Apoptosis is rapidly triggered by antisense depletion of MCL-1 in differentiating U937 cells.
Mcl-1 is a member of the Bcl-2 protein family, which has been shown to delay apoptosis in transfection and/or overexpression experiments. As yet no gene knockout mice have been engineered, and so there is little evidence to show that loss of Mcl-1 expression is sufficient to trigger apoptosis. U937 cells constitutively express the antiapoptotic protein Bcl-2; but during differentiation, in response to the phorbol ester PMA (phorbol 12 beta-myristate 13 alpha-acetate), Mcl-1 is transiently induced. The purpose of this investigation was to determine the functional role played by Mcl-1 in this differentiation program. Mcl-1 expression was specifically disrupted by chimeric methylphosphonate/phosphodiester antisense oligodeoxynucleotides to just 5% of control levels. The depletion of Mcl-1 messenger RNA (mRNA) and protein was both rapid and specific, as indicated by the use of control oligodeoxynucleotides and analysis of the expression of other BCL2 family members and PMA-induced tumor necrosis factor-alpha (TNF-alpha). Specific depletion of Mcl-1 mRNA and protein, in the absence of changes in cellular levels of Bcl-2, results in a rapid entry into apoptosis. Levels of the proapoptotic protein Bax remained unchanged during differentiation, while Bak expression doubled within 24 hours. Apoptosis was detected within 4 hours of Mcl-1 antisense treatment by a variety of parameters including a novel live cell imaging technique allowing correlation of antisense treatment and apoptosis in individual cells. The induction of Mcl-1 is required to prevent apoptosis during differentiation of U937 cells, and the constitutive expression of Bcl-2 is unable to compensate for the loss of Mcl-1. (Blood. 2000;96:1756-1763)
Moulding
D A
DA
School of Biological Sciences and the Department of Haematology, University of Liverpool, Liverpool, England. dale@liv.ac.uk
Giles
R V
RV
Spiller
D G
DG
White
M R
MR
Tidd
D M
DM
Edwards
S W
SW
D017209:Apoptosis / Q000235:genetics*; D001483:Base Sequence; D015153:Blotting, Western; D002454:Cell Differentiation / Q000187:drug effects* / Q000235:genetics; D016373:DNA, Antisense / Q000737:chemistry / Q000494:pharmacology*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D018613:Microscopy, Confocal; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D009363:Neoplasm Proteins / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000737:chemistry; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000187:drug effects / Q000235:genetics; D020298:U937 Cells
D016373:DNA, Antisense; C579094:Mcl1 protein, mouse; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D009363:Neoplasm Proteins; D009943:Organophosphorus Compounds; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; C032627:methylphosphonic acid; D013755:Tetradecanoylphorbol Acetate