Expression and activation of caspase-3/CPP32 in CD34(+) cord blood cells is linked to apoptosis after growth factor withdrawal.
Caspase-3/CPP32, a member of the interleukin-1 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. Reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot, and flow cytometry analysis were used in this study to determine the expression of caspase-3/CPP32 in CD34(+) CB cells during apoptosis. 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. These results demonstrate that caspase-3/CPP32 is involved in apoptosis of primitive CB CD34(+) cells but may not be the only mechanism involved.
Wang
L S
LS
Department of Microbiology/Immunology, Indiana University School of Medicine, Indianapolis, IN 46202-5254, USA.
Liu
H J
HJ
Xia
Z B
ZB
Broxmeyer
H E
HE
Lu
L
L
D018952:Antigens, CD34 / Q000032:analysis*; D017209:Apoptosis; D015153:Blotting, Western; D053148:Caspase 3; D020169:Caspases / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D016207:Cytokines / Q000008:administration & dosage*; D004789:Enzyme Activation; D005312:Fetal Blood / Q000166:cytology*; D005434:Flow Cytometry; D015870:Gene Expression; D006412:Hematopoietic Stem Cells / Q000201:enzymology; D006801:Humans; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018952:Antigens, CD34; D016207:Cytokines; D012333:RNA, Messenger; D011065:Poly(ADP-ribose) Polymerases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases