in MnSOD +/− Lck-Bax38/1 thymic tumors

Thymocyte and thymic tumor lysates were prepared from mice of the indicated genotypes. (A) Immunoblot analysis for MnSOD expression in three MnSOD +/+ control thymi and ten MnSOD +/−Lck-Bax38/1 thymic tumors is shown. (B) Quantification (Mean ± SD) of MnSOD protein expression normalized to Actin is shown. The means of these groups were not significantly different.

Figure 9. Model for Bax induced lymphoma formation

Previous studies suggest that ROS may promote malignant transformation by one of two routes either involving cellular proliferation or genomic instability. We hypothesized that Bax-induced lymphoma was mediated in part by alterations in ROS. The current study supports this hypothesis by demonstrating that MnSOD levels alter aneuploidy and the rate of tumor formation in Lck-Bax38/1 mice. Furthermore, since MnSOD had no impact on pre-malignant cellular proliferation in this model, the data suggest that the major route for ROS involvement in this model is via alterations in genomic instability (heavy arrows) as opposed to changes in mitogenic pathways (dotted arrows).