Predefining the second hit by crossing Eμ-myc/vav-bcl-2 and Eμ-myc/p53^+/− mice confirms a correlation between disease latency and second hit. (A) Representative immunoblots of splenocytes from Eμ-myc/vav-bcl-2 and Eμ-myc/p53^+/− mice (n = 2 each). The addition of etoposide is used as functional test of the DNA-damage pathway mediated by p53. After addition of etoposide, splenocytes from Eμ-myc/p53^+/− mice did not show enhanced p21 levels, indicating genetic alterations leading to loss of functional p53. Further, we detected increased levels of p53 in splenocytes from Eμ-myc/vav-bcl-2 mice after etoposide treatment, whereas no p53 could be detected in splenocytes derived from Eμ-myc/p53^+/− mice. (B) Representative immunoblots of splenocytes from Eμ-myc/p53^+/− and Eμ-myc/vav-bcl-2 mice (n = 4 and n = 3, respectively). As expected splenocytes from Eμ-myc/vav-bcl-2 showed higher expression levels of BCL-2. Splenocytes from Eμ-myc/vav-bcl-2 mice displayed regular levels of p53, whereas splenocytes derived from Eμ-myc/p53^+/− mice did not show any p53 protein. (C) Transplantation of splenocytes from Eμ-myc/vav-bcl-2 or Eμ-myc/p53^+/− mice into C57BL/6 recipients. Mice receiving cells from Eμ-myc/vav-bcl-2 showed massively enhanced survival (Eμ-myc/53^+/−, n = 16; Eμ-myc/vav-bcl-2 n = 19; P_{Eμ-myc/p53+/− vs Eμ-myc/vav-bcl-2} < .001). (D) Survival analysis for serially transplanted Eμ-myc/p53^+/− and Eμ-myc/vav-bcl-2 splenocytes. Cells from diseased primary mice that received a transplant were used for 1 subsequent transplantation. Serial transplantation did not result in altered survival time of the mice. (Eμ-myc/p53^+/− prim, n = 10; Eμ-myc/vav-bcl-2 prim, n = 10; Eμ-myc/p53^+/− sec, n = 8; Eμ-myc/vav-bcl-2 sec, n = 9; P_{Eμ-myc/p53+/− prim vs Eμ-myc/p53+/−sec} > .05; P_{Eμ-myc/vav-bcl-2 prim vs Eμ-myc/vav-bcl-2 sec} > .05.) Prim indicates primary transplantation; and sec, secondary transplantation.