Figure 4.

Development of leukemia in mouse no. 681 that received a transplant of TRKB- and BDNF-modified hematopoietic cells (32D). FACS analysis showing coexpression of TRKB and BDNF before transplantation (A) and in myeloid blasts recovered from the animal (B). (C) Peripheral blood smear showing marked leukocytosis consisting predominantly of immature myeloid cells. (D) Cytospin of BM showing myeloblasts with an abundant cytoplasm (×1000). (E,F) Diffuse myeloblastic infiltration in spleen and complete effacement of its normal structure (×100, ×1000). (G,H) Extensive infiltration of leukemic cells in liver, primarily in portal areas, but also diffusely in the sinusoid (×100, ×1000). (I) Constitutive activation of TRKB in leukemic cells and blocking of the TRKB/BDNF autocrine loop by anti–BDNF-neutralizingantibody and K252a. Leukemic cells were treated with either 20 μg/mL anti-BDNF antibody or 100 nM K252a for 12 hours. Total cell lysates (300 μg) from untreated and treated cells were immunoprecipitated with the anti-TRK (C-14), separated on SDS–polyacrylamide gel electrophoresis (PAGE), blotted, and probed with an anti-pTRK (E-6) antibody (top and middle panels with short and long exposition, respectively). The blot was stripped and reprobed with the anti-TRKB (H181) antibody. Arrows point to the phosphorylated forms of TRK. Note different expression pattern of human TRKB in human cells (only 145-kDa form in Figure 1) and rodent cells (both 145-kDa and 120-kDA forms) as previously described.⁵¹ (J) Antihuman BDNF antibody (10 μg/mL and 20 μg/mL) inhibited growth of leukemic cells in colony-forming assays. Results are presented as the average percentage of colonies formed in the presence of antibody (100% value derived from untreated control). Results presented are the mean plus or minus SD of 2 independent experiments.