Rapamycin induces apoptosis of primary malignant lymphoblasts. (A) Analysis of mitochondrial membrane potential (i), phosphatidyl-serine externalization (ii), and caspase 3 catalytic activity (iii) of cALL blasts cultured with rapamycin (50 ng/mL). After 6 hours of incubation, mitochondrial potential was analyzed by calculating the amount of JC-1 monomers by flow cytometry. Annexin V binding and caspase 3 activity were measured by flow cytometry and fluorometric assay, respectively, after 24 hours of incubation. The percentages on the bars indicate the amount of annexin V-positive cells. (B) Dose/response curve to rapamycin. Malignant lymphocytes from 5 different samples and peripheral B or T lymphocytes from a healthy donor were incubated with rapamycin at different concentrations. After 24 hours, the cells were harvested and apoptosis was evaluated by annexin V staining and flow cytometry. For B and T lymphocyte analysis, the whole PBMCs were acquired in flow cytometry, after which B or T cells were gated on the basis of CD3/SSc or CD20/SSc parameters, and the percentage of annexin V⁺ cells was calculated. Pt. indicates patient number. (C) Mean values ± standard deviation (SD) of rapamycin-induced apoptosis in responder samples. Apoptosis was evaluated by annexin V staining of ALL blasts, from the indicated patients, after 24 hours of incubation with or without rapamycin (100 ng/mL).