Figure 7.

Anti‐CD13 mAb MY7‐induced apoptosis is caspase dependent. A) U937 cells were treated for 24 h with IgG1 or MY7 (35 μg/ml) or etoposide (1 μM) or left untreated. Caspase‐3, caspase‐8, and caspase‐9 activities were determined using the substrates DEVD‐pNA, IETD‐pNA, and LEHD‐pNA, respectively. Release of pNA was measured at 405 nm. Data are expressed as a fold‐increase relative to the corresponding untreated samples (baseline values for caspase‐3, caspase‐8, and caspase‐9 activity were 1.3±0.2, 2.3±0.2, and 0.9±0.1 nmol pNA/60 min/100 μg protein at 37°C, respectively). Data are expressed as means ± sd from 3 assays. IgG1 did not alter the caspase activity profile. Inset: U937 cell lysates were examined for PARP‐1 expression by immunoblotting. B) U937 cells were incubated with IgG1 or MY7 (35 μg/ml) for 48 h after 1 h of pretreatment with Z‐VAD‐fmk (a broad‐spectrum caspase inhibitor), Ac‐LEHD‐CHO (a caspase‐9 inhibitor) or Z‐IETD‐fmk (a caspase‐8 inhibitor) (50 μM). Cell death was determined as described in Materials and Methods. Percentage of specific MY7‐mediated cell death was obtained by subtracting the percentage of baseline death in untreated cells or cells pretreated with caspase inhibitor from the percentage of death in the corresponding MY7‐treated cells. IgG1 did not alter the baseline levels of apoptosis. Values are expressed as means ± sd. Separate experiments were performed in duplicate and were repeated 3 times. ∗P < 0.05 vs. corresponding untreated cells.