CD44 ligation increases the p27 content in primary AML blasts. (A) Effect of CD44 ligation on p27 level in noncultured AML blasts. Enriched populations of AML blasts from 3 distinct AML patients were thawed, incubated for 4 hours at 37°C, treated with A3D8 or IgG1 (controls), and processed for Western blotting analysis of p27 as indicated in “Materials and methods.” These relative protein level values are shown in italics below the lanes. Results represent 1 representative experiment of 2. (B) Effect of CD44 ligation on growth of proliferating AML blasts. Enriched populations of AML blasts from 4 distinct AML patients were thawed, incubated for 4 hours at 37°C, and cultured for 1 week in the presence of thrombopoietin, FLT3-ligand, and stem cell factor to trigger their proliferation, as detailed in “Materials and methods.” Then, exponentially growing primary leukemic cells were seeded in 96-well culture plates in complete culture medium supplemented with the anti-CD44 mAb A3D8 (2.5 μg/mL) or IgG1 (controls) and cultured for 4 to 6 days at 37°C. Cell counts and viability were evaluated in triplicate by the trypan blue exclusion test during 4 days. Values are means ± SD calculated from 3 independent experiments. Data obtained with A3D8-treated cells are significantly different from those of controls (P < .05, Mann Whitney test). (C) Effect of CD44 ligation on p27 level of proliferating AML blasts. p27 and α-tubulin expression were analyzed by Western blotting with specific antibodies 0 hours and 24 hours after addition of A3D8 to proliferating AML blasts (as described in the legend to panel B). Results represent 1 representative experiment of 3.