ESAs did not demonstrate cytoprotective activities in nonhematopoietic cells. (A) A representative experiment in rat neonatal cardiac myocytes evaluating caspase-3/caspase-7 activity after treatment with increasing doses of rHuEpo and DA, positive control caspase inhibitor Z-VAD-FMK, and negative control inactive I-rHuEpo. Cells were pretreated for 2 hours with test article, and then apoptosis was induced with 150nM staurosporine in the presence of test article for 22 hours. Data are mean ± SEM and shown with Student t test for significance (n = 12 per group) with caspase-3/caspase-7 activity measured as relative fluorescence units (RFU) and relative to the vehicle control at 0. There was significant reduction of caspase-3/caspase-7 activity at all doses of Z-VAD-FMK (P < .001) but no significant reduction with any other treatment. (B) A representative experiment using primary human RPTECs, which were pretreated with DA at concentrations from 1.5 to 150 ng/mL or with media alone for 24 hours before the addition of cisplatin at a final concentration of 20μM. Control groups included pretreatment with DA without cisplatin treatment (white bars). Apoptosis was measured after 48 hours of cisplatin treatment using a caspase-3/caspase-7 assay. Data are mean ± SEM (n = 6 per group). Note the inability of DA at any concentration to inhibit cisplatin-induced caspase-3/caspase-7 activity. (C) Human neuronal cell line SH-SY5Y was pretreated with 200 U/mL rHuEpo for 0, 2, or 24 hours before induction of hypoxia (1.4%-1.6% O₂ for 24 hours). Some treatment groups were also starved of glucose for 24 hours during hypoxia and compared with normoxia. Cell viability was determined with a Cell Titer Glo luminescent assay (relative light units [RLU]) with mean ± SEM shown (n = 6 per group). Student t test was used to determine statistical significance. No significant increase in cell viability was observed with rHuEpo treatment.