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. 2012 Nov 27;109(48):19685-90.
doi: 10.1073/pnas.1206406109. Epub 2012 Nov 12.

A critical role for Mnt in Myc-driven T-cell proliferation and oncogenesis

Jason M Link  1 , Sara Ota, Zi-Qiang Zhou, Colin J Daniel, Rosalie C Sears, Peter J Hurlin
Affiliations

A critical role for Mnt in Myc-driven T-cell proliferation and oncogenesis

Jason M Link et al. Proc Natl Acad Sci U S A. .

Abstract

Mnt (Max's next tango) is a Max-interacting transcriptional repressor that can antagonize both the proproliferative and proapoptotic functions of Myc in vitro. To ascertain the physiologically relevant functions of Mnt and to help define the relationship between Mnt and Myc in vivo, we generated a series of mouse strains in which Mnt was deleted in T cells in the absence of endogenous c-Myc or in the presence of ectopic c-Myc. We found that apoptosis caused by loss of Mnt did not require Myc but that ectopic Myc expression dramatically decreased the survival of both Mnt-deficient T cells in vivo and Mnt-deficient MEFs in vitro. Consequently, Myc-driven proliferative expansion of T cells in vitro and thymoma formation in vivo were prevented by the absence of Mnt. Consistent with T-cell models, mouse embryo fibroblasts (MEFs) lacking Mnt were refractory to oncogenic transformation by Myc. Tumor suppression caused by loss of Mnt was linked to increased apoptosis mediated by reactive oxygen species (ROS). Thus, although theoretically and experimentally a Myc antagonist, the dominant physiological role of Mnt appears to be suppression of apoptosis. Our results redefine the physiological relationship between Mnt and Myc and requirements for Myc-driven oncogenesis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Control of thymocyte production and survival by Mnt and Myc. (A) Single-cell suspensions of thymocytes were cultured ex vivo for 24 h, and the percentage of mature thymocytes (CD4+CD8+) surviving (AnnexinVNEG7AADNEG) was assessed by FACS. Each dot represents cell survival from one thymus. (B) Thymocytes were counted, and the number of cells per thymus was normalized to an age-matched control thymus in the same experiment to avoid age-related changes in thymocyte number. Each dot represents thymus cellularity from one mouse; n represents the number of control thymi analyzed. (C) The plots are representative FACS plots from thymocytes stained with CD4 (x axis) and CD8 (y axis) and illustrate the relative dearth of mature thymocytes when Mnt and/or Myc are absent. The images in C are representative thymi from each mouse type and illustrate that the number of mature thymocytes correlates with thymus size.
Fig. 2.
Fig. 2.
Mnt is required for survival of ex vivo mitogen-stimulated T cells. (A) CD4+ splenic T cells from control and MntTcKO mice were cultured with 1µg/mL ConA and relative amounts of Myc and Mnt transcripts were assessed by quantitative RT-PCR using β-actin as a control. Amount of RNA is given as the percentage change from unstimulated (0hr) control cells. (B) Splenocytes were cultured for 48 h with the indicated concentrations of ConA, and the percentage of CD4+ cells surviving (AnnexinVNEG7AADNEG) was determined by FACS. The mean ± SD from four separate experiments is given. Conditions marked with an asterisk (*) have significantly (P < 0.05) lower survival than control cells at the same concentration of ConA. NS, not significant. (C) Splenocytes were cultured for 5 d with the indicated concentrations of ConA. The number of surviving CD4+ cells for each condition was calculated from the total number of cells generated and the percentage of live CD4+ cells in each culture. Fold expansion from unstimulated cells is plotted. Data are representative of five separate experiments. (D) CD4+ T cells were isolated from splenocytes by magnetic bead depletion, and 1 × 106 cells were cultured without or with 1.0 µg/mL ConA and with 10 µM BrdU for 48 h. FACS detection of BrdU incorporation was used to measure DNA synthesis and 7AAD fluorescence was used to measure cell cycle stage.
Fig. 3.
Fig. 3.
Myc-mediated tumorigenesis is dependent on Mnt. (A) The survival of mice with conditional, biallelic, ectopic expression of MycT58A in T cells is significantly increased when Mnt is conditionally deleted in T cells (P < 0.0001). (B) Examples of a thymoma found in TMycT58A mice and the lack of thymoma formation in MntTcKO+TMycT58A mice. (C) Hematoxylin/eosin (H&E) histology of thymi from the indicated mouse strains and ages. The dashed line demarcates the medulla region. (D) Mnt deficiency has a significant effect on survival of TMycT58A thymocytes. Thymocyte survival was assessed as in Fig. 1. Data are from at least three thymi per mouse type.
Fig. 4.
Fig. 4.
Mnt is required for MEF survival and tumorigenesis. (A, C, and E) Immortal WT and MntKO MEFs expressing combinations of ectopic MycWT, MycT58A, Bcl-2, or RasG12V were cultured for 24 h in complete media or in media deprived of serum (i.e., 0.1% FBS) and glutamine. The percentage of surviving (AnnexinVNEG7AADNEG) cells was determined by FACS. Data are the means (± SD) of four independent experiments. (B, D, and F) Immortal MEFs expressing MycWT, MycT58A, Bcl-2, or RasG12V were injected s.c. in nude mice at each shoulder per nude mouse and eight mice per cell type to test for tumorigenicity. The frequency of tumor formation observed is given below each photo. Mice injected with WT MEFs expressing MycWT, MycT58A Myc+Bcl-2, or Myc+RasG12V formed tumors of ∼2 cm2 within 3 wk and were killed according to Institutional Animal Care and Use Committee (IACUC) guidelines. Mice injected with MntKO MEFs expressing MycWT, MycT58A Myc+Bcl-2, or Myc+RasG12V failed to form tumors, even after more than 3 mo. This assay was repeated using two independent sets of immortal WT and MntKO MEFs (four injections of each set).
Fig. 5.
Fig. 5.
The survival defect in Mnt-deficient T cells and MEFs is ROS-dependent. (A) Splenocytes were cultured at low cell density (0.5 × 106 cells/well) with or without 50 µM 2-ME or at high cell density (2 × 106 cells/well) in the presence of 1 µg/mL ConA. The number of live CD4+ cells was determined before and after 5 d of culture, and the number of doublings is given on the y axis. (B) The relative amount of intracellular ROS was determined by the mean fluorescence intensity of CM-H2DCFDA–stained, live CD4+ T cells before and after 24 h of culture with media alone or 1 µg/mL ConA. (C) The relative amount of intracellular ROS was determined by the mean fluorescence intensity of CM-H2DCFDA–stained subconfluent immortal MEFs after 24 h of culture with or without 100 µg/mL BSO. (D) The percentage of surviving (7AADNEG) immortal MEFs was determined by FACS after 24 h of culture with a range of BSO concentrations. (E) MEFs were treated for 28 h with media only, 50 µM 2-ME, 5 mM GSH, or 5 mM NAC and exposed to 100 µM BSO for the final 24 h of culture. The percentage of surviving (7AADNEG) MEFs was determined by FACS. Error bars represent SD of two replicates. (F) Immortal MEFs (left y axis), thymocytes and CD4+ T cells (right y axis) were stained with the superoxide-specific dye MitoSox, and fluorescence was determined by FACS. Data are given as the fold change in mean fluorescence intensity from WT/control cells (represented by a horizontal black line). Each graph is representative of at least three experiments with nearly identical results. (G) Sorted CD4+ T cells were cultured for 3 d with 1 µg/mL ConA and 10 ng/mL IL-2 and then resuspended in fresh media either with or without 1 µg/mL ConA and 10 ng/mL IL-2. The rate at which cells changed the pH of fresh media (G) or consumed oxygen (H) was determined over time. Error bars represent SD of two replicates. RFU, relative fluorescence units.
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