Figure 3

In vitro sensitivity of MLL-AF9 AML cell lines to cytotoxic agents. Drug sensitivity tests were performed using short-term cell lines established from bone marrow of sick primary MLL-AF9 AML mice (Materials and methods section). (a) WT/MLL-AF9 AMLs (n=4) were treated with standard chemotherapeutic drugs cytarabine (300 ng/ml), etoposide (300 ng/ml), daunorubicin (50 ng/ml), bortezomib (5 nM) or BH3 mimetic ABT-737 (1 μg/ml), harvested at 4, 8, 16 and 24 h and analyzed by flow cytometry following staining with annexin V-Alexa Fluor 647 and propidium iodide (dying and dead cells are positive for annexin V or both markers). Results are expressed as percentage of dead and dying cells relative to that of cells cultured in parallel in medium alone. Value shown is mean±S.E.M. (b–f) AMLs of the indicated genotypes were cultured for 16 h in the presence of ABT-737 alone (b); or with cytarabine (c), etoposide (d), daunorubicin (e) or the proteasome inhibitor bortezomib (f), each alone (gray) or in combination with ABT-737 (black). The percentage of dead and dying cells is expressed relative to cells of the same genotype incubated in the absence of drug(s). Values represent mean±S.E.M. Data plotted for ABT-737 alone were pooled from all experiments (n=8 independent lines for WT and n=2–6 independent lines for other genotypes). The number of independent lines used for other drugs±ABT-737 were: WT n=5; bim^−/− n=3, except with bortezomib where n=1; puma^−/− n=3; noxa^−/− n=3; bmf^−/− n=4; puma^−/−noxa^−/− n=2; puma^−/−bim^−/− n=2; noxa^−/−bmf^−/− n=3. P-values were calculated using an unpaired t test with Welch’s correction. *P<0.05 between WT AMLs and the indicated BH3-only gene KO AMLs in response to a particular drug or combination; ^#P<0.05 for drug in combination with ABT-737 versus just the single drug alone for the same genotype