Negative effects of CD45 expression on t(8;21) leukemia cells and AE9a leukemogenesis. (A) Serial replating of fetal liver cells transduced with AE9a or AE9a/CD45. The y-axis represents the average number of colonies with SD of duplicates per 103 (plating number 1) or 104 (plating numbers 2 to 5) cells plated. Cells transduced with AE9a/CD45 had no colonies at the fourth and fifth platings. Similar results were obtained from 2 independent experiments with bone marrow Lin− cells. (B) Typical third-plating colony morphology of fetal liver cells transduced with the indicated retroviruses. Images were taken at room temperature using Nikon Eclipse TS100 microscope with the 2×/0.06 objective lens, Nikon DS-Fi1 digital camera and Nikon DS Camera Control Unit DS-U2 system (Nikon). (C) Wright-Giemsa staining of fetal liver cells transduced with the indicated retroviruses from the third plating. The AE9a picture is a montage of cells because of the low cell density on the cytospin slide. Images were taken at room temperature using Olympus BX51 microscope with the 20×/0.5 objective lens, the DP71 digital camera and the DP-BSW acquisition software (Olympus). (D) Kasumi-1 cells transduced with murine CD45 or the MIP control vector were labeled with CFSE and cultured for 42 hours. The acetate groups of CFSE are cleaved by intracellular esterases to yield the highly fluorescent carboxyfluorescein succinimidyl ester. The intensity of CFSE fluorescence decreases as cells undergo cell divisions. Cells were fixed in 0.2% paraformaldehyde immediately after labeling (red histograms) or after 42 hours of culture (green histograms) for flow cytometry. A clear reduction of CFSE fluorescence intensity was observed in the MIP transduced cells after 42 hours of culture, indicating that the cells had undergone cell divisions (left panel). However, the CFSE fluorescence intensity in the CD45 transduced Kasumi-1 cells remained the same after 42 hours of culture (right panel). (E) Annexin V staining of CD45 or MIP transduced Kasumi-1 cells after 42 hours of culture. The majority of CD45 transduced cells are positive for annexin V. (F) Survival curves showing the lifespan of MF-1 mice transplanted with AE9a (n = 9) or AE9a/CD45.1 (n = 9) transduced fetal liver cells. The P value was calculated using log-rank test. (G) The intensity of CD45.1 in leukemia cells from blood of 1 representative AE9a (antibody background staining; blue histogram) and 1 representative AE9a/CD45.1 (green histogram) leukemia mice at 12 weeks after transplantation compared with that in AE9a/CD45.1 transduced fetal liver cells right after sorting (red histogram).
