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Apaf-1XL is an inactive isoform compared with Apaf-1L
W N Fu1, S M Kelsey, A C Newland
1Department of Haematology/Oncology, St. Bartholomew's and Royal London School of Medicine and Dentistry, Turner Street, London, E1 2AD, United Kingdom.
Biochemical and Biophysical Research Communications
|March 27, 2001
Summary
A novel Apaf-1XL variant in K562 leukemia cells reduces caspase-9 binding, contributing to resistance against cytochrome c-induced apoptosis. This finding highlights a potential mechanism in apoptosis regulation.
Area of Science:
- Molecular Biology
- Cell Biology
- Apoptosis Research
Background:
- Apoptosis activating factor 1 (Apaf-1) is essential for initiating programmed cell death via the intrinsic pathway.
- Cytochrome c and dATP trigger Apaf-1 oligomerization and subsequent activation of caspase-9 and caspase-3.
- Differential sensitivity to apoptosis exists across various cell types, suggesting underlying molecular mechanisms.
Purpose of the Study:
- To investigate the molecular basis for differential resistance to cytochrome c-induced apoptosis in human leukemia cell lines.
- To identify potential alterations in Apaf-1 function contributing to apoptosis resistance.
- To elucidate the role of Apaf-1 variants in the regulation of caspase activation.
Main Methods:
- Comparative analysis of cytochrome c-induced caspase-9 and -3 activation in K562 and CEM/VLB(100) cell-free systems.
- Apaf-1 cDNA sequencing to identify genetic variations between resistant and sensitive cell lines.
- Immunoprecipitation assays to assess the binding affinity of Apaf-1 variants to caspase-9.
Main Results:
- K562 cells exhibited increased resistance to cytochrome c-induced caspase activation compared to CEM/VLB(100) cells.
- Apaf-1 cDNA sequencing revealed an 11 amino acid insert (Apaf-1XL) in K562 cells, located between the CARD and CED-4 domains.
- Apaf-1XL demonstrated reduced binding capacity to caspase-9 in cell-free reactions compared to the standard Apaf-1L protein.
Conclusions:
- The Apaf-1XL isoform, characterized by an insert between the CARD and CED-4 domains, contributes to apoptosis resistance in K562 cells.
- Reduced binding of Apaf-1XL to caspase-9 impairs the formation of the apoptosome complex, thereby inhibiting caspase activation.
- This study suggests that alternative splicing of Apaf-1 can modulate apoptosis sensitivity and provides insights into leukemia cell resistance mechanisms.