Expression and activation of caspase-3/CPP32 in CD34+ cord blood cells is linked to apoptosis after growth factor withdrawal

Objective. Caspase-3/CPP32, a member of the interleukin-l converting enzyme (ICE) family, is considered an executioner protease in mammalian cells during apoptosis, Although expression and activation of caspase-3/CPP32 protein have been studied in many tissues and leukemia cell lines, this has not been explored in primitive hematopoietic CD34(+) cells. In this study, we evaluated expression and activation of caspase-3/CPP32 protein in CD34(+) cells from cord blood (CB) during apoptosis induced by growth factor deprivation. Methods. Reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot, and now cytometry analysis were used in this study to determine the expression of caspase-3/CPP32 in CD34(+) CB cells during apoptosis, Results. Our results demonstrated that caspase-3/CPP32 mRNA was constitutively expressed at a very low level in freshly isolated CD34(+) cells. Expression of caspase-3/CPP32 mRNA and protein was upregulated when these cells were first expanded in suspension culture with growth factors for 3 days. However, only the 32 kDa inactive caspase-3/CPP32 proenzyme was detected in the freshly isolated CD34(+) cells and after 3 days expansion with cytokines, Within 12 hours after growth factor withdrawal from expanded cells caspase-3/CPP32 was activated and a cleavage 20 kDa protein was detected; a poly(ADP-ribose) polymerase (PARP) was cleaved by activated caspase-3/CPP32, Activation of caspase-3/CPP32 and apoptosis upon growth factor withdrawal were inhibited/reduced by the caspase inhibitors, z-VAD-fmk and DEVD-CHO. Conclusion. These results demonstrate that caspase-3/CPP32 is involved in apoptosis of primitive Cn CD34(+) cells but may not be the only mechanism involved. (C) 2000 International Society for Experimental Hematology, Published by Elsevier Science Inc.