title: Role of Smac in human leukaemic cell apoptosis and proliferation.
abstract: Smac (or DIABLO) is a recently identified, novel proapoptotic molecule, which is released from mitochondria into the cytosol during apoptosis. Smac functions by eliminating the caspase-inhibitory properties of the inhibitors of apoptosis proteins (IAP), particularly XIAP. In this study, we stably transfected both full-length (FL) and mature (MT) Smac genes into the K562 and CEM leukaemic cell lines. Both FL and MT Smac transfectants increased the sensitivity of leukaemic cells to UV light-induced apoptosis and the activation of caspase-9 and caspase-3. Purified cytosol from the mature Smac transfectants, or the addition of human recombinant Smac protein or N-7 peptide into nontransfected cytosol, showed an increased sensitivity to cytochrome c-induced activation of caspase-3. The mature Smac enhanced the susceptibility of both K562 and CEM cells to TRAIL-induced apoptosis. Overexpression of the mature Smac protein also inhibited proliferation, as detected by reduced colony formation and Ki-67 expression in leukaemic cells. Cell cycle analysis revealed that Smac transfectants displayed significant G0/G1 arrest and reduction in 5-bromo-2'-deoxyuridine (BrdU) incorporation. Smac sensitized human acute myeloid leukaemia blasts to cytochrome c-induced activation of caspase-3. However, Smac failed to overcome Apaf-1-deficiency-mediated resistance to cytochrome c in primary leukaemic blasts. In summary, this study reveals that Smac/DIABLO exhibits a potential role in increasing apoptosis and suppressing proliferation in human leukaemic cells. Importantly, it also indicates that it is crucial to evaluate the levels of Apaf-1 and XIAP proteins in patient samples before using Smac peptide therapy in the treatment of human leukaemia.
authors.affiliation: Department of Haematology/Oncology, St Bartholomew's, The Royal School of Medicine and Dentistry, London El 2AD, UK. L.jia@mul.ac.uk
authors.forename: Li
authors.initials: L
authors.lastname: Jia
authors.forename: Yasmeen
authors.initials: Y
authors.lastname: Patwari
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Kelsey
authors.forename: Srinivasa M
authors.initials: SM
authors.lastname: Srinivasula
authors.forename: Samir G
authors.initials: SG
authors.lastname: Agrawal
authors.forename: Emad S
authors.initials: ES
authors.lastname: Alnemri
authors.forename: Adrian C
authors.initials: AC
authors.lastname: Newland
mesh_terms: D017209:Apoptosis / Q000502:physiology* / Q000528:radiation effects; D051017:Apoptosis Regulatory Proteins; D053477:Apoptotic Protease-Activating Factor 1; D002352:Carrier Proteins / Q000235:genetics / Q000502:physiology*; D020169:Caspases / Q000378:metabolism; D002455:Cell Division / Q000502:physiology*; D003574:Cytochrome c Group / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007938:Leukemia / Q000201:enzymology / Q000473:pathology*; D024101:Mitochondrial Proteins / Q000235:genetics / Q000502:physiology*; D011506:Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured; D051636:X-Linked Inhibitor of Apoptosis Protein
chemical_list: C506592:APAF1 protein, human; D051017:Apoptosis Regulatory Proteins; D053477:Apoptotic Protease-Activating Factor 1; D002352:Carrier Proteins; D003574:Cytochrome c Group; C412403:DIABLO protein, human; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins; D011506:Proteins; D011994:Recombinant Proteins; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; D020169:Caspases