Reversible Tumorigenesis by MYC in Hematopoietic Lineages Dean W. Felsher, J.Michael Bishop  Molecular Cell  Volume 4, Issue 2, Pages 199-207 (August 1999) DOI: 10.1016/S1097-2765(00)80367-6

Figure 1 A Conditional Transgenic Model for MYC-Induced Tumorigenesis (A) Tetracycline regulatory system constructs. (B) Mice transgenic for both EμSR-tTA and tet-o-MYC conditionally express MYC protein in splenic lymphocytes. Western analysis was performed on spleen lymphocytes from transgenic mice in the absence (−) or the presence (+) of doxycycline in drinking water. Similar results were seen in four independent experiments. (C) Survival curves for transgenic mice. Open squares, mice transgenic for EμSR-tTA and tet-o-MYC; closed squares, mice transgenic for EμSR-tTA and tet-o-MYC with doxycycline treatment or either EμSR-tTA or tet-o-MYC alone. There were at least 20 animals per group. (D) Deceased animals had an enlarged thymus, spleen, liver, and mesenteric lymph nodes. Histology demonstrated invasion of (E) spleen (40×), (F) blood (100×), (G) kidney (40×), and (H) liver (20×). Molecular Cell 1999 4, 199-207DOI: (10.1016/S1097-2765(00)80367-6)

Figure 3 Inactivation of the MYC Transgene Resulted in Tumor Regression (A) Survival of transgenic mice with tumors that were then treated (closed squares, n = 20) or not treated (open squares, n = 5) with doxycycline. (B) Gallium imaging of control mouse (Control), a mouse moribund from tumor prior to doxycycline treatment (Pre-Rx), or 2 weeks after doxycycline treatment (Doxycycline). Identical results were seen in eight other mice. (C) Survival of transplant recipients of transgenic tumors treated with doxycycline when moribund with tumor burden. Open squares portray results with tumors transplanted directly into syngeneic hosts (tumors 966, 967, 1232, 1137, 1979, and 2263) or after in vitro passage for 2 months (tumors 1137 and 1232). Open circles portray results of tumor 966 after in vitro passage for 2 months. In all cases, transplanted tumors were inoculated into at least 12 syngeneic mice intraperitoneally, 107 cells per mouse. (D) MYC expression in relapsed tumors. Western analysis in the absence (−) or presence (+) of doxycycline treatment of tumor 966 and two doxycycline-resistant variants (R1 and R2). Molecular Cell 1999 4, 199-207DOI: (10.1016/S1097-2765(00)80367-6)

Figure 5 Kinetics of Tumor Regression Tumor 966 was transplanted into syngeneic mice, and when moribund, the mice were treated with doxycycline. Animals were sacrificed before treatment, or either 3 or 6 days after instituting treatment with doxycycline. (A–C) Histology of the spleen after 0, 3, or 6 days of doxycycline treatment. Histology of the gastrointestine 0 or 3 days after doxycycline treatment ([D] versus [E], 20×; [H] versus [I], 100×). (F and G) Tunel assay of a gastointestinal tumor, either 0 or 3 days after doxycycline treatment. (J and K) DAPI staining of a gastrointestinal tumor 0 and 3 days after doxycycline treatment. Three animals were analyzed per time point. Identical results were seen with tumor 967. Molecular Cell 1999 4, 199-207DOI: (10.1016/S1097-2765(00)80367-6)

Figure 2 Clonality of Tumors (A) Tumors exhibited clonal T cell receptor β chain rearrangements. Southern blot of HindIII-digested genomic DNA hybridized with probes to Cβ1 or Jβ2, as shown in the figure. Arrows at left indicate the position of germline fragments. Asterisks indicate rearranged fragments. (B) Tumor cells were surface positive for a single T cell receptor, Vβ. Tumors were analyzed by FACS analysis for the surface expression of T cell receptor Vβ2, 3, 4, 5.1 and 5.2, 6, 7, 8.1 and 8.2, 8.3, 9, 10b, 11, 12, 13, 14, and 17a (Pharmingen, TCR screening panel). Tumors were surface positive only for the indicated Vβ. Molecular Cell 1999 4, 199-207DOI: (10.1016/S1097-2765(00)80367-6)

Figure 4 Inactivation of the MYC Transgene Resulted in Sustained Tumor Regression in Transplant Recipients Tumor 966 was transplanted into syngeneic mice, and when moribund, the mice were treated with doxycycline for 2 weeks. (A) Prior to doxycycline treatment, mice exhibited enlargement of thymus, spleen, liver, and a gastrointestinal mass (see labels with arrows). (B) Doxycycline treatment resulted in the apparent absence of tumor in thymus, liver, spleen, and gastrointestine (see labeled arrows). Doxycycline treatment resulted in the histologic regression of tumors in liver ([C] versus [D], 20×) and kidney ([E] versus [F], 20×). Identical results were seen in five other mice. The same results were seen in syngeneic mice transplanted with tumor 967, 1137, 1232, 1979, or 2263. Molecular Cell 1999 4, 199-207DOI: (10.1016/S1097-2765(00)80367-6)

Figure 6 Inactivation of MYC Resulted in Differentiation (A) Relative cell number in the absence (open squares) or presence (closed squares) of doxycycline (tumor 966). Cell lines derived from tumor were grown in vitro in the presence or absence of doxycylcine and counted in triplicate every 2 days in the presence of trypan blue to determine the total number of viable cells. Data are expressed as the ratio between the mean number of viable cells and the total number of cells prior to instituting treatment with doxycycline. Identical results were seen with tumors 967, 1137, and 1232. (B) Cell cycle arrest with doxycycline treatment (tumor 966). FACS and PI staining are shown in the bottom two panels. BRDU incorporation and PI staining are shown in the top two panels. Identical results were seen with tumors 967, 1137, and 1232. (C) Differentiation of tumors after doxycycline treatment. Giemsa staining of cytospun preparations of tumor cells treated in vitro with doxycycline for 2 days is shown. T cell lymphoma (T cell) was tumor 967. Acute myeloid leukemia (AML) was tumor 1137. Identical results were seen with T cell lymphomas 966 and 1232, and acute myeloid leukemia 785. Molecular Cell 1999 4, 199-207DOI: (10.1016/S1097-2765(00)80367-6)