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Expression and genetic analysis of XIAP-associated factor 1 (XAF1) in cancer cell lines
W G Fong1, P Liston, E Rajcan-Separovic
1Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, Ontario, K1H 8M5, Canada.
Abstract:
X-linked inhibitor of apoptosis protein (XIAP) is a potent modulator of programmed cell death. XIAP specifically binds and inhibits the function of caspase-3, -7, and -9, key effector proteases of apoptosis. We recently isolated, by yeast two-hybrid screening, a novel 34-kDa zinc finger protein, XIAP-associated factor 1 (XAF1). Both the caspase inhibiting and the anti-apoptotic abilities of XIAP were found to be blocked by overexpressed XAF1. Here, we report the isolation and characterization of the human XAF1 gene. The xaf1 gene consists of seven exons spanning 18 kb. Fluorescence in situ hybridization analysis localized the xaf1 locus at 17p13.2, telomeric to the p53 gene. The xaf1 locus was further refined to YAC 746C10, approximately 3 cM distal to TP53. Microsatellite analysis of the xaf1 locus using the NCI 60 cell line panel revealed significantly decreased heterozygosity at all three polymorphic markers tested, suggesting that allelic loss of the xaf1 gene is prevalent in cancer cell lines. Examination of the same NCI cell line panel for xaf1 RNA expression demonstrated that cancer cell lines exhibited very low levels of mRNA relative to normal human liver. In contrast, XIAP mRNA levels were relatively high in the majority of cancer cell lines tested. We propose that a high level of XIAP to XAF1 expression in cancer cells may provide a survival advantage through the relative increase of XIAP anti-apoptotic function.
Insights
XIAP-associated factor 1 (XAF1) antagonizes XIAP's anti-apoptotic function. Loss of XAF1 and high XIAP expression in cancer may promote cell survival by increasing XIAP's inhibitory effects on apoptosis.
Area of Science:
- Molecular Biology
- Genetics
- Cancer Biology
Background:
- X-linked inhibitor of apoptosis protein (XIAP) is a key regulator of programmed cell death, inhibiting caspases-3, -7, and -9.
- XIAP-associated factor 1 (XAF1) was identified as a novel protein that antagonizes XIAP's function.
Purpose of the Study:
- To isolate and characterize the human XAF1 gene.
- To investigate the role of XAF1 in cancer by analyzing its gene locus, allelic loss, and RNA expression in cancer cell lines.
Main Methods:
- Yeast two-hybrid screening to identify XAF1.
- Fluorescence in situ hybridization (FISH) and YAC mapping to localize the xaf1 gene.
- Microsatellite analysis and RNA expression profiling of NCI 60 cancer cell lines.
Main Results:
- The human xaf1 gene spans seven exons and is located at chromosome 17p13.2.
- Allelic loss of the xaf1 gene is prevalent in cancer cell lines.
- Cancer cell lines show significantly lower XAF1 mRNA levels compared to normal liver, while XIAP mRNA levels are high.
Conclusions:
- The xaf1 gene is frequently altered in cancer, exhibiting allelic loss and reduced expression.
- A high XIAP to XAF1 expression ratio in cancer cells may confer a survival advantage by enhancing XIAP's anti-apoptotic activity.