Effect of signal transduction inhibitors on the thrombin-induced apoptosis.
The human tumor cells (U937) were incubated in triplicate in the wells of a 96-well microculture plate (1 × 104 cells/well) with or without the presence of thrombin (0.5 U/mL), and/or the indicated inhibitors for 16 hours. The microcultures were pulsed with 0.037 MBq (1 μCi) of 3H-thymidine for 8 hours, and the3H-thymidine uptake was determined using liquid scintillation counting. Each bar represents average CPM ± SE from triplicate microcultures. The star on the bar indicates that the inhibitor was able to significantly (P ≤ .05; Studentt test) block the thrombin-induced inhibition of proliferation/apoptosis. The letters A-I on the x-axis indicate the inhibitors used in the microcultures: (A) no thrombin; (B) 0.5 U/mL thrombin; (C) 0.5 μmol/L herbimycin A (an inhibitor of phospholipase D, tyrosine kinases, and anti-CD3 antibody mediated apoptosis); (D) 100 μmol/L genistein (a broad spectrum inhibitor of protein tyrosine kinases); (E) 10 μmol/L H-7 (a potent inhibitor of PKC); (F) 100 μmol/L HA-1004 (an inhibitor of CaM kinase II, myosin light chain kinase, and PKA); (G) 1 μmol/L wortmanin (an inhibitor of PI3-kinase, and phospholipase D); (H) 0.5 μg/mL cytochalasin D (an inhibitor of function of the cytoskeletal protein actin); and (I) 0.3 μM DEVD-CHO (a cell permeable inhibitor of caspases 3, 6, 7, 8, and 10). Microcultures C-I were incubated in the presence of thrombin (0.5 U/mL). All these inhibitors were from Calbiochem (San Diego, CA).
