Figure 6
The critical role for PML in suppression of Myc-driven lymphomas: elevation of PML and PML-NB in lymphoma cells and premalignant B-lymphoid cells from Eμ-myc/E6AP+/− mice. (A) Immunoblot analysis of the indicated proteins in extracts from Eμ-myc/E6AP+/− and control (E6AP+/+) Eμ-myc lymphomas. Probing for actin was used as a loading control. PML isoforms are indicated. (B) Immunofluorescence staining of PML nuclear bodies in lymphomas from Eμ-myc/E6AP+/− and control (E6AP+/+) Eμ-myc mice. Magnification ×1000. (C) Immunoblot analyses of the indicated proteins in premalignant B-lymphoid cells from 4-week-old Eμ-myc/E6AP+/− and control (E6AP+/+) Eμ-myc mice. Probing for actin was used as a loading control. (D) Immunoblot analyses of the indicated proteins in B lymphoid cells from 4-week-old E6AP+/− and control WT (E6AP+/+) mice. Probing for actin was used as a loading control. Lymphoma extract (A, lane 11) was used as a positive control (C). (E) PML loss accelerates tumor onset in Myc-expressing tumors. Eμ-myc transgenic mice were crossed to PML−/− mice. The resulting Eμ-myc mice that were either WT, Pml+/−, or Pml−/− were monitored for tumor onset by spleen as well as lymph node palpation and weekly blood smear analysis. The onset of lymphomas in both Eμ-myc/Pml+/− and Eμ–myc/Pml−/− mice was substantially accelerated compared with control Eμ-myc mice (P < .01). (F) Staining of tissue sections with hematoxylin and eosin revealed that Eμ-myc/Pml−/− lymphomas were highly invasive and infiltrated into the liver, but not kindey or lung. Magnification ×200. (G). HSCs derived from fetal livers of WT, p53−/−, and Pml−/− mice were retrovirally transduced with a MSCV-Myc construct coexpressing green fluorescent protein. The genetically modified stem cells were then used to reconstitute the hematopoietic system of lethally irradiated recipient (WT) animals, which were monitored for lymphoma onset by palpation, weekly blood smears, and whole body fluorescence imaging. Recipients of WT stem cells transduced with the Myc expression did not develop tumors over the observation period, whereas all of the recipients of Myc expression construct transduced p53−/− HSC developed very aggressive disease in a short time. Recipients of Myc expression construct transduced Pml−/− HSCs developed lymphoma more rapidly compared with the mice transplanted with the Myc expression construct transduced WT HSCs (P < .0003), again suggesting that PML can suppress Myc driven lymphoma development.

The critical role for PML in suppression of Myc-driven lymphomas: elevation of PML and PML-NB in lymphoma cells and premalignant B-lymphoid cells from Eμ-myc/E6AP+/− mice. (A) Immunoblot analysis of the indicated proteins in extracts from Eμ-myc/E6AP+/− and control (E6AP+/+) Eμ-myc lymphomas. Probing for actin was used as a loading control. PML isoforms are indicated. (B) Immunofluorescence staining of PML nuclear bodies in lymphomas from Eμ-myc/E6AP+/− and control (E6AP+/+) Eμ-myc mice. Magnification ×1000. (C) Immunoblot analyses of the indicated proteins in premalignant B-lymphoid cells from 4-week-old Eμ-myc/E6AP+/− and control (E6AP+/+) Eμ-myc mice. Probing for actin was used as a loading control. (D) Immunoblot analyses of the indicated proteins in B lymphoid cells from 4-week-old E6AP+/− and control WT (E6AP+/+) mice. Probing for actin was used as a loading control. Lymphoma extract (A, lane 11) was used as a positive control (C). (E) PML loss accelerates tumor onset in Myc-expressing tumors. Eμ-myc transgenic mice were crossed to PML−/− mice. The resulting Eμ-myc mice that were either WT, Pml+/−, or Pml−/− were monitored for tumor onset by spleen as well as lymph node palpation and weekly blood smear analysis. The onset of lymphomas in both Eμ-myc/Pml+/− and Eμ–myc/Pml−/− mice was substantially accelerated compared with control Eμ-myc mice (P < .01). (F) Staining of tissue sections with hematoxylin and eosin revealed that Eμ-myc/Pml−/− lymphomas were highly invasive and infiltrated into the liver, but not kindey or lung. Magnification ×200. (G). HSCs derived from fetal livers of WT, p53−/−, and Pml−/− mice were retrovirally transduced with a MSCV-Myc construct coexpressing green fluorescent protein. The genetically modified stem cells were then used to reconstitute the hematopoietic system of lethally irradiated recipient (WT) animals, which were monitored for lymphoma onset by palpation, weekly blood smears, and whole body fluorescence imaging. Recipients of WT stem cells transduced with the Myc expression did not develop tumors over the observation period, whereas all of the recipients of Myc expression construct transduced p53−/− HSC developed very aggressive disease in a short time. Recipients of Myc expression construct transduced Pml−/− HSCs developed lymphoma more rapidly compared with the mice transplanted with the Myc expression construct transduced WT HSCs (P < .0003), again suggesting that PML can suppress Myc driven lymphoma development.

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