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Apoptosis is rapidly triggered by antisense depletion of MCL-1 in differentiating U937 cells
D A Moulding1, R V Giles, D G Spiller
1School of Biological Sciences and the Department of Haematology, University of Liverpool, Liverpool, England. dale@liv.ac.uk
Abstract:
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)
Insights
Mcl-1 protein induction is essential to prevent apoptosis during U937 cell differentiation. Constitutive Bcl-2 expression cannot substitute for Mcl-1 to block cell death.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Mcl-1, a Bcl-2 family protein, delays apoptosis but its role in endogenous expression is unclear.
- Gene knockout studies for Mcl-1 are lacking, limiting evidence for its necessity in apoptosis.
- U937 cells express Bcl-2 and transiently induce Mcl-1 during phorbol ester (PMA) differentiation.
Purpose of the Study:
- To investigate the functional role of Mcl-1 during U937 cell differentiation.
- To determine if Mcl-1 induction is required to prevent apoptosis in this context.
Main Methods:
- Specific depletion of Mcl-1 using antisense oligodeoxynucleotides.
- Analysis of Mcl-1 mRNA and protein levels.
- Assessment of apoptosis using live cell imaging and other parameters.
- Monitoring expression of Bcl-2 family members (Bax, Bak) and TNF-alpha.
Main Results:
- Antisense-mediated Mcl-1 depletion reduced Mcl-1 to 5% of control levels.
- Mcl-1 depletion rapidly induced apoptosis in U937 cells.
- Bcl-2 expression did not prevent apoptosis upon Mcl-1 loss.
- Bak levels doubled, while Bax remained unchanged during differentiation.
Conclusions:
- Mcl-1 induction is necessary to prevent apoptosis during U937 cell differentiation.
- Constitutively expressed Bcl-2 cannot compensate for the loss of Mcl-1.
- Mcl-1 plays a critical role in regulating cell survival during differentiation.